Chemical synthesis of 23 kDa glycoprotein by repetitive segment condensation: A synthesis of MUC2 basal motif carrying multiple O-GalNAc moieties

Article abstract of DOI:10.1021/ja053711b

Peptide thioester corresponding to a MUC2 tandem repeat unit, which retains seven GalNAc moieties, was prepared by the Fmoc method followed by the low TfOH treatment to remove benzyl groups at the carbohydrate portions. The glycosylated peptide thioester was then consecutively joined by the activation of a thioester group by silver ions to obtain a MUC2 tandem repeat model composed of 141 amino acids with 42 GalNAc moieties.

Full text of DOI:10.1021/ja053711b

Published on Web 09/08/2005
Chemical Synthesis of 23 kDa Glycoprotein by Repetitive
Segment Condensation: A Synthesis of MUC2 Basal Motif
Carrying Multiple O-GalNAc Moieties
Hironobu Hojo,*,† Yoshiyuki Matsumoto,† Yuko Nakahara,† Emi Ito,‡
Yusuke Suzuki,‡ Minoru Suzuki,‡ Akemi Suzuki,‡ and Yoshiaki Nakahara*,†
Contribution from the Department of Applied Biochemistry, Institute of Glycotechnology,
Tokai UniVersity, Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan, and Sphingolipid
Expression Laboratory, RIKEN Frontier Research System, Wako 351-0198, Japan
Received June 6, 2005; E-mail: hojo@keyaki.cc.u-tokai.ac.jp
Abstract: Peptide thioester corresponding to a MUC2 tandem repeat unit, which retains seven GalNAc
moieties, was prepared by the Fmoc method followed by the low TfOH treatment to remove benzyl groups
at the carbohydrate portions. The glycosylated peptide thioester was then consecutively joined by the
activation of a thioester group by silver ions to obtain a MUC2 tandem repeat model composed of 141
amino acids with 42 GalNAc moieties.
Introduction
It is known that in the course of malignant transformation, the
synthesis of O-glycan becomes incomplete and short carbohy-
The carbohydrate on proteins, which is usually attached to
the peptide backbone via N- or O-linkage, has been shown to
play essential roles in many biological processes, such as protein
folding, cell-cell interaction, and tumor metastasis.1 However,
the details of these functions are not yet known, which mainly
derive from the microheterogeneity of the carbohydrate portion.
In the case of N-linked carbohydrate, the glycosylation usually
occurs at the consensus sequence of Asn-X-Ser/Thr. In addition,
the structure around the reducing end region is common to all
N-linked glycans. In contrast, the site of O-glycosylation, which
is typically found in the tandem repeat region of mucins, is
solely determined by the action of glycosyl transferases, which
cannot be predicted at present. Furthermore, the mucin-type
O-glycan has a more diverse structure compared to that of the
N-glycan and usually exists in a dense clustered form. Due to
these complex features, the functional and structural analysis
of mucins remains a difficult task.
drates, such as Tn (R-GalNAc), T (Gal-GalNAc), and sialyl-
Tn antigen, are highly expressed on mucins. Synthetic models
have been used to characterize the epitope structure of these
tumor-associated mucins13,14 and to develop efficient cancer
vaccines.15,16 Synthetic models have also been used as substrates
to analyze the specificity of various glycosyl transferases as
well as for structural characterization of mucins.17-24 Most of
these studies use glycopeptide models composed of a single
repeat sequence or a sequence within a repeat unit to eliminate
difficulties associated with the repeating character of mucins.
It has been made clear that the binding of monoclonal antibodies
(12) The synthesis of glycoprotein containing a mucin-like tandem repeat domain
using expressed protein ligation is also reported: Macmillan, D.; Bertozzi,
C. R. Angew. Chem., Int. Ed. 2004, 43, 1355-1359.
(13) Live, D. H.; Williams, L. J.; Kuduk, S. D.; Schwarz, J. B.; Glunz, P. W.;
Chen, X.-T.; Sames, D.; Kumar, R. A.; Danishefsky, S. J. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 3489-3493.
(14) Moller, H.; Serttas, N.; Paulsen, H.; Burchell, J. M.; Taylor-Papadimitriou,
J.; Meyer, B. Eur. J. Biochem. 2002, 269, 1444-1455.
To overcome these difficulties, various homogeneous gly-
copeptides have been synthesized as models of natural mucins.2-12
(15) Kuduk, S. D.; Schwarz, J. B.; Chen, X. T.; Glunz, P. W.; Sames, D.;
Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. J. Am. Chem. Soc.
1998, 120, 12474-12485.
(16) Ragupathi, G.; Coltart, D. M.; Willliams, L. J.; Koide, F.; Kagan, E.; Allen,
J.; Harris, C.; Glunz, P. W.; Livingston, P. O.; Danishefsky, S. J. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 13699-13704.
† Tokai University.
‡ RIKEN Frontier Research System.
(1) Dwek, R. A. Chem. ReV. 1996, 96, 683-720.
(2) Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.; Paulsen, H. Tetrahedron
Lett. 1991, 32, 5067-5070.
(17) Wu, W.-G.; Pasternack, L.; Huang, D.-H.; Koeller, K. M.; Lin, C.-C.; Seitz,
O.; Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 2409-2417.
(18) Naganagowda, G. A.; Gururaja, T. L.; Satyanarayana, J.; Levine, M. J. J.
Peptide Res. 1999, 54, 290-310.
(3) Bielfeldt, T.; Peters, S.; Meldal, M.; Bock, K.; Paulsen, H. Angew. Chem.,
Int. Ed. Engl. 1992, 31, 857-859.
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A.; Sherman, S. Biochemistry 2000, 39, 12076-12082.
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Trans. 1 1997, 2359-2368.
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Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662-2673.
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10.1021/ja053711b CCC: $30.25 © 2005 American Chemical Society

Synthesis of MUC2 Basal Motif with O-GalNAc Moieties
A R T I C L E S
Figure 1. A general scheme for the preparation of homogeneous tandem
repeat of mucins by the thioester method combined with benzyl-protection
strategy at the carbohydrate portion.
Figure 2. Synthetic route for the repeating unit of MUC2 carrying seven
GalNAcs 2. The asterisk denotes the protected GalNAc moiety.
created against tumor-associated mucins to the repeating
sequence of MUC1 becomes stronger as the number of repeating
unit increases,25,26 which means that the conformation and the
function of the tandem repeat are dependent on the length of
the peptide chain. Thus, if the repeating glycopeptide with
sufficient length can be prepared, such a model would clarify a
more accurate function and structure of mucins. In addition,
the model would be a candidate to create effective vaccines in
point of its stable conformation as well as of its sufficient size
for immunization without carrier proteins. We have been
establishing a facile method of glycoprotein synthesis based on
the thioester method,27,28 combining the benzyl-protection
strategy at the carbohydrate portion.29,30 Here, we report the
extension of this procedure for the preparation of MUC2 basal
structure carrying multiple O-GalNAc moieties at specific sites.
and thus their repeated introduction might yield much defective
peptides during the solid-phase synthesis. The sequence of the
repeating unit of MUC2 is an extreme case, as it retains five
consecutive Thr at around 10 residues from the C-terminal,
where the reactivity of the resin-bound peptide itself is decreased
because of the â-sheet formation.36 In addition, all benzyl groups
have to be completely removed from the dense clustered
carbohydrates. The second point is whether the repetition of
segment coupling proceeds efficiently. Although three consecu-
tive segment condensations by the thioester method have already
been achieved in the synthesis of many proteins,28 the reaction
here is more challenging because the repeating unit retains many
free hydroxyl groups at the carbohydrate portions, which
potentially receive O-acylation depending on reaction conditions.
If these requirements are fulfilled, this strategy will be generally
applicable for the synthesis of tandem repeated glycoproteins
and also for globular glycoproteins. Here, we demonstrate the
potential of our method by synthesizing the hexa-repeats of the
MUC2 unit carrying multiple Tn antigens at specific sites. This
appears to be the largest glycoprotein ever synthesized by a
fully chemical procedure.
Solid-Phase Synthesis of a Repeating Unit. The synthetic
route for the MUC2 unit is shown in Figure 2. The sequence of
this unit is reported to be PTTTPITTTTTVTPTPTPTGTQT.37
To avoid the epimerization of the C-terminal amino acid residue
during the segment coupling reaction, the original sequence was
designed to shift three residues so that the carboxyl terminal of
this sequence is made to be glycine. Among 14 potential
O-glycosylation sites within this sequence, we selected seven
sites and introduced Tn-antigen for this initial study. The
consecutive five Thr residues were included for the glycosylation
site for the mimicry of the dense cluster of carbohydrates of
mucins as well as for the synthetic challenge described above.
The glycosylated peptide thioester of the repeating unit was
prepared by the modified Fmoc strategy reported previously for
peptide thioester synthesis.34 First, C-terminal glycine was
introduced using Fmoc-Gly-SCH2CH2COOH by the 1,3-dicy-
clohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt)
method. The Fmoc group was then cleaved by the mixture of
1-methylpyrrolidine, hexamethyleneimine, and HOBt, which
effectively cleaves the Fmoc groups with the thioester linkage
remaining intact.38 The amino group was then reacted with the
second amino acid Tsoc-Thr(Bn)-OPfp 3, which is protected
Results and Discussion
Synthetic Strategy. Our synthetic strategy is shown in Figure
1. Fmoc-serine or threonine carrying various carbohydrates are
prepared based on our benzyl-protection strategy and are
introduced during the solid-phase synthesis of the peptide
thioester by the Fmoc method. After the chain assembly, the
resin is treated with TFA to deprotect the peptide portion and
is followed by “low trifluoromethanesulfonic acid (TfOH)”31
to remove the benzyl groups. The latter treatment has been
shown to deprotect various N- and O-linked carbohydrates, with
remaining glycosidic linkages intact.32-35 Then, the glycopeptide
thioester is consecutively joined by the thioester method to
obtain homogeneous tandem repeat structure.
To accomplish this strategy, there are two points which need
consideration. First is the preparation of a peptide thioester
carrying multiple carbohydrates. In mucins, Ser or Thr residues
often exist consecutively. The bulky glycosylated amino acids
generally retain reduced reactivity during the coupling reaction,
(25) Fontenot, J. D.; Tjandra, N.; Bu, D.; Ho, C.; Montelaro, R. C.; Finn, O. J.
Cancer Res. 1993, 53, 5386-5394.
(26) Karsten, U.; Serttas, N.; Paulsen, H.; Danielczyk, A.; Goletz, S. Glycobi-
ology 2004, 14, 681-692.
(27) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117.
(28) Aimoto S. Biopolymers 1999, 51, 247-265.
(29) Guo, Z.-W.; Nakahara, Y.; Nakahara, Y.; Ogawa, T. Angew. Chem., Int.
Ed. Engl. 1997, 36, 1464-1466.
(30) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1998,
309, 287-296.
(31) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J. Am. Chem. Soc. 1986, 108,
5242-5251.
(32) Takano, Y.; Habiro, M.; Someya, M.; Hojo, H.; Nakahara, Y. Tetrahedron
Lett. 2002, 43, 8395-8399.
(33) Takano, Y.; Kojima, N.; Nakahara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron
2003, 59, 8415-8427.
(34) Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara, Y.; Nabeshima, K.;
Toole, B. P.; Watanabe, Y. Tetrahedron Lett. 2003, 44, 2961-2964.
(35) Takano, Y.; Hojo, H.; Kojima, N.; Nakahara, Y. Org. Lett. 2004, 6, 3135-
3138.
(36) Kent, S. B. H. Annu. ReV. Biochem. 1988, 57, 957-989.
(37) Kim, Y. S.; Gum, J. R.; Brockhausen, I. Glycoconjugate J. 1996, 13, 693-
707.
(38) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-8672.
9
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A R T I C L E S
Hojo et al.
the success of this synthesis. However, a weak signal was also
observed at m/z 3871.2, which was derived from the des-Pro-
Thr peptide (calcd. for (M + Na)+: 3870.7). This contaminant
was overlapped with the desired peptide by this eluent system
and was only unveiled by a shallow gradient of acetonitrile in
20 mM AcONH4 (pH 5.7), as shown in Figure 3c. This fact
shows that in the case of a tandem repeat of mucins, where the
sequence is composed of limited amino acids with high
frequency, complete purification is difficult using a single
RPHPLC purification. If the stepwise synthesis was continued
to obtain two or three tandem repeat peptides, the separation of
the impurity could no longer be possible. Thus, in the synthesis
of the tandem repeat peptide, a segment coupling method is
essential. The impurity was removed by a second HPLC using
the buffer system and was desalted using aqueous acetonitrile
containing 0.1% TFA. The yield of peptide 2 was 5.9% based
on the amino groups on the initial resin.
Segment Condensation. Unit 2 was then condensed with
TQT-NH2 by the thioester method,27,28 as shown in Figure 4.
This condensation forms the original repeat sequence from the
C-terminus. Glycopeptide thioester 2 and TQT-NH2 were
dissolved in DMSO, and the thioester group was activated by
silver chloride. As shown in Figure 5, the reaction was almost
completed within 6 h without serious side reactions. The
following Fmoc removal was achieved by adding 5% piperidine
to the reaction mixture within 15 min. The crude peptide was
purified by HPLC, and the desired product 5 was obtained in
62% yield. Then, the coupling, piperidine treatment, and the
purification by gel filtration chromatography (GFC) were
repeated five more times by the thioester method. All coupling
reactions proceeded without serious side reactions within
6 h to obtain [TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]nTQT-
NH2 (n ) 2, 6; n ) 3, 7; n ) 4, 8; n ) 5, 9; n ) 6, 1), as
shown in Figure 5. Even in case of the last coupling reaction,
which yielded a glycoprotein of over 20 kDa carrying 42
GalNAc moieties of 1, the high efficiency of the coupling was
maintained. The yields of these coupling reactions after purifica-
tion by GFC were about 70% on average. The final purification
was achieved by RPHPLC, and the desired product 1 was
successfully obtained in 55% yield. The product was well
characterized by the amino acid analysis and ESI mass analysis,
as shown in Figure 6. These results demonstrate this method to
be highly efficient in the synthesis of a tandem repeat structure.
Figure 3. HPLC profile of glycopeptide thioester 2: (a) crude glycopeptide
2; (b) purified glycopeptide 2; (c) re-chromatogram of glycopeptide 2. The
asterisked peaks are derived from scavengers. Elution conditions of (a) and
(b): column, Mightysil RP-18 GP (4.6 × 150 mm) at a flow rate of 1 mL
min-1; eluent, A, 0.1% TFA, B, acetonitrile containing 0.1% TFA. Elution
conditions of (c): column, Mightysil RP-18 GP (4.6 × 150 mm) at a flow
rate of 1 mL min-1; eluent, A, 20 mM AcONH4 (pH 5.7), B, acetonitrile
containing 10% of solution A.
by a silyl carbamate group (Tsoc, N-triisopropylsilyloxycarbo-
nyl).39 Using acid fluoride,40 the third amino acid was then
condensed in the presence of a catalytic amount of the fluoride
ion. N-Deprotection by the fluoride ion generates a free amino
group, which is immediately acylated by the third amino acid
fluoride. Thus, the formation of diketopiperazine, which is a
well-known side reaction observed at the dipeptide stage, is
effectively prevented.39 If this precaution was not taken, 40%
of the defective peptide thioester, lacking the C-terminal two
amino acids, was formed in our previous study.34 Further
elongation of the peptide chain was carried out by the Fmoc
procedure using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate (HBTU) as a coupling reagent. The
carbohydrate portion was introduced using Fmoc-Thr carrying
benzyl-protected GalNAc 4 by the same activation method. After
the completion of the peptide chain assembly, the resin was
first treated with TFA (Reagent K41) to deprotect the peptide
portion. Then, the crude peptide was further treated with “low
TfOH”32-35 to remove the benzyl groups. Despite the introduc-
tion of five consecutive GalNAc moieties, and of the presence
of multiple benzyl groups, the desired peptide thioester carrying
seven GalNAcs was obtained in good purity, as demonstrated
by the HPLC data in Figure 3a. The crude product was purified
by HPLC using aqueous acetonitrile containing 0.1% TFA as
an eluent, as shown in Figure 3b. MALDI mass measurement
shows that the separated glycopeptide has a desired mass number
(m/z ) 4069.2, calcd for (M + Na)+: 4068.8), which supports
Conclusion
The peptide thioester carrying seven GalNAc moieties was
successfully prepared by the Fmoc procedure in high purity.
The obtained thioester was repeatedly condensed to the amino
component peptide with good yield and efficiency. These
successes demonstrate the general applicability of this procedure
for the synthesis of tandem repeated as well as globular
glycoproteins of 20 kDa. This method also enables us, in
principle, to synthesize glycopeptide thioesters carrying different
carbohydrates within a peptide sequence. Therefore, by con-
densing various heterogeneously glycosylated peptide thioesters
consecutively, the tandem repeat model having heterogeneous
carbohydrate between the repeating units can be synthesized.
Such glycopeptides will simulate the real mucin surfaces.
Together with homogeneous models these heterogeneous models
will contribute to the structural and functional analysis of natural
(39) Sakamoto, K.; Nakahara, Y.; Ito, Y. Tetrahedron Lett. 2002, 43, 1515-
1518.
(40) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401-5402.
(41) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res. 1990, 36,
255-266.
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Synthesis of MUC2 Basal Motif with O-GalNAc Moieties
A R T I C L E S
Figure 4. The synthesis of homogeneous tandem repeat model 1 by the consecutive segment coupling by the thioester method.
Materials and Methods
1H NMR spectra were recorded with a JEOL AL400 spectrometer
(400 MHz). Chemical shifts are expressed in parts per million downfield
from the signal for internal Me4Si for a solution in CDCl3. The Fmoc
deblocking reagent [1-methylpyrrolidine (25%), hexamethylene imine
(2%), HOBt (2%) in 1-methyl-2-pyrrolidinone (NMP)-DMSO (1:1)]
was prepared according to the previously reported procedure.38 RPHPLC
purification of glycopeptide thioester 2 and glycopeptide 5 was
performed using Mightysil RP-18 (10 × 250 mm, Kanto Chemical) at
a flow rate of 2.5 mL/min. Gel filtration chromatography of glyco-
peptides 6, 7, 8, 9, and 1 was performed by G3000PWXL (7.8 mm ×
300 mm, TOSOH) using 50% aqueous acetonitrile containing 0.1%
TFA as an eluent at a flow rate of 0.5 mL/min. Amino acid composition
was determined with a Lachrom amino acid analyzer (Hitachi, Tokyo)
after hydrolysis with 6 M HCl at 150 °C for 2 h in an evacuated sealed
tube. The amount of the peptide was calculated based on the amino
acid analysis data. ESI mass measurement was carried out on an Applied
Biosystems/MDS Sciex QSTAR pulsar i mass spectrometer. The
glycopeptide was sprayed at a concentration of 1-2 pmol/µL in water/
methanol (v/v) containing 0.1% formic acid or in water/methanol
containing 0.1% TFA. MALDI mass measurement was performed by
an Applied Biosystems/Voyager-DE Pro using dihydroxybenzoic acid
as a matrix.
Figure 5. RPHPLC profile of the crude peptide obtained by the segment
couplings. Elution conditions: column, Mightysil RP-18 GP (4.6 × 150
mm) at a flow rate of 1 mL min-1; eluent, A, 0.1% TFA, B, acetonitrile
containing 0.1% TFA. The arrows in (a)-(f) show peptides 5, 6, 7, 8, 9,
and 1, respectively, before Fmoc removal.
TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG-SCH2CH2CONH2, 2.
CLEAR-amide resin (290 mg, 0.35 mmol/g) was treated with the Fmoc
deblocking reagent for 5 and 15 min. After washing with NMP, Fmoc-
mucins. Along this line, further studies are currently being
undertaken, and the results will be presented in due course.
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A R T I C L E S
Hojo et al.
AcONH4 (pH 5.7) as an eluent and was desalted by RPHPLC using
the former solvent system to give purified glycopeptide 2 (2.4 mg, 0.59
µmol, 5.9% yield based on the amino groups on the initial resin).
MALDI-TOF mass: found m/z 4069.2, calcd for (M + Na)+ m/z
4068.8. Amino acid analysis: Thr13.09Glu1.08Pro4.66Gly1.25Val1Ile0.99
.
TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTGTQT-NH2, 5. Glyco-
peptide thioester 2 (4.0 mg, 1.0 µmol), TQT-NH2 (3.5 mg, 10 µmol),
and 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOOBt, 4.8
mg, 29 µmol) were dissolved in DMSO (100 µL) containing DIEA
(3.4 µL, 20 µmol). AgCl (0.4 mg, 3 µmol) was then added, and
the resulting mixture was stirred in the dark overnight. Piperidine (5
µL) was added, and the solution was kept in the dark for 1 h. The
product was precipitated by ether and purified by RPHPLC using
aqueous acetonitrile containing 0.1% TFA as an eluent to give
glycopeptide 5 (1.6 mg, 400 nmol, 40%). MALDI-TOF mass: found
m/z 4089.0, calcd for (M + Na)+ m/z 4088.9. Amino acid analysis:
Thr15.08Glu2.05Pro5.01Gly1Val0.99Ile0.96
.
[TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]2TQT-NH2, 6. Glyco-
peptide thioester 2 (2.0 mg, 500 nmol), glycopeptide 5 (1.6 mg, 400
nmol), and HOOBt (2.4 mg, 15 µmol) were dissolved in DMSO (30
µL) containing DIEA (1.7 µL, 9.8 µmol). AgCl (0.2 mg, 1.5 µmol)
was then added, and the resulting mixture was stirred in the dark
overnight. Piperidine (3 µL) was added, and the solution was kept in
the dark for 1 h. The product was precipitated by ether and purified by
gel filtration chromatography to give glycopeptide 6 (2.3 mg, 290 nmol,
73%). MALDI-TOF mass: found m/z 7812.2, calcd for (M + Na)+
m/z 7812.2. Amino acid analysis: Thr28.13Glu2.94Pro9.58Gly2Val1.99Ile1.93
.
[TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]3TQT-NH2, 7. Glyco-
peptide thioester 2 (1.6 mg, 400 nmol), glycopeptide 6 (2.2 mg, 280
nmol), and HOOBt (2.0 mg, 12 µmol) were dissolved in DMSO (30
µL) containing DIEA (1.4 µL, 8.1 µmol). AgCl (0.2 mg, 1.5 µmol)
was then added, and the resulting mixture was stirred in the dark
overnight. Piperidine (3 µL) was added, and the solution was kept in
the dark for 1 h. The product was precipitated by ether and purified by
gel filtration chromatography to give glycopeptide 7 (2.9 mg, 250 nmol,
89%). MALDI-TOF mass: found m/z 11536.5, calcd for (M + Na)+
Figure 6. ESI mass spectrum of 1. The desired peak in (b) accompanies
multiple Na+ added peaks, which might be derived from the fact that
GalNAc moieties tend to coordinate Na+.
Gly-SCH2CH2COOBt (0.2 mmol), which was prepared by mixing
Fmoc-Gly-SCH2CH2COOH (77 mg, 0.2 mmol), 1 M DCC/NMP (0.2
mL), and 1 M HOBt/NMP (0.2 mL) for 30 min at room temperature,
was added, and the reaction mixture was vortexed for 1 h. The resin
was washed with NMP and was treated with the Fmoc deblocking
reagent (2 and 5 min). After NMP washing, the resin was reacted with
Tsoc-Thr(Bn)-OPfp 3 (115 mg, 0.2 mmol) in tetrahydrofuran (THF)
for 15 min. The reaction was repeated with the same amount of fresh
Tsoc-Thr(Bn)-OPfp. The resin was washed with CH2Cl2. Fmoc-Pro-F
(2.2 mmol), prepared by mixing Fmoc-Pro (81 mg, 0.24 mmol), fluoro-
N,N,N′,N′-tetramethylformadinium hexafluorophosphate (58 mg, 0.22
mmol), and N,N-diisopropylethylamine (DIEA, 76 µL, 0.44 mmol) in
CH2Cl2, was then added. The reaction was initiated by adding 1 M
n-Bu4NF in THF (10 µL) to the reaction mixture. After vortexing for
1 h, the resin was washed with CH2Cl2 and NMP. The remaining amino
acids were introduced manually using Fmoc-amino acid (0.5 mmol),
0.45 M HBTU-HOBt (1.0 mL, 0.45 mmol), and DIEA (0.16 mL, 0.9
mmol), except Fmoc-Thr(GalNAc) 4, which was introduced in 0.2
mmol scale. The brief protocol is as follows. (1) NMP wash (1 min ×
6), (2) Fmoc removal (2 and 18 min), (3) NMP wash (1 min × 6), (4)
coupling (60 min), (5) NMP wash (1 min × 3), (6) capping by 10%
Ac2O, 5% DIEA in NMP (5 min). After completion of the chain
assembly, 480 mg of protected peptide resin was obtained. A part of
the resin (50 mg) was treated with Reagent K (0.75 mL) for 1 h at
room temperature. TFA was removed by the nitrogen stream, and the
peptide was precipitated with ether and washed with the same solvent
twice. The precipitate was further treated with low-TfOH (0.5 mL) for
2 h at -10 °C. The peptide was precipitated with ether, washed with
the same solvent twice, and dried in vacuo. The crude peptide was
purified by RPHPLC using aqueous acetonitrile containing 0.1% TFA
to obtain partially purified glycopeptide 2. The peptide was further
purified by RPHPLC using aqueous acetonitrile containing 20 mM
m/z 11533.0. Amino acid analysis: Thr39.87Glu4.05Pro14.73Gly3Val3.00
-
Ile3.04
.
[TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]4TQT-NH2, 8. Glyco-
peptide thioester 2 (1.3 mg, 320 nmol), glycopeptide 7 (2.9 mg, 250
nmol), and HOOBt (1.6 mg, 9.8 µmol) were dissolved in DMSO (30
µL) containing DIEA (1.1 µL, 6.3 µmol). AgCl (0.2 mg, 1.5 µmol)
was then added, and the resulting mixture was stirred in the dark
overnight. Piperidine (3 µL) was added, and the solution was kept in
the dark for 1 h. The product was precipitated by ether and purified by
gel filtration chromatography to give glycopeptide 8 (2.9 mg, 190 nmol,
77%). MALDI-TOF mass: found m/z 15255.3, calcd for (M + Na)+
m/z 15253.9. Amino acid analysis: Thr51.85Glu5.03Pro18.93Gly4Val3.99
-
Ile3.98
.
[TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]5TQT-NH2, 9. Glyco-
peptide thioester 2 (0.93 mg, 230 nmol), glycopeptide 8 (2.9 mg, 190
nmol), and HOOBt (1.1 mg, 6.7 µmol) were dissolved in DMSO (20
µL) containing DIEA (0.8 µL, 4.6 µmol). AgCl (0.2 mg, 1.5 µmol)
was then added, and the resulting mixture was stirred in the dark
overnight. Piperidine (2 µL) was added, and the solution was kept in
the dark for 1 h. The product was precipitated by ether and purified by
gel filtration chromatography to give glycopeptide 9 (2.7 mg, 144 nmol,
77%). MALDI-TOF mass: found m/z 18976.4, calcd for (M + Na)+
m/z 18974.3. Amino acid analysis: Thr64.25Glu5.70Pro24.06Gly5Val4.93
-
Ile5.00
.
9
13724 J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005

Synthesis of MUC2 Basal Motif with O-GalNAc Moieties
A R T I C L E S
[TQTPT(GalNAc)TTPIT(GalNAc)T(GalNAc)T(GalNAc)T-
(GalNAc)T(GalNAc)VTPT(GalNAc)PTPTG]6TQT-NH2, 1. Glyco-
peptide thioester 2 (0.73, 180 nmol), glycopeptide 9 (2.7 mg, 141 nmol),
and HOOBt (0.9 mg, 5.5 µmol) were dissolved in DMSO (20 µL)
containing DIEA (0.6 µL, 3.5 µmol). AgCl (0.1 mg, 0.7 µmol) was
then added, and the resulting mixture was stirred in the dark overnight.
Piperidine (2 µL) was added, and the solution was kept in the dark for
1 h. The product was precipitated by ether and purified by gel filtration
chromatography followed by RPHPLC using Protein-RP (4.6 × 150
mm, Yamamura Co., Ltd.) to give glycopeptide 1 (1.8 mg, 78 nmol,
55%). ESI mass: found m/z 22674.6, calcd for (M + H)+ m/z 22673.7.
Acknowledgment. This work was supported by a Grant-in-
Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan [(C) No.
16580093]. The authors also acknowledge Tokai University for
a grant-aid for high-technology research.
Supporting Information Available: Preparation of Tsoc-Thr-
(Bn)-OPfp 3 and Fmoc-Thr(GalNAc) 4. This material is
available free of charge via the Internet at http://pubs.acs.org.
Amino acid analysis: Thr80.70Glu7.07Pro29.09Gly6Val5.75Ile5.98
.
JA053711B
9
J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005 13725