Efficient sequential segment coupling using N-alkylcysteine-assisted thioesterification for glycopeptide dendrimer synthesis

Article abstract of DOI:10.1021/ol801340m

(Figure Presented) A highly pure MUC1-derived glycopeptide dendrimer of 22 kDa was prepared by a sequential segment coupling, achieved by an N-alkylcysteine (NAC)-assisted thioesterification. The glycopeptide having C-terminal NAC was prepared by the Fmoc method and converted to the thioester by 3-mercaptopropionic acid treatment. The thioester was condensed with a lysine trimer carrying NAC to afford tetramer. which was then converted to the thioester. Two tetramers were condensed with ethylenediamine to give the octameric glycopeptide dendrimer.

Full text of DOI:10.1021/ol801340m

ORGANIC
LETTERS
2008
Vol. 10, No. 16
3531-3533
Efficient Sequential Segment Coupling
Using N-Alkylcysteine-Assisted
Thioesterification for Glycopeptide
Dendrimer Synthesis
Chinatsu Ozawa, Hidekazu Katayama, Hironobu Hojo,* Yuko Nakahara, and
Yoshiaki Nakahara*
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai UniVersity,
Kanagawa 259-1292, Japan
hojo@keyaki.cc.u-tokai.ac.jp; yonak@keyaki.cc.u-tokai.ac.jp
Received June 13, 2008
ABSTRACT
A highly pure MUC1-derived glycopeptide dendrimer of 22 kDa was prepared by a sequential segment coupling, achieved by an N-alkylcysteine
(NAC)-assisted thioesterification. The glycopeptide having C-terminal NAC was prepared by the Fmoc method and converted to the thioester
by 3-mercaptopropionic acid treatment. The thioester was condensed with a lysine trimer carrying NAC to afford tetramer, which was then
converted to the thioester. Two tetramers were condensed with ethylenediamine to give the octameric glycopeptide dendrimer.
Mucins are large glycoproteins that cover the surface of
epithelial cells. It is well-known that the carbohydrate structure
of mucins alters during the course of malignancy.1 Typical
tumor markers, such as T (Gal-GalNAc), Tn (GalNAc), and
sialyl-T antigens, are highly expressed on mucins derived from
adenocarcinomas due to the incomplete elongation of the
carbohydrate chains. Thus, mucins could be potential clinical
targets for cancers by inducing effective antibodies against these
tumor markers. On the basis of this idea, conjugates composed
of a cancer-related glycopeptide and a carrier protein have been
prepared as vaccine candidates.2 In several cases, clinical trials
are underway. However, the conjugates have limitations, such
as the irrelevant immune response against the carrier proteins
and the ambiguous composition of the materials. These points
might be disadvantages if the conjugates are used as human
vaccines. Various fully synthetic glycopeptide conjugates have
also been examined as vaccine candidates.2a,b,d,f,3 The glyco-
peptide dendrimers, one of the glycopeptide conjugates, are
(2) (a) 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. (b) Kudryashov, V.; Glunz, P. W.; Williams, L. J.;
Hintermann, S.; Danishefsky, S. J.; Lloyd, K. O. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 3264–3269. (c) Ragupathi, G.; Coltart, D. M.; Williams,
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. (d) Keding, S. J.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 11937–11942. (e) Dziadek, S.; Kowalczyk, D.; Kunz, H. Angew.
Chem., Int. Ed. 2005, 44, 7624–7630. (f) Ragupathi, G.; Koide, F.;
Livingston, P. O.; Cho, Y. S.; Endo, A.; Wan, Q.; Spassova, M. K.; Keding,
S. J.; Allen, J.; Ouerfelli, O.; Wilson, R. M.; Danishefsky, S. J. J. Am.
Chem. Soc. 2006, 128, 2715–2725. (g) Wittrock, S.; Becker, T.; Kunz, H.
Angew. Chem., Int. Ed. 2007, 46, 5226–5230.
(1) Kim, Y. S.; Gum, J., Jr.; Brockhausen, I. Glycoconjugate J. 1996,
13, 693–707.
10.1021/ol801340m CCC: $40.75
Published on Web 07/19/2008
2008 American Chemical Society

particularly advantageous as they are self-immunogenic due
to their large size.4 Dendrimeric compounds can be prepared
by the convergent approach, in which the glycopeptide and
the branching unit are prepared separately and condensed.
However, when the number of glycopeptide chain increases,
the condensation reaction becomes difficult to proceed
completely. We recently applied a similar strategy for the
synthesis of a glycopeptide dendrimer composed of eight
glycopeptide chains.5 As with other cases, the reaction gave
a mixture of the desired product and imperfect dendrimers,
and we successfully isolated the desired glycopeptide den-
drimer in high purity using preparative gel-electrophoresis.
However, considering the general applicability of the method,
an establishment of a more general synthetic route for
glycopeptide dendrimer is desirable.
In this paper, we designed a new method for synthesizing a
highly pure glycopeptide dendrimer using the potential of the
post-solid-phase peptide synthesis (SPPS) thioesterification, in
which N-alkyl cysteine (NAC) at the C-terminus of the peptide
was used as an N- to S- acyl migratory device.6 The efficiency
of the method was demonstrated by the synthesis of the
glycopeptide dendrimer 1, which consisted of eight peptide
chains of the tandem repeat region of MUC11 carrying two
T-antigens (Scheme 1). First, the glycopeptide thioester 5 was
Scheme 1.
Novel Route for Glycopeptide Dendrimer Synthesisa
Figure 1. Synthesis of glycopeptide thioester 5: (a) synthetic route,
Thr* denotes threonine residue carrying benzyl-protected Gal-
GalNAc moiety. (b) RPHPLC profile of crude glycopeptides 6 and
7 and thioester 5. Elution conditions: column, Mightysil RP-18 GP
(4.6 × 150 mm, Kanto, Japan) at a flow rate of 1 mL /min; eluent,
A, 0.1% TFA, B, acetonitrile containing 0.1% TFA. The asterisked
peaks were derived from nonpeptidic components.
The glycine residue was quantitatively introduced after double
coupling at 50 °C, which was monitored by the amino acid
(3) (a) Palcic, M. M.; Li, H.; Zanini, D.; Bhella, R. S.; Roy, R.
Carbohydr. Res. 1998, 305, 433–442. (b) Jezek, J.; Velek, J.; Veprek, P.;
Velkova, V.; Trnka, T.; Pecka, J.; Ledvina, M.; Vondrasek, J.; Pisacka, M.
J. Pept. Sci. 1999, 5, 46–55. (c) Glunz, P. W.; Hintermann, S.; Schwarz,
J. B.; Kuduk, S. D.; Chen, X.-T.; Williams, L. J.; Sames, D.; Danishefsky,
S. J.; Kudryashov, V.; Lloyd, K. O. J. Am. Chem. Soc. 1999, 121, 10636–
10637. (d) Lo-Man, R.; Bay, S.; Vichier-Guerre, S.; Deriaud, E.; Can-
tacuzene, D.; Leclerc, C. Cancer Res. 1999, 59, 1520–1524. (e) Komba,
S.; Werdelin, O.; Jensen, T.; Meldal, M. J. Pept. Sci. 2000, 6, 585–593. (f)
Lo-Man, R.; Vichier-Guerre, S.; Bay, S.; Deriaud, E.; Cantacuzene, D.;
Leclerc, C. J. Immunol. 2001, 166, 2849–2854. (g) Vichier-Guerre, S.; Lo-
Man, R.; BenMohamed, L.; Deriaud, E.; Kovats, S.; Leclerc, C.; Bay, S. J.
Peptide Res. 2003, 62, 117–124. (h) Lo-Man, R.; Vichier-Guerre, S.; Perraut,
R.; Deriaud, E.; Huteau, V.; BenMohamed, L.; Diop, O. M.; Livingston,
P. O.; Bay, S.; Leclerc, C. Cancer Res. 2004, 64, 4987–4994. (i) Buskas,
T.; Ingale, S.; Boons, G.-J. Angew. Chem., Int. Ed. 2005, 44, 5985–5988.
(j) Dziadek, S.; Hobel, A.; Schmitt, E.; Kunz, H. Angew. Chem., Int. Ed.
2005, 44, 7630–7635. (k) Veprek, P.; Hajduch, M.; Dzubak, P.; Kuklik,
R.; Polakova, J.; Bezouska, K. J. Med. Chem. 2006, 49, 6400–6407. (l)
Cremer, G.-A.; Bureaud, N.; Piller, V.; Kunz, H.; Piller, F.; Delmas, A. F.
ChemMedChem. 2006, 1, 965–968. (m) Ingale, S.; Buskas, T.; Boons, G.-
J. Org. Lett. 2006, 8, 5785–5788. (n) Ingale, S.; Wolfert, M. A.; Gaekwad,
J.; Buskas, T.; Boons, G.- J. Nat. Chem. Biol. 2007, 3, 663–667. Also see
recent reviews: (o) Fortier, S.; Touaibia, M.; Lord-Doufour, S.; Galipeau,
J.; Roy, R.; Annabi, B. Glycobiology 2008, 18, 195–204. (p) Niederhafner,
P.; Reinis, M.; Sebestik, J.; Jezek, J. J. Pept. Sci. 2008, 14, 556–587.
aConditions: (a) (1) 5% aq MPA, 2 d, (2) RPHPLC, 20% from the
loading of C-terminal Gly on resin; (b) AgCl, HOOBt, DIEA, DMSO,
overnight; (c) (1) 10% 1,2-ethanedithiol-TFA, 1 h, (2) 5% aq MPA
containing 6 M urea, 4 d, (3) GFC, (4) RPHPLC, 17% (total of b and c);
(d) (1) ethylenediamine, AgCl, HOOBt, DIEA, DMSO, overnight, (2) GFC,
76%.
synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) method
combined with the NAC method as shown in Figure 1. To
prepare the peptide resin 8, Fmoc-N-ethyl-S-trityl cysteine
(Fmoc-(Et)Cys(Trt)) was introduced into CLEAR amide resin.
Then, a glycine, the C-terminal amino acid in the MUC1
sequence, was introduced using Fmoc-Gly activated by O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HATU) and N,N-diisopropylethylamine (DIEA).
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analysis of the acid-hydrolyzed resin sample. The remaining
amino acids were introduced by a peptide synthesizer (Applied
Biosystems, 433A) using the FastMoc protocol, which uses
piperidine in 1-methyl-2-pyrrolidinone (NMP) for the Fmoc
removal and O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) for the activation of amino acids,
except that Fmoc-Thr carrying the benzyl-protected Gal-GalNAc
moiety (T-antigen)7 activated by HBTU was introduced manually.
The obtained resin 8 was treated with Reagent K (aq TFA, phenol,
thioanisole, 1,2-ethanedithiol)8 to achieve deprotection of the
peptide part. After precipitation by diethyl ether, the crude peptide
was further treated with low-acidity TfOH9 to remove benzyl
groups of the carbohydrate portion. As expected, the peptide was
in an equilibrium between the amide 7 and thioester 6 forms as
shown in Figure 1. The peptide was then dissolved in a 5%
3-mercaptopropionic acid (MPA) solution to obtain the glycopep-
tide thioester 5. After 2 d, the NAC-containing peptides were almost
converted to the thioester 5 without significant decomposition of
the carbohydrate and the thioester portions as shown in Figure 1b.
The yield of the purified glycosylated peptide thioester 5 was 20%
based on the initial loading amount of Gly on the resin. This yield
was significantly higher than those of glycosylated peptide thioesters
prepared by modified Fmoc strategy using a resin, which im-
mobilizes a peptide via a thioester linkage.9b,c This result
demonstrated the practical applicability of the NAC method
for glycopeptide thioester synthesis. The lysine trimer
carrying NAC 4, in which the N-S acyl transfer activity is
blocked by the Trt group, was synthesized by the Fmoc SPPS
method using 2-chlorotrityl alcohol resin (see the Supporting
Information).
Next, the glycopeptide dendrimer 1 was synthesized by the
thioester method10 according to Scheme 1. The glycopeptide
thioester 5 and dendrimer core 4 were dissolved in DMSO
containing 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine
(HOOBt) and DIEA. AgCl was then added to initiate the
coupling reaction. The reversed-phase (RP)HPLC analysis of
the solution indicated that the thioester 5 was almost consumed
within 6 h and the new peak corresponding to the desired
tetramer 3 appeared. The tetramer 3 was precipitated by ether
and used in the next reaction without further purification.
The Trt group of the C-terminal NAC residue was removed
by TFA containing 5% 1,2-ethanedithiol, and thioesterification
was carried out by 5% aq MPA treatment. The reaction
proceeded without significant side reactions within 4 d. Then
the desired tetrabranched thioester 2 was isolated by RPHPLC
in high purity. Finally, the thioester 2 was condensed with
ethylenediamine by the thioester method to obtain the octamer.
After isolation by gel filtration chromatography (GFC), the
isolated product was analyzed by SDS-PAGE, MALDI-TOF
mass (Figure 2), and amino acid composition analyses, which
demonstrated that the product 1 was obtained in high purity.
Figure 2. (a) MALDI-TOF mass spectrum of 1 and (b) SDS-PAGE
of the glycopeptide dendrimer. Key: lane 1, m.w. standard; lane 2,
coupling mixture for the preparation of the product 1; lane 3,
purified tetramer 2; lane 4, purified octamer 1.
The NAC-assisted thioesterification reaction proved to be an
efficient method to obtain highly pure glycopeptide thioester
in good yield. The consecutive application of the NAC method
to the peptide and tetrameric dendrimer successfully gave a
highly pure octabranched glycopeptide dendrimer with molec-
ular weight larger than 20 kDa, which will be a candidate for
unambiguous cancer vaccines. In this sequential coupling
method, the final stage is a condensation of the two tetra-
branched dendrimers. Thus, the purification was easily achieved
by the GFC, due to the large difference in molecular weight
between the product and the starting material. This strategy can
realize the stepwise introduction of tetramer to dendrimer core,
which would achieve the preparation of dendrimers having
different components, such as B and T cell epitopes, and
glycopeptides and fluorescent tag. These molecules are useful
for the induction of a more efficient immune response and for
the trace of the cellular uptake of the dendrimers. Along this
line, the synthesis of these dendrimers is now in progress.
(4) (a) Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5409–5413.
(b) Sadler, K.; Tam, J. P. J. Biotechnol. 2002, 90, 195–229.
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Akahane, T.; Nakahara, Y. Tetrahedron 2007, 63, 9685–9690.
(6) (a) Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y.
Tetrahedron Lett. 2007, 48, 25–28. (b) Hojo, H.; Onuma, Y.; Akimoto, Y.;
Nakahara, Y.; Nakahara, Y. Tetrahedron Lett. 2007, 48, 1299.
(7) Nakamura, K.; Ishii, A.; Ito, Y.; Nakahara, Y. Tetrahedron 1999,
55, 11253–11266.
Acknowledgment. This work was partly supported by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sport, Sciences and Technology of Japan.
We thank Tokai University for a grant-in-aid for high-
technology research. We also thank the Japan Society for the
Promotion of Science for a Grant-in-Aid for Creative Scientific
Research (No. 17GS0420).
(8) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res.
1990, 36, 255–266.
(9) (a) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J. Am. Chem. Soc.
1986, 108, 5242–5251. (b) Hojo, H.; Haginoya, E.; Matsumoto, Y.;
Nakahara, Y.; Nabeshima, K.; Toole, B. P.; Watanabe, Y. Tetrahedron Lett.
2003, 44, 2961–2964. (c) Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.;
Suzuki, Y.; Suzuki, M.; Suzuki, A.; Nakahara, Y. J. Am. Chem. Soc. 2005,
127, 13720–13725.
Supporting Information Available: Synthetic procedures
for glycopeptide thioester 5, dendrimer core 4, and glyco-
peptide dendrimers 2 and 1. This material is available free
of charge via the Internet at http://pubs.acs.org.
(10) (a) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117.
(b) Aimoto, S. Biopolymer (Pept. Sci.) 1999, 51, 247–265.
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