A highly convergent chemical synthesis of conformational epitopes of rhamnogalacturonan II

Article abstract of DOI:10.1002/anie.200701750

(Chemical Equation Presented) Upon careful tuning of the reactivity of glycosyl donors and acceptors, tetra- and hexasaccharide fragments (see structure) of the B chain of rhamnogalacturonan II (RGII) were prepared in a convergent and stereoselective mann

Full text of DOI:10.1002/anie.200701750

Communications
DOI: 10.1002/anie.200701750
Carbohydrate Synthesis
A Highly Convergent Chemical Synthesis of Conformational Epitopes
of Rhamnogalacturonan II**
Yu Rao and Geert-Jan Boons*
Recent estimates indicate that more than 2000 genes encode
proteins involved in the biosynthesis and remodeling of the
polysaccharide-rich primary cell wall of plants.[1] The func-
tions of only a handful of these gene products are known.[2,3]
Furthermore,there is data to support the hypothesis that
carbohydrate epitopes change during plant development;
however,detailed knowledge about these processes is lacking.
It is critical that these deficiencies in knowledge be addressed
urgently because advances in plant sciences and genetics will
provide researchers with tools to develop the next generation
of agroenergy and agromaterial crops,which will be higher-
yielding and tailored for modern biorefinery operations.[4]
Monoclonal antibodies are attractive tools for monitoring
changes in the composition and organization of the primary
plant wall at the cellular and subcellular level.[5] To date,only
a small number of such monoclonal antibodies have been
generated for which the binding specificities have been
Scheme 1. Targeted hexasaccharide 1 and tetrasaccharide 2.
characterized. Synthetic chemistry can provide well-defined
plant-derived oligosaccharides,[6–8] which can be employed for
the production of monoclonal antibodies. In particular,the
highly complex pectins are prime targets for this purpose. For
example,rhamnogalacturonan II (RGII) is a structurally
highly complex pectic oligosaccharide of the primary cell
wall of higher plants[9] and is composed of four structurally
different oligosaccharides referred to as side chains A–D,
which are attached to a linear a(1!4)-linked d-GalpA
polysaccharide backbone. These polysaccharides are cross-
linked covalently through borate diesters (1:2) between two
apiofuranosyl residues of the A side chain. The resulting
three-dimensional pectic network contributes to the mechan-
ical properties of the primary cell wall and is required for
normal plant growth and development. Indeed,changes in
cell-wall properties that decrease the borate cross-linking of
pectin lead to symptoms such as dwarfism.[10]
plant species,such as Arabidopsis,lack the b-l-Araf-(1!3)-l-
Rhamp moiety of 1.[9] In particular,we were interested in
whether the presence of this disaccharide moiety would affect
the ring conformation of the crowded a-l-Arap residue.[11]
The oligosaccharides were substituted with an artificial
aminopropyl spacer to facilitate conjugation to carrier
proteins,as required for immunization and ELISA.
The chemical synthesis of 1 was challenging owing to the
presence of a number of unusual monosaccharide moieties,
the crowdedness around the central arabinopyranoside
moiety,and the presence of 12,- cis-linked 2-O-methyl-a-l-
fucoside and b-l-arabinofuranoside residues,which are
difficult to introduce in a stereoselective manner.[12–15] Ini-
tially,we envisaged that hexasaccharide 1 could be prepared
in a highly convergent manner from disaccharides 13, 14,and
15 (see Scheme 3),which in turn could be prepared from
monosaccharides 3–8 (Scheme 2).[16–20]
Thus,we planned to obtain the core 2- O-methyl-a-l-
Fucp-(1!2)-b-d-Galp unit by glycosylation of the fucosyl
donor 3 with the spacer-containing galactoside 4a or 4b,
followed by regioselective opening of the benzylidene ring to
give the glycosyl acceptors 12 and 13. Compounds 12 and 13
would then be coupled with disaccharide 14 (see Scheme 3),
which we intended to prepare by the chemoselective glyco-
sylation of trichloroacetimidate 5 with thioglycoside 6. The
attractiveness of the latter glycosylation approach is that the
thioglycoside functionality of 6 is stable under the conditions
used for the activation of trichloroacetimidate 5. However,
the resulting thioglycosyl disaccharide 14 can be employed as
a glycosyl donor by activating the anomeric thiophenyl
As part of a program to generate well-defined monoclonal
antibodies against fragments of primary plant cell walls,we
report herein the chemical synthesis of hexasaccharide 1
(Scheme 1),which is derived from the B side chain of RGII.
The simpler tetrasaccharide 2 was also prepared,as several
[*] Dr. Y. Rao, Dr. G.-J. Boons
Complex Carbohydrate Research Center
The University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Fax: (+1)706-542-4412
E-mail: gjboons@ccrc.uga.edu
[**] This research was supported by the NSF (Grant No. DBI 0421683).
Supporting information for this article, including synthetic proce-
dures and 1H and 13C NMR spectra for all compounds is available
on the WWW under http://www.angewandte.org or from the author.
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Scheme 2. Building blocks for the synthesis of oligosaccharides 1 and 2.
Bn=benzyl, Bz=benzoyl, Lev=levulinoyl.
moiety with a thiophilic promoter.[21] The Lev ester of the
Arap moiety of the tetrasaccharide could then be removed
and the resulting alcohol coupled with disaccharide 15 to give
a fully protected hexasaccharide. The preparation of disac-
charide 15 also exploits the selective activation of a trichlor-
oactimidate functionality in the presence of a thioglycoside.
Finally,we planned to introduce the b-arabinofuranoside
residue stereoselectively by employing the conformationally
constrained glycosyl donor 7. The 3,5-O-(di-tert-butylsilyl)
protecting group of this compound locks the five-membered
ring in an E3 conformation,in which the approach of a reagent
Scheme 3. Reagents and conditions: a) NIS, TfOH, 4-Š MS, CH2Cl2/
Et2O (1:4), ꢀ208C; b) 1. NaOMe, MeOH, RT; 2. NaH, BnBr, TBAI,
DMF, 08C; c) NaCNBH3, THF, 2m HCl, Et2O, 4-Š MS, RT; d) TMSOTf,
4-Š MS, CH2Cl2, ꢀ708C. DMF=N,N-dimethylformamide, MS=molec-
ular sieves, NIS=N-iodosuccinimide, TBAI=tetrabutylammonium
iodide, Tf=trifluoromethanesulfonyl, TMS=trimethylsilyl.
from the a face is blocked as a result of steric hindrance by (Scheme 4). Unfortunately,NIS/TfOH-promoted glycosyla-
H2.[22]
Thus,NIS/TfOH-promoted glycosylation
tion of 12 with 14 in dichloromethane at 08C did not lead to
of 3 with 4a the formation of tetrasaccharide 16,and the reaction was not
[23]
in a mixture of dichloromethane and diethyl ether gave improved by varying the solvent,temperature,and promoter.
disaccharide 9 in an excellent yield of 85% but in a The reactivity of the hydroxy group at C4 of 12 is probably too
disappointing a/b ratio of approximately 4:1 (Scheme 3). low as a result of its axial orientation and the presence of the
Fortunately,the use of the less reactive glycosyl acceptor 4b, neighboring deactivating electron-withdrawing benzoyl ester
which has an electron-withdrawing benzoyl ester group at C2 functionality. We envisaged that the reactivity of the glycosyl
rather than an activating benzyl ether,led to disaccharide 10 acceptor could be improved by replacing the electron-with-
in excellent yield almost exclusively as the a anomer. drawing benzoyl ester group with a benzyl ether group.
Furthermore,this compound could be converted readily
Indeed,the coupling of 13 with 14 in the presence of NIS/
into 11 by treatment with sodium methoxide in methanol TfOH as the thiophilic activator gave tetrasaccharide 17 in
followed by benzylation with benzyl bromide and sodium 71% yield. The Lev ester group of 17 was removed selectively
hydride in DMF. The benzylidene acetals of 10 and 11 were by treatment with hydrazinium acetate in a mixture of
opened regioselectively by using NaCNBH3 and HCl in dichloromethane and methanol to give the glycosyl acceptor
diethyl ether to give the glycosyl acceptors 12 and 13, 18,the coupling of which with the thioglycosyl donor 15 led to
respectively.
the formation of hexasaccharide 21 in an unacceptable yield
Disaccharide 14 was prepared in 91% yield by the of 8%. The glycosylation is probably poor-yielding as a result
TMSOTf-mediated glycosylation[24] of trichloroacetimidate of steric crowding around the C2 hydroxy group of the
5 with thioglycoside 6. The a anomer of 14 was formed arabinopyranosyl moiety.
exclusively as a result of neighboring-group participation by
We expected the stepwise introduction of the monosac-
the acetyl ester functionality at C2 in 5. Disaccharide 15 was charide units of the b-l-Araf(1!3)-a-l-Rhap moiety to be
obtained mainly as the b anomer in 84% yield by coupling 7 more efficient. Thus,NIS/TfOH-promoted glycosylation of 18
with 8a in the presence of a catalytic amount of TMSOTf. The with 8b gave pentasaccharide 19 in 60% yield. Next,the Lev
b-anomeric configuration of 15 was confirmed by the ester group in 19 was removed under standard conditions,and
observed chemical shifts for H1 (dH = 5.15 ppm, J1,2
5.4 Hz) and C1 (dC = 97.8 ppm). Fortunately,no aglycon
=
the resulting glycosyl acceptor 20 was coupled with the
conformationally constrained arabinofuranoyl donor 7 to give
transfer was observed in either glycosylation. (Aglycon hexasaccharide 21 in 92% yield. Careful examination of the
transfer occasionally occurs when trichloroacetimidates are NMR spectroscopic data for this compound revealed that
coupled with thioglycosyl acceptors.)
With disaccharides 12–15 in hand,we focused our
attention on the assembly of the hexasaccharide
only the b anomer had been formed. An NIS/TfOH-pro-
moted glycosylation of a 3,5-O-(di-tert-butylsilyl)-protected
1-thioarabinofuranoside with 20 gave 21 in good yield but
1
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Scheme 4. Reagents and conditions: a) NIS, TfOH, 4-Š MS, CH2Cl2, ꢀ208C; b) NH2NH2·AcOH, CH2Cl2/MeOH, RT; c) TMSOTf, 4-Š MS, CH2Cl2,
ꢀ708C; d) 1. TBAF, THF, RT; 2. NaOMe/MeOH, RT; 3. Pd/C, tBuOH/AcOH/H2O, RT; e) 1. NaOMe/MeOH, RT; 2. Pd/C, tBuOH/AcOH/H2O, RT.
TBAF=tetrabutylammonium fluoride.
with slightly reduced anomeric selectivity.[22] Preactivation of
the 1-thioarabinofuranoside with Ph2SO/Tf2O followed by the
addition of 20 led to the formation of 21 with decreased
anomeric selectivity.[25]
Finally, 21 was deprotected to give 1 by a three-step
procedure that involved the removal of the di-tert-butylsilyl
protecting group by treatment with TBAF in THF,saponifi-
cation of the acetyl and benzoyl ester groups with sodium
methoxide in methanol,and subsequent catalytic hydro-
genolysis over Pd/C to remove the benzyl ether groups and
convert the azido moiety into an amine. Tetrasaccharide 2 was
obtained readily from 17 by a standard three-step depro-
tection sequence.
1
Table 1: Comparison of the 13C and H NMR spectroscopic data for the
Arap rings of 1 and 2.[a]
1
2
C1 [ppm]
H1 [ppm]
JH1,H2 [Hz]
JH2,H3 [Hz]
JH3,H4 [Hz]
104.5
4.90
3.0
4.5
5.0
101.3
4.49
8.0
10.0
3.0
JH4,H5 [Hz]
5.0
11.0
4.0
a
JH4,H5 [Hz]
5.0
b
[a] The spectra were recorded in D2O at 258C.
Careful examination of the chemical shifts and coupling
constants in the 1H and 13C NMR spectra of compounds 1 and
2 revealed conformational epitopes of the arabinopyranosyl
moiety (Table 1 and Figure 1). Thus,the large coupling
constants JH1,H2 and JH2,H3 between vicinal hydrogen atoms
and the relatively small values for JH4,H5 and JH4,H5 of the
RGII may be biologically significant,as available data suggest
that there is considerable structural variability among the
RGII B side chains of different plants as a result of the
presence or absence of substituents linked to C2 and/or C3 of
the Arap moiety.[9] It may well be possible that the con-
formation of the Arap moiety affects the cross-linking of
a
b
arabinopyranosyl moiety of tetrasacchar-
4
ide 2 are in agreement with a regular C1
conformation. However,the values JH1,H2
and JH2,H3 for compound 1 were found to be
much smaller and JH4,H5 larger than the
b
equivalent values for 2. These data indicate
that the arabinopyranoside ring in 1 adopts
1
a distorted C4 conformation. Unfavorable
steric interactions of the trisubstituted
arabinopyranosyl moiety are probably alle-
viated somewhat in this conformation.
The difference in the conformation of a
disubstituted (as in compound 2) and a
trisubstituted (as in compound 1) arabino-
pyranosyl moiety of the B side chain of
Figure 1. Proposed conformations of hexasaccharide 1.
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[3] Arabidopsis Genome Initiative, Nature 2000, 408,796.
RGII by borate esters,which is an essential process for
normal plant growth. Additional research will be required to
determine the importance of the conformational flexibility of
the RGII side chain to the cross-linking process. It is to be
expected that antibodies that recognize conformational
epitopes of the side chain of RGII will be valuable tools for
such studies.
[4] A. J. Ragauskas,C. K. Williams,B. H. Davison,G. Britovsek,J.
Cairney,C. A. Eckert,W. J. Frederick,J. P. Hallett,D. J. Leak,
C. L. Liotta,J. R. Mielenz,R. Murphy,R. Templer,T. Tscha-
plinski, Science 2006, 311,484.
[5] J. P. Knox, Int. Rev. Cytol. 1997, 171,79.
[6] N. A. Jones,S. A. Nepogodiev,C. J. MacDonald,D. L. Hughes,
R. A. Field, J. Org. Chem. 2005, 70,8556.
[7] A. L. Chauvin,S. A. Nepogodiev,R. A. Field, J. Org. Chem.
2005, 70,960.
[8] M. A. J. Buffet,J. R. Rich,R. S. McGavin,K. B. Reimer,
Carbohydr. Res. 2004, 339,2507.
[9] M. A. OꢀNeill,T. Ishii,P. Albersheim,A. G. Darvill, Annu. Rev.
Plant Biol. 2004, 55,109.
[10] M. A. OꢀNeill,S. Eberhard,P. Albersheim,A. G. Darvill, Science
2001, 294,846.
[11] J. N. Glushka,M. Terrell,W. S. York,M. A. OꢀNeill,A. Gucwa,
A. G. Darvill,P. Albersheim,J. H. Prestegard, Carbohydr. Res.
2003, 338,341.
[12] G. J. Boons, Contemp. Org. Synth. 1996, 3,173.
[13] G. J. Boons, Tetrahedron 1996, 52,1095.
[14] A. V. Demchenko, Synlett 2003,1225.
[15] T. L. Lowary, Curr. Opin. Chem. Biol. 2003, 7,749.
[16] H. Amer,A. Hofinger,M. Puchberger,P. Kosma, J. Carbohydr.
Chem. 2001, 20,719.
[17] D. J. Lefeber,J. P. Kamerling,J. F. G. Vliegenthart, Chem. Eur. J.
2001, 7,4411.
In conclusion,we have shown that an oligosaccharide with
a highly dense architecture that contains a number of unusual
1,2-cis glycoside residues can be prepared in a highly
convergent and stereoselective manner by careful tuning of
the reactivity of the glycosyl donors and acceptors of the
saccharide building blocks. For the stereoselective installment
of the a-linked 2-O-methylfucoside unit,it was essential to
employ a glycosyl acceptor of reduced reactivity. The b-linked
arabino-l-furanoside unit could be introduced by employing a
conformationally constrained arabinosyl donor locked in a
conformation that blocks its a face. Furthermore,it was
necessary to maximize the reactivity of the galactoside
residue as a glycosyl acceptor by benzylation of the hydroxy
group at C3. A stepwise approach to the installation of the b-
l-Araf-(1!3)-a-l-Rha unit at the crowded C2 hydroxy group
of Arap was more efficient than the use of a disaccharide
building block. Finally,the use of quasiorthogonal trichlor-
oacetimidates and thioglycosides minimized protecting-group
manipulations during the assembly of the hexasaccharide.
[18] J. H. Wang,J. Li,D. Tuttle,J. Y. Takemoto,C. W. T. Chang, Org.
Lett. 2002, 4,3997.
[19] V. Pozsgay, Carbohydr. Res. 1992, 235,295.
[20] J. S. Sun,X. W. Han,B. Yu, Carbohydr. Res. 2003, 338,827.
[21] H. Yamada,T. Harada,H. Miyazaki,T. Takahashi, Tetrahedron
Lett. 1994, 35,3979.
Received: April 19,2007
Published online: July 6,2007
[22] X. M. Zhu,S. Kawatkar,Y. Rao,G. J. Boons, J. Am. Chem. Soc.
2006, 128,11948.
[23] G. H. Veeneman,S. H. van Leeuwen,J. H. van Boom, Tetrahe-
dron Lett. 1990, 31,1331.
[24] R. R. Schmidt,W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994,
50,21.
Keywords: carbohydrate synthesis · conformation analysis ·
glycosylation · oligosaccharides · pectins
.
[1] N. Carpita,M. Tierney,M. Campbell, Plant Mol. Biol. 2001, 47,
[25] D. Crich,C. M. Pedersen,A. A. Bowers,D. J. Wink,
J. Org.
1.
Chem. 2007, 72,1553.
[2] R. Perrin,C. Wilkerson,K. Keegstra, Plant Mol. Biol. 2001, 47,
115.
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