Synthesis and solution-phase conformation of the RG-I fragment of the plant polysaccharide pectin reveals a modification-modulated assembly mechanism

Article abstract of DOI:10.1021/ja9090963

The syntheses of pure RG-I fragments of key plant matrix biomolecule pectin using a counterintuitive late-stage convergent cis-glycosylation has allowed detailed analyses of their solution-phase conformations, metal binding affinities, pKa values, self-assembly equilibria, and diffusional kinetics. These reveal a striking, right-handed 31-helix that provides an effective and repeating lateral display of putative liganding carboxylates. Moreover, these heteropolymeric structures allow units as short as tetrasaccharides to self-assemble through carbohydrate-carbohydrate interactions that are induced by the presence of Ca(II), a known dynamic trigger in planta. These self-assembly properties can be switched simply by the addition or removal of a single methyl group in this repeating unit through methyl (de)esterification, another known dynamic trigger in planta. Together, the combined effect of Ca(II) and methylation revealed here suggests a concerted molecular basis for these two major dynamic modifications in planta.

Full text of DOI:10.1021/ja9090963

Published on Web 01/25/2010
Synthesis and Solution-Phase Conformation of the RG-I Fragment of the Plant
Polysaccharide Pectin Reveals a Modification-Modulated Assembly Mechanism
Eoin M. Scanlan,† Mukram M. Mackeen,† Mark R. Wormald,‡ and Benjamin G. Davis*,†
Chemistry Research Laboratory, Department of Chemistry, UniVersity of Oxford, Mansfield Road, Oxford OX1 3TA, U.K., and
Glycobiology Institute, Department of Biochemistry, Oxford UniVersity, South Parks Road, Oxford OX1 3QU, U.K.
Received October 26, 2009; E-mail: Ben.Davis@chem.ox.ac.uk
Pectins are complex polysaccharides in which four primary
domains have been described: homogalacturonan (HGA), rham-
nogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and
xylogalacturonan (XGA).1,2 Modifications of the carbohydrate
backbones of RG-I and HGA allow the plant cell to react to
changing conditions such as aging and host-pathogen interactions.3
Two important modifications involve methyl esterification of the
galacturonic acid residues and the formation of calcium bridges
between pectin chains.2 The HGA region of pectin is highly methyl-
esterified, especially as it emerges from production in the Golgi
apparatus. These regions are then de-esterified in the plant cell wall
by the action of plant pectinmethylesterases (PMEs). Although
heavily studied for the HGA region of pectin,4 methyl esterification
of galacturonic acid (GalA) has also been reported in RG-I.5 These
modifications alter the 3D structure of the pectic matrix and may
play a fundamental role in cell growth and defense mechanisms.3
Here we report the synthesis and detailed conformational study of a
synthetically pure fragment of RG-I (1) and its methyl ester RG-I-OMe
(2) and the effect of esterification upon calcium binding. Experimental
3D structures determined by NMR spectroscopy reveal that a single methyl
ester critically and dramatically modulates calcium binding and the
subsequent biophysical properties of this key plant polysaccharide.
disaccharide 7, which was employed directly as a disaccharide donor
unit for assembly of tetrasaccharide 10 and hexasaccharide 14 (Scheme
1). Disaccharide acceptor 9 was prepared by coupling of rhamnosyl
thioglycoside 5 and galactosyl acceptor 6 followed by selective
deprotection of the C-2′ acetyl of 8. The key (2 + 2) coupling between
7 and 9 proceeded smoothly, giving tetrasaccharide 10 in 83% yield.
Despite the relative cis-1,2 configuration, 10 was formed as the desired
R-anomer with apparently complete stereoselectivity. Primary (OH-
6, OH-6′′) and secondary (OH-2′′) hydroxyl groups were revealed in
one pot upon treatment with freshly prepared sodium methoxide.
Selective oxidation of OH-6 and OH-6′′ to carboxylate using sequential
TEMPO/NaClO2 and NaOCl successfully converted Gal residues to
GalA. Finally, following benzyl esterification to aid purification, careful
control of the deprotection conditions allowed selective access to both
1 and 2 from 13. Thus, treatment under catalytic hydrogenation
conditions furnished methyl ester 2 as the major product when MeOH
was used as the solvent and the free acid 1 in THF/H2O solvent. The
potential for iterative extension of the pectin chain was demonstrated
through (4 + 2) glycosylation to give 14.
Scheme 1a
Figure 1. RG-I tetrasaccharide unit 1 and corresponding methyl ester 2.
Analysis of the RG-I structure (Figure 1) initially suggested
stereoselective oligomer assembly6-10 through a late-stage coupling
of an L-rhamnosyl (L-Rha)-terminated glycosyl donor fragment to a
D-GalA-terminated acceptor fragment (assembly a in Figure 1); this
logic was suggested by a typically strong preference in L-rhamnosy-
lation for the required R-L-Rha linkage in RG-I, which contrasts with
the typically poor selectivity in the formation of the cis linkage11 of
R-D-GalA. However, despite the synthesis of a variety of appropriate
building blocks [see the Supporting Information (SI)], their use in
convergent disaccharide-to-disaccharide (2 + 2) coupling surprisingly
failed. Consequently, counter to traditional glycosylation logic,11 we
assembled RG-I via late-stage R-cis-galactosylation (assembly b in Figure
1) using a latent/quasiorthogonal donor-acceptor assembly strategy.12-15
Monosaccharide building blocks 3-6 were readily prepared from
the parent sugars L-rhamnose and D-galactose (see the SI). Coupling
of the rhamnosyl trichloroacetimidate donor 3 and the galactosylth-
ioglycoside acceptor 4 (itself a latent donor) furnished the R-linked
a Reagents and conditions: (a) TMSOTf, DCM, 0 °C, 65%; (b) NIS,
TMSOTf, DCM, 0 °C, 75%; (c) NaOMe, MeOH/THF, rt, 84%; (d) NIS,
TMSOTf, DCM, 0 °C, 83%; (e) NaOMe, MeOH/THF, rt, 78%; (f) TEMPO,
NaClO2, NaOCl (two stages), 0 °C to rt; (g) Cs2CO3, BnBr, DMF, rt, 72%
over two steps; (h) Pd/C, H2, MeOH, rt, 44%; (i) Pd/C, H2, THF/H2O, rt, 62%;
(j) NIS, TMS·OTf, DCM, 0 °C, 68%.
The conformations, carboxylate pKa values, and calcium binding
properties of these synthetic samples of 1 and 2 were investigated by NMR
† Department of Chemistry.
‡ Department of Biochemistry.
9
7238 J. AM. CHEM. SOC. 2010, 132, 7238–7239
10.1021/ja9090963  2010 American Chemical Society

C O M M U N I C A T I O N S
spectroscopy. The pKa values of the single acid residue in 2 and the two
carboxylic acid residues in 1 were 3.6, indicating no interactions between
the two titratable groups in 1. The value of 3.6 is similar to those previously
reported for isolated pectin and polygalacturonic acid.16
Conformations of 1 and 2 in both water and 800 mM Ca2+(aq)
were determined using cross-linkage rotating-frame nuclear Overhauser
effects (ROEs) and molecular modeling. These revealed very similar
arrangements for 1 and 2 (Figure 2) in which the two carboxy groups
are placed on opposite sides, consistent with the pKa results.
Figure 3. Working model for switching by methyl (de)esterification 1 a
2/(a) a (b) in heteropolymeric sugar unit self-assembly. Two very different
Ca2+-binding modes of (b) RG-I 1 and (a) RG-I-OMe 2 are interconverted.
based on homopolymeric structures (e.g., homogalacturonic acid) and
a requirement of ∼14 residues.26 Our results show that through
heteropolymeric units it is possible to form discrete oligomers for a
much shorter pectin chain based on the RG-I-OMe tetrasaccharide.
Moreover, the nonesterified RG-I tetrasaccharide displays multimeric
interactions between chains initiated by monocomplexation with Ca2+.
A switch between these two dramatically different states is modulated
simply by methyl (de)esterification, a known2,5 dynamic postbiosyn-
thetic regulatory modification in plants.
In summary, syntheses of the RG-I tetrasaccharide and its methyl ester
RG-I-OMe have revealed the first experimental conformational structures.
These show that a single methyl ester generates significant differences in
biophysical behavior, without altering the conformation, through the mo-
dulation of self-assembly. The combined effect of Ca(II) and methylation
revealed here suggests a concerted molecular basis for two major dynamic
modifications (Ca binding and pectin methyl esterification)2 in planta.
Figure 2. Conformation of 2 in the absence (a) and presence (b) of Ca2+
.
(c) Ca2+ binding site in 2. (d) Model of the helical structure of RG-I pectin.
A pioneering molecular modeling study carried out on the RG-I
disaccharide linkages of RhaR1-4GalA and GalAR1-2Rha suggested
three potential conformers for each linkage.17 Our conformational
experimental results are consistent with only one of the three
conformers for each linkage. Interestingly, this is the high-energy
conformer [(φ, ψ) ) (-40°, -10°)] in the relaxed potential maps for
the GalAR1-2Rha linkage and not one of the predicted low-energy
conformers. The tetrasaccharide model was then used to build an 18-
mer model that showed threefold symmetry (31) and a pitch of 22 Å
containing two repeating units per helical turn (Figure 2d). This right-
handed glycan helix therefore provides an effective and repeating lateral
display of putative liganding carboxylates.
Supporting Information Available: Experimental details, extended
synthetic discussion, characterization data, and spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Ridley, B. L.; O’Neill, M. A.; Mohnen, D. Phytochemistry 2001, 57, 929.
(2) Caffall, K. H.; Mohnen, D. Carbohydr. Res. 2009, 344, 1879.
(3) Protsenko, M. A.; Buza, N. L.; Krinitsyna, A. A.; Bulantseva, E. A.;
Korableva, N. P. Biochemistry (Moscow) 2008, 73, 1053.
(4) Goldberg, R.; Prat, R.; Morvan, C. Carbohydr. Polym. 1994, 23, 203.
(5) Rihouey, C.; Morvan, C.; Borissova, I.; Jauneau, A.; Demarty, M.; Jarvis,
M. Carbohydr. Polym. 1995, 28, 159.
(6) In regard to synthetic studies on pectin regions (RG-I/II, HGA), see the SI
for an extended discussion and refs 7-10 for leading examples of protected
RG-I oligosaccharide precursors/analogues.
(7) Rich, J. R.; McGavin, R. S.; Gardner, R.; Reimer, K. B. Tetrahedron:
Asymmetry 1999, 10, 17.
(8) Maruyama, M.; Takeda, T.; Shimizu, N.; Hada, N.; Yamada, H. Carbohydr.
Res. 2000, 325, 83.
(9) Reiffarth, D.; Reimer, K. B. Carbohydr. Res. 2008, 343, 179.
(10) Nemati, N.; Karapetyan, G.; Nolting, B.; Endress, H.-U.; Vogel, C.
Carbohydr. Res. 2008, 343, 1730.
(11) Demchenko, A. V. Synlett 2003, 1225.
(12) Boons, G. J. Tetrahedron 1996, 52, 1095.
(13) Boons, G. J.; Grice, P.; Leslie, R.; Ley, S. V. Tetrahedron Lett. 1993, 34, 8523.
(14) Boons, G. J.; Isles, S. Tetrahedron Lett. 1994, 35, 3593.
(15) Cao, S. D.; Gan, Z. H.; Roy, R. Carbohydr. Res. 1999, 318, 75.
(16) Ralet, M. C.; Dronnet, V.; Buchholt, H. C.; Thibault, J. F. Carbohydr.
Res. 2001, 336, 117.
(17) Cros, S.; Garnier, C.; Axelos, M. A.; Imberty, A.; Perez, S. Biopolymers
1996, 39, 339.
Calcium binding to 2 showed a single binding curve with a KD value
of 145 mM at pH 5.6. The presence of 800 mM calcium had no effect on
the observed pattern of ROEs. The pattern of chemical shift changes
resulting from calcium addition was consistent with a single binding site
on 2 (Figure 2b). Calcium binding was also associated with a decrease in
the diffusion coefficient, D (Figure 3), as measured by DOSY spectros-
copy, consistent with dimer formation to produce a neutral complex.
Calcium binding to 1 is a more complex multievent process. There
is an initial relatively18-20 high affinity binding with a KD of 15
mM21-23 that is not accompanied by a decrease in diffusion coefficient;
this is followed by further, more complex changes in chemical shift
that can be approximated by a 1.6 M KD binding curve and are
associated with a significant change in diffusion. For the higher-affinity
binding, the 10-fold increase in binding relative to 2 is consistent only
with interaction of the bound calcium with both carboxylate groups
of 1, which required a conformational change to bring these two groups
to the same side of the molecule, resulting in a neutral monomer
complex (“monocomplex”). The lower affinity binding is then associ-
ated with aggregation. The unsaturated binding curve resembles that
for the previously reported multichain association of polygalacturonic
(18) Typical sugar-Ca KD values are >20 mM (see refs 19 and 20).
(19) Gould, R. O.; Rankin, A. F. J. Chem Soc. D 1970, 489.
(20) Angyal, S. J. AdV. Carbohydr. Chem. Biochem. 1989, 47, 1.
(21) Intracellular [Ca2+] levels in planta reach up to ∼100 mM (see refs 22 and 23).
(22) Tuteja, N.; Sopory, S. K. Plant Signal. BehaV. 2008, 3, 525.
(23) White, P. J.; Broadley, M. R. Ann. Bot. 2003, 92, 487.
(24) Rinaudo, M.; Ravanat, G.; Vincedon, M. Makromol. Chem. 1980, 181, 1059.
(25) Grant, G. T.; Morris, E. R.; Rees, D. A. FEBS Lett. 1973, 32, 195.
(26) Powell, D. A.; Morris, E. R.; Gidley, M. J.; Rees, D. A. J. Mol. Biol. 1982,
155, 517.
acid in the presence of Ca2+
summarized in Figure 3.
.
24 This Ca-binding model for 1 and 2 is
Rees25 and colleagues have proposed the “egg-box model” for the
occurrence of dimers and multichain association during Ca2+ binding
JA9090963
9
J. AM. CHEM. SOC. VOL. 132, NO. 21, 2010 7239