Investigating bacterial N-linked glycosylation: Synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine

Article abstract of DOI:10.1021/ja054265v

The chemical synthesis and biological activity of undecaprenyl pyrophosphate bacillosamine (Und-PP-Bac), an obligatory intermediate in the asparagine-linked glycosylation pathway of Campylobacter jejuni, are reported. The key transformation involves the c

Full text of DOI:10.1021/ja054265v

Published on Web 09/14/2005
Investigating Bacterial N-Linked Glycosylation: Synthesis and Glycosyl
Acceptor Activity of the Undecaprenyl Pyrophosphate-Linked Bacillosamine
Eranthie Weerapana, Kerney Jebrell Glover, Mark M. Chen, and Barbara Imperiali*
Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received June 28, 2005; E-mail: imper@mit.edu
Asparagine-linked protein glycosylation has long been considered
a unique feature of the eukaryotic kingdom; however, recent studies
have revealed the presence of N-linked glycoproteins in Campylo-
bacter jejuni, a gram-negative bacterium that is involved in human
gastroenteritis.1 The process of N-linked glycosylation in C. jejuni
displays significant similarities to the analogous process in eukary-
otes.2 A series of glycosyltransferases act sequentially to assemble
a glycan donor on a polyisoprenyl carrier (undecaprenyl pyrophos-
phate, Und-PP), which is transferred to the asparagine side chain
of proteins at the N-X-S/T consensus sequence.3 In C. jejuni, the
glycan donor has been identified as the Und-PP-linked heptasac-
charide GalNAc-R1,4- GalNAc-R1,4-(Glcâ1,3)-GalNAc-R1,4-Gal-
NAc-R1,4-GalNAc-R1,3-Bac-R1,PP-Und (1), where Bac is the
unusual sugar bacillosamine (2,4-diacetamido-2,4,6-trideoxyglu-
cose) that is only found in specific bacterial systems (Figure 1).4
Figure 1. Structure of the undecaprenyl-linked heptasaccharide.
benzyl 2-acetamido-2-deoxy-â-D-galactopyranoside 4.7 The C-4
Bioinformatic and mutagenesis data suggest that the five glyco-
syltransferases (Pgl C, A, J, H, and I located in the pgl gene cluster)
are responsible for the biosynthesis of the heptasaccharide.5
To study these glycosyltransferases in vitro, large quantities of
highly pure undecaprenyl-linked glycan substrates are essential.
These polyisoprene-linked compounds are only present in very small
quantities, making purification from native sources an unrealistic
endeavor. Furthermore, this is complicated by the fact that these
strains of bacteria are extremely pathogenic, thus requiring special-
ized handling. Employing chemical synthesis, we can access the
desired milligram quantities of the Und-PP-linked glycans for
biochemical studies of the glycosyltransferases. Here we report the
synthesis of Und-PP-Bac (2) (Scheme 1), a key intermediate in
the Pgl pathway, and initial efforts to investigate the activity of
the Pgl glycosyltransferases in vitro using this synthetic substrate.
The synthesis of 2 involves the coupling of undecaprenyl
phosphate to bacillosamine6 phosphate 3, which is synthesized from
azido functionality was installed by a selective benzoylation of C-3
and C-6 followed by triflation and subsequent displacement with
sodium azide at C-4 resulting in intermediate 5. To deoxygenate
at C-6, the iodo functionality was introduced to afford 6, which
was reduced concurrently with the C-4 azide by catalytic hydro-
genation. Subsequent acetylation of both the amino and hydroxyl
functionalities resulted in intermediate 7. At this stage, to improve
the solubility properties of the intermediate and render it compatible
with subsequent transformations, it was necessary to exchange the
C-3 acetyl-protecting group for a benzoyl group to afford 8.
Deprotection of the anomeric hydroxyl followed by phosphorylation
with tetrabenzyl pyrophosphate afforded the phosphorylated bacil-
losamine derivative 3. Phosphorylation with tetrabenzyl pyrophos-
phate proceeds with >16:1 selectivity in favor of the R-phosphate.
However, upon deprotection, a minor (<15%) impurity in the
Scheme 1. Synthesis of Und-PP-Baca
a Reagents and conditions: (a) benzoyl chloride, pyridine, -40 f 0 °C, 70%; (b) Tf2O, CH2Cl2/pyridine, 0 °C f rt; (c) NaN3, DMF, rt, 62% over two
steps; (d) NaOMe, MeOH, rt, 80%; (e) TsCl, pyridine, 0 °C f rt, 75%; (f) NaI, MeCN, 80 °C, 70%; (g) H2, Pd(OH)2/C, DIPEA, MeOH, 32 °C; (h) Ac2O,
pyridine, rt, 65% over two steps; (i) NaOMe, MeOH, 90%; (j) BzCl, pyridine, 60%; (k) H2, Pd(OH)2/C, MeOH, 32 °C, 86%; (l) LiHMDS, -68 °C; then
[(BnO)2P(O)]2O, -68 f 0 °C, 50%; (m) H2, Pd/C, MeOH, 99%; (n) carbonyl diimidazole, DMF; then undecaprenyl phosphate, 50%; (o) NaOMe, MeOH,
99%.
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10.1021/ja054265v CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. (a) Hydrolysis and 2-aminobenzamide labeling. (b) HPLC trace
of 2AB-labeled GalNAc-Bac (fluorescence detection). * Denotes major
product peak. (c) MALDI-MS of disaccharide (peak at 592.27 denotes
sodium adduct of expected peak).
Figure 2. (a) Reaction catalyzed by PglA. (b) Radioactive assay for PglA
activity with Und-PP-Bac, 2. DPM (disintegrations per minute) denotes
radioactive counts in the organic phase.
has recently been used to investigate the glycosyltransferases
Pgl J, H, and I, resulting in the chemoenzymatic synthesis of the
Und-PP-heptasaccharide 1.10 Access to Und-PP-Bac removes a
significant obstacle in the study of bacterial N-linked glycosylation
and paves the way for biophysical and biochemical analysis of the
process. These studies will provide an important foundation for
studies targeted at understanding the more complex, yet analogous,
process of N-linked glycosylation in eukaryotic systems.
product is observed, which cannot be separated by chromotography.
The minor product may be due to epimerization at the anomeric
center.
Undecaprenyl phosphate was synthesized from undecaprenol
using phosphoramidite chemistry as previously reported.8 The
coupling to bacillosamine phosphate 3 was effected using activation
with 1,1′-carbonyldiimidazole. In the final step, the C-3 benzoyl
ester was removed with sodium methoxide, resulting in the first
chemical synthesis of Und-PP-Bac (2).
Acknowledgment. This research was supported by the NIH
In vivo mutagenesis studies have shown that PglA is the
glycosyltransferase that catalyzes the transfer of a GalNAc residue
from UDP-GalNAc to 2 to form GalNAc-R1,3-Bac-R1-PP-Und (9)
(Figure 2a).9 To validate PglA activity in vitro, Und-PP-Bac (2)
was used as the cosubstrate with UDP-GalNAc for purified PglA,
which was cloned and overexpressed in Escherichia coli. The
preliminary enzyme activity assay involved monitoring the transfer
of radiolabeled GalNAc from aqueous-soluble UDP-[3H]-GalNAc
to organic-soluble [3H]-GalNAc-Bac-PP-Und in the presence of
purified PglA (Figure 2b). The significant increase in radioactivity
in the organic extract upon addition of 2 confirms that PglA accepts
this synthetic substrate very efficiently.
The disaccharide product from this enzymatic reaction was
characterized by acidic hydrolysis of the saccharide from the
undecaprenyl pyrophosphate carrier to yield 10, followed by
labeling of the reducing terminus with 2-aminobenzamide (2AB)
via a reductive amination to afford 11. The resulting 2AB-labeled
disaccharide 11 was isolated on a GlykoSepN normal-phase column
and analyzed by MALDI-MS (Figure 3).
The synthetic route outlined herein provides access to milligram
quantities of Und-PP-Bac, which is the first membrane-associated
substrate in the C. jejuni N-linked protein glycosylation pathway.
The availability of this substrate has further enabled validation of
the enzymatic activity of the glycosyltransferase PglA, revealing
that Und-PP-Bac serves as the glycosyl acceptor with UDP-N-acetyl
galactosamine as the glycosyl donor in the enzyme-catalyzed
transformation. The disaccharide obtained from the PglA reaction
(GM39334) and postdoctoral fellowship (GM65699) to K.J.G.
Supporting Information Available: Experimental procedures and
product characterization for all new compounds synthesized. Cloning
and expression of PglA and procedures for assaying activity. This
material is available free of charge via the Internet at http://pubs.acs.org.
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