Isoprenoid phosphonophosphates as glycosyltransferase acceptor substrates

Article abstract of DOI:10.1021/ja500622v

Glycosyltransferases that act on polyprenol pyrophosphate substrates are challenging to study because their lipid-linked substrates are difficult to isolate from natural sources and arduous to synthesize. To facilitate access to glycosyl acceptors, we assembled phosphonophosphate analogues and showed these are effective substrate surrogates for GlfT1, the essential product of mycobacterial gene Rv3782. Under chemically defined conditions, the galactofuranosyltransferase GlfT1 catalyzes the formation of a tetrasaccharide sequence en route to assembly of the mycobacterial galactan.

Full text of DOI:10.1021/ja500622v

Communication
pubs.acs.org/JACS
Isoprenoid Phosphonophosphates as Glycosyltransferase Acceptor
Substrates
Mario A. Martinez Farias,† Virginia A. Kincaid,‡ Venkatachalam R. Annamalai,† and Laura L. Kiessling*,†,‡
‡
†Department of Chemistry and Department of Biochemistry, University of WisconsinMadison, Madison, Wisconsin 53706,
United States
S
* Supporting Information
ABSTRACT: Glycosyltransferases that act on polyprenol
pyrophosphate substrates are challenging to study because
their lipid-linked substrates are difficult to isolate from
natural sources and arduous to synthesize. To facilitate
access to glycosyl acceptors, we assembled phosphono-
phosphate analogues and showed these are effective
substrate surrogates for GlfT1, the essential product of
mycobacterial gene Rv3782. Under chemically defined
conditions, the galactofuranosyltransferase GlfT1 catalyzes
the formation of a tetrasaccharide sequence en route to
assembly of the mycobacterial galactan.
ipid-linked oligosaccharide pyrophosphates serve as
1
Lacceptors for a large class of glycosyltransferases. Progress
in understanding these enzymes is hindered by the difficulty of
isolating these biosynthetic acceptors from natural sources and
the challenge of their chemical synthesis.2,3 Although some
Figure 1. Top: Replacement of the allylic phosphate in 1 (blue) with a
acceptors have been generated through astutely orchestrated
syntheses,4−8 the lability of the allylic pyrophosphoryl group
narrows the range of transformations that can be applied to
their synthesis. We envisioned addressing these barriers by
synthesizing a glycosyltransferase acceptor in which the
pyrophosphoryl group was replaced with a phosphonophos-
phate group (Figure 1, top). This modification should mimic
key features of the acceptor, while augmenting its hydrolytic
stability and synthetic accessibility.9 The impetus for testing
these surrogates arose from our investigation of galactan
biosynthesis.
The galactan, a critical component of the mycobacterial cell
wall, is composed of galactofuranose (Galf), the energetically
disfavored 5-membered ring isomer of galactose.10,11 Galf has
not been identified in any mammalian glycans, rendering the
enzymes that mediate Galf incorporation potential antimyco-
bacterial targets.12,13 The essential14 Mycobacterium tuberculosis
gene Rv3782 encodes a protein termed GlfT1. GlfT1 appears to
be a galactofuranosyltransferase that primes galactan assembly.
Its assigned role is to promote the elongation of decaprenyl-
linked L-Rha-α-(1,3)-D-GlcNAc pyrophosphate 1 by one to
three Galf residues (Figure 1, bottom).15 The resulting lipid-
linked oligosaccharide is a substrate for the related galactofur-
anosyltransferase GlfT2.13 GlfT2 can process synthetic
substrates bearing even a single galactofuranose residue.16 In
principle, therefore, GlfT1 needs only to catalyze the addition
of one Galf residue to its substrate (Figure 1). Still, experiments
with membrane extracts or cell lysates suggested that GlfT1
phosphonate (blue) and (2Z,6Z)-farnesyl lipid affords surrogate 2.
Bottom: The product of GlfT1 (denoted with the question mark) is
elongated by GlfT2 to afford the mycobacterial galactan (n = 10−20).
could catalyze the addition of multiple Galf residues.15,17,18 We
sought to assess the enzyme products under chemically defined
conditions, which required obtaining active GlfT1 and a
suitable acceptor.
The predicted GlfT1 acceptor 1 is a demanding synthetic
target; therefore, we considered which of its features would be
required for function. An analysis of literature suggested that
the pyrophosphate group would be important.19−21 Consistent
with this analysis, we found that O-alkyl disaccharides lacking a
pyrophosphate linkage, such as 12-phenoxydodec-2-enyl Rha-
α-(1,3)-GlcNAc S6, were not elongated by purified, recombi-
nant GlfT1.22 Attempts to synthesize substrates analogous to 1
were complicated by the lability of the allylic pyrophosphate.
We therefore considered a modification that would increase the
stability of intermediates en route to the target glycosyl
acceptors. Phosphonophosphates can serve as pyrophosphate
analogues, and their increased stability suggested that they
could be more readily synthesized. We therefore targeted
surrogate 2 (Figure 1, top) with the goal of ascertaining
whether this compound could function as a GlfT1 acceptor.
Received: January 20, 2014
© XXXX American Chemical Society
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a
Scheme 1. Synthesis of GlfT1 Acceptor Surrogate 2
a
The convergent route shown was also used to prepare 8 and 9.
In selecting compound 2, we recognized the potential risk of
generating a phosphonophosphate that is not isosteric with the
pyrophosphate. Nevertheless, this risk by the increased
synthetic accessibility of compound 2 and because enzymes,
such as farnesyltransferases, bind lipid-substituted pyrophos-
phate and truncated phosphonophosphate derivatives sim-
ilarly.23 Because the related glycosyltransferase GlfT2 is
influenced by the acceptor lipid substituent,13 our synthetic
route was designed to access acceptors with diverse lipid groups
(Scheme 1). The decaprenol substituent was replaced with a
(2Z,6Z)-farnesyl group, as this shorter lipid substituent can
simplify substrate handling.24 Glycosyltransferases can be
sensitive to the polyprenol lipid alkene and its geometry,4,7,25
so we preserved the isoprenyl alkene geometry closest to the
oligosaccharide. We reasoned that if compound 2 was inactive,
we could modify our route to identify a suitable substrate.
The route to acceptor surrogate 2 began with the
glycosylation of compounds 3 and 4. The stereochemical
outcome of the glycosylation was controlled by using the α-
selective silylated L-rhamnosyl donor 3.26 Activation of this
“super armed” donor27 with N-iodosuccinimide and silver
trifluoromethanesulfonate provided the desired α-glycoside.
The silyl protecting groups were exchanged for acetate groups,
as unmasking the former late in the synthetic route was
problematic. This protecting group change facilitated purifica-
tion and characterization of the acylated intermediates.
bearing a C10 isoprenyl (neryl) lipid carrier as well as surrogate
9 bearing only a monosaccharide were generated. The stability
of the allylic phosphonophosphate derivatives facilitated their
synthesis and subsequent analysis.
The ability of the acceptor surrogates to serve as substrates
was tested using purified GlfT1. Recombinant His6-tagged
Mycobacterium smegmatis GlfT1 was produced in M. smegmatis
mc2155 and isolated by affinity chromatography. Exposure of
disaccharide 2 to GlfT1 in the presence of donor sugar UDP-
Galf31 afforded oligosaccharide products (Figure 2), indicating
The α-phosphoryl group was installed using a phosphityla-
tion−oxidation sequence.28 Hydrogenolysis afforded the
anomeric lactol, which was exposed to dibenzyl N,N-
diisopropylphosphoramidite in the presence of 1H-tetrazole.29
The intermediate glycosyl phosphite was oxidized in situ to
yield a phosphotriester. The benzyl protecting groups were
removed by reductive hydrogenation in the presence of an
amine base to afford phosphomonoester 6 in good yield, with
9:1 α:β selectivity favoring the desired anomer.
A critical transformation was joining disaccharide 6 with lipid
7 to form the phosphonophosphate. Activation of the
phosphoryl group of either coupling partner through formation
of the phosphoryl chloride or phosphoryl imidazolide could
result in self-coupling side products or product instability under
the reaction conditions. Ultimately, Moffatt−Khorana con-
ditions30 were effective. The electrophilic component, C15
isoprenyl phosphonomorpholidate 7, was prepared in four
steps from (2Z,6Z)-farnesol (see Supporting Information).
Disaccharide 6 and morpholidate 7 were exposed to 1H-
tetrazole, and the product was saponified to afford
phosphonophosphate 2. Though the yield was modest, the
transformation was highly reproducible. We exploited this
convergent synthetic route to produce acceptors to probe the
structural features required for enzymatic activity. Surrogate 8
Figure 2. Top: Representative MALDI-TOF mass spectrum obtained
from a reaction mixture of compound 2, UDP-Galf, and GlfT1. Masses
corresponding to +2 and +3 Galf residues were observed. Bottom:
Corresponding +2 and +3 products from elongation of acceptor 2.
The linkage pattern shown is in agreement with that of endogenous
galactan (see ref 10).
that compound 2 is an effective substrate. Mass spectrometry
analysis indicated that the major product is extension of the
acceptor by +2 Galf units. NMR data for the isolated product
were consistent with those of a related tetrasaccharide32 (see
Supporting Information). A product extended by +3 Galf units
is also observed. GlfT1 consumes nearly all of the acceptor to
produce these oligosaccharides.
The +1 product was not observed. This result is consistent
with processing of the natural substrate by microsomal
preparations of GlfT1.17 These results indicate that the
phosphonophosphate acceptor emulates the natural substrate.
The absence of the +1 product is notable given that the
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Communication
polymerase GlfT2 can elongate an acceptor with a single Galf
residue.16 We postulate that the ability of GlfT2 to generate a
polymer with faithfully alternating β-(1,5) and β-(1,6) linkages
arises from GlfT1-catalyzed formation of the +2 Galf
disaccharide. In this way, GlfT1 sets the register for
polymerization by GlfT2.13,33,34 The +2 disaccharide product
may also lead to more efficient polymerization by GlfT2. We
previously showed that GlfT2 exhibits a kinetic lag phase when
polymerizing a lipid-linked Galf disaccharide.13 The lag phase
was abrogated with a substrate bearing a Galf tetrasaccharide, as
this oligosaccharide presumably fills the monomer subsites
during polymerization.33 Thus, the tetrasaccharide product
generated by the action of GlfT1 should be processed rapidly
by GlfT2.
The identity of the lipid carrier can affect a substrate’s ability
to serve as a glycosyl acceptor. We therefore analyzed the
efficiency of elongation of substrates bearing different lipids by
quantifying the amount of UDP released upon GlfT1 addition.
Specifically, the assay employed couples UDP production to the
luciferase/luciferin reaction, wherein UDP production by GlfT1
is related linearly to increases in luminescence (Figure 3, top).35
data highlight their distinct roles in galactan assembly.
Specifically, GlfT1 does not efficiently process O-alkyl
disaccharides, but it does elongate lipid-linked pyrophosphate
or phosphonophosphate acceptors substituted with the
disaccharide L-Rha-α-(1,3)-D-GlcNAc by two to three Galf
residues. Unlike the polymerase GlfT2, GlfT1 does not
generate longer polymers and its substrate preference appears
to be more narrowly defined. Indeed, GlfT2 is a relatively
promiscuous carbohydrate polymerase that can act on varied
truncated lipid-linked acceptors.16,36 The high specificity of
GlfT1 and its ability to append at least two Galf residues should
yield substrates that can be rapidly polymerized by GlfT2 to
afford polysaccharides of defined sequence. These data
highlight the key role of GlfT1 in controlling galactan
biosynthesis.
Our studies of GlfT1 highlight the utility of the
phosphonophosphate-containing acceptor substrates. The
substitution of a phosphonophosphate for a pyrophosphate
can facilitate the synthesis of complex glycosyltransferase
acceptors. Glycosyltransferases in prokaryotes, eukaryotes, and
archaea that act on pyrophosphate-linked acceptors are
ubiquitous, including enzymes that mediate peptidoglycan
assembly6,37 and that generate O-38,39 and N-linked5 glycans.
We anticipate that the approach described herein will facilitate
characterization of glycosyltransferase activity of the large
family of enzymes that act on pyrophosphate-containing
acceptors.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Experimental procedures and H, 31P, and 13C NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
kiessling@chem.wisc.edu
■
Figure 3. Top: Glycosyltransferase activity promotes UDP release,
which is monitored using a luminescence assay. Bottom: Relative
output of UDP-Galf turnover by GlfT1 with acceptors 2, 8, and 9.
Notes
The authors declare no competing financial interest.
As expected, no GlfT1 activity was observed with mono-
saccharide 9, highlighting that GlfT1 requires a substrate
bearing the disaccharide L-Rha-α-(1,3)-D-GlcNAc (Figure 3,
bottom). The difference between C10-linked 8 and C15-linked 2
was pronounced. GlfT1 treatment produces almost 5 times as
much UDP in the presence of acceptor 2 than with 8. These
data indicate that acceptor 2, with its longer (2Z,6Z)-farnesyl
lipid, is a superior enzyme substrate. We used this assay to
compare the kinetics of elongation by GlfT1 and determined an
apparent Km of 86 25 μM for acceptor 2 and an apparent
ACKNOWLEDGMENTS
■
This research was supported by the NIH (R01-AI063596).
M.A.M.F. thanks NIGMS for a fellowship (F31 GM101953),
V.R.A. thanks NIGMS for a postdoctoral fellowship (F32
GM084475), and V.A.K. thanks the NSF (DGE-1256259). We
thank S. A. Goueli and H. Zegzouti (Promega Co.) for reagents
(Figure 3), M. R. Levengood for preliminary GlfT1 assays, D.
A. Wesener for purified M. smegmatis mc2155 genomic DNA,
and M. S. DeCanio for assistance generating the GlfT1
expression vector.
Vmax of 1.53
0.16 μM/min. We previously found that the
lipid substituent is important for the binding and processing of
acceptors by GlfT2,13 and our results suggest the lipid is also an
important component for GlfT1 acceptor substrates.
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