Chemoenzymatic Synthesis of C6-Modified Sugar Nucleotides to Probe the GDP- d -Mannose Dehydrogenase from Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acs.orglett.9b00967

The chemoenzymatic synthesis of a series of C6-modified GDP-d-Man sugar nucleotides is described. This provides the first structure-function tools for the GDP-d-ManA producing GDP-d-mannose dehydrogenase (GMD) from Pseudomonas aeruginosa. Using a common C6 aldehyde functionalization strategy, chemical synthesis introduces deuterium enrichment, alongside one-carbon homologation at C6 for a series of mannose 1-phosphates. These materials are shown to be substrates for the GDP-mannose pyrophosphorylase from Salmonella enterica, delivering the required toolbox of modified GDP-d-Mans. C6-CH₃ modified sugar-nucleotides are capable of reversibly preventing GDP-ManA production by GMD. The ketone product from oxidation of a C6-CH₃ modified analogue is identified by high-resolution mass spectrometry.

Full text of DOI:10.1021/acs.orglett.9b00967

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Chemoenzymatic Synthesis of C6-Modified Sugar Nucleotides To
Probe the GDP‑D‑Mannose Dehydrogenase from Pseudomonas
aeruginosa
Sanaz Ahmadipour,† Giulia Pergolizzi,‡ Martin Rejzek,‡ Robert A. Field,‡ and Gavin J. Miller*,†
†Lennard-Jones Laboratory, School of Chemical and Physical Sciences, Keele University, Keele, Staffordshire ST5 5BG, United
Kingdom
‡Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom
S
* Supporting Information
ABSTRACT: The chemoenzymatic synthesis of a series of C6-modified GDP-D-Man sugar nucleotides is described. This
provides the first structure−function tools for the GDP-D-ManA producing GDP-D-mannose dehydrogenase (GMD) from
Pseudomonas aeruginosa. Using a common C6 aldehyde functionalization strategy, chemical synthesis introduces deuterium
enrichment, alongside one-carbon homologation at C6 for a series of mannose 1-phosphates. These materials are shown to be
substrates for the GDP-mannose pyrophosphorylase from Salmonella enterica, delivering the required toolbox of modified GDP-
D-Mans. C6-CH3 modified sugar-nucleotides are capable of reversibly preventing GDP-ManA production by GMD. The ketone
product from oxidation of a C6-CH3 modified analogue is identified by high-resolution mass spectrometry.
lginate constitutes the major component of the
Aexopolysaccharide coating within mucoidal Pseudomonas
aeruginosa infections, which are particularly deleterious for
cystic fibrosis patients,1 contributing to the biofilm environ-
ment and augmenting the antibiotic resistance profile of the
bacterium. The molecular genetics and regulation of alginate
biosynthesis2 have been well-characterized in this human
pathogen.3 However, the biological chemistry of the under-
pinning biosynthetic polymer production and modification
processes is still insufficiently understood. Pivotal to this
biosynthetic pathway is GDP-D-mannose dehydrogenase
Figure 1. (a) Oxidation of GDP-D-Man 1 to GDP-D-ManA 2, which
(GMD), which is the enzyme required to form the alginate
is the feedstock sugar nucleotide for alginate biosynthesis. R= H or
Ac. (b) Proposed oxidative conversion of 1 to 2, using a key active site
cysteine residue.
sugar nucleotide feedstock, GDP-D-ManA (Figure 1a).4,5
Since there is no corresponding enzyme in humans, specific
inhibition of GMD could be envisaged as a tactic to stop
alginate production and interfere with biofilm formation in
residue, with the oxidation of C6 aldehyde 3 to thioester 5
(intermediated by thiohemiacetal 4), followed by hydrolysis to
give 2.
chronic mucoid P. aeruginosa infections. GMD is a member of
a small group of NAD+-dependent four-electron-transfer
dehydrogenases, which includes UDP-glucose dehydrogenase
(UGD),6,7 and converts GDP-D-Man 1 to GDP-D-ManA 2.
The GMD-catalyzed oxidation step is proposed to have four
discrete steps (Figure 1b), coordinated by an active site Cys268
Received: March 19, 2019
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.9b00967
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In order to investigate this mechanism of action for GMD
and to lay a foundation for the development of potential
inhibitors, we report herein the design, synthesis, and
preliminary evaluation of a series of C6-modified GDP-D-
Man tools (Figure 2). Modified sugar nucleotide analogues
Scheme 1. Synthesis of Diastereomerically Pure C6-Methyl
Mannose 1-Phosphates 9 and 10 from Common Aldehyde
Precursor 6
Figure 2. Strategy to GDP-D-Man tools, modified at C6 from a
common aldehyde intermediate.
have valuable potential as probes to study glycosyltransferases
and other enzymes that use these activated glycosylating agents
as substrates.8,9 Therefore, the synthesis (both enzymatic and
chemical) of natural and non-natural sugar nucleotides is a
topic of continuing interest and challenge.10,11
separated into amounts of each required anomeric 1-phosphate
at a later stage.
Mixture 7 proved difficult to separate by silica gel
chromatography, and, therefore, we opted to continue a single
synthetic route toward 9 and 10, first completing benzylation
of the C6-OH. Following this reaction, the C6 stereochemistry
We selected targets with either deuterium or small alkyl
group (Me) modifications at C6 of the parent pyranose ring to
enable: (i) probing of the GMD active site space for non-native
substrate binding, (ii) establishing evidence for a ketone or
thiohemiketal intermediate in the oxidation mechanism (in
place of 3 or 4), and (iii) capability to assess the
diastereoselectivity of proton abstraction during the oxidation
of 1 to 3. D-Mannose systems diastereoselectively deuterated at
C6 have previously provided important chemical tools,
illustrated by the synthesis of ADP-[6″-D]-D,D-Hep for
elucidating the mechanism of ADP-L-glycero-D-manno-heptose
6-epimerase.12 Furthermore, pyranose C6 homologation using
small alkyl groups has provided important mechanistic probes
for the study of UDP-glucose dehydrogenase, which is the
enzyme responsible for the oxidative conversion of UDP-Glc
to UDP-GlcA.6
1
was assigned by comparison to H and 13C NMR data of
known 6S-methyl-tetrabenzyl thioglycoside reported by
Davis,14 who confirmed the stereochemistry at C6 using
nuclear Overhauser effect (nOe) analysis of a 4,6 benzylidene
derivative. Our data for the mixture of C6 diastereoisomers
showed a strong match (C1 δ 85.6 vs 85.5 ppm reported; H1 δ
5.83 (d, J = 1.7 Hz) vs 5.81 ppm reported) and showed the 6S
diastereoisomer to be the minor component in our mixture
(data for the 6R diastereoisomer was distinct from 6S (C1 δ
85.8; H1 δ 5.62 (d, J = 1.9 Hz)).
We next completed anomeric 1-phosphate installation (in
protected form) using dibenzylphosphate (DBP) as the
acceptor under thioglycoside activation conditions (NIS/
1
AgOTf) in satisfactory yield (63%, two steps). H and 31P
NMR confirmed the presence of the anomeric phosphate with
the characteristic doublet of doublets observed for H1 coupling
to H2 and 31P (3JH1−H2 = 2.0 Hz, 3JH1−P = 6.1 Hz for 10). This
approach then allowed a late-stage chromatographic resolution
of the diastereomeric material 8S/8R before a final hydro-
genolysis to deliver free anomeric phosphates 9 and 10.
The final deprotection reaction required some optimization
to align the solubility differences of the organic starting
material and the aqueous soluble glycosyl 1-phosphate. We
evaluated several alternative solvent systems, finally settling on
a 6:2:1 mixture of EtOH:THF:5% aqueous NaHCO3 (to
provide the sodium salt form of the phosphate). A catalyst
loading of 0.2 mol equivalents of 1:1 w/w Pd(OH)2/C:Pd/C
per OBn group was also established. We found that the use of
pressures above that of a balloon of hydrogen (up to 10 bar in
a Parr vessel) and increasing the temperature of the reaction
(up to 55 °C from ambient) provided no immediate increase
in the rate of completion, rather leading to mixtures of
products and incomplete hydrogenolysis, which was then
susceptible to anomeric degradation as the reaction continued.
Our optimized conditions consistently delivered the depro-
tected glycosyl 1-phosphates in good yields (vide infra).
For the synthesis of monodeuterated 6R and 6S mannose 1-
phosphate targets 16 and 17, we started from known uronate
We hypothesized that, starting from D-mannose, a suitably
protected C6 aldehyde thioglycoside donor would serve as the
common material to access a series of C6-modified glycosyl 1-
phosphates and sugar nucleotide targets (Figure 2). Therefore,
we began our synthesis from D-mannose, accessing the
required C6 aldehyde 6, following established procedures on
a multigram scale (see Figure S1 in the Supporting Information
(SI)). Using MeMgBr, we first sought to evaluate the addition
of small alkyl groups to 6 (Scheme 1), to enable one-carbon
homologation.
However, this reaction yielded significant amounts (up to
1
80% by crude H NMR) of a competing C4−C5 elimination
product. In order to reduce the basicity of the organometallic
reagent (reactions using reduced equivalents of Grignard did
not prevent the elimination), transmetalation of MeMgBr with
CeCl3 afforded the corresponding organocerium13 reagent in
situ. Subsequent treatment of 6 with this material suppressed
competing elimination and delivered the target secondary
alcohol 7 on a multigram scale as a mixture of C6
diastereoisomers (diastereomeric ratio (dr) of 4:6) in good
yield (86%). While the observed diastereoselectivity for this
nucleophilic addition was low, it was, for our intended
purposes, beneficial, because it allowed the material to be
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ester 1115 (Scheme 2), reasoning that a diastereoselective
reduction of deuterated aldehyde 13 would be an effective
13C−2H coupling of 23.1 Hz. Having completed their
stereochemical assignments, both 14 and 15 were converted
through to their respective 1-phosphates 16 and 17 using the
previously established procedures (Scheme 2). From thio-
glycoside 12, we also synthesized a doubly C6-deuterated
mannose 1-phosphate 18.
Scheme 2. Synthesis of a C6-Deuterated Mannose 1-
Phosphates 16−18
These materials, along with 9 and 10, completed a series of
regio-defined and stereo-defined mannose 1-phosphate chem-
ical tools, which were next evaluated as substrates for a
recombinant mannose pyrophosphorylase (from Salmonella
enterica18) in converting them to their respective GDP
conjugates. Glycosyl 1-phosphates 9 and 10 and 16−18 were
individually incubated with GDP-mannose pyrophosphorylase
at 37 °C. TLC monitoring of the reactions at 18 h indicated
that the majority of the glycosyl 1-phosphates had been
consumed and the reactions were stopped. The sugar
nucleotide products 19−23 were then purified by strong
anion exchange chromatography and C18 high-performance
liquid chromatography (HPLC) to deliver the final, pure
materials (see Table 1, as well as Figure S4 in the Supporting
Information).
The GDP-D-Man pyrophosphorylase showed good substrate
promiscuity toward non-native phosphates 9 and 10 and 16−
18, rapidly converting all but 6R-Me derivative 9 and delivered,
following purification, milligram quantities of the required
sugar nucleotides in good yields (53%−64%). Glycosyl 1-
phosphate 9 showed a slower conversion by TLC and HRMS
strategy and negate the higher costs and availability associated
with chiral, deuterated reducing agents.
Reduction of 11 with NaBD4 to doubly C6-deuterated
pyranose 12 proceeded in good yield (81%), furnishing
material for a multigram scale oxidation, which was completed
using the Parikh−Doering method16 to give deuteroaldehyde
13 in 92% crude yield. With 13 in hand, we completed
independent diastereoselective reductions using either (R)-
(+)-Alpine-Borane or (S)-(−)-Alpine-Borane to access the
respective 6R and 6S deuterated thioglycosides 14 and 15. To
confirm the diastereomeric identity of these alcohols, we
synthesized small amounts of their 1,6-anhydro derivatives,
which enabled nOe experiments to establish their 6-position
configuration (Figure 3).
a
Table 1. Enzymatic Synthesis of C6-Modified GDP-D-Man
Derivatives 19−23
Figure 3. Synthesis of 1,6-anhydro derivatives from 14 and 15 to
facilitate stereochemical assignment at C6 (red/blue arrows represent
the observed nOe; green arrow represents the direction of view for
Newman projection).
For the H6 endo compound, (derived from 15), we observed
an nOe from H6 to H4 (and H5) along with transfer from H4 to
H6 (see Figure S3 in the Supporting Information).
Comparatively for H6 exo (derived from 14), we observed
only transfer from H6 to H5. From the one-dimensional (1D)
1H NMR data, a very small 3J H6−5 coupling of 0.8 Hz for the
H6 endo inferred that the dihedral angle between these two
protons was approaching 90°, further supporting our assign-
ment and matching 1D NMR data reported previously by
Meguro.17 2D-HSQC data showed the expected correlation
between H6 and C6 for both diastereoisomers, with a 1J
a
See Figure S4 in the Supporting Information for the general
b
enzymatic procedure. Synthesized chemically.
C
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analyses (see Figures S4 and S6 in the Supporting
Information), even after several small-scale repeats and a
prolonged 38 h reaction time. This analogue was instead
synthesized chemically, using GMP-morpholidate, to ensure
that sufficient material could be isolated to characterize and
complete the series. The substrate scope for the phosphorylase
suggests a relaxed specificity at C6 of mannose, possibly
inferring that it is not involved in binding to the enzyme. This
is consistent with observations made by Lowary and co-
workers,18 using the same pyrophosphorylase for the synthesis
of 6-deoxy and 6-OMe GDP-D-Mans.
Sugar nucleotides 19−23 were incubated with GMD from P.
aeruginosa, and the colorimetric release of NADH was
monitored at 460 nm. Figure 4a illustrates the relative activity
of each of probes 19−23 over the first 5 min of incubation. As
expected, the two C6-homologated species, 19 and 20, showed
a reduced ability to produce NADH, compared to GDP-D-
mannose 1. Although 19 and 20 can theoretically only produce
1 equiv of NADH (compared to 2 equiv for 1), they fall short
of achieving that level of conversion. This is consistent with the
expected keto-sugar product serving as an inhibitor of GMD
activity. For the reaction of GMD with 20, we obtained direct
evidence of the ketone oxidation product via high-resolution
mass spectrometry (HRMS).
While this finding is subject to further evaluation, it is
evident that the ketone is not tightly bound: spiking 60 min
incubations that contained GMD and 19 and 20 with GDP-D-
mannose 1 led to renewed NADH production (Figure 4b).
C6-deuterated analogues 21−23 were processed by the
enzyme to a comparable level to 1, as expected: further kinetic
evaluation of these materials is underway.
In conclusion, we have developed a first series of sugar
nucleotide probes to target the GDP-D-Man dehydrogenase
from P. aeruginosa, using a chemoenzymatic approach.
Strategic chemical modifications at C6 of the pyranose ring
(deuteration and one-carbon homologation) followed by
enzymatic pyrophosphorylation were effected. Access to
these materials will enable a more-detailed mechanistic
investigation of GMD and other important biochemical
enzymes that utilize GDP-D-Man, including nucleotidylyl-
transferases, glycosyltransferases, and phosphorylases.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b00967.
1
Experimental procedures, characterization data, and H,
13C, and 31P NMR spectra for all new compounds,
alongside enzymatic methods (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: g.j.miller@keele.ac.uk.
ORCID
Gavin J. Miller: 0000-0001-6533-3306
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Engineering and Physical Research Council (EPSRC) are
thanked for project grant funding (No. EP/P000762/1) to
G.J.M. at Keele. We also thank the EPSRC UK National Mass
Spectrometry Facility (NMSF) at Swansea University, Dr.
Richard Darton (Keele) for running nOe spectra, Prof. Peter
Tipton (University of Missouri) for providing the GMD
plasmid, and Professor Todd Lowary (University of Alberta)
for providing the GDP-mannose pyrophosphorylase clone.
Work at the JIC is supported by the Biotechnology and
Biological Science Research Council (BBSRC) Institute
Strategic Program on Molecules from NatureProducts and
Pathways (No. BBS/E/J/000PR9790) and the InnovateUK
IBCatalyst [Nos. BB/M02903411 and EP/N033167/10].
Figure 4. (a) Initial rates of NADH production over 5 min for sugar
nucleotides 19−23. GMD (50 μg/mL), GDP sugars (50 μM), NAD+
(200 μM), the activity is a mean standard error of three repeats. A
negative control experiment was run with no GMD. Data are not
normalized for a maximum of 1 equiv NADH produced by 19 and 20.
(b) GMD function with probes 19 and 20 over 120 min, 50 μM
spiking with 1 at 60 min.
D
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