Zwitterionic pyrrolidene-phosphonates: Inhibitors of the glycoside hydrolase-like phosphorylase: Streptomyces coelicolor GlgEI-V279S

Article abstract of DOI:10.1039/c7ob00388a

We synthesized and evaluated new zwitterionic inhibitors against glycoside hydrolase-like phosphorylase Streptomyces coelicolor (Sco) GlgEI-V279S which plays a role in α-glucan biosynthesis. Sco GlgEI-V279S serves as a model enzyme for validated anti-Tuberculosis (TB) target Mycobacterium tuberculosis (Mtb) GlgE. Pyrrolidine inhibitors 5 and 6 were designed based on transition state considerations and incorporate a phosphonate on the pyrrolidine moiety to expand the interaction network between the inhibitor and the enzyme active site. Compounds 5 and 6 inhibited Sco GlgEI-V279S with K_i = 45 ± 4 μM and 95 ± 16 μM, respectively, and crystal structures of Sco GlgE-V279S-5 and Sco GlgE-V279S-6 were obtained at a 3.2 ? and 2.5 ? resolution, respectively.

Full text of DOI:10.1039/c7ob00388a

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Zwitterionic pyrrolidine-phosphonates: Inhibitors of the glycoside
hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S
Sri Kumar Veleti†‡, Cecile Petit†, Jared J. Lindenberger‡, Donald R. Ronning* and Steven J.
Sucheck*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We synthesized and evaluated new zwitterionic inhibitors against glycoside hydrolase-like phosphorylase Streptomyces
coelicolor (Sco) GlgEI-V279S which plays a role in α-glucan biosynthesis. Sco GlgEI-V279S serves as a model enzyme for
validated anti-tuberculosis (TB) target Mycobacterium tuberculosis (Mtb) GlgE. Pyrrolidine inhibitors 5 and 6 were designed
based on transition state considerations and incorporate a phosphonate on the pyrrolidine moiety to expand the interaction
network between the inhibitor and the enzyme active site. Compounds 5 and 6 inhibited Sco GlgEI-V279S with Ki = 45 ± 4
µM and 95 ± 16 µM, respectively, and crystal structures of Sco GlgE-V279S-5 and Sco GlgE-V279S-6 were obtained at a 3.2
Å and 2.5 Å resolution, respectively.
www.rsc.org/
V279S variant diffract to higher resolution in comparison to Mtb
GlgE.11, 12 Therefore, we have used Sco GlgEI-V279S to evaluate
substrate analogues13, 14 and transition state-like inhibitors.15
Introduction
Tuberculosis (TB) is caused by the bacterium Mycobacterium
tuberculosis (Mtb). TB kills more people every year than any
other infectious disease. The World Health Organization
reported that in 2015 about 10.4 million individuals fell ill and
1.4 million deaths resulted from TB.1 In order to eradicate TB,
there is a need to identify new compounds and drug targets
Sco GlgEI is a phosphorylase that catalyzes a reversible
glycosyl transfer from a saccharide donor substrate to
17
phosphate.16,
The mechanism is a double displacement
mechanism consisting of two inverting steps with an
intermediate β-glycosyl enzyme intermediate.10, 18 During the
first glycosylation step, the acid/base E423 side chain
protonates the glycosidic oxygen. Protonation facilitates
phosphate leaving group departure and, at the same time, the
nucleophile D394 attacks at the anomeric carbon leading to the
formation of a covalent β-glycosyl-enzyme intermediate.11 In
the second step, the acid/base residue deprotonates a
nucleophile to attack at the anomeric carbon with positive
charge accumulation on the anomeric carbon and endocyclic
oxygen. The D394 nucleophile has been unambiguously
assigned by trapping studies.10 The release of phosphate in the
mechanism is facilitated by E423, which deprotonates the
incoming acceptor α-1,4-glucan.10
which act faster and show activity in drug resistant Mtb strains.2,
3
Kalscheuer et al. have reported a four-step pathway in Mtb that
involves conversion of trehalose to α-1,6-branched-α-1,4-
glucan.4-7 The third step is catalyzed by GlgE which is an α-
maltose1-phosphate
(M1P):(1→4)-α-D-glucan
4-α-D-
maltosyltransferase (EC2.4.99.16)4, 5 and is a member of the
CAZy glycoside hydrolase (GH)-like phosphorylase GH13_3
family.8 Deletion of GlgE leads to rapid buildup of toxic levels of
M1P which elicits pleiotropic stress in the cell leading to cell
death in a matter of two weeks.4 This data combined with the
fact that there is no equivalent enzyme in the human proteome
suggests GlgE may be an advantageous anti-TB drug target.9
Streptomyces coelicolor GlgE isoform I (Sco GlgEI) is a close
structural homologue of Mtb GlgE possessing 53 % sequence
identity with the Mtb H37Rv GlgE.10-12 Through creation of the
Sco GlgEI-V279S variant, there is 100 % identity in the active site
residues of these two homologues, and crystals of the Sco GlgEI-
Department of Chemistry and Biochemistry and School of Green Chemistry and
Engineering, The University of Toledo, 2801 W. Bancroft Street, Toledo, Ohio
43606, United States. donald.ronning@utoledo.edu, steve.sucheck@utoledo.edu
† Contributed equally to the manuscript
‡ Current Addresses: S.K.V.: National Institute of Allergy and Infectious Diseases 33
North Dr. Bethesda, MD 20814. J.J.L.: Center for Retrovirus Research and College of
Pharmacy, The Ohio State University, Columbus, Ohio 43210
Figure 1 Proposed transition state for the first step in the
mechanism of GH-like phosphorylase Sco GlgEI (left). Designed
zwitterionic pyrrolidine-phosphonates based on transition state
considerations (right). n = 1 or 2.
Electronic Supplementary Information (ESI) available: 1H and 13C NMR spectra for
new compounds (10, 15, 16, 17, 5 and 6), data collection and refinement statistics.
See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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In the first step of the reaction, the transition-state involves studies, we explore introduction of a phosphoVnieawteArtioclenOntlhinee
protonation and charge accumulation on the anomeric nitrogen of compounds (
) to mimicDtOhIe: 10r.i1n0g39c/Ch7aOrgBe0,03r8in8Ag
5-6
exocyclic oxygen. As the carboxylate of D394 attacks the geometry and departing phosphate charge and enzyme
anomeric carbon the atom rehybridizes with sp2 character stabilizing contacts that can develop in the transition state. The
between the anomeric carbon and endocyclic oxygen. These spacing between the nitrogen and phosphate was varied to
geometric requirements distort the pyranose ring from a explore optimal charge separation. The work is also relevant to
4
ground state chair conformation to a strained H3 half chair N-phosphonate analogues which have shown activity against
conformation.10, 19 The GH13 family also has a second conserved bacterial transglycosylases.42 In addition, we compare our
aspartate residue (D480 for GlgEI). This residue is postulated to results to the X-ray structure of Sco GlgEI-V279S bound to MCP
form hydrogen bonds with the C-2 and C-3 hydroxyl groups in (Figure 2) which was designed as a non-hydrolysable mimic of
the transition state.20-22 The proposed interactions, charges, M1P.12, 13
and conformations for the first transition state are illustrated in
Figure 1.
Results and discussion
It has been postulated that GHs bind transition states with
extraordinary affinity23, 24 and there is now an extensive body of
The plan for the synthesis of pyrrolidine-based phosphonates
and was to start with previously prepared polyhydroxlyated
pyrrolidine ( ), shown in Figure 2. We envisioned subjecting
5
literature on inhibitors that are suggested to mimic GH
transition states.25-28 In these studies, we prepared iminosugars
that are protonated at physiological pH and mimic the positive
charge that develops on the anomeric carbon and endocyclic
oxygen expected for a late transition state. This is in contrast to
early transition state GH inhibitors which mimic the initial
protonation of the leaving group oxygen.29 The 6-membered
polyhydroxylated piperidine iminosugars, represented by
nojirimycin 130 and 1-deoxynojirimycin 231 are classical GH
inhibitors.32-34 These compounds mimic the ring size of the
substrate and the charge that develops in a late transition state
that is stabilized by the nucleophile in the active site.
Polyhydroxylated pyrrolidines also show potent GH inhibitory
activity. Fleet et al. prepared 1,4-dideoxy-l,4-imino-D-mannitol
6
4
pyrrolidine 415 to a Kabachnik-Fields reaction43 to access the
one carbon phosphonate. We also considered the use of an aza-
Micheal reaction44 with compound 10 or possibly a reductive
amination42 with compound 15 to access the two carbon
homologues (Scheme 1).
To prepare the required phosphonate building blocks,
available diethyl vinylphosphonate
phosphonate 11 were deprotected with TMSBr to afford the
phosphonic acid and the phosphonic acid 12, respectively
(Scheme 1). Both and 12 were converted to the chloride
derivative and subjected to benzylation with benzyl alcohol to
afford dibenzyl vinylphosphonate 10 and dibenzyl
(7
)
and available
8
8
(
)
(DIM)
3 in 1984, which is the first example of this type of
allylphosphonate (14),45 respectively. The functional group
manipulation provided benzyl protected phosphonates for a
planned single step global deprotection. Ozonolysis of alkene 14
provided aldehyde 15, which could be used directly without any
further purification.
inhibitor.35 This initial work has been followed by many related
examples.36-41 The pyrrolidines mimic both the shape and
charge of the half-chair transition state (Figure 1). Previously,
we designed 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-
mannitol (4) as a transition state-like mimic of Sco GlgEI-V279S
based on this knowledge.15 The pyrrolidine
inhibitory activity, which afforded the X-ray crystallographic
studies with bound in the catalytic site of Sco GlgEI-V279S and
4 had moderate
4
showing the nitrogen atom within hydrogen bonding distance
of the D394 carboxylate.12 In the current
Scheme 1 Synthesis of zwitterionic pyrrolidine-phosphonates.
Reagents and conditions: (a) TMSBr, CH2Cl2; (b) oxalyl chloride,
cat. DMF, 6 h, DCM; (c). pyridine, BnOH, THF (over all 53%); (d)
ozonolysis, DCM; (e) HP(O)(OBn)2, CH2O, 60 °C (56%); (f) 10%
Pd(OH)2 on carbon, MeOH, quantitative; (g) Et3N, H2O. (h)
MeOH, NaCNBH3.
Figure
hydrolases (1-3) and designed inhibitors of Sco GlgEI-V279S
MCP 4-6).
2 Natural and designed inhibitors for glycoside
(
,
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Figure 4 A) Sco GlgEI-V279S in complex with
calculated while omitting is contoured at 3σ with
the enzyme active site. B) Sco GlgEI-V279S in complex with
Fo-Fc omit map calculated while omitting is contoured at 3σ
with bound in the enzyme active site. The red spheres
5
. Fo-Fc omit map
5
5
bound in
6
.
6
6
represent water molecules. -1 and -2 subsites are labeled.
Table S1). Despite being resolved at a 3.2 Å resolution, the Fo-
Fc omit map of inhibitor
compound in the active site (Figure 4A). The Fo-Fc omit map of
inhibitor , however, lacks strong density for the ethylene linker
5 indicated the presence of the
6
resulting in poor resolution of the phosphonate moiety (Figure
4B). The high B factors of the ethyl phosphonate moiety reflects
this and, taken together, suggest only weak interactions
between the ethyl phosphonate moiety and the enzyme active
site. An ordered water molecule, however, is observed in both
structures at the same position, leading to the entrance of the
active site and 3.3 Å and 4.0 Å away from the phosphonate
Figure 3. A. Ki determination of
determination of
rates with and without inhibitor. Error bars are calculated from
the result of reactions performed in triplicate.
5
with Sco GlgEI-V279S. B. Ki
i
6
with Sco GlgEI-V279S. Vo /Vo are steady-state
moiety of the inhibitors in ScoGlgEI-V279S-
V279S- complexes, respectively.
5 and ScoGlgEI-
6
Looking at the interactions between the enzyme and the
compounds, all the interactions previously seen in the -2 subsite
are conserved in both structures.12 Despite a slight shift of
Thus, compound
4 was subjected to Kabachnik-Fields
reaction43 by aqueous formaldehyde treatment followed by
addition of dibenzyl phosphite at reflux to yield 16 as shown in
Scheme 1.42 Compound 16 was globally debenzylated by
treating with 10% Pd(OH)2/C-H2 in methanol to afford the target
inhibitor 5, all five hydrogen bonded interactions previously
reported in the -1 subsite between maltose-C-phosphate and
the enzyme are retained.12 The shift resulted, however, in a
weakened ionic interaction between the nitrogen of the
pyrrolidine ring and the nucleophile D394 (Figure 5A and 5B).
molecule
aminophosphonates by an aza-Micheal reaction.44 While our
attempts to synthesize using this approach in combination
with vinylphosphonate 10 were unsuccessful, subjecting
5. Odinets et al. have reported the synthesis of β-
6
Comparatively, the complex ScoGlgEI-V279S-6 features a shift in
the 5-hydroxymethyl of the pyrrolidine ring toward the D480
resulting in a strong bidentate interaction between the side
chain of D480 and the pyrrolidine ring (Figure 5C and 5D), an
interaction that has been reported to be characteristic of GH13
family proteins.20-22 The shift also allowed for the ionic
interaction between the nitrogen of the pyrrolidine ring and the
nucleophile D394 to be retained. Additionally, the ethylene
linker displaced the phosphonate moiety resulting in a loss of
interactions between it and the side chains of N395 and E423
allowing for the formation of a new ionic interaction between
the carbonyl oxygen of the N395 side chain and the nitrogen of
the pyrrolidine ring. E423 further stabilizes this new interaction
by forming a hydrogen bonded network between its side chain
and the N395 side chain and backbone nitrogen. The loss of
these two hydrogen bonded interactions with the phosphonate
confirms its destabilization, further highlighted by the lack of
density of the side chains of E423, Y357 and K355 with distances
of 2.9 Å and 3.7 Å between the
molecule 4 to NaCNBH3 in the presence of aldehyde 15 yielded
alkylated amine 17 in 56% yield,42 which was cleanly
debenzylated using 10% Pd(OH)2/C-H2 in methanol.
Inhibition studies on ScoGlgEI-V279S were performed by
coupling GlgEI phosphorylase activity on M1P to a purine
nucleoside phosphorylase (PNP) activity as previously
described.13, 46 Using this assay (Figure 3), we determined the Ki
for compound 5 and 6 against Sco GlgEI-V279S to be Ki = 45 ± 4
µM and Ki = 95 ± 16 µM, respectively. Thus, the one carbon
phosphonate
phosphonate
5
was 2-fold more potent than the two carbon
6
. The one carbon phosphonate was also 2-fold
5
more potent than our previously reported transition state-like
inhibitor
Ki = 102 ± 7 μM.15 In spite of this improvement, we
4
conclude that both of the new targets are suboptimal transition
state mimics based on μM affinity.
Compounds
5 and 6 were co-crystallized with Sco GlgEI-
V279S and X-ray diffraction studies produced structures to 3.2
Å and 2.5 Å resolution, respectively (Supporting Information,
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Figure 5 A) Interactions between Sco GlgEI-V279S and the
pyrrolidine ring of . B) Interactions between Sco GlgEI-V279S
and the phosphonate moiety of . C) Interactions between Sco
GlgEI-V279S and the pyrrolidine ring of . D) Interactions
between Sco GlgE-V279S and the phosphonate moiety of
Both red spheres in panel B and D represent water molecules.
5
Figure 6 A) Superimposed active site of Sco GlgEI-V279S in
5
complex with
of Sco GlgEI-V279S in complex with MCP (PDB: 4U31) and
Superimposed active site of Sco GlgEI-V279S- and Sco GlgEI-
E423A-maltoheptaose (PDB: 5CVS). B) Superimposed active site
of Sco GlgEI-V279S- and Sco GlgEI-E423A-maltoheptaose (PDB:
5CVS). The red spheres in panel C) and D) are water molecules.
4
(PDB: 4U2Y) and
5
. B) Superimposed active site
6
6
. C)
6
.
5
6
phosphonate and Y357 and K355, respectively, versus 2.6 Å and
3.4 Å in the ScoGlgEI-V279S- complex.
When comparing ScoGlgEI-V279S-
(Figure 6A) and Sco GlgEI-V279S- and Sco GlgEI-V279S-MCP
5
4
and Sco GlgEI-V279S-5
As previously noted, the pyrrolidine-based inhibitor
4
6
mimics the oxocarbenium transition state and the half-chair
conformation with its 5-membered ring and positively charged
(Figure 6B), a shift of the side chains of N352, Y357 and K355
can be observed. Similarly, N352 displayed complete lack of
nitrogen. Compound
4
had a Ki = 102 ± 7 μM.15 To improve the
density at 1 σ in Sco GlgEI-V279S-
assumes the withdrawn position it takes in both Sco GlgEI-
V279S- and Sco GlgEI-V279S-maltoheptaose (PDB: 5CVS) to
5 suggesting that N352
affinity of our inhibitor the active site, a phosphonate moiety
was added to emulate the first transition state of the SN2
mechanism previously proposed by the literature or an early
dissociative SN1-like transition state. The transition state was
6
accommodate the substrate without interacting with either the
phosphonate or acceptor molecule. Additionally, the presence
of maltoheptaose unraveled the need of the glucose in the -1
and -2 subsite to be shifted toward the entrance of the active
site, which resulted in residues Y357, N395 and K355 also
shifting (Figure 6C and 6D). Specifically, Y357 is displaced by 1 Å
to interact with both the endocyclic and hydroxymethyl oxygens
of the glucose molecule bound in the +1 subsite instead of the
mimicked using a methylene linker between
phosphonate moiety. The Ki dropped to 45 ± 4 µM, which is a 2-
fold improvement over . Despite the modest resolution, 3.2 Å,
of the determined X-ray crystal structure, inhibitor was clearly
4 and the amended
4
5
resolved in the active site (Figure 4A) and all hydrogen bonded
interactions previously observed in both the -2 and -1 subsite of
Sco GlgEI-V279S-
4 were conserved with a weakened ionic
phosphonate in ScoGlgEI-V279S-
5 and Sco GlgEI-V279S-6 or the
bridging oxygen between the -1 and +1 subsite. Finally, the side
chain of N395, while interacting with the phosphonate moiety
interaction between E394 and the pyrrolidine ring. (Figure 5A
and 5B). The additional interactions between the enzyme and
phosphonate of
5 are thought to be responsible for the
of 5 and the nitrogen of the pyrrolidine ring of 6, is rotated 180°
improvement in the Ki. It is noteworthy to mention that out of
all 5 hydrogen bonded interactions observed, three side chains,
K355, N352 and Y357 exhibit poor density at 1 σ, which suggests
that despite the presence of the phosphonate moiety, the
entrance in the active site remains dynamic, likely to
as to allow its carbonyl oxygen to interact with the O3’ of the
glucose located in the +1 subsite and its amine with the
endocyclic oxygen of the glucose in the -1 subsite. Also of
interest, the water molecule consistently observed guarding the
entrance of the active site is positioned at the same place as the
methylhydroxyl of the glucose positioned in the +2 subsite. It
has been shown that, when designing an oxocarbenium
intermediate inhibitor, extending it further out of the active site
to block the entrance, results in more potent inhibitors.47
accommodate the incoming
Sco GlgE-V279S- complex showed a complete lack of
α-glucan acceptor molecule.
6
density for the ethylene linker between the phosphonate and
pyrolidine ring (Figure 4B), suggesting high dynamicity of the
linker, resulting in poor density of the phosphonate. When
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looking at the interactions between
interactions between
6
and the active site, all better inhibitor when compared to previously reported
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DOI: 10.1039/C7OB00388A
4
and the active site were conserved, inhibitors. The data from these ongoing studies provides
including the strong ionic interaction between D394 and the valuable insight into the development of even more potent
pyrrolidine ring (Figure 5C). However, only two weak phosphorylase inhibitors.
interactions were observed between the phosphonate and the
side chains of K355 and Y357, which exhibit no density at 1σ.
The high dynamicity and minimal interactions between the
Experimental
enzyme and the phosphonate are likely the reason for the
higher Ki of 95 ± 16 µM, showing little to no improvement
General methods
All fine chemicals such as 10% palladium on activated carbon,
diethyl allylphosphonate, diethyl vinylphosphonate,
bromotrimethylsilane, benzyl alcohol, pyridine, 37%
compared to the Ki of our previous inhibitor, 4. These results
may suggest that the enzyme is not stabilizing the phosphate as
it dissociates and leaves the active site. Since the side chains of
K355 and Y357 showed high dynamicity in these structures,
these residues are suggested to guide the acceptor molecule
into the active site allowing for E423 to act as nucleophile. The
presence of a water molecule positioned consistently at the
entrance of the active site and where the hydroxymethyl of the
glucose positioned in the +2 subsite of Sco GlgE-E423A-
maltoheptaose strengthens this hypothesis (Figure 6C and 6D).
The side chain of K355 consistently exhibits no density in the
previously mentioned complexes, we therefore postulate that
it is guiding acceptor substrate molecules and products in and
out of the active site, alternatively opening and capping the
active site. Using these new data, the next generation of our
inhibitors could exploit newly observed hydrogen bonding
formaldehyde in water, dibenzyl phosphite, benzoyl cyanide, N-
iodosuccinamide, silver trifluoromethanesulfonate
and
anhydrous solvents such as anhydrous methanol and N,N-
dimethylformamide were purchased and used without
purification. TLCs (silica gel 60, f254) were visualized under UV
light or by charring (5% H2SO4 – MeOH). Flash column
chromatography was performed on silica gel (230-400 mesh)
using solvents as received. 1H NMR were recorded either on 600
MHz spectrometer in CDCl3 using residual CHCl3 as internal
references, respectively. 13C NMR were recorded on the 150
MHz in CDCl3 using the triplet centered at δ 77.27 as internal
reference. GCOSY method was used to confirm the NMR peak
assignment. High resolution mass spectrometry (HRMS) was
performed on a micro mass Q-TOF2 instrument. An EnzChek®
Phosphate Assay Kit was purchased for use in the Sco GlgEI-
V279S inhibition assay.
interactions in the complexes made with
5, 6 and the
maltoheptaose aiming for tighter binding. This would be
particularly important when extending inhibitors beyond the
active site, which has been shown to improve affinity and
selectivity in similar enzyme systems.47 Exploiting the
interactions between the consistently observed water molecule
as well as the maltoheptaose and the enzyme could be used to
improve upon our inhibitors. Another option would be to
investigate the array of sp2 and sp3 hybridization alterations that
the anomeric carbon undergoes during the reaction29 as well as
mimicking the protonation of the exocyclic oxygen during the
first step of the reaction by using a six-membered ring
possessing a substituted nitrogen in place of the exocyclic
oxygen.
Dibenzyl vinylphosphonate (10).48 To a stirred solution of
7
(1.60 g, 9.74 mmol) in dichloromethane (20.0 mL) was added
bromotrimethylsilane (5.30 mL, 40.0 mmol) at room
temperature. The mixture was stirred for 9 h at the same
temperature and then concentrated to afford a residue. To an
ice-cooled, stirred solution of this residue in dichloromethane
(20.0 mL) was slowly added oxalyl chloride (3.00 mL, 35.0 mmol)
and DMF (a few drops). After being stirred for 6 h at room
temperature, the mixture was concentrated to give a residue.
To a stirred solution of this residue in tetrahydrofuran (30.0 mL)
was added benzyl alcohol (2.30 mL, 22.0 mmol) and pyridine
(1.80 mL, 22.0 mmol) at -21oC. After the mixture was stirred for
30 min at the same temperature, stirring was continued for 6 h
at room temperature. The mixture was poured into saturated
aqueous potassium bisulfate and extracted with ethyl acetate.
The combined extracts were washed with brine and dried over
magnesium sulfate. Removal of the solvent gave a residue,
which was purified by flash column chromatography
(hexane/ethyl acetate = 10:1-1:1) to give 10 as pale yellow oil;
yield: 53% (1.50 g); silica gel TLC Rf = 0.3 (1:1 ethyl acetate-
hexanes); 1H NMR (CDCl3, 600 MHz): δ 4.95 (m, 4H, -CH2Ph),
5.97-6.25 (m, 3H, Alkene H’s), 7.23-7.26 (m, 10H, Aromatic H’s);
13C NMR (150 MHz, CDCl3): δ 67.4, 67.4, 124.9, 126.1, 127.9,
128.4, 128.6, 136.0, 136.1, 136.2 ppm; 31P NMR (162 MHz,
CDCl3): δ 19.3 ppm. Mass spectrum (ESI-MS), m/z = 311.2
(M+Na)+, C16H17NaO3P requires 311.27. Data matches
reference.
Conclusions
Our initial inhibitor, compound
with a Ki of 102 ± 7 μM. Based on our previously reported crystal
structure, the synthesized zwitterionic pyrrolidine-
4 exhibited enzyme inhibition
phosphonates inhibitors were designed to better fill the
available space between the nitrogen atom of the inhibitor and
E423. Firstly, the tertiary ammonium moiety was anticipated to
be protonated at physiologic pH, hence mimicking the partial
positive charge that can develop at the anomeric carbon in
either of the two transition states. Secondly, the phosphonate
moiety provided additional enzyme interactions that could be
present in the first transition state, without allowing for
nucleophilic attack due to lack of an acceptable leaving group.
The coupled effect of stabilized charge, by D394, at the
anomeric site and the additional hydrogen-bonded interactions
Dibenzyl allylphosphonate (14).45 To a stirred solution of 11
(1.60 g, 10.0 mmol) in dichloromethane (20.0 mL) was added
with the phosphonate moiety made target molecules
5 a 2-fold
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bromotrimethylsilane (5.30 mL, 40.0 mmol) at room (29 mg); yield: 76% (350 mg); [α]D23 = -14.0° (c = 1, CHCl3); silica
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1
DOI: 10.1039/C7OB00388A
temperature. The solution was stirred for 9 h and concentrated gel TLC Rf = 0.45 (3:7 ethyl acetate-hexanes); H NMR (CDCl3,
to afford residue. The residue was taken up in 600 MHz): δ 3.41-3.44 (m, 2H, H-1, H-a), 3.58-3.61 (m, 2H, H-4,
a
dichloromethane (20.0 mL) and cooled on ice. To the cold H-b), 3.63-3.68 (m, 4H, H-e, H-f, H-g, H-h), 3.70-3.77 (m, 2H, H-
solution oxalyl chloride (3.00 mL, 35.0 mmol) and DMF (a few 6a, H-3), 3.83-3.98 (m, 4H, H-2, H-5’, H-6b, H-2’), 4.25 (t, 1H, J =
drops) was slowly added. The solution was allowed to warm to 5.7 Hz, H-3’), 4.35 (dd, 1H, J1,2 = 5.52 Hz, J1,3 = 7.62 Hz, H-4’), 5.21
room temperature and stirred for 6 h. The solution was (d, J = 3.9 Hz, 1H, H-1’); 13C NMR (150 MHz, CDCl3): δ 57.5, 57.9,
concentrated to dryness and taken up in tetrahydrofuran (30.0 59.1, 61.2, 62.9, 64.0, 70.1, 71.7, 73.3, 73.4, 74.4, 81.3, 98.9
mL) and cooled to -21oC. Benzyl alcohol (2.30 mL, 22.0 mmol) ppm; 31P NMR (162 MHz, CDCl3): δ 13.3 ppm. Mass spectrum
and pyridine (1.80 mL, 22.0 mmol) were added to the solution. (HRMS), m/z = 418.1124 (M-H)-,C13H26NO12Prequires 418.1114.
The solution was stirred at this temperature for 30 min, allowed
Dibenzyl
((3-hydroxy-2,5-bis(hydroxymethyl)-(3-O-α-D-
to warm to room temperature, and stirred an additional 6 h. The glucopyranosyl)pyrrolidin-1-yl)ethyl)phosphonate (17). To a
mixture was poured into saturated aqueous potassium bisulfate solution of 14 (150mg, 490 µmol) in dichloromethane (1.0 mL)
and extracted with ethyl acetate. The combined extracts were was passed ozone at -78 oC until the solution turned light blue.
washed with brine and dried over magnesium sulfate. Removal The reaction was quenched with dimethyl sulfide (91 µL, 1.2
of the solvent afforded a residue that was purified by flash mmol). The resulting solution was poured into water and
column chromatography (hexane/ethyl acetate = 10:1-1:1) to extracted multiple times with dichloromethane. The combined
give 14; yield: 50% (1.52 g); silica gel TLC Rf = 0.45 (1:1 ethyl extracts were dried over anhydrous sodium sulfate and
1
acetate-hexanes); H NMR (CDCl3, 600 MHz): δ 2.55 (dd, J = 7.3, concentrated to afford a colorless oil of crude dibenzyl (2-
22.0 Hz, 2H, -CH2), 4.90-5.11 (m, 6H, =CH2, -CH2Ph), 5.70 (m, 1H, oxoethyl)phosphonate (15) which was in the next step without
=CH), 7.22-7.27 (m, 10H, Aromatic H’s); 13C NMR (150 MHz, further purification. 2,5-Dideoxy-3-O-α-D-glucopyranosyl-2,5-
CDCl3): δ 31.6, 32.5, 67.4, 67.4, 77.0, 77.2, 77.4, 120.2, 120.4, imino-D-mannitol
4 (45.0 mg, 0.138 mmol) was taken up in
127.0, 127.9, 128.5, 136.3 ppm; 31P NMR (162 MHz, CDCl3): δ methanol (1 mL). This solution was added to aldehyde 15 and
29.2 ppm. Mass spectrum (ESI-MS), m/z = 325.20 (M+Na)+, stirred 10 min at room temperature. To the solution was added
C17H19NaO3P requires 325.30. Data matches reference.
Dibenzyl ((3-hydroxy-2,5-bis(hydroxymethyl)-(3-O-α-D- followed by overnight stirring (~16 h). The solution was
glucopyranosyl)pyrrolidin-1-yl)methyl)phosphonate (16). 2,5- concentrated to dryness and dissolved in a minimal amount of
Dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (45.0 water and loaded on a C18 column. The column was eluted with
sodium cyanoborohydride (17.3 mg, 0.276 mmol) in one portion
4
mg, 0.138 mmol) was added to aqueous formaldehyde (2 mL, a solution of H2O-MeOH (1:1). The purified product was
37% CH2O in water) and dibenzylphosphite (30.0 µL, 0.138 evaporated under reduced pressure to obtain 17 as colorless
mmol). The solution was heated to 60 oC for 3 h and the solvent solid; yield: 56% (47.0 mg); [α]D23 = -14.0° (c = 1, CHCl3); 1H NMR
removed under reduced pressure. The residue was dissolved in (CDCl3, 600 MHz): δ 2.14-2.26 (m, 2H, H-g, H-h), 3.03-3.43 (m,
a minimal amount of water and loaded on a C18 column. The 4H, H-1, H-4, H-e,H-f), 3.62 (m, 10H, H-a, H-b, H-c, H-d, H-2’, H-
column was eluted with a solution of H2O-MeOH (1:1). The 3’, H-4’, H-5’, H-6a’, H-6b’), 5.01-5.14 (m, 5H, -CH2Ph, H-1’),
purified product was concentrated under reduced pressure to 7.36-7.42 (m, 10H, Aromatic H’s); 13C NMR (150 MHz, CDCl3): δ
obtain 16 as a viscous oil; yield: 56% (46.0 mg); [α]D23 = -14.0° (c δ 23.9, 24.8, 39.8, (47.1, 47.3, 47.4, 47.6, 47.8, 48.0; D-
1
= 1, H2O); H NMR (CDCl3, 600 MHz): δ 3.23-3.34 (m, 2H, H-1, H- methanol), 58.7, 59.2, 61.3, 66.6, 67.1, 67.4, 67.6, 68.7, 70.3,
4), 3.35-3.78 (m, 11H, H-a, H-b, H-c, H-d, H-2’, H-3’, H-4’, H-5’, 71.9, 72.8, 84.3, 98.1, 127.6, 127.6, 127.8, 127.9, 128.1, 128.2,
H-6,6’, H-4), 3.83 (dd, 1H, J = 3.6, 19.8 Hz, H-e, H-f), 3.57 (t, 1H, 128.2, 128.3, 136.2, 136.2, 136.3, 136.4 ppm; 31P NMR (162
J = 9.6 Hz, H-6a’), 3.67 (m, 1H, H-4’), 3.86 (dd, 2H, J = 9.0, 29.0 MHz, CDCl3): δ 29.1 ppm. Mass spectrum (HRMS), m/z =
Hz, Ha, Hb), 5.02-5.08 (m, 5H, H-1’, -CH2Ph), 7.31-7.34 (m, 10H, 614.2359 (M+H)+, C28H40NO12P requires 614.2366.
Aromatic H’s); 13C NMR (150 MHz, CDCl3): δ 55.3, 56.4,
((3-hydroxy-2,5-bis(hydroxymethyl)-(3-O-α-D-
59.5,59.5, 61.2, 67.8, 67.8, 70.2, 70.2, 71.1, 72.7, 73.5, 75.6, glucopyranosyl)pyrrolidin-1-yl)ethyl)phosphonic acid (6).
83.9, 98.3, 127.3, 127.5, 127.6, 127.8, 127.8, 127.8, 127.9, Palladium (10%) on carbon (catalytic amount) was added to a
127.1, 128.2, 128.3, 136.2, 136.2 ppm; 31P NMR (162 MHz, solution of compound 17 (47.0 mg, 0.07 mmol) in methanol
CDCl3): δ 26.4 ppm. Mass spectrum (HRMS), m/z = 600.2231 (500 µL). The mixture was stirred at ambient temperature under
(M+H)+, C27H38NO12P requires 600.2210.
hydrogen atmosphere for 24 h. The mixture was filtered
through Celite® followed by rinsing the Celite® with methanol.
((3-hydroxy-2,5-bis(hydroxymethyl)-(3-O-α-D-
glucopyranosyl)pyrrolidin-1-yl)methyl)phosphonic acid (5). The filtrate was concentrated under reduced pressure and the
Palladium (10%) on carbon (catalytic amount) was added to a residue purified by gel permeation column chromatography on
solution of compound 16 (45.0 mg, 0.07 mmol) in methanol (0.5 Sephadex LH-20 (H2O) to obtain compound
6 as a colorless
mL). The reaction mixture was stirred at ambient temperature solid; yield: quantitative (32.0 mg); [α]D23 = -14.0° (c = 1, CHCl3);
under hydrogen atmosphere for 24 h. The mixture was filtered 1H NMR (CDCl3, 600 MHz): δ 2.02-2.16 (m, 2H, H-a, H-b), 3.37-
through Celite® followed by rinsing the Celite® with methanol. 3.42 (m, 2H, H-1, H-c), 3.55-3.58 (m, 2H, H-4, H-d), 3.60-3.65 (m,
The filtrate was concentrated and the residue purified by gel 2H, H-g, H-h), 3.66-3.71(m, 2H, H-e, H-f), 3.72-3.83(m, 2H, H-6a,
permeation column chromatography on Sephadex LH-20 (H2O) H-3), 3.84-3.86 (m, 1H, H-6b), 4.01-4.03 (dd, 1H, J1,2 = 4.92 Hz,
to obtain pure compound
5
as a white solid; yield: quantitative
J
1,3 = 10.32 Hz, H-2’), 4.05-4.08 (m, 2H, H-2, H-5’), 4.33-4.34 (t,
6 | J. Name., 2012, 00, 1-3
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1H, J = 3.36 Hz, H-3’), 4.41-4.42 (dd, 1H, J1,2 = 3.36 Hz, J1,3 = 5.7
Hz, H-2’), 5.15 (d, J = 3.7 Hz, H-1’); 13C NMR (150 MHz, CDCl3): δ
58.6, 58.7, 67.6, 68.8, 68.8, 69.2, 69.2, 69.9, 70.4, 70.8, 70.9,
71.1, 74.9, 97.7 ppm; 31P NMR (162 MHz, CDCl3): δ 18.8 ppm.
Mass spectrum (HRMS), m/z = 434.1433 (M+H)+, C14H28NO12P
requires 434.1427.
G. Besra, S. Bornemann and W. R. Jacobs, Nat
View Article Online
Chem Biol, 2010, 6, 376-384D.OI: 10.1039/C7OB00388A
A. D. Elbein, I. Pastuszak, A. J. Tackett, T. Wilson
and Y. T. Pan, J Biol Chem, 2010, 285, 9803-9812.
G. Chandra, K. F. Chater and S. Bornemann,
Microbiol, 2011, 157, 1565-1572.
5.
6.
Sco GlgEI-V279S inhibition studies
7.
8.
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M. Coutinho and B. Henrissat, Nucleic Acids Res,
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K. Syson, C. E. M. Stevenson, A. M. Rashid, G.
Saalbach, M. H. Tang, A. Tuukkanen, D. I.
Svergun, S. G. Withers, D. M. Lawson and S.
Bornemann, Biochemistry, 2014, 53, 2494-2504.
K. Syson, C. E. M. Stevenson, M. Rejzek, S. A.
Fairhurst, A. Nair, C. J. Bruton, R. A. Field, K. F.
Chater, D. M. Lawson and S. Bornemann, J Biol
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The Kis for inhibitors against Sco GlgEI-V279S were determined
using EnzChek® Phosphate Assay Kit (Molecular Probes) as part
of a coupled enzyme assay as previously described.15 Using this
assay, we determined a Ki for compound
5 and 6 against Sco
9.
GlgEI-V279S. Assays were performed at 22 °C in a 96 well format
on a Spectra max 340PC (Molecular Devices). All stock solutions
of 1mM MESG were prepared and stored in dH2O at -20 °C.
Phosphate release, catalyzed by Sco GlgE-V279S, was
monitored by the production of 2-amino-6-mercapto-7-methyl-
purine in a coupled assay catalyzed by purine nucleoside
phosphorylase. The reaction was monitored at 5 s intervals for
30 min. Each reaction consisted of 1 mM MESG, 0.2 U of PNP,
20X reaction buffer (1.0 M Tris-HCl, 20 mM MgCl2, pH 7.5,
containing 2 mM sodium azide), glycogen as maltosyl acceptor,
50 nm GlgE, 250 µM M1P and varied concentration of inhibitors
10.
11.
12.
13.
14.
15.
5
and
6. Inhibition was determined by comparing the relative
rate of the reaction performed with inhibitor against a reaction
i
i
that contained no inhibitor (Vo /Vo), where Vo and Vo are steady-
state rates with and without inhibitor, respectively. The
equilibrium dissociation constants (Ki) for inhibitor
5 and 6 for
Sco GlgE found to be 45.03 ± 4.01 µM and 95.98 ± 16.02 µM
respectively. Ki was obtained by fitting the data into Eq 1 using
Prism.
i
KM + [S]
KM [I]
Vo
Vo
=
KM + [S] +
Ki
(Eq 1)
KM is the Michaelis-Menten constant for M1P; [S] and [I] are the
concentrations of M1P and inhibitor, respectively.
16.
17.
18.
19.
20.
21.
22.
23.
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Acknowledgements
The work was support by a grant from National Institution of
Health (Grant No. AI105084) to S.J.S. and D.R.R.
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