Synthesis of a poly-hydroxypyrolidine-based inhibitor of Mycobacterium tuberculosis GlgE

Article abstract of DOI:10.1021/jo501481r

(Chemical Equation Presented). Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis (Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to iden

Full text of DOI:10.1021/jo501481r

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Open Access on 08/19/2015
Synthesis of a Poly-hydroxypyrolidine-Based inhibitor of
Mycobacterium tuberculosis GlgE
Sri Kumar Veleti, Jared J. Lindenberger, Sandeep Thanna, Donald R. Ronning, and Steven J. Sucheck*
Department of Chemistry and Biochemistry, School of Green Chemistry and Engineering, The University of Toledo, 2801 West
Bancroft Street, Toledo, Ohio 43606, United States
S
* Supporting Information
ABSTRACT: Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis
(Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to identify new
antituberculosis drug targets. Mtb GlgE is a maltosyl transferase involved in α-glucan biosynthesis. Mutation of GlgE in Mtb
increases the concentration of maltose-1-phosphate (M1P), one substrate for GlgE, causing rapid cell death. We have designed
2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (9) to act as an inhibitor of GlgE. Compound 9 was synthesized using
a convergent synthesis by coupling thioglycosyl donor 14 and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-β-D-
fructopyranose (23) to form disaccharide 24. A reduction and intramolecular reductive amination transformed the intermediate
disaccharide 24 to the desired pyrolidine 9. Compound 9 inhibited both Mtb GlgE and a variant of Streptomyces coelicolor (Sco)
GlgEI with Ki = 237 27 μM and Ki = 102 7.52 μM, respectively. The results confirm that a Sco GlgE-V279S variant can be
used as a model for Mtb GlgE. In conclusion, we designed a lead transition state inhibitor of GlgE, which will be instrumental in
further elucidation of the enzymatic mechanism of Mtb GlgE.
M1P to generate linear α-glucans. Subsequently, α-1,6 branches
are introduced into the α-glucan by GlgB.6
INTRODUCTION
■
Tuberculosis (TB) is a dreadful infectious disease whose
causative agent is Mycobacterium tuberculosis (Mtb).1 Strepto-
mycin was first used to cure TB in the late 1940s. For a short
time, streptomycin gave the world hope that TB could be
completely eradicated. However, TB has reemerged as a major
global health threat due to poverty, co-infection with HIV, long
treatment times, and poor drug compliance practices. This has
led to the emergence of extensively drug-resistant TB (XDR-
TB), which is considered almost untreatable.1 Hence, the
discovery of novel antitubercular agents is urgently needed.
Trehalose (α-D-glucopyranosyl-(1 → 1)-α-D-glucopyrano-
side) is a pivotal metabolite in mycobacteria used for carrying
mycolic acid during biosynthesis of the mycolyl arabinogalactan
and trehalose-6,6′-dimycolate (TDM).2 Following the mycolyl
transfer reaction, trehalose is transferred back into the
cytoplasm from the periplamic space and is modified in the
production of various trehalolipids and sulfolipids.3,4 Trehalose
is also converted into a branched α-glucan. The conversion
process involves four enzymes: trehalose synthase (TreS),
maltokinase (Pep2), GlgE, and GlgB. The process involves the
isomerization of trehalose to maltose (4-O-α-D-glucopyranosyl-
D-glucose) by TreS, and then α-maltose-1-phosphate (M1P) is
produced by phosphorylation of maltose by Pep2.5 GlgE uses
Kalscheuer et al. reported that absence of GlgE led to self-
poisoning by the accumulation of phosphosugar M1P, directed
by a self-amplifying feedback response leading to cell death.6
Cell death was not due to absence of glucan in the cell wall and
capsule, but rather it was due to accumulation of M1P to a
lethal concentration, which caused cell death. GlgE is essential
for survival of the pathogen, and the absence of a human
homologue substantiates GlgE as a new drug target.6
Syson et al. reported the structure of Streptomyces coelicolor
(Sco) GlgE isoform-I, which indicated that GlgE belongs to the
glycosyl hydrolase family GH13.7 Recently, Syson et al. trapped
Sco GlgE with 2-deoxy-2-fluoro-α-maltosyl fluoride, which
provided additional evidence for a double-displacement
mechanism proceeding through a transition state as shown in
Scheme 1.8 The first step of the reaction is nucleophilic attack
of Asp418 on the anomeric center of M1P to generate a β-
maltosyl enzyme intermediate. The enzyme intermediate is
attacked by a glucan to extend the chain. It is predicted that the
anomeric oxygen on the phosphate moiety is protonated by
Received: July 10, 2014
© XXXX American Chemical Society
A
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Scheme 1. Proposed α-Retaining Double-Displacement Mechanism of GlgE (Mtb GlgE Numbering)
acid/base Glu447. Glutamate447 subsequently deprotonates
the incoming glucan. Literature supports the mechanism
showing retention of configuration of M1P and its conversion
to α-1,4-glycosidic linkage.6 However, until now attempts to
prepare a transition-state inhibitor have not been reported.
The replacement of the endocyclic oxygen atom in
carbohydrates with a nitrogen atom has been found in natural
products that inhibit glycosyl hydrolases in the micromolar
range.9 Nojirimycin (5-amino-5-deoxy-D-glucose, 1) and 1-
deoxynojirimycin (1, 5-dideoxy-1, 5-imino-D-glucitol, 2)
(Figure 1) are classical examples of such compounds. Poly-
hydroxypyrolidines 3 and 4 represent 5-membered ring
compounds related to monosaccharides that also act as potent
inhibitors for many glucosidases or glycoside hydrolases.9 The
hydroxyl groups present on these compounds resemble the
glycan moiety of the substrates. In all of these examples, the
nitrogen atom has the ability to become protonated to maintain
a positive charge at physiological pH that permits strong
interactions with a carboxylate group found in the active site of
this enzyme class. The carboxylate is predicted to stabilize the
positive charge that develops in the transition state, and it is
notable that the 5-membered aza-sugars 3 and 4 are more
potent inhibitors, with Ki = 0.18 μM and Ki = 0.33 μM,
respectively, than 6-membered aza-sugars 1 and 2, Ki = 6.3 μM
and Ki = 12.6 μM, respectively, against yeast α-glucosidase.9
According to Wong et al., nojirimycin-type azasugars can mimic
only the transitory charge generated during the hydrolysis of
glucosides, while the pyrolidine-type aza sugars also mimic the
shape of the postulated flattened half-chair transition state
(Figure 1).10,11 Wong et al. also show that five-membered aza
sugars were potent inhibitors of α-L-rhamnosidase (glycoside
hydrolase family) from Penicillium decumbens.10 For example,
compounds 5−8 are good inhibitors of pyranose-based α-L-
rhamnosidase with Ki values 0.14, 5.5, 11.5, and 46 μM,
respectively. Keeping in mind that five-membered ring poly-
hydroxypyrolidine have been shown to exhibit very good
activity against α-glucosidases, we became interested in other
modified analogues of these molecules as transition-state
inhibitors for GlgE and designed inhibitor 9.
RESULTS
■
Synthesis. In order to obtain the target molecule 9, we
designed a convergent synthesis. We synthesized the
compounds p-methylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-
glucopyranoside (14) (Scheme 2) and 5-azido-3-O-benzyl-5-
deoxy-1,2-O-isopropylidene-β-D-fructopyranose (23) (Scheme
3) separately. Subsequently, the two glycosides 14 and 23 were
coupled together to afford disaccharide 24, which was subjected
Figure 1. Poly-hydroxypyrrolidine-based inhibitor 9 and illustration of
expected binding interactions in the enzyme active site.
B
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a
anhydride followed by glycosylation with p-thiocresol and
boron trifluoride diethyl etherate to generate a peracetylated
thioglycosyl donor 11 with 85% yield (Scheme 2). Subsequent
Scheme 2
́
deacetylation under Zemplen conditions afforded tetraol 13,
which was then benzylated to afford the desired p-
methylphenyl-2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyrano-
side (14).
a
Synthesis of 5-Azido-3-O-benzyl-5-deoxy-1,2-O-isopropy-
Reagents and conditions: (i) Ac2O, pyridine, 4-(dimethylamino)-
lidene-β-D-fructopyranose (23).13 Yan
́
ez et al. and Tatibouet
̃
̈
pyridine, rt (86%); (ii) BF3.Et2O, p-thiocresol, CH2Cl2, 0 °C − rt
(85%); (iii) NaOMe, MeOH, rt (95%); (iv) NaH, BnBr, N,N-
dimethylformamide, rt (85%).
et al. have reported the generation of key intermediate 23 by
following a protection and deprotection strategy on D-
(+)-fructose 15.13,14 D-Fructose (15) was protected with
acetone to give 1,2:4,5-di-O-acetonide (16). Alcohol 16 was
benzylated at the OH-3 position to afford protected D-
(+)-fructose (17). Selective deprotection of the 4,5-O-
isopropylidene group was achieved with 60% acetic acid to
produce diol 18. Initially, controlled Garegg conditions were
employed on 18 to give selective iodination at OH-5 as
reported by Rollin et al. However, the major product was
eliminated product as shown in the cited literature.15 Therefore,
a
Scheme 3
as reported by Yanez and co-workers, 18 was subjected to
́
̃
selective benzoylation using dibutyltin oxide to afford products
19 and 20 in 38% and 52% yields, respectively. The reaction of
20 with modified Garegg’s conditions caused the substitution of
the hydroxyl group at C-5 with iodine by inversion of
configuration to give the corresponding 4-O-benzoyl-3-O-
benzyl-5-deoxy-5-iodo-1,2-O-isopropylidene-α-L-sorbopyranose
(21).13 Compound 21 was subjected to an SN2 substitution
reaction with sodium azide in N,N-dimethylformamide to give
product 22 with 90% yield. Finally, the deprotection of the
a
Reagents and conditions: (i) 0.5% H2SO4, acetone, rt (59%); (ii)
NaH, BnBr, DMF, rt (86%); (iii) 60% aq AcOH, 55 °C (76%); (iv)
(a) n-Bu2SnO, MeOH; (b) BzCl, Et3N, dioxane, 0 °C (19 = 38%) (20
= 52%) (1:1.3); (v) I2, Ph3P, imidazole, toluene (90%); (vi) NaN3,
N,N-dimethylformamide, 100 °C (91%); (vii) NaOMe, MeOH, rt
(98%).
benzoyl group at OH-4 was achieved using Zemplen
́
conditions
to afford 23 (Scheme 3).
Synthesis of 2,5-Dideoxy-3-O-α-D-glucopyranosyl-2,5-
imino-D-mannitol (9).16 Coupling of compound 14 and 23
using classical glycosylation conditions with N-iodosuccinamide
and trimethylsilyl trifluoromethanesulfonate yielded disacchar-
ide 24 with 76% yield with the α-anomer as a single product.17
The deprotection of 1,2-O-acetonide was achieved using 60%
trifluoroacetic acid to afford diol 25. Compound 25 was first
subjected to reduction and intramolecular reductive amination
using methanol, ethanol, and ethyl acetate as solvents in 1 N
hydrochloric acid (HCl) and yielded the desired product mixed
with methyl- and ethyl-substituted products, respectively, as
confirmed by ESI-MS. Presumably, aldehydes formed from
trace oxidation of the respective solvents participated in a
tandem reductive amination process to produce the observed
masses. These undesired reductive-amination products were
avoided by replacing the alcoholic solvents with a solution of
toluene/water/HCl (1:1:1). The use of H2 gas at 100 psi also
helped to afford a polyhydroxypyrolidine 9 in a clean
transformation (Scheme 4). To access M1P for working on
inhibition studies we followed our reported procedure.18
Inhibition Studies. Two different GlgE homologues were
studied using the EnzChek Phosphate Assay Kit (Molecular
Probes). This assay couples the phosphate produced from GlgE
activity to the phosphorylase activity of purine nucleoside
phosphorylase, which is based on work originally described by
Webb.19 Briefly, purine nucleoside phosphorylase (PNP)
converts the substrate 2-amino-6-mercapto-7-methylpurine
riboside (MESG) to ribose-1-phosphate and 2-amino-6-
mercapto-7-methylpurine (Figure 2).19 Conversion of MESG
to 2-amino-6-mercapto-7-methylpurine shifts absorbance from
330 to 360 nm, respectively. The assay is described in Figure 2.
to global deprotection, rearrangement, and reductive amination
to afford poly-hydroxypyrolidine 9 as shown in (Scheme 4).
Synthesis of p-Methylphenyl 2,3,4,6-Tetra-O-benzyl-1-
thio-β-D-glucopyranoside (14).12 To synthesize the thioglyco-
syl donor 14, D-glucose (10) was peracetylated with acetic
a
Scheme 4
a
Reagents and conditions: (i) N-iodosuccinamide, TMSOTf, 0 °C,
CH2Cl2 (76%); (ii) 60% aq TFA, rt (85%); (iii) 5% palladium on
activated carbon, toluene/water (1:1), 1 N HCl, 100 psi, quantitative.
C
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relative rate of the reaction performed with inhibitor against a
reaction that contained no inhibitor (Vi′/ Vi), where Vi′ and Vi
are steady-state rates with and without inhibitor, respectively.
The equilibrium dissociation constant (Ki) for inhibitor 9 for
Mtb GlgE found to be 237 μM
27 and Sco GlgEI-V279S
found to be 102 μM 7.52. These values are near the reported
Mtb GlgE Km of 250 μM 5.6
DISCUSSION
■
We have initiated efforts to develop transition-state inhibitors
for GlgE by replacing of the endocyclic oxygen atom of
glycosides with a nitrogen atom by considering the structures of
natural products that inhibit glycoside hydrolases. We anticipate
the resulting secondary ammonium moiety will mimic that
partial positive charge accumulation at the anomeric carbon of
the true substrate and form a strong interaction with the
Asp418 nucleophile conserved in this enzyme class. The
confluence of these interactions would therefore promote
reasonable inhibition of all GlgE homologues. We were able to
synthesize 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-
mannitol (9) by following a convergent glycosylation strategy
followed by in situ deprotection and intramolecular reductive
Figure 2. GlgE-coupled enzyme assay utilizing PNP; (a) M1P and
glycogen (n = a number of maltose units) serve as substrates for GlgE;
(b) PNP consumes Pi and MESG to form 2-amino-6-mercapto-7-
methylpurine.
Using this assay, we determined a Ki for compound 9 for Mtb
GlgE and the Sco GlgEI-V279S variant, which was designed
using multiple sequence alignments to better emulate the Mtb
GlgE maltosyl donor site (Figures 3 and 4). Assays were
1
amination. The H, 13C NMR and optical rotation data are in
accordance with the literature values.16 We evaluated the Ki for
both Mtb GlgE and Sco GlgEI-V279S using a PNP-based
coupled enzyme assay we developed. This assay has the
advantage of being performed in continuous mode. We also
tested whether 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-
D-mannitol (9) would inhibit the coupling enzyme PNP and no
inhibition was observed (Supporting Information, Figure S-21).
Since compound 9 inhibited both Mtb GlgE and Sco GlgEI-
V279S to a similar degree, this confirms that Sco GlgEI-V279S
can be used as a surrogate for Mtb GlgE due to its similarity to
the Mtb GlgE maltosyl donor site, enzymatic activity, laboratory
stability and ease of crystallization. We are currently in the
process of addressing the detailed mode of interaction of 2,5-
dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (9) with
Sco GlgEI-V279S via X-ray crystallography. The data from
those ongoing studies are expected to provide valuable insight
concerning the development of more potent transition-state
inhibitors for Mtb GlgE.
Figure 3. Ki determination of 9 with Mtb GlgE. Vi′/Vi are steady-state
rates with and without inhibitor. Error bars are calculated from the
result of reactions performed in triplicate.
CONCLUSION
■
We have synthesized a 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-
imino-D-mannitol (9). We evaluated the inhibitory activity of 9
against Mtb GlgE and Sco GlgEI-V279S. The Ki values were 237
27 μM for Mtb GlgE and Ki 102 7.52 μM for Sco GlgEI-
V279S, respectively. We have identified a lead transition state
inhibitor against GlgE enzyme. Compound 9 has been
successfully cocrystallized with Sco GlgEI-V279S and the X-
ray diffraction data will be published elsewhere. The data will
help us develop an improved class of transition state-based
GlgE inhibitors which may lead to the discovery of new
therapies to treat TB.
Figure 4. Ki determination of 9 with Sco GlgEI-V279S. Vi′/Vi are
steady-state rates with and without inhibitor. Error bars are calculated
from the result of reactions performed in triplicate.
performed under atmospheric pressure at 22 °C in a 96 well
format on a Spectra max 340PC (Molecular Devices). All 1
mM MESG stock solutions were prepared and stored in dH2O
at −20 °C. Phosphate release, catalyzed by Mtb GlgE, was
monitored by the production of 2-amino-6-mercapto-7-
methylpurine 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 (0.01 U), 250 μM M1P, and varied concentration
of inhibitor 9. Inhibition was determined by comparing the
EXPERIMENTAL SECTION
■
Materials and Methods. All fine chemicals such as D-(+)-glucose
monohydrate, benzyl bromide, acetic anhydride, p-thiocresol, benzoyl
chloride, dibutyltin oxide, 5% palladium on activated carbon,
trifluoroacetic acid, sodium azide, and anhydrous solvents such as
anhydrous methanol and N,N-dimethylformamide were purchased and
used without purification. TLC (silica gel 60, f254) was visualized under
UV light or by charring (5% H2SO4−MeOH). Flash column
D
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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 600 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. EnzChek Phosphate Assay Kit was purchased for use in
the Mtb GlgE inhibition assay.
3.50 (t, J = 12 Hz, 2H), 3.64−3.81 (m, 4H), 4.55−4.92 (m, 8H),
7.04−7.52 (m, 20H) ppm.
1,2:4,5-Di-O-isopropylidene-β-D-fructopyranose (16).22 To a
suspension of D-(+)-fructose (15) (10.0 g, 55.5 mmol) and acetone
(200 mL) was added concentrated sulfuric acid (0.97 mL). The
reaction mixture was stirred for 2 h at room temperature. The reaction
was cooled to 0 °C and was quenched with NaOH (3.0 g) in H2O (28
mL). After evaporation of the acetone, the residue was extracted with
dichloromethane. The organic phase was separated, washed with water
and brine, and then dried over anhydrous MgSO4. After filtration and
removal of solvents under reduced pressure, the residue was
recrystallized with ether/hexane to afford the product as white solid
16: yield 59% (8.5 g); silica gel TLC Rf = 0.41 (1:1 ethyl acetate−
1,2,3,4,6-Penta-O-acetyl-D-glucopyranose (11).20,21 D-
(+)-Glucose (10) (3.00 g, 16.6 mmol) was suspended in dry pyridine
(6.71 mL, 83.2 mmol) and acetic anhydride (7.8 mL, 83.0 mmol), and
a catalytic amount of 4-(dimethylamino)pyridine was added. The
solution was stirred at ambient temperature for 16 h. The reaction
mixture was diluted with ethyl acetate and washed successively with 1
N HCl (30 mL) and saturated aq NaHCO3 (60 mL). The resulting
organic phase was dried (anhydrous Na2SO4) and filtered, and the
filtrate concentrated under reduced pressure to give product 11: yield
86% (5.6 g); silica gel TLC Rf = 0.5 (1:1 acetone−hexane); 1H NMR
(CDCl3, 600 MHz) δ 2.02 (d, J = 12 Hz, 9H), 2.10 (d, J = 12 Hz, 6H),
3.83 (dd, J = 1.8 Hz, 6 Hz, 1H), 4.10 (d, J = 6 Hz, 1H), 4.28 (d, J = 12
Hz, 1H), 5.11−5.26 (m, 3H), 5.70 (d, J = 12 Hz, 1H) ppm.
1
hexane); H NMR (CDCl3, 600 MHz) δ 1.36 (s, 3H), 1.41 (s, 3H),
1.49 (s, 3H), 1.56 (s, 3H), 2.11 (d, J = 6.0 Hz, 1H), 3.71 (dd, J = 6.0,
18 Hz, 2H), 3.79 (d, J = 12 Hz, 1H), 3.92 (dd, J = 1.8 Hz,13.2 Hz,
1H), 4.25 (dd, J = 1.2 Hz, 7.8 Hz, 1H), 4.35 (d, J = 2.4 Hz, 1H), 4.62
(dd, J = 3 Hz, 8.4 Hz, 1H) ppm.
3-O-Benzyl-1,2:4,5-O-isopropylidene-β-D-fructopyranose
(17).22 To a solution of 16 (4.0 g, 15.7 mmol) in dry N,N-
dimethylformamide (10 mL) was added sodium hydride (524 mg, 31.5
mmol) at room temperature. The solution was stirred for 0.5 h, and
then benzyl bromide (1.3 mL, 31.5 mmol) was added. The reaction
was further stirred for 1 h at room temperature, diluted with water, and
then extracted with diethyl ether. The combined extracts were washed
with water and brine, dried over anhydrous MgSO4, and concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel with 1:9 acetone−hexane to give 3-O-
benzyl-1,2-O-isopropylidene-β-D-fructopyranose (17) as an oily
product: yield 86% (4.76 g); silica gel TLC Rf = 0.34 (3:7 acetone−
4-Methylphenyl 2,3,4,6-Tetra-O-acetyl-β-D-thioglucopyrano-
side (12).20,21 Peracetylated glucose (11) (2.0 g, 5.1 mmol) was
dissolved in dichloromethane (20 mL). To the solution were added by
p-thiocresol (955 mg, 7.60 mmol) and boron trifluoride−diethyl
etherate (0.7 mL, 5.6 mmol) at 0 °C. The resulting solution was stirred
at ambient temperature under nitrogen atmosphere. The reaction was
monitored by TLC and appeared to be complete after 16 h. The
reaction mixture was diluted with dichloromethane (14 mL) and
successively washed with saturated aq NaHCO3 (16 mL), 10% aq
NaCl (8 mL), and water (16 mL). The resulting organic phase was
dried (anhydrous Na2SO4) and filtered, and the filtrate was
concentrated under reduced pressure to give product 12. The product
was purified by silica gel flash column chromatography by eluting with
1:9 acetone−hexane. The product fractions were combined,
concentrated, and dried in vacuum to afford a white crystalline
product to give product 12: yield 85% (1.9 g); silica gel TLC Rf = 0.61
(2:8 acetone−hexane); 1H NMR (CDCl3, 600 MHz) δ 2.0 (d, J = 12
Hz, 6H), 2.09 (s, 6H), 2.35 (s, 3H), 3.69 (dd, J = 1.8 Hz, 6 Hz, 1H),
4.19 (m, 2H), 4.63 (d, J = 6 Hz, 1H), 4.92−5.03 (m, 2H), 5.21 (d, J =
12 Hz, 1H), 7.12−7.40 (m, 4H) ppm.
1
hexane); H NMR (CDCl3, 600 MHz) δ 1.33 (s, 3H), 1.41 (s, 3H),
1.43 (s, 3H), 1.55 (s, 3H), 3.61 (dd, J = 12.0 Hz, 30 Hz, 2H), 3.74 (d, J
= 12 Hz, 1H), 3.91 (d, J = 6 Hz, 1H), 4.25 (d, J = 6 Hz, 1H), 4.45−
4.68 (m, 2H), 7.28−7.34 (m, 5H) ppm.
3-O-Benzyl-1,2-O-isopropylidene-β-D-fructopyranose (18).22
A solution of benzyl ether 17 (1.0 g, 3.2 mmol) in 60% acetic acid in
water was stirred overnight at 55 °C. After evaporation of solvent, the
residue was purified by flash chromatography on silica gel with 1:9
acetone−hexane to give the diol 18 as a white solid: yield 76% (675
1
mg); silica gel TLC Rf = 0.25 (3:7 ethyl acetate−hexane); H NMR
(CDCl3, 600 MHz) δ 1.30 (s, 3H), 1.51 (s, 3H), 3.18 (d, J = 6 Hz,
1H), 3.48 (d, J = 6 Hz, 1H), 3.72 (d, J = 12.0 Hz, 1H), 3.73−4.05 (m,
2H), 4.57 (d, J = 12 Hz,1H), 4.67 (d, J = 12 Hz, 1H), 7.31−7.38 (m,
5H) ppm.
4-Methylphenyl 1-Thio-β-D-glucopyranoside (13).20,21 Com-
pound 12 (800 mg, 1.76 mmol) was deacetylated by dissolving in dry
methanol followed by addition of a catalytic amount of sodium metal
until the solution reached pH 9. The reaction was monitored for
completion using TLC. The reaction was neutralized by adding
Amberlite IRA-118H H+ resin until the pH reached 7. Then resin was
filtered away and the filtrate was concentrated under reduced pressure
5-O-Benzoyl- and 4-O-Benzoyl-3-O-benzyl-1,2-O-isopropyli-
dene-β-D-fructopyranose (19 and 20).13 To a stirred solution of
18 (500 mg, 1.61 mmol) in anhydrous methanol (5 mL) was added
dibutyltin oxide (441 mg, 1.77 mmol). The suspension was heated for
7 h under reflux and then concentrated to afford the 3,4-
dibutylstannylene derivative as a solid foam. The material was dried
overnight under vacuum. The foam was taken up in triethylamine
(0.24 mL, 1.77 mmol) in anhydrous dioxane (5 mL) and cooled (0
°C). To the solution was added a solution of benzoyl chloride (0.21
mL, 1.77 mmol) in the same solvent (1 mL) by dropwise addition.
The reaction was allowed to warm to room temperature and was
stirred for an additional 4 h. TLC revealed the presence of two new
compounds of higher mobility. Methanol (5 mL) was added, and after
30 min the mixture was concentrated and the residue was subjected to
a flash chromatography with 1:9 acetone-hexane to afford first pure 19:
yield 38% (253 mg); silica gel TLC Rf = 0.26 (3:7 acetone−hexane);
1H NMR (CDCl3, 600 MHz) δ 1.47 (s, 3H), 1.51 (s, 3H), 3.79 (d, J =
1
to afford the compound 13: yield 95% (0.48 g); H NMR (CDCl3,
600 MHz): δ 2.42 (s, 3H), 3.31−3.52(m, 4H), 3.79 (dd, J = 6 Hz, 12
Hz, 1H), 3.98 (d, J = 12 Hz, 1H), 4.64 (d, J = 10.2 Hz, 1H), 7.23−7.59
(m, 4H) ppm.
p-Methylphenyl 2,3,4,6-Tetra-O-benzyl-1-thio-β-D-glucopyr-
anoside (14).12 A solution of compound 13 (462 mg, 1.61 mmol) in
dry N,N-dimethylformamide (10 mL) was cooled to 0 °C. The
solution was treated dropwise with a suspension of sodium hydride
(60% dispersion in mineral oil) (452 g, 11.3 mmol). Benzyl bromide
(1.14 mL, 9.66 mmol) was added dropwise over 30 min and the
solution stirred at room temperature for 16 h. The reaction was
poured over ice and extracted with diethyl ether (10 mL). The
combined organic layers were washed with brine. The resulting
organic phase was dried (anhydrous Na2SO4) and filtered, and the
filtrate concentrated under reduced pressure to obtain a product. The
product was purified by silica gel flash column chromatography by
eluting with 1:9 acetone−hexane. The product fractions were
combined, concentrated, and dried in vacuum to afford a yellow
solid product 14: yield 85% (1.87g); silica gel TLC Rf = 0.61 (8:2
12.0 Hz, 1H), 3.90 (dd, J = 1.8 Hz, 12 Hz, 1H), 4.0 (dd, J = 1.8 Hz, 13
Hz, 1H), 4.12−4.14 (m, 2H), 4.32 (dd, J = 6 Hz, 12 Hz, 1H), 4.78 (d,
J = 12 Hz, 1H), 4.93 (d, J = 12 Hz, 1H), 5.44 (m, 1H), 7.36−8.08 (m,
10H) ppm; a second elution gave 20; yield: 52% (350 mg); silica gel
1
TLC Rf = 0.36 (3:7 acetone-hexane); H NMR (CDCl3, 600 MHz) δ
1.44 (s, 3H), 1.54 (s, 3H), 3.81 (dd, J = 2 Hz, 12.0 Hz, 1H), 4.01 (m,
2H), 4.13 (m, 1H), 4.66 (d, J = 12 Hz, 1H), 4.87 (d, J = 12 Hz, 1H),
5.54 (d, J = 6 Hz, 1H), 7.29−8.11 (m, 10H) ppm.
1
hexane−ethyl acetate); H NMR (CDCl3, 600 MHz) δ 2.32 (s, 3H),
E
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4-O-Benzoyl-3-O-benzyl-5-deoxy-5-iodo-1,2-O-isopropyli-
dene-α-L-sorbopyranose (21).13 To a solution of triphenylphos-
phine (430 mg, 1.64 mmol), imidazole (223 mg, 3.28 mmol), and
iodine (416 mg, 1.64 mmol) in dry toluene was added compound 20
(340 mg, 0.82 mmol). The solution was heated at 110 °C for 2 h. TLC
revealed a compound higher mobility than starting material. The
reaction was cooled, washed successively with 10% aq sodium
thiosulfate and brine, and then concentrated. The concentrated
residue was subjected to a flash chromatography with 1:9 acetone−
hexane to afford compound 21: yield 90% (0.38 g); silica gel TLC Rf =
128.1, 128.5, 138.0, 138.3, 138.7 ppm; mass spectrum (HRMS), m/z =
880.3793 (M + Na)+, C50H55N3O10 requires 880.3785.
2,3,4,6-Tetra-O-benzyl-1-(5-azido-3-O-benzyl-5-deoxy-β-D-
fructopyranose)-α-D-glucopyranoside (25). A solution of com-
pound 24 in 60% aqueous trifluoroacetic acid was stirred at room
temperature for 30 min. The solution was concentrated and repeatedly
coevaporated with toluene. The residue was purified by silica gel flash
column chromatography by eluting with 1:9 ethyl acetate−hexanes.
The product fractions were combined, concentrated, and dried in
vacuum to afford a yellow solid product 25: yield 85% (1.87 g); [α]23
D
1
0.5 (3:7 acetone−hexane); H NMR (CDCl3, 600 MHz) δ 1.42 (s,
= 10.1 (c = 1, CHCl3); silica gel TLC Rf = 0.61 (8:2 hexanes−ethyl
1
3H), 1.52 (s, 3H), 3.61 (d, J = 6 Hz, 1H), 3.86 (d, J = 12 Hz, 1H),
3.90−3.94 (m, 2H), 4.11 (m, 2H), 4.47 (d, J = 12 Hz, 1H), 4.65 (d, J =
6 Hz, 1H), 5.83 (m, J = 9 Hz, 1H), 7.13−8.11 (m, 10H) ppm.
5-Azido-4-O-benzoyl-3-O-benzyl-5-deoxy-1,2,-O-isopropyli-
dene-β-D-fructopyranose (22).13 A stirred solution of 21 (275 mg,
0.52 mmol) and sodium azide (171 mg, 2.62 mmol) in dry N,N-
dimethylformamide (10 mL) was heated at 100 °C for 24 h. TLC
revealed a slightly slower running compound in comparison to starting
material. The reaction was cooled, diluted with water, and then
extracted with diethyl ether. The combined extracts were washed with
water and brine, dried over anhydrous MgSO4, and concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel with 1:9 acetone−hexane to give
compound 22 as a crystalline product: yield 91% (0.23 g); silica gel
acetate); H NMR (CDCl3, 600 MHz) δ 3.36 (dd, 2H, J = 6, 72 Hz,
H-6a, H-6b), 3.51 (dd, 1H, J = 3.6, 7.2 Hz, H-2′), 3.62 (m, 4H’s, H-5,
H-4, H-a, H-6′), 3.70 (d, 1H, J = 18 Hz, H-3′), 3.96 (d, 1H, J = 18 Hz,
Ha), 4.05 (m, 3H, H-3′, H-4, H-6′), 4.12 (m, 1H, H-5′), 4.4−5.0 (m,
10H’s, −CH2Ph), 5.01 (d, 1H, J = 3.6 Hz, H-1′), 7.07- 7.33 (m, 25H,
aromatic H’s); 13C NMR (150 MHz, CDCl3) δ 55.3, 57.1, 60.9, 61.5,
65.2, 65.2, 68.1, 69.0, 71.5, 71.6, 73.1, 73.4, 73.6, 73.7, 74.2, 74.9, 75.2,
76.0, 77.0, 77.2, 77.9, 78.9, 79.7, 80.2, 81.8, 82.3, 96.9, 98.1, 100.4,
127.9, 128.5, 128.8, 137.0, 137.3, 137.8, 137.9, 138.5, 138.7 ppm; mass
spectrum (HRMS), m/z = 840.3472 (M + Na)+, C47H51N3O10
requires 840.3469.
2,5-Dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol
(9).16 Palladium (5%) on carbon (catalytic amount) was added to a
solution of compound 25 (90.0 mg, 0.08 mmol) in toluene (0.5 mL)
and water (0.5 mL). The reaction mixture was stirred at ambient
temperature under hydrogen atmosphere (98 psi) for 24 h. The
catalyst was filtered away, and reaction mixture was washed with
methanol. The filtrate was concentrated and the residue purified by gel
permeation column chromatography on Sephadex LH-20 (H2O) to
obtain pure compound 9 as a crystalline solid: yield quantitative (27
mg); [α]23 = +48.5 (c = 0.1, H2O) [lit.16 [α]20 = +49.5 (c = 0.1,
1
TLC Rf = 0.73 (3:7 acetone−hexanes); H NMR (CDCl3, 600 MHz)
δ 1.44 (s, 3H), 1.53 (s, 3H), 3.78 (dd, J = 6 Hz, 12 Hz, 1H), 3.99 (dd,
J = 12 Hz, 18 Hz, 2H), 4.05 (d, J = 6 Hz, 1H), 4.15 (dd, J = 6 Hz, 12
Hz, 1H), 4.27 (m, 1H), 4.67 (d, J = 12 Hz, 1H), 4.88 (d, J = 12 Hz,
1H), 5.66 (dd, J = 6 Hz, 12 Hz, 1H), 7.29−8.14 (m, 10H) ppm.
5-Azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-β-D-fruc-
topyranose (23).15 The compound 22 (0.21 g, 0.47 mmol) was
debenzoylated by being dissolved in dry methanol followed by
addition of a catalytic amount of sodium metal until the solution
reached pH 9. The reaction was monitored for completion using TLC.
The reaction was neutralized by adding Amberlite IRA-118H H+ resin
until the pH reached 7. The resin was filtered away, and the filtrate
concentrated under reduced pressure and purified by flash
chromatography on silica gel with 1:9 acetone−hexane to afford the
compound 23: yield 98% (0.15 g); silica gel TLC Rf = 0.42 (3:7
acetone−hexanes); 1H NMR (CDCl3, 600 MHz) δ 1.43 (s, 3H), 1.48
(s, 3H), 3.67 (d, J = 12 Hz, 1H), 3.94−4.05 (m, 5H), 4.19 (dd, J = 6
Hz, 12 Hz, 1H), 4.76 (d, J = 12 Hz, 1H), 4.84 (d, J = 12 Hz, 1H),
7.31−7.38 (m, 5H) ppm.
D
D
H2O)]; 1H NMR (CDCl3, 600 MHz) δ 3.32−3.93 (m, 12 H, Ha, Hb,
H-1, H-4, Ha’, Hb’, H-2′, H-3′, H-4′, H-5′, H-6a,b), 4.20 (t, 1H, J = 6
Hz, H-2), 4.30 (t, 1H, J = 6 Hz, H-3), 5.19 (d, 1H, J = 3.84 Hz, H-1′);
13C NMR (150 MHz, CDCl3) δ 57.1, 58.3, 60.3, 62.0, 63.1, 69.2, 70.8,
72.4, 72.5, 73.5, 80.4, 98.1 ppm; mass spectrum (HRMS), m/z =
326.1463 (M + H)+, C12H23NO9 requires 326.1451.
Protein Expression and Purification. The glgE (Rv1327c) gene
from M. tuberculosis strain H37Rv was PCR amplified using the
following primers: 5′- CAC CAT ATG AGT GGC CGG GCA AT-3′
and 5′- AAA GGA TCC TCA CCT GCG CAG CA- 3′. The product
was placed between the NdeI and BamHI cut sites of a modified pET-
28 plasmid (EMD Biosciences). The resulting pDR28-glgE encodes a
recombinant GlgE enzyme possessing an N-terminal polyhistidine tag.
The gene encoding the Sco GlgEI-V279S variant was placed between
the NdeI and XhoI cut sites of pET32 (EMD Biosciences) resulting in
plasmid pET32-Sco-V279S. The recombinant protein expressed by this
plasmid possesses a C-terminal polyhistidine tag. The sequences of
both plasmids were confirmed by DNA sequencing.
The pDR28-glgE or pET32-Sco-V279S plasmid was used to
transform T7 Rosetta cells. The bacterial cells were cultured at 37
°C in Luria Broth to an O.D. of 1.2−1.6 at 600 nm. Protein expression
was induced by the addition of IPTG to a final concentration of 1 mM.
Cells were harvested by centrifugation after incubating for 24 h at 16
°C. Pelleted cells were resuspended in buffer A containing 5 mM
imidazole, 500 mM NaCl, 20 mM Tris pH 7.5, 10% glycerol, and 5
mM β-mercaptoethanol. Lysozyme (10 μM) and DNaseI (100 μM)
were added to the cell resuspension and incubated for 1 h on ice prior
to lysis by sonication. The resulting suspension was centrifuged at
15000g for 30 min. The supernatant was applied to a 5 mL HiTrap
Talon Crude column (GE Healthcare) previously equilibrated with
buffer A. Proteins were eluted from the column by applying a linear
gradient of imidizole from 5−500 mM over 20 column volumes.
Fractions containing GlgE were pooled and applied to a Hi-Load
Superdex 200 size-exclusion column for further purification. GlgE was
eluted from the column isocratically with a buffer containing 150 mM
NaCl, 20 mM Tris pH 7.5, and 0.3 mM TCEP. Fractions containing
only GlgE were subsequently pooled. The purity of the protein was
2,3,4,6-Tetra-O-benzyl-1-(5-azido-3-O-benzyl-5-deoxy-1,2-
O-isopropylidene-β-D-fructopyranose)-α-D-glucopyranoside
(24). A mixture of p-methylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-
glucopyranoside (14) (185 mg, 0.280 mmol) and 5-azido-3-O-benzyl-
5-deoxy-1,2,-O-isopropylidene-β-D-fructopyranose (23) (80.0 mg, 0.23
mmol) was dissolved in dichloromethane (5 mL). Powdered 4 Å
molecular sieves were added (200 mg) to the solution, and the mixture
was stirred under N2 at 0 °C 1 h. N-Iodosuccinamide (80.3 mg, 0.350
mmol) and trimethylsilyl triflate (0.017 mL, 0.097 mmol) were added.
After being stirred for 10 min, the reaction mixture was neutralized
with triethylamine, diluted with dichloromethane, and filtered through
Celite. The filtrate was washed successively with saturated sodium
thiosulfate, water, and brine. The filtrate was dried over sodium sulfate
(anhydrous), filtered, concentrated to dryness, and purified by flash
chromatography by eluting with 1:9 acetone−hexane to give
compound 24: yield 76% (0.35 g); [α]23 = −14.0 (c = 1, CHCl3);
D
silica gel TLC Rf = 0.45 (3:7 ethyl acetate−hexanes); 1H NMR
(CDCl3, 600 MHz) δ 1.44 (s, 3H, CH3), 1.48 (s, 3H, CH3), 3.49 (dd,
1H, J = 3.6, 9.6 Hz, H-2′), 3.57 (t, 1H, J = 9.6 Hz, H-6a′), 3.67 (m,
1H, H-4′), 3.86 (dd, 2H, J = 9.0, 29.0 Hz, Ha, Hb), 3.97 (dd, 2H, J =
12, 84 Hz, H-6a,b), 4.06 (m, 2H, H-3′, H-6′b), 4.14 (m, 1H, H-5′),
4.44−5.15 (m, 10H, −CH2Ph), 4.97 (d, 1H, J = 3.6 Hz, H-1′), 7.15−
7.31 (m, 25H, aromatic H’s), 4.85−4.37 (m, 14H, −CH2Ph), 5.40 (d, J
= 3.6 Hz, H-1′), 7.32- 7.39 (m, 35H, aromatic H’s); 13C NMR (150
MHz, CDCl3) δ 26.2, 27.2, 61.4, 61.9, 69.0, 71.6, 71.9, 73.5, 73.7, 74.3,
75.3, 75.8, 79.9, 79.9, 80.7, 81.9, 100.4, 106.0, 112.3, 127.5, 127.6,
F
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The Journal of Organic Chemistry
Featured Article
confirmed using SDS-PAGE and concentrated using ultrafiltration. Sco
GlgEI-V279S was purified using the same protocol.
(7) Syson, K.; Stevenson, C. E.; Rejzek, M.; Fairhurst, S. A.; Nair, A.;
Bruton, C. J.; Field, R. A.; Chater, K. F.; Lawson, D. M.; Bornemann,
S. J. Biol. Chem. 2011, 286, 38298−38310.
Inhibition Studies. Assays were performed under atmospheric
pressure at 22 °C in a 96-well format on a Spectra max 340PC
(Molecular Devices). All 1 mM MESG stock solutions were prepared
and stored in dH2O at −20 °C. Phosphate release catalyzed by Mtb
GlgE was monitored using a coupled assay utilizing purine nucleoside
phosphorylase at 5 s intervals for 10 min. For each reaction, a master
mix was made consisting of 1 mM MESG (20 μL), 0.2 U of PNP (1
μL), 20× reaction buffer (1.0 M Tris−HCl, 20 mM MgCl2, pH 7.5,
containing 2 mM sodium azide) (5 μL), glycogen (0.5 μL), 50 nm
GlgE (5 μL), 250 μM M1P (3.125 μL), and 40 mM stock solution of
2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (9) inhibi-
tor in dH2O and was added for each corresponding stock solution to
reach the desired inhibitor concentration (0.0−1.5 mM) and varied
volumes of dH2O for an overall reaction volume of 100 μL. Inhibitor 9
was not added to the positive control, and the negative control lacked
GlgE. Inhibition was determined by comparing the relative rate of the
reaction performed with inhibitor against a reaction that contained no
inhibitor (Vo′/Vo), where Vo′ and Vo are steady-state rates with and
without inhibitor, respectively. The equilibrium dissociation constant
(Ki) for inhibitor was obtained by fitting the data into the following
equation using Prism
(8) Syson, K.; Stevenson, C. E.; Rashid, A. M.; Saalbach, G.; Tang,
M.; Tuukkanen, A.; Svergun, D. I.; Withers, S. G.; Lawson, D. M.;
Bornemann, S. Biochemistry 2014, 53, 2494−2504.
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Tetrahedron 2006, 62, 10277−10302.
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370.
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̃
1440−1447.
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̈
Eur. J. Org. Chem. 2011, 2011, 2286−2292.
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̈
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1997, 38, 225−226.
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(17) Castelli, R.; Overkleeft, H. S.; van der Marel, G. A.; Codee, J. D.
Org. Lett. 2013, 15, 2270−2273.
(18) Veleti, S. K.; Lindenberger, J. J.; Ronning, D. R.; Sucheck, S. J.
Bioorg. Med. Chem. 2014, 22, 1404−1411.
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266.
′
V
Km + [S]
Km + [S] +
i
=
K
m[I]
Ki
V
i
where Km is the Michaelis−Menten constant for M1P and [S] and [I]
are the concentrations of M1P and inhibitor, respectively.
(21) Hansen, S. G.; Skrydstrup, T. Eur. J. Org. Chem. 2007, 2007,
3392−3401.
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60, 564−577.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for new compounds are available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: steve.sucheck@utoledo.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by a DeArce Memorial Fund
grant to S.J.S. from the University of Toledo and a grant from
the National Institutes of Health (Grant No. AI105084) to
S.J.S. and D.R.R.
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dx.doi.org/10.1021/jo501481r | J. Org. Chem. XXXX, XXX, XXX−XXX