Synthesis and in Vitro Characterization of Trehalose-Based Inhibitors of Mycobacterial Trehalose 6-Phosphate Phosphatases

Article abstract of DOI:10.1002/cbic.201800551

α,α′-Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose-related biochemical processes potential targets for chemotherapy. The trehalose 6-phosphate synthase (TPS)/trehalose 6-phosphate phosphatase (TPP) pathway, also known as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of α,α′-trehalose derivatives in the forms of the 6-phosphonic acid 4 (TMP), the 6-methylenephosphonic acid 5 (TEP), and the 6-N-phosphonamide 6 (TNP). These non-hydrolyzable substrate analogues of TPP were examined as inhibitors of Mtb, Mycobacterium lentiflavum (Mlt), and Mycobacterium triplex (Mtx) TPP. In all cases the compounds were most effective in inhibiting Mtx TPP, with TMP [IC₅₀=(288±32) μm] acting most strongly, followed by TNP [IC₅₀=(421±24) μm] and TEP [IC₅₀=(1959±261) μm]. The results also indicate significant differences in the analogue binding profile when comparing Mtb TPP, Mlt TPP, and Mtx TPP homologues.

Full text of DOI:10.1002/cbic.201800551

Accepted Article
Title: Synthesis and in vitro characterization of trehalose-based
inhibitors of mycobacterial trehalose 6-phosphate phosphatases
Authors: Sunayana Kapil, Cecile Petit, Victoria N. Drago, Donald R.
Ronning, and Steven J. Sucheck
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Synthesis and in vitro characterization of trehalose-based
inhibitors of mycobacterial trehalose 6-phosphate phosphatases
Sunayana Kapil‡[a], Cecile Petit‡[b], Victoria N. Drago , Donald R. Ronning* and Steven J. Sucheck*
[a]
[a]
[a]
Dedicated to Professor Chi-Huey Wong on his 70th birthday
Abstract: α,α’-Trehalose plays roles in the synthesis of several cell
wall components involved in pathogenic mycobacteria virulence. Its
absence in mammalian biochemistry makes trehalose-related
biochemical processes potential targets for chemotherapy. The
Antigen 85 Complex (Ag85) transfers[14] the mycolic acids to the
cell wall arabinogalactan (AG) or to a second molecule of TMM to
form trehalose dimycolate (TDM) a key virulence factor.[15] Free
trehalose produced by the action of Ag85s on TMM is salvaged
by the action of the ABC transporter LpqY-SugABC.[ It is also
known that Mtb can enter an antibiotic resistant state under the
stress of low oxygen.[16] Intriguingly, trehalose has been linked to
the ability of Mtb to adapt its metabolism under these
6a]
trehalose-6-phosphate
synthase
(TPS)/trehalose-6-phosphate
phosphatase (TPP) pathway, also referred to as the OtsA/OtsB2
pathway, is the major pathway involved in the production of trehalose
in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for
Mtb survival. We describe the synthesis of 6-phosphonic acid 4 (TMP), conditions.[17] Thus, compounds interfering with the various
6
-(methylene)phosphonic acid 5 (TEP) , and 6-N-phosphonamide 6
TNP) derivatives of α,α’-trehalose. These non-hydrolyzable substrate
analogs of TPP were examined as inhibitors of Mtb, M. lentiflavum
Mlt), and M. triplex (Mtx) TPP. In all cases the compounds inhibited
trehalose-producing pathways may offer new approaches for
more effective treatments against Mtb.
(
(
Mtx TPP most strongly, with TMP (IC50 = 288 ± 32 μM) inhibiting most
strongly, followed by TNP (IC50 = 421 ± 24 μM) and TEP (IC50 = 1959
±
261 μM). The results also indicate significant differences in the
analog binding profile when comparing Mtb TPP, Mlx TPP, and Mtx
TPP homologs.
Introduction
α,α'-Trehalose (1) is an essential disaccharide present in plants,[1]
some bacteria,[2] fungi,[3] parasitic nematodes,[4] and insects;[5]
however, it is absent in mammals. Depending on context,
trehalose can serve as an energy source, metabolic regulator,
anti-desiccant, and cell wall component.[2] The lack of mammalian
trehalose synthesis, import, or processing pathways makes those
pathways potential targets for chemotherapy in Mycobacterium
tuberculosis (Mtb).[6] In Mtb, three pathways for synthesizing
trehalose are known,[6b] they are the TreY-TreZ, TreS and the
OtsA/OtsB pathways (Figure 1). The OtsA/OtsB pathway is
considered most prevalent in replicating Mtb.[7] OtsA catalyzes the
formation of trehalose-6-phosphate (2, T6P) from UDP-glucose or
ADP-glucose and glucose-6-phosphate.[8] OtsB has two
homologues, OtsB1 and OtsB2, and only OtsB2 has phosphatase
activity and is essential for Mtb growth.[7, 9] The phosphate moiety
of T6P is removed by OtsB2 to yield trehalose. The OtsA/OtsB
pathway is also referred to as the trehalose-6-phosphate
synthase (TPS)/trehalose-6-phosphate phosphatase (TPP)
pathway and TPP is used in place of OtsB2 hereafter.
Figure 1. Routes for the synthesis and recycling of trehalose, as well as its
conversion to TMM/TDM in Mtb. a. TreY–TreZ pathway; b. TreS pathway; c.
OtsA/OtsB pathway.
Recently, 6-sulfate, 6-(methylene)phosphonate, and 6-
(fluoromethylene)fluorophosphonate derivatives of α,α'-trehalose
were reported active (K
i
=130-480 μM) against Mtb TPP[18] while
Trehalose has several fates related to virulence and cell wall
synthesis. These include conversion into intracellular α-glucan,
sulfolipid-1, diacyltrehalose, and polyacyltrehalose. [10] Trehalose
is also modified with mycolic acids by polyketide synthase 13
aryl-D-glucopyranoside 6-sulfates were also reported as mimics
of T6P with activity against TPP.[ Based on the activity of these
α,α'-trehalose derivatives against Mtb TPP, we developed
heptabenzyl α,α'-trehalose derivative 3 and synthetic routes to
access 6-phosphonic acid 4 (TMP), 6-(methylene)phosphonic
acid 5 (TEP), 6-N-phosphonamide 6 (TNP), and a 6-oxirane 7
derivatives of 6-deoxy-α,α'-trehalose (Figure 2). The library of 6-
19]
[11]
(
Pks13) to form mono-α-alkyl β-ketoacyl trehalose (TMMk).
[
12]
TMMk is reduced to trehalose monomycolate (TMM) by CmrA
which is transported to the cell wall by MmpL3 (Figure 1).[13] The
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deoxy-α,α'-trehalose derivatives was evaluated for inhibition
against TPP homologs encoded by Mtb, Mlt and Mtx. Validamycin
A (8) was included due to topological similarity with α,α'-trehalose
and for known inhibitory activity against E. coli TPP.[20] Therefore,
the Mlt and Mtx TPP homologs were included in this study
because of the high sequence identity with the Mtb TPP, 72% and
derivative 9 in 70% yield by treatment with triphenylphosphine in
the presence of iodine and imidazole (Scheme 1). Iodide 9 was
subjected to Michaelis–Arbuzov conditions by treatment with
trimethyl phosphite to afford 6-phosphonate derivative 10 in 58%
yield. The phosphonate ester 10 was deprotected with
bromotrimethylsilane (TMSBr) to afford the 6-phosphonic acid
derivative 11. The latter was debenzylated by hydrogenolysis with
71%, respectively, and the 100% identity in catalytic residues.
Additionally, the relative paucity of available Mtb TPP structural
information suggested protein structural dynamics that hinder
crystallization. The Mlt and Mtx TPP homologs lack two large
loops indicated in the Mtb TPP sequence and are being pursued
as TPP surrogates to afford structural determination of
mycobacterial TPP enzymes. Finally, the Mtb, Mlt, and Mtx TPP
20% Pd(OH)
phosphonate TMP.
2 2
on carbon under 1 atm. of H to afford 6-
enzymes in this study exhibit K
μM,[21] respectively.
M
values of 640,[7] 130,[21] and 82
Scheme 1. Synthesis of 6-phosphonic acid-α,α'-trehalose derivative TMP.
The target 6-(methylene)phosphonic acid analog TEP was also
accessible through intermediate 3. Intermediate 3 was first
subjected to a Swern oxidation to afford aldehyde 12 in 62% yield
(
1
Scheme 2).[18] Aldehyde 12 was converted to the phosphonate
3 in 49% yield via a Horner–Wadsworth–Emmons reaction
utilizing tetramethyl methylenediphosphonate and sodium hydride.
The phosphonate ester 13 was deprotected with
Figure 2. 6-deoxy-α,α'-trehalose derivatives prepared from heptabenzyl α,α'-
trehalose derivative 3 and Validamycin A (8) which shows topological similarities
with trehalose.
bromotrimethylsilane (TMSBr) to afford the 6-(vinylphosphonic
acid) derivative 14. The latter was debenzylated by
hydrogenolysis with 20% Pd(OH) on carbon under 1 atm. of H
2 2
to afford 6-(methylene)phosphonic acid TEP. The route is a minor
modification of reported work[18] which used tetraethyl
methylenediphosphonate in the Horner–Wadsworth–Emmons
reaction along with other modifications in reagents and reaction
sequence.
Results
Chemistry studies. Prior research has shown that 6-sulfate, 6-
(
methylene)phosphonate
TEP,
and
6-
(
fluoromethylene)phosphonate derivatives of α,α'-trehalose
[
18]
possessed inhibitory activity against Mtb TPP;
however, no
microbiological evaluation was reported. That data, in addition to
the know growth inhibition data of 6-modified derivatives of α,α'-
trehalose against mycobacteria,[14b, 22] prompted us to investigate
the synthesis of additional 6-modified derivatives. In the current
work we focused on synthesis and study of 6-phosphonate α,α'-
trehalose TMP, an alternative route to 6-(methylene)phosphonate
analogue TEP, a 6-N-phosphonamide analogue TNP, and an 6-
oxirane analogue 7 due to similarity with reported Mtb TPP
inhibitors (Figure 2).[18]
Scheme 2. Synthesis of 6-(methylenephosphonic acid)-α,α'-trehalose
derivative TEP.
The target 6-phosphonate analog TMP was accessible through a
heptabenzyl α,α'-trehalose intermediate 3[22c] prepared by our
reported route.[22d] The route allows gram scale access to
unsymmetrical 6-modified α,α'-trehalose analogues. In order to
access 6-phosphonate TMP, heptabenzyl α,α'-trehalose
derivative 3 was converted to the 6-iodo-6-deoxy-α,α'-trehalose
The target 6-phosphoramidic acid analog TNP was accessible
through iodide intermediate 9.
Iodide 9 was subjected
nucleophilic substitution with sodium azide to afford azide 15 in
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8
2% (Scheme 3). Azide 15 was reduced to amine 16 via a
the weakest inhibition for each enzyme, which is consistent with
the expected enzyme mechanism. The presence of an ethyl
moiety between the phosphonate and trehalose moiety is
sufficiently large as to prevent appropriate simultaneous
coordination of these two components of the inhibitor. The
inhibitory activity of TMP and TNP exhibited IC50 values lacking a
consistent pattern. TNP was the most potent inhibitor against the
Mtb TPP, while TMP exhibited the lowest IC50 values for both the
Mlt and Mtx TPP homologs. Compound 7 (data not shown)
showed no inhibition. For comparison, Validamycin A (8) was
also tested as an inhibitor of the mycobacterial TPPs as it had
been previously shown to completely inhibit E. coli TPP at a
concentration of 25 μM.[20] Interestingly, Validamycin A exhibited
unexpectedly high IC50 values for the mycobacterial TPPs (Figure
Staudinger reaction reduction in good yield. The 6-amino-α,α'-
trehalose derivative 16 was treated with dibenzylphosphoryl
chloride to afford dibenyzl phosphoramidate 17. Global
deprotection of the benzyl groups with 10% Pd on carbon under
1
2
atm. of H yielded 6-phosphoramidic acid analog TNP.
3, J, K, and L). Neither the structure of the E. coli TPP nor the
T6P-bound Mtb TPP have been deposited in the PDB, so it is
difficult to rationalize the observed difference between the E. coli
TPP and mycobacterial TPPs in response to Validamycin A. One
difference, which merits further investigation, may lie in the lack
of an N-terminal domain in E. Coli TPP. The structure of an
inactive Cryptococcus neoformans TPP with T6P bound shows
how both the essential Mg2+ is coordinated by the conserved
Aspartate residues and how the phosphate moiety of T6P
interacts with the Mg2 as well as conserved Histidine and Lysine
active site residues.[25] However, the residues in both the catalytic
domain and the cap domain of C. neoformans TPP that interact
directly with the glucose moieties of trehalose are not highly
conserved in TPP homologs. It is also shown that structural
changes in C. neoformans TPP are stimulated by binding of T6P.
Therefore, it is likely that the sequence differences and structural
dynamics lead to differential inhibition by Validamycin A.
Scheme 3. Synthesis of 6-(phosphoramidic acid)-α,α'-trehalose derivative TNP.
The target oxirane analog 7 was likewise accessible through
iodide intermediate 9. Iodide 9 was subjected to nucleophilic
substitution with trimethylsilylacetylene followed by deprotection
of the silyl group with tetrabutylammonium fluoride (TBAF) to
afford compound 18 in 48% yield over 2 steps (Scheme 4).
Compound 18 was subjected to Birch reduction to reduce the
benzyl groups and convert the alkyne to an alkene. The resulting
hydroxyl groups were protected by acetylation to afford
compound 19 in 40% yield.[23] The alkene 19 was treated with
meta-chloroperoxybenzoic acid (mCPBA) to afford epoxide 20 as
a mixture of diastereomers in 59% yield. Deprotection of the
+
3 2
acetyl groups of 20 was achieved with NEt /MeOH/H O (1:3:1) to
afford oxirane analog 7.[24]
Discussion
TPP is an essential enzyme for survival and virulence of Mtb. It
catalyzes the second reaction in the de novo trehalose
biosynthesis pathway. Since trehalose is not produced by
mammals, they possess no homologues of TPP rendering it an
intriguing drug target. Since enzymes evolve to strongly bind their
transition-states, initial inhibitory studies on any enzyme typically
attempt to mimic the expected transition states. In the case of TPP,
formation of trehalose occurs through an associative-two-step
mechanism (Figure 4).[26]
During the first step of the reaction, the nucleophilic aspartate,
Asp153/120 (numbering corresponds with Mtb TPP/Mlt TPP
n
residue numbering: Asp in Figure 4), launches an attack on the
Scheme 4. Synthesis of 6-oxiranyl-α,α'-trehalose derivative 7.
phosphoryl group of T6P, resulting in the formation of a penta-
coordinate trehalose-phosphoaspartyl enzyme intermediate.
Then acting as a general acid, Asp155/122 (Aspn+2 in Figure 4)
protonates trehalose promoting formation of the phosphoaspartyl
intermediate and release of free trehalose. The proposed
pentacoordinate phosphorous transition states and intermediates
are challenging to emulate in stable organic compounds.
Therefore, we have employed phosphonate compounds as
substrate analogues for the initial inhibitory studies.
Enzyme inhibition studies. To assess the ability of non-
hydrolysable mimics of the natural TPP substrate, T6P, to inhibit
TPP, TMP, TEP, TNP, 7 and Validamycin A (8) were evaluated.
These non-hydrolysable mimics were tested against the TPP
homologs encoded by Mtb, Mlt and Mtx. All of the inhibitory curves
and resulting IC50 values are given in Figure 3. Few general trends
of inhibition for the three homologs were observed. TEP exhibited
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Figure 3. TPP Inhibition studies. A, B and C; TPP inhibition curves for TNP. D. E, F; TPP inhibition curves for TEP. G, H, and I; TPP inhibition curves for TMP. J,
K, L; TPP inhibition curves for Validamycin A. M; IC50 values of TNP, TEP, TMP and Validamycin A against Mtb TPP, Mlt TPP and Mtx TPP.
n
Figure 4. Associative-two-step mechanism of TPP. A. First step of the reaction. B. Second step of the reaction. Asp corresponds to the nucleophile. Aspn+2
corresponds to the general acid/general base.
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A magnesium co-factor, required for this reaction to occur, affords
the correct positioning of the substrate phosphoryl group and for
neutralization of the negative charges forming on the transition-
state. The three non-hydrolysable phosphorus-based compounds
were made to mimic the T6P substrate and assess the impact of
length and atom character of the chain connecting trehalose and
the phospho-mimic. While the TMP IC50 against the Mtx TPP and
Mtb TPP homologs was only 1.8- to 2.8-fold worse than the
The inhibitory results show that the TEP analogue with the longer
linker and lacking a hydrogen bond acceptor significantly
weakens affinity by any TPP homologue. In contrast, the lone pair
of electrons on the nitrogen atom of the aminophosphonate
moiety in the TNP analogue may interact with the magnesium co-
factor to enhance binding. In contrast, the Mlt TPP and Mtx TPP
exhibited a different pattern of response to the inhibitors,
particularly in regards to the inhibition by TMP. While this lack of
correlation is troubling, it is well documented that TPP enzyme
active sites undergo a conformational change upon binding
substrate and structural dynamics can have a profound effect on
substrate or inhibitor affinity.[ In this context, and in regards to
our results regarding the lack of inhibitory effect of Validamycin A
on mycobacterial TPP, in contrast to E. coli TPP, suggest that the
N-terminal domain of mycobacterial TPP might play an important
role in substrate binding. In this prospect, the shorter N-terminal
domains of both Mlt TPP and Mtx TPP, in comparison to the N-
M
determined T6P K , respectively, TEP exhibited an IC50 that was
3.4- to 135-fold worse than the determined T6P K
M
. The third
inhibitor, TNP, was synthesized to test the role for the O-6 oxygen
of T6P binding by TPP. The IC50 of TNP suggests binding affinity
similar to the Mtb TPP binding of the T6P substrate. When
compared to the inhibitory activities of TMP and TEP against Mtb
TPP, this suggests enhanced affinity due to the presence of a
hydrogen bond acceptor and donor at position 6 which has an
important role in enzyme recognition. Compound 7, on the other
hand, appeared unable to interact with any of the enzymes. The
moderate inhibition data is consistent with other studies that
describe T6P analogs designed to mimic phosphate.[18-19] One
potential path forward to improve inhibition may be to mimic
phosphate with α-hydrodroxy- or α-amino- phosphonates. These
bidentate moieties may have the potential to bind magnesium ions
and improve inhibitory potential. In addition, α-hydrodroxy- or α-
amino- groups may make up for the loss of the putative H-bond
acceptor absent in the current phosphonate series. It is expected
25]
terminal
domain
of Mtb TPP,
would
explain
such
discrepancies. This suggests that structural changes in Mtb TPP
due to substrate analogue binding may be distinct from changes
occurring in the Mlx TPP and Mtx TPP homologues, particularly
in the case of TMP binding. Resolution of this discrepancy will
follow structural determination of TPP/inhibitor complexes and
complementary molecular dynamic studies.
that the compounds in the current series could potentially be Experimental Section
imported in Mtb by LpqY-SugABC (Figure 1) which recognizes
trehalose and some of its derivatives.[14b, 22] For example, it has
General Methods
been shown that Mtb could take-up extracellular trehalose
All chemicals and solvents were purchased from Fisher Scientific,
Acros Organics, Alfa Aesar or Sigma-Aldrich. Solvents were dried
by distillation, other standard procedures and through a solvent
purification system by passing HPLC grade solvent through
activated alumina and copper columns. All reactions were carried
out at room temperature under nitrogen atmosphere using a
nitrogen balloon unless mentioned in procedure. Reactions were
monitored by TLC (silica gel, f254) under UV light or by charring
(5% sulfuric acid-methanol) and the purification was performed by
flash column chromatography on silica gel (230-400 mesh) using
the solvent system specified, solvents were used without
including
a
fluorescein-containing trehalose probe and
[
22a]
incorporate the materials into growing bacilli.
The same
investigators showed that 6-fluoro-6-deoxy-α,α'-trehalose and 6-
bromo-6-deoxy-α,α'-trehalose possessed activity (MIC 200
μg/mL) against Mtb.[22a]
For future studies, it would be interesting to investigate how these
new compounds might effect trehalose production, Mtb growth or
recovery from dormancy, Ag85 activity, TMM/TDM production, or
biofilm formation. Related compounds, like 6-azido-6-deoxy-α,α'-
trehalose, are known to inhibit growth of M. aurum (MIC 200
μg/mL) as well as inhibit Ag85C activity, synthesis of TMM, TDM,
and reduced cell wall–bound mycolic acids and TMM export. [14b]
While a library of N,N-dialkylamino and 6,6-bis(sulfonamido)
analogs of α,α'-trehalose was shown active (MIC 16-128 μg/mL)
against Mtb H37Ra.[22b] A second 6,6-bisalkyl library of α,α'-
trehalose derivatives was found active against M. smegmatis.[22c]
In addition, 6,6-bis(α-ketoesters/amides) analogs of α,α'-
trehalose have been reported to be active against Ag85C[22d] and
1
13
purification for chromatography. H NMR and C NMR were
recorded on a Bruker Avance III 600 MHz spectrometer using
31
CDCl or D O as internal references. P NMR were recorded on
3
2
a VXRS 400 MHz using CDCl or D O as solvents. High resolution
3
2
mass spectrometry was performed on a Waters SYNAPT HRMS
nano ESI-MS instrument and low-resolution mass spectrometry
was performed on an ESquire-LC-MS.
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-
6
-deoxy-, 6-fluoro-6-deoxy-, and 6-azido-6-deoxy-α,α'-trehalose
glucopyranosyl-(1→1)-α-D-glucopyranoside (9). To a solution of
3 (0.950 g, 0.977 mmol, 1eq.) in tetrahydrofuran (10 mL) at 0 °C
was added triphenylphosphine (0.307 g, 1.17 mmol, 1.2 eq.),
imidazole (0.166 g, 2.44 mmol, 2.5 eq.) and iodine (0.297 g, 1.17
mmol, 1.2 eq.). The reaction was refluxed for 3 h. After completion,
water (10 mL) was added to the reaction flask and organic layer
was extracted with ethyl acetate (15 mL). The organic layer was
were shown to inhibit (MIC 50-100 μM) M. smegmatis biofilm
formation.[
applications of the target compounds.
22e]
Collectively, these studies suggest several possible
Conclusions
2 2 3
washed successively with Na S O (10 mL X 3) and water (10 mL
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X 3). The ethyl acetate was dried over anhydrous Na
filtered. Purification was performed by flash column
chromatography on silica gel (5% ethyl acetate-hexanes) to afford
2
SO
4
and
away through a plug of Celite® 545 that was washed with 20%
methanol-dichloromethane. The filtrate and washings were
concentrated to dryness to afford product TMP as a colorless
1
a colorless viscous liquid (0.768 g, 70% yield): R
f
= 0.35 (20%
): δ 7.38-7.24
m, 33H, aromatic), 7.13 (dd, 2H, J = 7.4, 1.9 Hz, aromatic), 5.27
d, 1H, J = 3.5 Hz, H-1), 5.23 (d, 1H, J = 3.5 Hz, H-1’), 4.98 (m,
H, benzylic), 4.85 (m, 3H, benzylic), 4.72 (m, 5H, benzylic), 4.56
d, 1H, J = 12.1 Hz, benzylic), 4.47 (d, 1H, J = 10.8 Hz, benzylic),
.40 (d, 1H, J = 12.1 Hz, benzylic), 4.14 (m, 1H, H-5’), 4.09 (t, 1H,
solid (0.030 g, quantitative yeild): H NMR (600 MHz, D
2
O): δ 5.24
ethyl acetate-hexanes); 1H NMR (600 MHz, CDCl
(d, 1H, J = 3.5 Hz, H-1), 5.02 (d, 1H, J = 3.8 Hz, H-1’), 3.94 (m,
1H, H-5), 3.75-3.53 (m, 7H, H-2, H-2’, H-4, H-4’, H-5’, H-6a’, H-
6b’), 3.33 (t, 1H, J = 9.4 Hz, H-3’), 3.15 (t, 1H, J = 9.4 Hz, H-3),
3
(
(
3
(
4
2.12 (m, 1H, H-6a), 1.75 (m, 1H, H-6b); 13C NMR (150 MHz, D
δ 92.93, 92.88, 74.32, 74.22, 72.25, 72.05, 71.11, 70.91, 69.59,
2
O):
68.03, 34.82; 31P NMR (162 MHz, D
O): δ 30.78 (s, 1-P); HRMS
2
+
J = 9.3 Hz, H-3), 4.03 (t, 1H, J = 9.4 Hz, H-3’), 3.69 (m, 2H, H-4’,
H-5), 3.62 (dd, 1H, J = 9.6, 3.6 Hz, H-2’), 3.52 (m, 2H, H-6a’, H-
(ESI): calculated for C12
H23PO13, 429.0774 [M+Na] ; observed,
+
m/z = 429.0774 [M+Na] .
4
3
), 3.41 (m, 2H, H-6b’, H-4), 3.25 (dd, 1H, J = 10.9, 4.5 Hz, H-6a),
.12 (dd, 1H, J = 10.8, 2.9 Hz, H-6b); 13C NMR (150 MHz, CDCl
):
3
2,2',3,3',4,4',6'-Hepta-O-benzyl-6-carbaldehyde-α-D-
δ 139.03, 138.81, 138.52, 138.44, 138.35, 138.30, 138.02, 128.74, glucopyranosyl-(1→1)-α-D-glucopyranoside (12). Oxalyl chloride
1
1
9
7
9
28.68, 128.63, 128.62, 128.62, 128.60, 128.22, 128.20, 128.14,
28.14, 128.11, 127.95, 127.89, 127.86, 127.80, 127.60, 94.61,
4.24, 82.06, 81.89, 81.40, 79.81, 79.68,77.92, 75.90, 75.85,
5.68, 75.35, 73.74, 73.34, 73.01, 70.97, 68.92, 68.30, 66.12,
(0.07 mL, 0.80 mmol, 4 eq.) was slowly added to a solution of
dimethyl sulfoxide (0.11 mL, 1.60 mmol, 8 eq.) in dichloromethane
(5 mL) maintained at -78 °C. The solution was allowed to stir at
that temperature for 20 min. To this solution was added 3 (0.20 g,
0.020 mmol, 1eq.) dissolved in 2 mL of dichloromethane. This
solution was allowed to stir for 1 hour at -78 °C. Triethylamine
.59; HRMS (ESI): calculated forC61
H
63IO10, 1105.3364 [M+Na]+;
+
observed, m/z = 1105.3331 [M+Na] .
(
0.45 mL, 3.2 mmol, 16 eq.) was added and the solution was
2
,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-
stirred and additional 20 min. The reaction was allowed to warm
to room temperature over 30 min. dichloromethane (5 mL) was
added to the solution followed by successive washings with
α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside (10). Trimethyl
phosphite (10 mL) was added to compound 9 (0.500 g, 2.16
mmol) and the reaction was refluxed for 36 h. After the completion, saturated NH
4
Cl solution (10 mL X 3) and saturated NaCl solution
excess trimethyl phosphite was evaporated under reduced
pressure. The product was purified by flash column
chromatography on silica gel (5% ethanol-choroform) to afford the
(10 mL X 3). The organic layer was dried over anhydrous Na SO
and concentrating under reduced pressure. The residue was
purified by flash column chromatography on silica gel (40% ethyl
2
4
product as a colorless solid (0.202 g, yield 58%): R
f
= 0.30 (8%
acetate-hexanes) to afford the product as a viscous liquid (0.12 g,
1
1
ethanol-chloroform); H NMR (600 MHz, CDCl
3
): δ 7.38-7.14 (m,
62% yeild): R
f
= 0.37 (30% ethyl acetate-hexanes); H NMR (600
35H, aromatic), 5.47 (d, 1H, J = 3.5 Hz, H-1), 5.26 (d, 1H, J = 3.5
MHz, CDCl
3
): δ 9.36 (s, 1H, H-6), 7.40-7.13 (m, 35H, aromatic),
Hz, H-1’), 4.97 (m, 4H, benzylic), 4.84 (d, 3H, J = 10.8 Hz,
benzylic), 4.71 (m, 3H, benzylic), 4.65 (d, 1H, J = 11.2 Hz,
benzylic), 4.55 (d, 1H, J = 12.3 Hz, benzylic), 4.49 (d, 1H, J = 11
Hz, benzylic), 4.43 (d, 1H, J = 12.1 Hz, benzylic), 4.30 (m, 1H, H-
5.23 (d, 1H, J = 3.5 Hz, H-1), 5.15 (d, 1H, J = 3.7 Hz, H’-1), 5.02
(dd, 2H, J = 10.9, 5.6 Hz, benzylic), 4.87 (m, 5H, benzylic), 4.73
(d, 1H, J = 11.7 Hz, benzylic), 4.67 (m, 3, H-5, benzylic), 4.57 (m,
3H, benzylic), 4.48 (d, 1H, J = 10.6 Hz, benzylic), 4.41 (d, 1H, J =
12.1 Hz, benzylic), 4.15 (m, 1H, H-5’), 4.10 (t, 1H, J = 9.6 Hz, H-
3), 4.02 (t, 1H, J = 9.4 Hz, H-3’), 3.70 (t, 1H, J = 9.6 Hz, H-4’),
3.60 (dd, 1H, J = 9.7, 3.7 Hz, H-2’), 3.57-3.51 (m, 3H, H-2, H-4,
H-6a’), 3.40 (dd, 1H, J = 10.6, 1.8 Hz, H-6b’); 13C NMR (150 MHz,
5
6
), 4.11 (m, 3H, H-3, H-3’, H-5’), 3.66 (m, 2H, H-2, H-2’), 3.61 (m,
H, 2X-OCH ), 3.55 (m, 1H, H-4’), 3.42 (t, 1H, H-4), 2.12 (m, 1H,
3
1
3
H-6a), 1.88 (m 1H, H-6b); C NMR (150 MHz, CDCl
3
): δ 139.11,
1
1
1
7
7
1
38.87, 138.84, 138.68, 138.58, 138.33, 138.14, 128.62, 128.61,
28.58, 128.55, 12853, 128.48, 128.17, 128.10, 128.01, 127.76,
27.73, 92.55, 92.14, 82.18, 82.09,82.03, 81.50, 81.49, 79.81,
9.73, 78.01, 75.82, 75.71, 75.17, 75.15, 73.70, 73.27, 72.92,
0.83, 68.66, 66.92, 66.88, 66.11, 52.46, 52.41, 52.32, 52.28,
3
CDCl ): δ 197.98, 138.93, 138.60, 138.35, 137.99, 137.95, 137.91,
137.55, 128.71, 128.64, 128.61, 128.58, 128.54, 128.51, 128.38,
128.32, 138.23, 128.20, 128.10, 128.05, 127.92, 127.87, 127.83,
127.78, 127.66, 95.29, 94.66, 81.97, 81.73, 79.50, 78.85, 78.51,
5.52; 31P NMR (162 MHz, CDCl
): δ 32.13 (s, 1-P); LRMS (ESI):
77.78, 76.00, 75.81, 75.38, 74.85, 73.70, 73.40, 72.90, 71.05,
3
+
+
calculated for C63
H69PO13, 1087.4 [M+Na] ; observed, m/z =
68.20; LRMS (ESI): calculated for C61
observed, m/z = 993.4 [M+Na]+.
H
62
O
11, 993.4 [M+Na] ;
1
087.5 [M+Na]+.
6-Deoxy-6-(phosphonic acid)-α,α'-trehalose (4, TMP). To
a
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6E-(dimethyl
solution of 10 (0.080 g, 0.075 mmol) in dichloromethane (4 mL)
was added bromotrimethylsilane (129 µL, 0.977 mmol, 13 eq.)
dropwise and the resulting solution stirred for 2 h at room
temperature. A solution of methanol-water (1.1:1.8 mL) was
added followed by concentration to dryness to afford
phosphonicacid 11. The residue was dissolved in 3 mL of
tetrahydrofuran-ethanol (1:4) and 20% Pd(OH) /C (80 mg) was
2
added. The suspension was stirred overnight at room
temperature under 1 atm. of hydrogen. The catalyst was filtered
vinylphosphonate)-α,α’-trehalose (13). Sodium hydride, 60%
dispersion in oil, (7 mg, 0.30 mmol, 3 eq.) was added to a solution
of tetramethyl methylenediphosphonate (0.03 mL, 0.15 mM, 1.5
eq.) in tetrahydrofuran (3 mL) maintained at 0 °C. The mixture
was stirred for 30 min. A solution of aldehyde 12 (0.10 g, 0.10
mmol, 1eq.) in 1 mL of tetrahydrofuran was added dropwise to the
anion. The solution was allowed to warm to room temperature and
stirred for another 2 h. The solvent was evaporated and the
residue purified by flash chromatography on silica gel (5%
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methanol-dichloromethane) to afford 13 as a colorless viscous
H-5), 4.02 (m, 2H, H-3’, H-3), 3.68 (m, 1H, H-4’), 3.61 (m, 1H, H-
liquid (0.054 g, 49% yeild):
dichloromethane); H NMR (600 MHz, CDCl
R
f
=
0.48 (10% methanol-
): δ 7.38-7.23 (m,
2), 3.56 (m, 1H, H-2’), 3.49 (m, 2H, H-6a’, H-6b’), 3.37 (m, 1H, H-
4), 3.18 (m, 2H, H-6a, H-6b); C NMR (150 MHz, CDCl ): δ
3
1
13
3
3
6
=
4
3H, aromatic), 7.24-7.13 (m, 2H, aromatic), 6.96-6.88 (m, 1H, H-
), 5.94 (m, 1H, H-7), 5.23 (d, 1H, J = 3.7 Hz, H-1), 5.1 (d, 1H, J
3.7 Hz, H-1’), 5.0 (dd, 2H, J = 10.8, 2.2 Hz, benzylic), 4.86 (m,
H, benzylic), 4.75 (m, 1H, H-5), 4.72-4.76 (m, 2H, benzylic), 4.57
m, 4H, benzylic), 4.47 (d, 1H, J = 10.8 Hz, benzylic), 4.39 (d, 1H,
J = 12.1 Hz, benzylic), 4.15 (m, 1H, H-5’), 4.09 (t, 1H, J = 9.06 Hz,
H-3), 4.03 (t, 1H, J = 9.06 Hz, H-3’), 3.66 (m, 6H, H-4’, 2X-OCH ),
.56 (m, 2H, H-2 H-2’), 3.51 (m, 1H, H-6a’), 3.38 (dd, 1H, J = 10.6,
138.83, 138.66, 138.29, 138.16, 138.06, 137.79, 128.49, 128.43,
128.38, 128.37, 128.03, 128.01, 127.93, 127.90, 127.89, 127.73,
127.66, 127.62, 127.57, 127.47, 127.38, 94.50, 94.01, 81.82,
81.46, 79.50, 79.36, 78.30, 77.69, 77.26, 77.04, 76.83, 75.62,
(
75.21, 75.13, 73.53, 72.96, 72.78, 70.74, 70.35, 68.10, 51.08;
+
HRMS (ESI): calculated for C61
observed, m/z = 1020.4424 [M+Na] .
H
63IO10, 1020.4411 [M+Na] ;
+
3
4
1
1
3
.8 Hz, H-6b’), 3.25 (dd, 1H, J = 10.1, 9.2 Hz, H-4); C NMR (150
): δ 149.17, 138.57, 138.35, 138.01, 137.76, 137.67,
37.50, 137.36, 128.28, 128.18, 127.75, 127.39, 127.15, 116.68,
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-amino-α,α’-trehalose
(16). Triphenylphosphine (0.236 g, 0.900 mmoL, 3 eq.) and water
(0.027 g, 1.50 mmoL, 5 eq.) were added to compound 15 (0.300
g, 0.300 mmol, 1 eq.) dissolved in tetrahydrofuran (8 mL). The
solution was stirred for 12 h and concentrated under reduced
pressure. Ethyl acetate (10 mL) and water (10 mL) were added to
the residue and the organic layer separated and dried over
MHz, CDCl
3
1
1
7
7
15.42, 94.30, 93.63, 81.64, 81.58, 81.19, 78.98, 78.60, 77.41,
5.48, 75.40, 75.31, 74.87, 73.27, 72.71, 72.59, 70.45, 70.31,
0.17, 67.86, 52.10; 31P NMR (162 MHz, CDCl
3
): δ 32.42 (s, 1-P);
H
69PO13, 1099.4373 [M+Na]+;
HRMS (ESI): calculated for C64
observed, m/z = 1099.4370 [M+Na] .
+
2 4
anhydrous Na SO . The solvent was removed under reduced
pressure and the residue subjected to flash column
6
-Deoxy-6-(methylenephosphonic acid)-α,α’-trehalose (5, TEP).
Bromotrimethylsilane (83.4 µL, 0.652 mmol, 13 eq.) was added
dropwise to solution of 13 (0.09 g, 0.05 mmol) in
dichloromethane (4 mL) and the resulting solution was stirred for
h at room temperature. The solution was quenched with 2 mL
chromatography on silica gel (10% acetone-hexanes) to afford 16
as a white solid (0.290 g, quantitative yield): R
f
= 0.21 (30%
1
a
acetone:hexanes); H NMR (600 MHz, CDCl
3
): δ 7.67 (m, 9H,
aromatic), 7.52 (m, 4H, aromatic), 7.44 (m, 9H, aromatic), 7.37-
7.29 (12H, aromatic), 7.14 (dd, 2H, J = 7.7, 1.5 Hz, aromatic), 5.22
(d, 1H, J = 3.7 Hz, H-1), 5.20 (d, 1H, J = 3.7 Hz, H-1’), 5.01 (t, 2H,
J = 10 Hz, benzylic), 4.88 (m, 3H, benzylic), 4.83 (d, 1H, J = 10.8
Hz, benzylic), 4.72 (m, 5H, benzylic), 4.64 (m, 1H, J = 11.2 Hz,
benzylic), 4.55 (d, 1H, J = 12.3 Hz, benzylic), 4.48 (d, 1H, J =
10.8 Hz, benzylic), 4.39 (d, 1H, J = 12.1 Hz, benzylic), 4.18 (dd,
1H, J = 12.1, 2.2 Hz, H-5’), 4.08 (td, 2H, J = 9.3, 7.1 Hz, H-3, H-
3’), 4.00 (m, 1H, H-5), 3.7 (t, 1H, J = 9.7 Hz, H-4’), 3.63 (dd, 1H,
J = 9.7,3.7 Hz, H-2’), 3.53 (m, 2H, H-6a’, H-4), 3.41 (m, 2H, H-6b’,
H-4), 2.81 (dd, 1H, J = 13.7, 2.8 Hz, H-6a), 2.66 (dd, 1H, J = 13.8,
2
of methanol-water (1.1:1.8) followed by concentration to dryness.
To the solution of benzylated disaccharide 14 was taken up in 3
mL of tetrahydrofuran-ethanol (1:4) and 20% Pd(OH) /C (50 mg)
2
was added. The mixture was stirred overnight under hydrogen (1
atm.). The catalyst was filtered off through Celite® 545 and
washed with 20% methanol-dichloromethane. The combined
filtrate and washings were concentrated to dryness to afford TEP
as viscous syrup (0.019 g, quantitative yield): H NMR (600 MHz,
D O): δ 5.04 (s, 2H, H-1, H-1’), 3.74-3.64 (m, 6H, H-6a’, H-6b’, H-
2
1
5, H-5’, H-3, H-3’), 3.55-3.50 (m, 2H, H-2, H-2’), 3.32 (t, 1H, J =
9.6 Hz, H-4’), 3.16 (t, 1H, J = 9.4 Hz, H-4), 1.98 (m, 1H, H-6a),
1
.84 (m, 1H, H-6b), 1.59 (m, 2H, H-7a, H-7b); 13C NMR (600 MHz,
5.3 Hz, H-6b); 13C NMR (150 MHz, CDCl
3
): δ 138.66, 138.63,
138.16, 138.04, 138.01, 137.62, 132.67, 131.98, 131.93, 131.87,
131.79, 131.76, 128.37, 128.29, 128. 21, 128.16, 127.97, 127.81,
127.77, 127.77, 127.70, 127.51, 127.47, 127.36, 127.33, 127.29,
127.27, 127.19, 93.94, 93.57, 81.67, 81.56, 79.54, 79.28, 78.08,
77.58, 75.41, 75.34, 74.87, 74.70, 73.32, 72.81, 72.60, 71.71,
2
D O): δ 93.39, 93.11, 73.14, 72.43, 72.36, 72.08, 71.61, 71.50,
3
1
71.11, 70.88, 69.60, 60.41, 24.33; P NMR (162 MHz, D
2
O): δ
27.29 (s, 1-P); HRMS (ESI): calculated for C13 29PO13, 443.0930
H
+
+
[
M+Na] ; observed, m/z = 443.0946 [M+Na] .
70.46, 67.97, 42.34; LRMS (ESI): calculated for C61H63IO10, 972.5
+
+
[
M+H] ; observed, m/z = 972.9 [M+H] .
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-azido-α,α’-trehalose
(
15). Sodium azide (0.114 g, 1.76 mol, 5 eq.) was added to a
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-(dibenyzl
phosphoramidate)-α,α’-trehalose (17). Dibenzylphosphoryl
solution of iodide 9 (0.380 g, 351 mmol, 1eq.) in DMF (8 mL). The
mixture was heated to reflux for 24 h. The solution was diluted
with ethyl acetate and washed with saturated NaCl solution (10
mL X 3). The organic layer was dried over anhydrous Na SO ,
2 4
filtered, and the solvent evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
chloride (0.045 g, 0.154 mmol, 3 eq.) dissolved in 2 mL of
dichloromethane was added to compound 16 (0.050 g, 0.051
mmoL, 1eq.) dissolved in 5 mL of dichloromethane. Triethylamine
(0.051 g, 0.51 mmoL, 10 eq.) was added and the solution stirred
for 6 h. The solution was concentrated under reduced pressure
and the residue subjected to flash column chromatography on
(
8% ethyl acetate-hexanes) to afford 15 as a colorless viscous
syrup (0.300 g, 86% yield):
acetate:hexanes); H NMR (600 MHz, CDCl
R
f
=
0.33 (20% ethyl
silica gel (8% acetone-hexanes) to afford a 17 as aviscous syrup
1
1
1
3
): δ H NMR (600
(0.033 g, 52% yield): R
f
=0.32 (30% acetone:hexanes); H NMR
MHz, CDCl
.22 (d, 2H, J = 3.5 Hz, H-1, H-1’), 4.99 (dd, J = 10.8, 7.3 Hz, 2H,
benzylic), 4.85 (m, 4H, benzylic), 4.71 (m, 4H, benzylic), 4.62 (s,
H, benzylic), 4.55 (m, 2H, benzylic), 4.46 (d, 1H, J = 10.6 Hz,,
benzylic), 4.38 (d, 1H, J = 12.1 Hz, benzylic), 4.16 (m, 1H, H-5’,
3
): 7.32 (m, 33H, aromatic), 7.12 (m, 2H, aromatic),
(600 MHz, CDCl
3
): δ 7.3 (m, 43H, aromatic), 7.14 (m, 2H,
5
aromatic), 5.12 (d, 1H, J = 3.7 Hz, H-1), 5.10 (d, 1H, J = 3.7 Hz,
H-1’), 4.97 (m, 6H, benzylic), 4.81 (m, 5H, benzylic), 4.63 (m, 6H,
benzylic), 4.53 (m, 1H, benzylic), 4.45 (m, 1H, benzylic), 4.38 (m,
1H, benzylic), 4.12 (m, 1H, H-5’), 4.01 (m, 3H, H-3, H-3’, H-5’),
1
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3
3
1
1
1
1
1
7
6
1
.65 (m, 1H, H-4’), 3.56 (m, 1H, H-2’), 3.50 (m, 2H, H-5, H-6a’),
64 10
68.63, 68.24, 21.24; LRMS (ESI): calculated for C63H O ,
1003.4 [M+Na] ; observed, m/z = 1003.9 [M+Na] .
+
+
.44 (m, 2H, H-2), 3.37 (m, 1H, H-6b’), 3.08 (m, 1H, H-4), 2.93 (m,
1
3
3
H, H-6a), 2.70 (m, 1H, H-6b); C NMR (150 MHz, CDCl ): δ
38.96, 138.91, 138.46, 138.35, 138.23, 138.21, 137.96, 128.70,
28.65, 128.62, 128.56, 128.54, 128.51, 128.43, 128.40, 128.23,
28.14, 128.06, 128, 127.96, 127.91, 127.86, 127.72, 127.59,
27.56, 127.52, 94.38, 93.91, 79.55, 79.32, 77.87, 77.83, 75.73,
5.66, 75.25, 75.03, 73.66, 72.97, 72.93, 70.83, 68.30, 68.24,
2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-deoxy-6-vinyl-α,α’-trehalose
(19). Compound 18 (0.245 g, 0.250 mmol) was dissolved in 2 mL
of tetrahydrofuran in a three-neck flask equipped with a dry ice
condenser. The flask wwas then set in a dry ice-acetone bath.
Ammonia gas at was passed through the condenser until several
milliliters of ammonia collected. A small piece of sodium metal
was added to the solution, which immediately turned turquois in
color. The ammonia was refluxed at -33 °C for 15 minutes. The
reaction was quenched with 5 mL of methanol. The ammonia was
evaporated and the remaining liquids removed under reduced
pressure and the residue further dried under high vacuum for 12
h. To the dried residue was added pyridine (3 mL), acetic
anhydride (3 mL) and a catalytic amount of DMAP. The solution
was stirred for 12 h and then the solvent was concentrated under
reduced pressure. The residue was subjected to flash column
chromatography on silica gel (5% ethyl acetate-hexanes) to afford
3
1
8.20, 68.14, 68.11, 41.53; P NMR (162 MHz, D
2
O): δ 10.89 (s,
-P); HRMS (ESI): calculated for C61
H
63IO10, 1232.5289 [M+H]+;
+
observed, m/z = 1232.5276 [M+H] .
6-(phosphoramidic acid)-α,α’-trehalose (6, TNP). A catalytic
amount of 20% Pd(OH) /C was added to a solution of benzylated
2
disaccharide derivative 17 (0.020 g, 0.016 mmol) dissolved in 3
mL of tetrahydrofuran-ethanol (1:4). The mixture was stirred
overnight under hydrogen (1 atm.). The catalyst was filtered away
through a plug of Celite® 545 and washed with 20% methanol-
dichloromethane. The combined filtrate and washings were
concentrated to dryness afford TNP as a viscous syrup (0.006 g,
19 as an amorphous solid (0.095 g, 59% yield): R
f
= 0.33 (30%
1
1
quantitative yield): H NMR (600 MHz, D
2
O): δ 5.12 (d, 1H, J = 3.5
ethyl acetate:hexanes); H NMR (600 MHz, CDCl
3
): δ 5.72 (m, 1H,
Hz, H-1), 5.07 (d, 1H, J = 3.5 Hz, H-1’), 3.87 (m, 1H, H-5), 3.82
m, 1H, H-6a’), 3.75 (m, 4H, H-3, H-3’, H-5’, H-6b’), 3.64 (m, 1H,
H-4’), 3.55 (m, 2H, H-2, H-2’), 3.33 (m, 1H, H-4), 3.25 (m, 1H, H-
H-7), 5.48 (m, 2H, H-3, H-3’), 5.27 (d, 2H, J = 3.7 Hz, H-1, H-1’),
5.01 (m, 7H, H-2, H-2’, H-4, H6a,b, H8a,b), 4.23 (m, 1H, H-6a’),
4.04 (m, 3H, H-4’, H-5’, H-6b’), 3.38 (m, 1H, H-5), 2.06 (m, 21H,
(
a), 3.03 (m, 1H, H-6b); 13C NMR (150 MHz, D
2.46, 72.14, 71.40, 70.99, 70.66, 69.53, 68.09, 60.36, 40.44; 31
O): δ 4.62 (s, 1-P); HRMS (ESI): calculated for
2 3 3
O): δ 93 .52, 93.35, 7XCH ): δ 170.67, 170.06, 169.95,
); 13C NMR (150 MHz, CDCl
6
7
P
169.70, 169.69, 169.65, 169.61, 132.39, 118.48, 91.94, 71.78,
70.10, 69.74, 69.10, 68.56, 68.07, 61.78, 35.45, 29.72, 20.75,
NMR (162 MHz, D
23PO13, 422.1064 [M+H] ; observed, m/z = 422.1052 [M+H] .
2
+
+
20.64; HRMS (ESI): calculated for C61H
63IO10, 647.2187 [M+H]+;
C
12
H
observed, m/z = 647.2193 [M+H]+.
2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose
(
18). n-Butyllithium (0.415 mL, 2 M) was slowly added to a
2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-deoxy-6-oxiranyl-α,α’-trehalose
(20). Compound 19 (0.050 g, 0.077 mmol, 1 eq.) was dissolved in
dichloromethane (3 mL) and 3-chloroperoxybenzoic acid (0.066 g,
0.387 mmol, 5 eq.) was added. The solution was stirred for an
hour at room temperature and the solvent was was evaporated
under reduced pressure. The reside was subjected to flash
column chromatography on silica gel (10% ethyl acetate-
hexanes) to afford 20 as an amorphous solid (0.030 g, 59% yield):
solution of trimethylsilylacetylene (118 µL, 0.831 mmol, 3 eq.) in
tetrahydrofuran (3 mL) at -78 °C. The solution was allowed to
warm to 0 °C and was strirred for 30 min. Afterward, the solution
was allowed to warm to room temperature over 50 min. The
solution of anion was added to another round bottom flask
containing iodide 9 (0.300 g, 0.277 mmol, 1 eq). To the resulting
solution was added hexamethylphosphoramide (0.768 mL, 4.43
mmol, 16 eq.). The solution changed to brown in color and was
R
f
= 0.42 (30% ethyl acetate:hexanes); 1H NMR (600 MHz,
CDCl ): δ 5.51 (m, 3H), 5.32 (m, 4H), 5.08 (m, 7H), 4.92 (t, 1H, J
stirred for 12
h
at room temperature. The solution was
3
concentrated under reduced pressure and the residue subjected
to flash column chromatography on silica gel (5% ethyl acetate-
= 9.7 Hz), 4.23 (dt, 2H, J = 12.6, 6.3 Hz), 4.06 (m, 5H), 2.98 (m,
1H), 2.78 (m, 2H) 2.43 (td, 2H, J = 5.5, 2.7 Hz), 2.17 (m, 6H,
3 3 3
2XCH ), 2.08 (m, 36H, 12XCH ): δ
); 13C NMR (150 MHz, CDCl
hexanes) to afford 18 as a colorless viscous syrup (0.095 g, 35%
1
yield): R
f
= 0.73 (30% ethyl acetate-hexanes); H NMR (600 MHz,
170.69, 170.11, 170.10, 170.02, 169.98, 169.95, 169.94, 169.87,
169.87, 169.79, 196.73, 169.65, 169.64, 169.62, 92.29
(anomeric), 92.11 (anomeric), 91.75 (anomeric), 91.34
(anomeric), 72.20, 71.85, 70.24, 70.13, 70.05, 70.00, 69.95,
69.92, 69.40, 69.22, 68.62, 68.17,68.09, 67.49, 67.38, 61.85,
CDCl
3
): δ 7.26-7.15 (m, 33H, aromatic), 7.06 (dd, 2H, J = 7.5 Hz,
.8, aromatic), 5.17 (dd, 2H, J = 8.9, 3.6 Hz, H-1, H-1’), 4.93 (dd,
H, J = 10.8, 6.2 Hz, benzylic), 4.8 (m, 4H, benzylic), 4.63 (m, 5H,
1
2
benzylic), 4.48 (d, 1H, J = 12.1 Hz, benzylic), 4.39 (d, 1H, J = 10.6
Hz, benzylic), 4.32 (d, 1H, J = 12.1 Hz, benzylic), 4.09 (m, 2H, H-
48.27, 48.18, 20.65; LRMS (ESI): calculated for C61H63IO10, 685.2
+
+
5, H-5’), 3.97 (td, 2H, J = 9.3, 7.7 Hz, H-3, H-3’), 3.62 (t, 1H, J =
9.7 Hz, H-3), 3.53 (ddd, 3H, J = 9.5, 7.6, 3.6 Hz, H-4’, H-2, H-2’),
3.45 (dd, 1H, J = 10.7, 3.2 Hz, H-6a’), 3.31 (dd, 1H, J = 10.6, 1.8
[M+Na] ; observed, m/z = 685.3 [M+Na] .
6-deoxy-6-oxiranyl-α,α’-trehalose (7). Compound 20 (0.044 g,
0.066 mmol) was dissolved in 9 mL of a mixture of triethylamine-
methanol-water (2:6:1). The solution was stirred for an hour in
Hz, H-6b’), 2.37 (m, 1H, H-6a), 2.25 (dt, 1H, J = 17, 3.3 Hz, H-6b),
.95 (t, 1H, J = 2.6 Hz, H-7 alkyne); 13C NMR (150 MHz, CDCl
):
1
3
δ 139.03, 138.90, 138.49, 138.45, 138.30, 137.97, 128.59, 128.53, dark. The solvent was evaporated under reduced pressure and
1
(
7
28.17, 128.13, 127.59, 127.54, 94.54 (anomeric), 94.46
anomeric), 81.96, 81.67, 80.46, 80.20, 79.72, 79.52, 77.82,
5.83, 75.80, 75.59, 75.28, 73.66, 72.95, 72.87, 71.05, 70.82,
subjected to flash column chromatography on silica gel using
ethyl acetate-isopropanol-water (6:3:1) as an eluent to afford 7 as
an viscous syrup (0.012 g, 50% yield): R = 0.45 (6:3:1 ethyl
f
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1
Y=100/(1+(XHillSlope)/(IC50HillSlope)) as implemented in Prism 7.0
(GraphPad). All measurements were performed in quadruplicate.
acetate-isopropanol-water); H NMR (600 MHz, D
2
O): δ 5.05 (m,
2
2
H, anomeric), 3.87 (m, 1H), 3.72 (m, 5H), 3.54 (m, 2H), 3.33 (m,
H), 3.19 (m, 1H), 3.09 (m, 2H), 1.79 (m, 2H). C NMR (150 MHz,
O): δ 93.54, 93.38, 93.21, 93.04, 73.24, 73.19, 72.53, 72.50,
1
3
D
2
7
6
3
3
2.41, 72.11, 71.97, 71.38, 71.07, 71.05, 70.92, 70.29, 69.62,
9.59, 69.52, 69.16, 60.42, 51.44, 50.44, 48.28, 46.71, 46.59,
Acknowledgements
3.63, 23.19, 20.40, 8.15; HRMS (ESI): calculated for C61H63IO10,
+
+
69.1397 [M+H] ; observed, m/z = 369.1219 [M+H] .
This work was supported in part by a grant from the National
Institutes of Health (Grant No. AI105084) to S.J.S. and D.R.R.
Protein expression and purification. The otsB2 gene, encoding
for the Mtb (UniProt code: P9WFZ5) trehalose-6-phosphate
phosphatase, TPP, was amplified using Mtb genomic DNA and
inserted into a modified pET-28 plasmid. Codon optimized genes
encoding the TPP orthologs from Mlt (UniProt code:
A0A0E4GVV3) and Mtx (UniProt code: A0A024JUE5) were
purchased from IDT Inc., amplified and inserted into modified
pET-32 plasmids. The resulting pDR28-otsB2, pDR32-Mlt-otsB2
and pDR32-Mtx-otsB2 plasmids were used in this study to encode
and produce recombinant TPP possessing N-terminal
polyhistidine tags. The sequences of the expression plasmids
mentioned above were confirmed by DNA sequencing (MWG
Operon). These plasmids were used to transform E. coli T7
Rosetta. The cells were then cultured at 37 °C in Luria Broth to an
O.D.600 nm of 0.6. Gene expression was then induced by adding
IPTG to a final concentration of 1 mM followed by a 24-hour
incubation at 16 °C. The bacteria were harvested by
centrifugation and resuspended with buffer A (20 mM Tris pH 7.5,
Notes and references
‡
S. Kapil and C. Petit contributed equally to the manuscript.
[
a] S. Kapil, C. Petit, V.N. Drago, Prof. Dr. D. R. Ronning and
Prof. Dr. S.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
E-mail: steve.sucheck@utoledo.edu;
donald.ronning@utoledo.edu
[b] Dr. C. Petit
EMBL Hamburg
c/oDESY, Building 25A, Notkestraß
e85, 22603 Hamburg, Germany
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Keywords: trehalose • trehalose 6-phosphate phosphatase •
enzyme inhibitors • Mycobacterium tuberculosis • carbohydrates
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‡
‡
Text for Table of Contents
Sunayana Kapil , Cecile Petit , Victoria
N. Drago, Donald R. Ronning* and
Steven J. Sucheck*
Page No. – Page No.
Synthesis and in vitro
characterization of trehalose-based
inhibitors of mycobacterial trehalose
6-phosphate phosphatases
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