Synthesis of C-glycoside analogues of β-galactosamine-(1→4)-3-O-methyl-d-chiro-inositol and assay as activator of protein phosphatases PDHP and PP2Cα

Article abstract of DOI:10.1016/j.bmc.2009.12.056

The glycan β-galactosamine-(1-4)-3-O-methyl-d-chiro-inositol, called INS-2, was previously isolated from liver as a putative second messenger-modulator for insulin. Synthetic INS-2 injected intravenously in rats is both insulin-mimetic and insulin-sensitizing. This bioactivity is attributed to allosteric activation of pyruvate dehydrogenase phosphatase (PDHP) and protein phosphatase 2Cα (PP2Cα). Towards identification of potentially metabolically stable analogues of INS-2 and illumination of the mechanism of enzymatic activation, C-INS-2, the exact C-glycoside of INS-2, and C-INS-2-OH the deaminated analog of C-INS-2, were synthesized and their activity against these two enzymes evaluated. C-INS-2 activates PDHP comparable to INS-2, but failed to activate PP2Cα. C-INS-2-OH was inactive against both phosphatases. These results and modeling of INS-2, C-INS-2 and C-INS-2-OH into the 3D structure of PDHP and PP2Cα, suggest that INS-2 binds to distinctive sites on the two different phosphatases to activate insulin signaling. Thus the carbon analog could selectively favor glucose disposal via oxidative pathways.

Full text of DOI:10.1016/j.bmc.2009.12.056

Bioorganic & Medicinal Chemistry 18 (2010) 1103–1110
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of C-glycoside analogues of b-galactosamine-(1?4)-3-O-methyl-D-
chiro-inositol and assay as activator of protein phosphatases PDHP and PP2C
a
Sunej K. Hans a,b, Fatoumata Camara a,b, Ahmad Altiti a,b, Alejandro Martín-Montalvo d,
David L. Brautigan e, Douglas Heimark c, Joseph Larner c, Scott Grindrod f, Milton L. Brown f,
David R. Mootoo a,b,
*
a Department of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10065, United States
b The Graduate Center, CUNY, 365 5th Avenue, New York, NY 10016, United States
c Allomed Pharmaceuticals, Charlottesville Virginia 22908, Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908-0735, United States
d Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, 41013 Seville, Spain
e Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, United States
f Georgetown University Medical Center, Department of Neuroscience, Research Building EP07, 3970 Reservoir Road, Washington, DC 20057, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The glycan b-galactosamine-(1-4)-3-O-methyl-D-chiro-inositol, called INS-2, was previously isolated
Received 6 August 2009
Revised 15 December 2009
Accepted 17 December 2009
Available online 4 January 2010
from liver as a putative second messenger–modulator for insulin. Synthetic INS-2 injected intravenously
in rats is both insulin-mimetic and insulin-sensitizing. This bioactivity is attributed to allosteric activa-
tion of pyruvate dehydrogenase phosphatase (PDHP) and protein phosphatase 2C
a (PP2Ca). Towards
identification of potentially metabolically stable analogues of INS-2 and illumination of the mechanism
of enzymatic activation, C-INS-2, the exact C-glycoside of INS-2, and C-INS-2-OH the deaminated analog
of C-INS-2, were synthesized and their activity against these two enzymes evaluated. C-INS-2 activates
Keywords:
C-Glycoside
Insulin mediator–modulator
Pyruvate dehydrogenase
Protein phosphatase
PDHP comparable to INS-2, but failed to activate PP2C
tases. These results and modeling of INS-2, C-INS-2 and C-INS-2-OH into the 3D structure of PDHP and
PP2C , suggest that INS-2 binds to distinctive sites on the two different phosphatases to activate insulin
a. C-INS-2-OH was inactive against both phospha-
a
signaling. Thus the carbon analog could selectively favor glucose disposal via oxidative pathways.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
oxidative glucose disposal (i.e., PDH), and non-oxidative glucose
disposal (glycogen synthase, GS).5 Both PDH and GS are classical
Soluble inositol glycans have been proposed to operate as insu-
lin messengers–modulators in concert with the insulin receptor
tyrosine kinase signaling system to control intracellular oxidative
and non-oxidative glucose disposal.1 By analogy with the PIP3 sig-
naling pathway, the insulin receptor is thought to regulate, via het-
targets of activation by insulin via dephosphorylation.6,7 When
docked into the X-ray crystal structure of PP2Ca, INS-2 bound to
an allosteric pocket adjoining the catalytic pocket that was occu-
pied by a phosphopeptide substrate.4 INS-2 enhanced reaction
with the phosphopeptide substrate, but not with the small mole-
cule substrate p-nitrophenyl phosphate. A point mutation D163A
2
erotrimeric G proteins, specifically Gq, the GPI-phospholipase
cleavage of a glycolipid to generate diacylglycerol and inositol gly-
cans as messengers–modulators. In earlier studies, we isolated
INS-2, 1 from beef liver as a novel inositol glycan pseudo-disaccha-
ride (Fig. 1). The INS-2 structure was determined by 2D NMR and
confirmed by chemical synthesis.3 Synthetic INS-2 was insulin-mi-
metic and insulin-sensitizing when injected in vivo, and when
added to living cells.3 In biochemical assays, INS-2 activated two
related Mg2+ dependent protein phosphatases, mitochondrial
pyruvate dehydrogenase phosphatase (PDHP) and a Ser/Thr pro-
in PP2C
no loss of catalytic activity.4 Thus a mechanism of allosteric activa-
tion of PP2C
was proposed.4
a completely eliminated allosteric activation by INS-2, with
a
OH
HO
HO
OH
O
HO
MeO
X
Y
HO
OH
tein phosphatase, PP2Ca 3,4
These protein phosphatases, respec-
.
1 X = O; Y = NH2
2 X = CH2; Y = NH2
3 X = CH2; Y = OH
tively dephosphorylate the rate limiting enzymes of intracellular
* Corresponding author. Tel.: +1 212 772 4356; fax: +1 212 772 5332.
E-mail address: dmootoo@hunter.cuny.edu (D.R. Mootoo).
Figure 1. (1) INS-2, (2) C-INS-2 and (3) C-INS-2-OH.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.056

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S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
To further define the structure–activity relationship of INS-2,
R
we synthesized C-INS-2, 2, the C-glycoside of 1 in which the glyco-
sidic oxygen is replaced with a methylene and C-INS-2-OH, 3, the
corresponding C-glycoside in which the 2-amino substituent is re-
placed with a hydroxyl group. These analogues were compared to
OTBDPS
OH
O
O
OTBDPS
O
O
+
7
a
O
MeO
O
O
b
INS-2 for activation of the two protein phosphatases, PP2C
a and
O
X
SPh
8
O
PDHP. C-Glycoside probes like 2 and 38 may provide insight on
the chemical and conformational requirements for activity,9 and
are of special interest as possible therapeutic agents that are stable
to hydrolysis.10–12
SPh
X = O
9
10 X = CH2
c
OTBDPS
OTBDPS
O
O
O
e, f
O
3
2
d
2. Synthesis
O
O
R
R
OH
C-Glycoside 3 was prepared using our protocol for b-C-galacto-
sides and the aminated analog 2 was obtained from an intermedi-
ate en route to 3.13 Accordingly, di-O-isopropylidene pinitol 4 was
transformed to the thiocarbonate 5, which when subjected to the
Keck radical allylation procedure gave the equatorial C-propenyl
derivative 6 in greater than 90% stereoselectivity as determined
by 1H NMR.14 (Scheme 1). Oxidative processing of 6 led to the
C-branched-ethanoic acid 7. Acid 7 and known 1-thio-1,2-O-iso-
propylidene acetal 8 were next subjected to the C-glycosidation
sequence (Scheme 2). Thus, DCC mediated esterification on 7 and
8, followed by Tebbe olefination of the derived ester 9 afforded
enol ether 10. In the key step, treatment of 10 with methyl triflate
in the presence of 2,6-di-tert-butyl-4-methylpyridine (DTBMP),
provided glycal 11 in 77% yield. Hydroboration of 11 afforded
C-pseudodisaccharide 12 as a single diastereomer. Removal of
the isopropylidene protecting groups provided C-INS-2-OH 3. For
the synthesis of 2, alcohol 12 was first oxidized to ketone 13 using
PCC. Treatment of 13 with O-methylhydroxylamine afforded a
mixture of oximes 14. Attempted reduction of 14 with standard
hydride reducing agents produced complex mixtures with low
yield of the desired hydroxylamine ether 15.15,16 The optimal con-
ditions were found to be Bu3SnH in the presence of BF3ꢀOEt2, which
afforded 15 in 82% isolated yield, and a minor compound (less than
5%) for which the physical data supported the axial isomer.17
Cleavage of the silylether in 15 followed by methoxylamine reduc-
tion and cleavage of acetal protecting groups provided C-INS-2 in
73% overall yield from 15. The structures of 2 and 3 were con-
firmed by 2D NMR and HRMS analysis (Supplementary data).
12
11
g
OTBDPS
O
OTBDPS
O
O
O
e, j, f
73%
i
O
O
R
R
X
NHOMe
13 X = O
15
h
14 X = NOMe
Scheme 2. Assembly of C-INS-2 and C-INS-2-OH. Reagents and conditions: (a) DCC,
CH2Cl2, DMAP, 89%; (b) Tebbe reagent, toluene/THF, pyridine, ꢁ78 °C?rt, 75%; (c)
MeOTf, DTBMP, CH2Cl2, MS 4 Å, 77%; (d) BH3ꢀMe2S, THF; then Na2O2, 76%; (e)
nBu4NF, THF; (f) HCl in ether, CH3OH, 2 steps, 92%,; (g) PCC, NaOAc, florisil, MS 4 Å,
CH2Cl2, 95%; (h) NH2OMeꢀHCl, NaOAc, THF/MeOH/H2O, 86%; (i) Bu3SnH, BF3ꢀOEt2,
CH2Cl2, ꢁ20 °C, 82%; (j) Na, NH3, ꢁ78 °C?rt, THF, 78%.
ously shown, Mn2+ alone and INS-2 showed phosphate release as-
sayed by dose-dependent increase in PP2Ca activity, while the C-
glycosides 2 and 3 were inactive. As shown in Figure 2B, with the
pNPP substrate, as previously shown,4 only Mn2+ increased the cat-
alytic activity of PP2Ca, not INS-2 or its analogues.
As shown in Figure 3, INS-2, C-INS-2, C-INS-2-OH were assayed
Mn2+ for their activation of PDHP. Only INS-2 and C-INS-2 acti-
vated PDHP significantly, and activation was seen only in the pres-
ence of Mn2+, relative to control. The C-INS-2-OH analog was
inactive in the presence or absence of Mn2+. The results were com-
bined from two separate sets of experiments with two separate
chemically synthesized samples. These data show equal activation
of PDHP by INS-2 and its carbon analog, C-INS-2. Thus while C-INS-
2-OH was inactive with both phosphatases, there was a marked
3. Enzyme assays
dichotomy with C-INS-2 which activated PDHP, but not PP2Ca.
PP2Cawas assayed using two separate substrates with and with-
out added INS-2 analogues. p-Nitrophenyl phosphate (pNPP) as sub-
4. Computer modeling
strate assayed the activity of the enzyme catalytic center and this
activity is unaffected by the allosteric site for INS-2. PP2Ca activity
with an octapeptide-phosphate substrate (RRRRPp-TPA) was sensi-
tive to the allosteric site regulating the active site.4 As seen in Figure
2A, with the octapeptide substrate and Malachite green, as previ-
Docking of INS-2 and its analogues was accomplished using the
FlexX flexible docking suite in the SYBYL shell.4,18 The crystal struc-
ture for PP2Ca (1ASQ) and PDHP1 (2PNQ) were acquired from the
Protein Data Bank. The allosteric sites for the two proteins were de-
tailed through a residue selection followed by an 8 Å radius selec-
tion. Compound structures were then prepared in the SYBYL shell
and minimized using the conjugate gradient method with an end-
point of 0.01 kcal. The top 30 conformations of each molecule were
then analyzed using G-score, D-score, PMF-score as previously de-
scribed.4 Multiple scoring functions were then combined for anal-
ysis by the C-score function to rate the conformations.19 In each
case the top scoring two to three conformations of the molecule
overlap well, but then the scores of the conformations drop off
quickly and the position of the molecule is less reproducible (Sup-
plementary data). Putative hydrogen bonds were analyzed through
the SYBYL shell by the addition of hydrogen atoms to the protein
structure with the Biopolymer function.
O
O
O
O
O
O
a
MeO
R
MeO
HO
O
O
4
5 R = OC(=S)OPh
6 R = CH2CH=CH2
7 R = CH2COOH
b
c
Scheme 1. Synthesis of the C-inositol segment. Reagents and conditions: (a)
PhOCSCl, toluene, pyridine, DMAP, 96%; (b) allyltributyltin, toluene, AIBN, 110 °C,
56%; (c) (i) O3, MeOH/CH2Cl2, ꢁ78 °C then Ph3P rt, 85%; (ii) NaClO2, CH3CN/H2O,
NaH2PO4, 75%.
Docking of INS-2 and C-INS-2 into the X-ray crystal structure of
PP2Ca revealed very different orientations and conformations (with

S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
1105
Figure 2. Protein phosphatase 2Ca activity in response to added INS-2, C-INS-2-OH and C-INS-2. The recombinant GST-PP2C fusion protein was assayed as described in the
experimental section, using either phosphopeptide (A) or p-nitrophenyl phosphate (B) as the substrate. Samples were assayed by triplicate and the average values plotted SD
as a function of the concentration of added compounds. (A) The PP2C activity using a phosphopeptide as substrate adding increasing concentrations of MnCl2, INS-2, C-INS-2-
OH or C-INS-2. (B) The PP2C activity using the p-nitrophenyl phosphate as a substrate adding various concentrations of MnCl2, INS-2, C-INS-2-OH or C-INS-2.
tinal hydrolytic breakdown.8,10 A similar strategy was imagined for
INS-2, a natural inositol glycan pseudo-disaccharide, initially iso-
lated from liver and then chemically synthesized and shown to
be an insulin-mimetic and sensitizing agent.3
INS-2, in vitro activates two phosphoprotein phosphatases,
mitochondrial PDHP and cytosolic PP2Ca 3,4
both members of the
,
same PPM family and with ꢂ20% amino acid sequence identity.21
Both have been crystallized and X-ray structures determined.21,22
By computer modeling, we have previously reported that INS-2
docked into an allosteric site on PP2Ca adjacent to the catalytic
site. With a point mutant D163A in the allosteric site, we showed
a specific loss of allosteric activation of the enzyme with INS-2,
but with full retention of catalytic activity measured with a non-
peptide substrate.4 Thus we were able to propose a mechanism
of allosteric action. By occupying the allosteric site, INS-2 would
hydrogen bond to Asp 243 and thus prevent Asp 243 from interfer-
ing with positioning of the phosphopeptide substrate at the cata-
lytic site.4
Figure 3. PDHP assay of INS-2, C-INS-2 and C-INS-2-OH 5 mM Mn2+. Activity is
expressed as change in absorbance at 340 nm. Mean + sem (n = 6) *p <0.016; **p
<0.004 versus control.
In the present study, C-INS-2, the C-glycoside analog of INS-2
was found to be inactive on PP2C
the molecular basis for the difference in activity of INS-2 and
C-INS-2 on PP2C , the binding of the two ligands to the X-ray crys-
tal structure of PP2C was modeled (Fig. 4A). Significant differ-
a (Fig. 3). To get insight into
respect to the intersaccharide torsions) for the two glycans (Fig. 4A
a
and B).11 The intersaccharide torsions
U, W for INS-2 and C-INS-2
a
are (270, 64) and (300, 210), respectively.20 In particular, Asp 243,
which is proximal to the catalytic site (vide infra), interacts with dif-
ferent residues on the two ligands, the sugar 2-amino and 3-hydro-
xyl on INS-2, and the sugar 4- hydroxyl and the inositol 2- and 3-
hydroxyls on C-INS-2 (Fig. 4A and B and Supplementary data). The
orientation of INS-2 suggests that Asp 243 is moved toward the allo-
steric pocket and away from the catalytic site. In contrast, bound C-
INS-2 does not appear to have a significant effect on the position of
Asp 243. However, both INS-2 and C-INS-2 dock very similarly into
the crystal structure for PDHP1. The respective intersaccharide tor-
sions are (244, 60) and (240, 60) and both ligands appear to pull Glu
ences in their binding suggest that INS-2 but not C-INS-2 pulls
Asp 243 into the allosteric site and away from the catalytic pocket,
thereby providing a possible explanation for the inactivity of
C-INS-2. In contrast to their activity on PP2C
showed similar activity on PDHP1. This result was somewhat sur-
prising because PDHP1 and PP2C are related enzymes. We there-
a, INS-2 and C-INS-2
a
fore modeled the binding of INS-2 and C-INS-2 to the recently
published 3D structure of PDHP1 (Fig. 4B) and compared the
results with those for PP2C
similarly to PDHP1 and in a way that suggests they both pull
Glu-351 (the comparable residue to Asp 243 on PP2C ), away from
a (Fig. 4A). INS-2 and C-INS-2 bound
a
351 (the comparable acidic residue to Asp 243 in PPC2
a) from the
the catalytic pocket. Interestingly, the bound conformations of
both ligands on PDHP1 are particularly high energy rotamers with
respect to the intersaccharide torsions, whereas PP2Ca selects rel-
active site (Fig. 4B and Supplementary data).
When C-INS-2-OH was docked into the X-ray crystal structures
of PP2C (Fig. 5A) and PDHP1(Fig. 5B), it bound to both enzymes
a
atively low energy conformations. The selection of high energy
conformers by PDHP1 may be a reflection of an intrinsically higher
binding affinity with this enzyme. Such conformational variations
in bound glycans are influenced by both glycan structure and
receptor architecture and have previously been noted.11,23,24
C-INS-2-OH was inactive with both phosphatases and its inactivity
is consistent with the absence of an appropriately placed amino
group that contributes to pulling Asp 243 or Glu 351 toward the
but not in a fashion that would be expected to pull the acidic res-
idue (either Asp 243 or Glu 351) away from the catalytic pocket.
5. Discussion
Carbon in place of oxygen bridges in carbohydrates are com-
monly synthesized to produce analogues that are resistant to intes-

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S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
Figure 4. (A) INS-2 and C-INS-2 docked in silico into the allosteric site of PP2Ca. H-bonds to the amino group indicated in pink. For the enzyme to be fully active Asp 243
moves to open up the catalytic site. Note the significant difference in position of Asp 243 with the two glycans. INS-2 pulls the acidic residue Asp 243 toward the allosteric
pocket and away from the catalytic site favoring hydrolysis. C-INS-2 still binds to this region, but does not pull Asp 243 towards the allosteric pocket, and is inactive. (B) INS-2
and C-INS-2 docked in silico into the allosteric site of PDHP1. H-bonds to the amino group indicated in pink. Both are similarly positioned to pull the acidic residue Glu 351
towards the allosteric pocket and away from the catalytic site favoring hydrolysis.
Figure 5. (A) C-INS-2-OH docked in silico into the allosteric site of PP2C
a. Note that C-INS-2-OH still binds but is blocked from pulling Asp 243 toward the allosteric pocket.
(B) C-INS-2-OH docked in silico into the allosteric site of PDHP1. Note again that C-INS-2-OH still binds but is blocked from pulling Glu 351 toward the allosteric pockets.
allosteric site. (Fig. 5A and B). However, it is also possible that the
20-amino group may function to chelate Mg2+ or Mn2+ in conjunc-
tion with an OH on the pinitol. This provides an alternative possi-
ble mechanism for the inactivity of C-INS-2-OH.
It is of interest that INS-2 alone activates PP2C
addition of Mn2+ for activating PDHP1. PP2C
is purified and crys-
tallized in the presence of Mn
2+ 22 Two binding sites in a bimetallic
center are well established. In contrast, the PDHP1catalytic subunit
a, but requires
a
.

S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
1107
is isolated in the presence of Mg2+ 21
.
A Mg2+ bimetallic center has
were dried (Na2SO4) and concentrated in vacuo. FCC of the residue
gave 5 (11.5 g, 95%). Rf = 0.43 (10% ethyl acetate/petroleum ether).
1H NMR (CDCl3) d 2.40 (s, 6H, 2 ꢃ CH3–C), 2.60 (two s, 6H, 2 ꢃ CH3-
C), 3.40 (dd, 1H, J = 7.7, 10.9 Hz, H-3), 3.58 (s, 3H, CH3O), 4.39 (t, 1H,
J = 7.7 Hz, H-2), 4.48–4.51 (m, 3H. H-1, H-5, H-6), 5.60 (dd, 1H,
J = 7.1, 10.9 Hz, H-4), 7.16 (d, 2H, J = 8.4 Hz, Ar-H), 7.31 (t, 1H,
J = 7.4 Hz, Ar-H), 7.44 (t, 2H, J = 7.9 Hz, Ar-H); 13C NMR (CDCl3) d
25.4 (CH3–C), 25.5 (CH3–C), 27.5 (CH3–C), 27.8 (CH3–C), 60.2
(CH3O), 75.4, 75.5, 76.0, 78.4, 80.4, 82.5, 109.5 (OCO),
110.0(OCO), 122.0 (Ar), 126.6 (Ar), 129.5 (Ar), 153.6 (Ar), 195.0
(C@S). HRMS (ESI) m/z calcd for C20H27O7S (M+H)+ 411.1472, found
411.1471.
been established. However, the PDHP1 catalytic subunit is known
to interact with its regulatory subunit, which also has a low affinity
Mg2+ binding site that is inhibitory. We propose that in the case of
PP2Ca
, sufficient Mn2+ is present bound to the bimetallic center of
the enzyme to chelate to added INS-2 to activate the enzyme. In
contrast, INS-2 and C-INS-2 need addition of Mn2+ to activate
PDHP1 and the effect is possibly to displace loosely bound Mg2+
in the regulatory subunit.21
6. Conclusion
The relative activity of the inositol glycan INS-2 and its C-glyco-
7.3. 4-Deoxy-4-C-(30-propenyl)-1,2,5,6-di-O-isopropylidene-3-O-
methyl-D-chiro-inositol 6
side analog C-INS-2 on PP2C
implicated in insulin modulation was evaluated. Both glycans acti-
vated PDHP similarly, but PP2C was only activated by INS-2. This
a and PDHP, two enzymes that are
a
A solution of 5 (4.17 g, 10.2 mmol), allyltributyltin (9.35 mL,
30.6 mmol), AIBN (0.33 g, 2.04 mmol) and toluene (18 mL) was
heated at reflux for 4 h at which point TLC showed complete con-
sumption of the starting material. The solution was concentrated
in vacuo. FCC of the residue afforded 6 (1.70 g, 56%). Rf = 0.64
(10% ethyl acetate/petroleum ether). 1H NMR (5% CDCl3/C6D6) d
1.40 (two s, 6H, 2 ꢃ CH3–C), 1.51 (two s, 6H, 2 ꢃ CH3–C), 1.79–
1.87 (m, 1H, H-4), 2.54–6.67 (m, 2H, CH2–30), 3.02 (dd, 1H, J = 6.8,
J = 12.4 Hz, H-3), 3.54 (s, 3H, CH3O), 4.06 (dd, 1H, J = 6.9, 9.2 Hz,
H-5), 4.15 (t, 1H, J = 6.3 Hz, H-6), 4.21 (t, 1H, J = 7.1 Hz, H-2), 4.30
(t, 1H, J = 6.6 Hz, H-1), 5.20 (d, 1H, J = 10.1 Hz, 1/2 ꢃ =CH2), 5.27
(d, 1H, J = 17.1 Hz, 1/2 ꢃ @CH2); 6.10 (m, 1H, –CH@) 13C NMR (5%
CDCl3/C6D6) d 25.1 (CH3–C), 25.2 (CH3–C), 27.8 (CH3–C), 27.8
(CH3–C), 32.7 (C-30), 41.5 (C-4), 58.4 (CH3O), 75.9, 77.2, 77.8,
79.2, 81.1, 108.7 (OCO), 108.9 (OCO), 117.3 (@CH2), 135.2
(–CH@).
result suggests that the inositol glycan binding sites on the two en-
zymes are distinct. Preliminary modeling experiments appear to
support this conclusion. What possible benefit may be derived from
these results? Since PDHP is a selective activator of mitochondrial
pyruvate oxidation, C-INS-2, in contrast to INS-2, would selectively
activate one of two pathways of glucose disposal, glucose oxidation,
bypassing the other pathway, glycogen synthesis. Such a selective
tool would be useful in investigating insulin and insulin-mimetic
agents for partitioning of intracellular carbon disposal between oxi-
dation and glycogen synthesis. Presently this may be done with
inhibitors of oxidation or of glycogen synthesis, which tend to be
non-specific. In terms of a potential therapeutic agent, selective glu-
cose disposal via oxidation without glycogen storage may be bene-
ficial against insulin resistance and diabetes for disposal of carbon
overload. For future studies, in the context of the advancement of
analogues of INS-2 as potential clinical agents, it will be of interest
to determine whether C-INS-2 does escape intestinal breakdown
and manifests insulin-mimetic effects in vivo.
7.4. 4-Deoxy-4-C-(20-ethanoic acid)-1,2,5,6-di-O-isopropylidene-
3-O-methyl-D-chiro-inositol 7
7. Experimental
Alkene 6 (1.35 g, 4.55 mmol) was dissolved in a 5/1 mixture of
CH2Cl2/MeOH (24 mL). The solution was cooled to ꢁ78 °C and trea-
ted with a stream of O3 in O2 until TLC indicated complete disap-
pearance of the starting material. The reaction was then purged
with nitrogen, and Ph3P (2.64 g, 9.09 mmol) was added. The mix-
ture was warmed to rt, stirred for 1 h at this temperature, and con-
centrated under reduced pressure. FCC of the residue gave the
derived aldehyde (1.15 g, 85%). Rf = 0.16 (10% ethyl acetate/petro-
leum ether). 1H NMR (CDCl3) d 1.30 (two s, 6H, 2 ꢃ CH3–C), 1.51
(two s, 6H, 2 ꢃ CH3–C), 2.09–2.18 (m, 1H, H-4), 2.48 (m, 1H, H-
20), 2.23 (dd, 1H, J = 4.7, 16.2 Hz, H-20), 3.15 (dd, 1H, J = 6.7,
12.1 Hz, H-3), 3.45 (s, 3H, CH3O), 4.04 (dd, 1H, J = 6.5, 9.7 Hz, H-
5), 4.21 (m, 2H, H- 2, 6), 4.36 (dd, 1H, J = 4.7, 6.7 Hz, H-1), 9.65
(d, 1H, J = 2.1 Hz, CH@O); 13C NMR (CDCl3) d 25.3 (CH3–C), 25.4
(CH3–C), 27.8 (2 ꢃ CH3–C), 39.1 (CH2), 43.9 (C-4), 59.1 (CH3O),
76.4, 76.8, 76.9, 80.2, 80.5, 109.3 (2 ꢃ OCO), 200.9 (C@O). HRMS
(ESI) m/z calcd for C15H24NaO6 (M+Na)+ 323.1468, found 323.1468.
To a solution of aldehyde from the previous step (1.4 g,
4.67 mmol) in a 5:1 mixture of CH3CN/H2O (85:17 mL) at 0 °C,
was added NaH2PO4ꢀ3H2O (6.46 g, 46.8 mmol), and a solution of
NaClO2 (0.51 g, 5.62 mmol) in H2O (85 mL) and H2O2 (0.55 mL).
The mixture was stirred at 0 °C until TLC indicated the disappear-
ance of the starting material. Solid Na2SO3 was then added to the
reaction. The reaction mixture was diluted with water and the or-
ganic phase was extracted with ether. The combined extract was
dried (Na2SO4), the solvent removed under reduced pressure, and
the residue purified by FCC to give 7 (1.10 g, 75%). Rf = 0.16 (25%
ethyl acetate/petroleum ether). 1H NMR (CDCl3) d 1.35 (s, 3H,
CH3–C), 1.38, (s, 3H, CH3–C), 1.45 (s, 3H, CH3–C), 1.53 (s, 3H,
CH3–C), 2.05 (m, 1H, H-4), 2.52 (dd, 1H, J = 7.0, 15.7 Hz, H-20),
7.1. General-synthesis
Unless otherwise stated, all reactions were carried out under a
nitrogen atmosphere in oven-dried glassware using standard syr-
inge and septa technique. 1H and 13C NMR spectra were obtained
on a Varian Unity Plus 500 (500 MHz) spectrometer. Chemical shifts
are relative to the deuterated solvent peak or the tetramethylsilane
(TMS) peak at (d 0.00) and are in parts per million (ppm). Assign-
ments for selected nuclei were determined from 1H COSY experi-
ments. Unless otherwise stated, thin layer chromatography (TLC)
was done on 0.25 mm thick precoated silica gel HF254 aluminum
sheets. Chromatograms were observed under UV (short and long
wavelength) light, and were visualized by heating plates that were
dipped in a solution of ammonium(VI) molybdate tetrahydrate
(12.5 g) and cerium(IV) sulfate tetrahydrate (5.0 g) in 10% aqueous
sulphuric acid (500 mL). Flash column chromatography (FCC) was
performed using Silica Gel 60 (230–400 mesh) and employed a step-
wise solvent polarity gradient, correlated with TLC mobility.
7.2. 1,2,5,6-Di-O-isopropylidene-3-O-methyl-4-O-phenylcarbo-
nothioyl-D-chiro-inositol 5
Phenyl chlorothionoformate (8.75 mL, 64.7 mmol) was added
dropwise to a suspension of alcohol 43 (8.05 g, 29.4 mmol) and
DMAP (0.72 g, 5.87 mmol), in dry toluene (120 mL) at rt. Pyridine
(12.0 mL, 147 mmol) was then added and the resulting suspension
was stirred for 2 h at rt. The reaction mixture was then washed
with HCl (0.1 M) and saturated NaHCO3, and the organic extracts

1108
S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
2.71 (dd, 1H, J = 4.3, 15.7 Hz, H-20), 3.16 (dd, 1H, J = 7.3, 12.2 Hz, H-
3), 3.54 (s, 3H, CH3O), 4.14 (dd, 1H, J = 6.4, 9.9 Hz, H-5), 4.25 (m, 2H,
H- 2, 6), 4.38 (dd, 1H, J = 4.7, 6.7 Hz, H-1); 13C NMR (CDCl3) d 25.4
(2 ꢃ CH3–C), 27.7 (CH3–C), 27.8 (CH3–C), 33.5 (CH2CO), 39.7 (C-4),
59.5 (CH3O), 76.4, 76.5, 76.7, 80.3, 80.4, 109.3 (2 ꢃ OCO), 176.9
(C@O).
7.7. 4-Deoxy-4-C-(2,6-anhydro-7-O-tertbutyldiphenylsilyl-4,5-O-
isopropylidene-1,3-dideoxy- -galacto-hept-2-enit-1-C-yl)-1,2,5,
6-di-O-isopropylidene-3-O-methyl- -chiro-inositol 11
D
D
A mixture of enol ether 10 (5.22 g, 6.49 mmol), 2,6-di-tert-bu-
tyl-4-methylpyridine (20.0 g, 97.3 mmol), and freshly activated,
powdered 4 Å molecular sieves (15.3 g) in anhydrous CH2Cl2
(200 mL), was stirred for 15 min at rt, under an atmosphere of ar-
gon, then cooled to 0 °C. Methyl triflate (9.50 mL, 84.4 mmol) was
then introduced, and the mixture warmed to rt, and stirred for an
additional 18 h, at which time, triethylamine (15 mL) was added.
The mixture was diluted with ether, washed with saturated aque-
ous NaHCO3 and brine, dried (Na2SO4), filtered and evaporated in
vacuo. FCC of the residue provided recovered 10 (1.22 g) and 11
(2.66 g, 77% based on recovered 10) as a light yellow oil. Rf = 0.58
(basic alumina, 10% ethyl acetate/petroleum ether). 1H NMR
(C6D6) d 1.29 (s, 9H, (CH3)3C), 1.30 (s, 3H, CH3–C), 1.34 (s, 3H, CH3–
C), 1.50 (s, 3H, CH3–C), 1.56 (s, 3H, CH3–C), 1.66 (s, 3H, CH3–C),
2.16 (m, 2H, H-4, 1/2 ꢃ CH2C@), 2.89 (br d, 1H, J = 10.2 Hz, 1/
2 ꢃ CH2C@), 3.07 (dd, 1H, J = 6.9, 12.0 Hz, H-3), 3.56 (s, 3H, CH3O),
4.10 (t, 1H, J = 6.9 Hz, H-5), 4.25, 4.33, 4.36 (m, m, t, J = 6.4 Hz, 3H,
2H, 1H resp. H-1, H-2, H-6, H-60, CH2–70), 4.47 (d, 1H, J = 6.4 Hz, H-
50), 4.73 (dd, 1H, J = 2.5, 6.4 Hz, H-40), 4.94 (d, 1H, J = 2.5 Hz, H-30),
7.31–7.41 (m, 6H), 7.87 (m, 4H); 13C NMR (C6D6) d 19.3 (Me3C–Si),
25.2 (CH3–C), 25.4 (CH3–C), 26.8 ((CH3)3C), 27.0 (CH3–C), 27.8
(CH3–C), 28.0 (CH3–C), 28.4 (CH3–C), 34.1 (CH2C@), 39.8 (C-4), 58.5
(CH3O), 63.8 (CH2O), 70.1, 72.0, 75.9, 80.0, 81.1, 100.5 (C-30), 108.5
(OCO), 108.8 (OCO), 110.0 (OCO), 128.5, 129.8, 133.6 (two signals),
135.8 (two signals), 157.1. HRMS (ESI) m/z calcd for C39H54O9NaSi
(M+Na)+ 717.3429, found 717.3422.
7.5. Monothioacetal ester 9
DCC (0.50 g, 2.42 mmol) was added at 0 °C to a mixture of acid 7
(0.64 g, 2.02 mmol), alcohol 813 (1.23 g, 2.42 mmol), and DMAP
(74.0 mg, 0.61 mmol) in dry dichloromethane (10 mL). The reac-
tion mixture was warmed to rt and stirred for 6 h. The mixture
was then diluted with ether and filtered. The filtrate was succes-
sively washed with 0.1 N aqueous HCl and brine, dried (Na2SO4),
filtered, and evaporated in vacuo. FCC of the residue gave 9
(1.45 g, 89%) as a colorless oil. Rf = 0.38 (10% ethyl acetate/petro-
leum ether). 1H NMR (CDCl3) d 1.05 (s, 9H, (CH3)3C), 1.26 (s, 3H,
CH3–C), 1.27 (s, 3H, CH3–C), 1.28 (s, 3H, CH3–C), 1.56 (two s, 6H,
2 ꢃ CH3–C), 2.13 (m, 1H, H-4), 2.34 (dd, 1H, J = 7.9, 16.5 Hz, 1/
2 ꢃ CH2C@O), 2.69 (dd, 1H, J = 3.8, 15.4 Hz, 1/2 ꢃ CH2C@O), 3.06
(dd, 1H, J = 7.1, 12.2 Hz, H-3), 3.20 (s, 3H, CH3O), 3.80 (dd, 1H,
J = 7.3, 10.0 Hz, 1/2 ꢃ CH2O), 3.85 (dd, 1H, J = 6.0, 9.9 Hz, 1/
2 ꢃ CH2O), 4.17 (dd, 1H, J = 6.6, 9.8 Hz, H-5), 4.19 (m, 2H, H-2, 6),
4.29 (t, J = 7.0 Hz, H-1), 4.42 (dd, 1H, J = 2.4, 7.7 Hz, H-20), 5.25
(m, H, H-30), 5.50 (d, 1H, J = 6.8 Hz, H-10), 7.24–7.34 (m, 3H, ArH),
7.37–7.49 (m, 6H, ArH), 7.56 (m, 2H, ArH), 7.67–7.74 (m, 4H,
ArH); 13C NMR (CDCl3) d 20.0 (Me3C–Si), 25.3 (CH3–C), 25.5
(CH3–C), 26.2 (CH3–C), 26.7 ((CH3)3C), 27.3 (CH3–C), 27.7 (CH3–C),
27.9 (CH3–C), 34.5 (CH2CO), 39.9 (C-4), 59.0 (CH3O), 62.0 (CH2O),
70.8, 76.5, 76.6, 78.9, 80.4, 80.5, 84.4 (OCS), 109.3 (2 ꢃ OCO),
111.5 (OCO), 127.5, 127.8, 127.9, 128.9, 129.7 (two signals),
132.3, 133.1 (two signals), 133.7, 135.6, 135.7, 171.4 (C@O). HRMS
(ESI) m/z calcd for C44H58O10SSiNa (M+Na)+ 829.3420, found
829.3423.
7.8. 4-Deoxy-4-C-(2,6-anhydro-7-O-tertbutyldiphenylsilyl-4,5-O-
isopropylidene-1-deoxy-D-galacto-D-glycero-heptit-1-C-yl)-1,2,5,
6-di-O-isopropylidene-3-O-methyl-D-chiro-inositol 12
BH3ꢀMe2S (1.1 mL, 1 M solution, mmol) was added at 0 °C to a
solution of glycal 11 (0.191 g, 0.275 mmol) in anhydrous THF
(8 mL) under an atmosphere of argon. The mixture was warmed
to rt and stirred for an additional 1 h. At that time the solution
was recooled to 0 °C and treated with a mixture of 3 N NaOH
(2 mL) and 30% aqueous H2O2 (2 mL) for 30 min. The solution
was then diluted with ether and washed with saturated aqueous
NaHCO3 and brine, dried (Na2SO4), filtered and evaporated under
reduced pressure. FCC of the residue provided 12 (0.149 g, 76%).
Rf = 0.51 (25% ethyl acetate/petroleum ether). 1H NMR (CDCl3) d
1.06 (s, 9H, (CH3)3C)), 1.32 (s, 3H, CH3–C), 1.34 (s, 3H, CH3–C),
1.40 (s, 3H, CH3–C), 1.44 (s, 3H, CH3–C), 1.51 (s, 3H, CH3–C), 1.54
(s, 3H, CH3–C), 1.75 (m, 1H, 1/2 ꢃ CH2), 2.01 (m, 2H, H-4, 1/
2 ꢃ CH2), 2.42 (d, 1H, J = 3.4 Hz, OH), 3.01 (dd, 1H, J = 6.3,
12.2 Hz, H-3), 3.45 (m, 2H), 3.49 (s, 3H, CH3O), 3.86 (m, 3H), 4.03
(t, 1H, J = 5.4 Hz, H-30), 4.07 (m, 2H), 4.20 (m, 2H), 4.39 (dd, 1H,
J = 1.8, 5.4 Hz, H-40), 7.35–7.46 (m, 6H), 7.67–7.75 (m, 4H); 13C
NMR (CDCl3) d 19.2 (Me3C–Si), 25.3 (CH3–C), 25.4 (CH3–C), 26.4
(CH3–C), 26.7 ((CH3)3C), 27.7 (CH3–C), 27.8 (CH3–C), 28.4 (CH3–C),
32.7 (CH2-10), 37.5 (C-4), 58.7 (CH3O), 62.7 (CH2O), 73.8, 74.8,
75.5, 76.1, 77.2, 79.1, 80.1, 80.3 (two signals), 109.2 (OCO), 109.4
(OCO), 109.6 (OCO), 127.6, 127.7, 129.6, 133.2, 133.4, 135.6 (two
signals). HRMS (ESI) m/z calcd for C39H56O10NaSi (M+Na)+
735.3534, found 735.3537.
7.6. Monothioacetal enol ether 10
To a mixture of ester 9 (0.592 g, 0.735 mmol), and pyridine
(0.3 mL) in anhydrous 3:1 toluene/THF (10:5 mL), was added under
an argon atmosphere at ꢁ78 °C, the Tebbe reagent (3.7 mL, 0.5 M
in THF). The reaction mixture was warmed to rt and stirred at this
temperature for 1 h. The mixture was then slowly poured into 1 M
aqueous NaOH at 0 °C, and the resulting suspension extracted with
ether. The combined organic phase was washed with brine, dried
(Na2SO4), filtered and concentrated in vacuo. FCC of the residue
on basic alumina provided 10 (0.44 g, 75%) as light yellow oil.
Rf = 0.49 (basic alumina, 10% ethyl acetate/petroleum ether). 1H
NMR (CDCl3) d 1.07 (s, 9H, (CH3)3C), 1.29 (s, 3H, CH3–C), 1.35 (s,
6H, 2 ꢃ CH3–C), 1.48 (s, 3H, 2 ꢃ CH3–C), 1.50 (s, 3H, CH3–C), 1.51
(s, 3H, CH3–C), 1.86 (m, 1H, H-4), 2.31 (dd, 1H, J = 5.4, 14.4 Hz, 1/
2 ꢃ CH2C@), 2.45 (dd, 1H, J = 5.4, 14.4 Hz, 1/2 ꢃ CH2C@), 3.14 (dd,
1H, J = 6.9, 12.5 Hz, H-3), 3.34 (s, 3H, CH3O), 3.81 (d, 1H,
J = 2.2 Hz, 1/2 ꢃ @CH2), 3.85–3.93 (m, 3H, CH2O, 1/2 ꢃ CH2C@),
3.97 (t, 1H, J = 6.3 Hz, H-6), 4.10 (dd, 1H, J = 7.9, 8.9 Hz, H-5), 4.16
(m, 2H, H- 1, 2), 4.25 (m, 1H, H-30), 4.49 (dd, 1H, J = 3.2, 8.0 Hz,
H-20), 5.49 (d, 1H, J = 7.0 Hz, H-10), 7.25–7.34 (m, 4H, ArH), 7.37–
7.48 (m, 4H, ArH), 7.51–7.58 (m, 2H, ArH), 7.67–7.73 (m, 5H,
ArH); 13C NMR (CDCl3) d 19.1 (Me3C–Si), 25.2 (CH3–C), 25.3
(CH3–C), 26.1 (CH3–C), 26.7 ((CH3)3C), 27.4 (CH3–C), 27.8
(2 ꢃ CH3–C), 34.3 (CH2C@), 39.3 (C-4), 58.6 (CH3O), 61.1 (CH2O),
74.2, 76.0, 76.9, 79.6, 79.7, 81.0, 83.7, 84.3, 109.3 (OCO), 111.3
(OCO), 112.0 (OCO), 127.3, 127.7, 127.8, 129.0, 129.7, 129.8,
131.8 (two signals), 133.0, 133.3, 134.1, 135.6, 159.4 (OC@CH2).
HRMS (ESI) m/z calcd for C45H60O9NaSSi (M+Na)+ 827.3625, found
827.3625.
7.9. C-INS-2-OH 3
To
a solution of TBDPS protected alcohol 12 (0.065 g,
0.09 mmol) in dry THF (1 mL), was added TBAF (0.18 mL,
0.18 mmol) at rt. The reaction was stirred for 5 h at rt. The mixture
was then diluted with saturated aqueous NaHCO3 and extracted

S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
1109
with ether. The combined organic phase was dried (Na2SO4) and
concentrated in vacuo. FCC of the residue afforded the derived pri-
mary alcohol (0.041 g, 95%) as a colorless oil. The product (0.040 g,
0.09 mmol) was dissolved in dry methanol (3 mL). The pH of the
solution was adjusted to 2–3 by addition of a 2 M solution of HCl
in anhydrous ether, and the mixture stirred for 18 h. The solution
was then diluted with methanol and evaporated under reduced
pressure. The dilution-evaporation procedure was repeated three
times and the residue dried in vacuo to afford 3 (0.029 g, 97%) as
a clear gum. Rf = 0.45 (50% methanol/ethyl acetate). 1H NMR
(D2O) d 1.68–1.78 (m, 1H, H-10a), 1.98 (m, 1H, H-4), 2.05 (m, 1H,
H-10b), 3.23 (t, 1H, J = 9.6 Hz, H-3), 3.33 (t, 1H, J = 9.5 Hz, H-30),
3.43 (s, 3H, CH3O), 3.45 (m, 1H, H-20), 3.49–3.67 (m, 4H, H- 60, 40,
CH2–70), 3.75 (dd, 1H, J = 3.1, 9.6 Hz, H-2), 3.89–3.79 (m, 3H, H-1,
J = 7.0 Hz, H-1), 4.23 (t, 1H, J = 7.0 Hz, H-2), 4.35 (t, 1H, J = 7.0 Hz,
H-5), 4.53 (dd, 1H, J = 1.7, J = 7.8 Hz, H-50), 4.78 (d, 1H, 7.8 Hz, H-
40), 5.09 (d, 1H, J = 9.5 Hz, H-20), 7.36–7.48 (m, 6H), 7.68–7.75 (m,
4H); 13C NMR (CDCl3) d 19.2 (Me3C–Si), 25.3 (2 ꢃ CH3–C), 25.4
(CH3–C), 26.0 (CH3–C), 26.8 ((CH3)3C), 27.8 (CH3–C), 27.9 (CH3–C),
34.0 (C-10), 38.6 (C-4), 58.4 (CH3O), 62.3 (CH3ON), 62.5 (CH2-70),
72.5, 74.2, 75.7, 76.7, 77.1, 77.5, 78.9, 79.5, 109.2 (OCO), 109.5
(OCO), 111.2 (OCO), 127.6, 127.7, 133.4, 133.7, 135.7, 135.8,
156.1 (C@N). HRMS (ESI) m/z calcd for C40H57NO10NaSi (M+Na)+
762.3643, found 762.3643.
7.12. C-Pseudodisaccharide methoxylamine 15
To a mixture of oxime 14 (0.092 g, 0.124 mmol), and Bu3SnH
(0.065 mL, 0.248 mmol) in CH2Cl2 (3 mL), was added at ꢁ20 °C,
BF3ꢀOEt2 (0.015 mL, 0.124 mmol). The mixture was maintained at
this temperature for 2 h, then diluted with saturated aqueous NaH-
CO3 and extracted with CH2Cl2. The combined organic phase was
dried (Na2SO4) and concentrated in vacuo. FCC of the residue affor-
ded 15 (0.075 g, 82%) as a colorless oil. Rf = 0.81 (25% ethyl acetate/
petroleum ether). 1H NMR (CDCl3) d 1.06 (s, 9H, (CH3)3C), 1.33 (s,
3H, CH3–C), 1.35 (s, 3H, CH3–C), 1.38 (s, 3H, CH3–C), 1.46 (s, 3H,
CH3–C), 1.49 (s, 3H, CH3–C), 1.51 (s, 3H, CH3–C), 1.82 (ddd, 1H,
J = 1.7, 10.0, 11.8 Hz, H-10a), 2.05 (m, 2H, H-10b, H-4), 2.59 (dd,
1H, J = 8.1, 9.7 Hz, H-30), 3.05 (dd, 1H, J = 6.1, 12.3 Hz, H-3), 3.49
(s, 3H, CH3O), 3.56 (s, 3H, CH3O), 3.73 (dt, 1H, J = 1.7, 9.9 Hz, H-
20), 3.84 (m, 2H, CH2-70), 3.91 (dd, 1H, J = 7.5, 9.0 Hz, H-60), 4.06
(t, 1H, J = 6.5 Hz, H-6), 4.13 (dd, 1H, J = 7.2, 8.6 Hz, H-5), 4.19 (m,
2H, H-1, 2), 4.40 (m, 2H, H-40, 50), 6.01 (br s, 1H, NH), 7.31–7.51
(m, 6H, ArH), 7.71–7.82 (m, 4H, ArH); 13C NMR (CDCl3) d 19.2
(Me3C–Si), 25.3 (2 ꢃ CH3–C), 26.4 (CH3–C), 26.8 ((CH3)3C), 27.8
(CH3–C), 27.9 (CH3–C), 28.5 (CH3–C), 33.2 (C-10), 37.8 (C-4), 58.5
(CH3O), 62.3 (CH3O), 63.0 (CH2–O), 65.7 (CH–N), 72.9, 73.2, 73.7,
75.9, 77.4, 79.1, 79.9, 80.4, 109.2 (OCO), 109.3 (OCO), 109.4
(OCO), 127.6, 127.7, 129.6, 133.5, 133.6, 135.5, 135.6. HRMS (ESI)
m/z calcd for C40H59NO10SiNa (M+Na)+ 764.3688, found 764.3688.
13
5, 50), 3.89 (t, 1H, J = 3.3 Hz, H-6); C NMR (CDCl3) d 28.8 (C-10),
37.9 (C-4), 58.5 (CH3O), 61.4 (C-70), 69.1 (C- 1/5/5’), 69.9 (C- 1/5/
50), 71.2 (C-2), 71.3 (C-30), 71.8 (C-6), 72.0 (C- 1/5/50), 73.9 (C-40),
77.8 (C-20), 78.2 (C-60), 79.9 (C-3). HRMS (ESI) m/z calcd for
C14H26NaO10 (M+Na)+ 377.1425, found 377.1425.
7.10. C-Pseudodisaccharide ketone 13
To a mixture of PCC (0.401 g, 1.87 mmol), florisil (0.996 g), so-
dium acetate (0.153 g, 1.87 mmol), freshly activated, powdered
4 Å molecular sieves (0.996 g) and Celite (0.996 g) in dry dichloro-
methane (15 mL), was added dropwise a solution of alcohol 12
(0.322 g, 0.466 mmol) in dry dichloromethane (5 mL). The reaction
mixture was stirred at rt until TLC indicated total consumption of
the starting material. The mixture was then diluted with ether
and filtered through a column of florisil. The filtrate was evapo-
rated in vacuo and the residue purified by FCC to afford ketone
13 (0.315 g, 95%) as colorless oil. Rf = 0.51 (25% ethyl acetate/petro-
leum ether). 1H NMR (CDCl3) d 1.07 (s, 9H, (CH3)3C)), 1.30 (s, 3H,
CH3–C), 1.35 (s, 3H, CH3–C), 1.42 (s, 3H, CH3–C), 1.45 (2s, 6H,
2 ꢃ CH3–C), 1.51 (s, 3H, CH3–C), 1.80 (ddd, 1H, J = 3.8, 10.5,
14.4 Hz, H-1a0), 1.90 (m, 1H, H-4), 2.21(ddd, 1H, J = 2.5, 9.2,
14.4 Hz, H-10b), 3.01 (dd, 1H, J = 6.3, 12.1 Hz, H-3), 3.49 (s, 3H,
CH3O), 3.93 (m, 2H, CH2–70), 4.12 (m, 3H, H-1, 5, 60), 4.20 (m, 2H,
H-2, 6), 4.26 (dd, 1H, J = 3.8, 9.2 Hz, H-20), 4.47 (d, 1H, J = 5.8 Hz,
H-40), 4.77 (dd, 1H, J = 1.7, 5.8 Hz, H-50), 7.32–7.56 (m, 6H, ArH),
7.67–7.99 (m, 4H, ArH); 13C NMR (CDCl3) d 19.2 (Me3C–Si), 25.3
(CH3–C), 25.4 (CH3–C), 26.1 (CH3–C), 26.8 ((CH3)3C), 27.2 (CH3–C),
27.7 (CH3–C), 27.9 (CH3–C), 29.8 (C-10), 38.3 (C-4), 58.9 (CH3O),
62.5 (C-70), 76.7, 77.1, 77.2, 77.5, 77.8, 79.0, 79.1, 80.0, 80.3,
109.2 (OCO), 109.4 (OCO), 110.8 (OCO), 127.7 (two signals),
128.3, 129.7, 133.2, 133.3, 135.5, 135.6, 204.6 (C@O). HRMS (ESI)
m/z calcd for C39H54O10SiNa (M+Na)+ 733.3393, found 733.3393.
7.13. C-INS-2-hydrochloride
To a solution of silylether 15 (0.033 g, 0.046 mmol) in dry THF
(1 mL), was added TBAF (0.09 mL, 0.09 mmol) at rt. The reaction
mixture was stirred for 5 h at rt then diluted with saturated aque-
ous NaHCO3 and extracted with ether. The combined organic phase
was dried (Na2SO4) and concentrated in vacuo. FCC of the residue
afforded the primary alcohol derivative (0.022 g, 95%) as a colorless
oil. Rf = 0.20 (50% ethyl acetate/petroleum ether). 1H NMR (CDCl3) d
1.35 (s, 3H, CH3–C), 1.36 (s, 3H, CH3–C), 1.37 (s, 3H, CH3–C), 1.50 (s,
3H, CH3–C), 1.51 (s, 3H, CH3–C), 1.53 (s, 3H, CH3–C), 1.80 (dd, 1H,
J = 3.9, 10.8, 13.9 Hz, H-10a), 2.10 (m, 2H, H-10b, H-4), 2.44 (dd,
1H, J = 1.9, 9.4 Hz, OH), 2.66 (t, 1H, J = 7.7 Hz, H-30), 3.10 (dd, 1H,
J = 6.5, 12.3 Hz, H-3), 4.06 (t, 1H, J = 7.1 Hz, H-6), 4.12 (t, 1H,
J = 6.9 Hz, H-5), 4.17 (dd, 1H, J = 1.8, 5.5 Hz, H-1), 4.21 (t, 1H,
J = 7.4 Hz, H-50), 4.25 (t, 1H, J = 4.2 Hz, H-2), 4.39 (dd, 1H, J = 5.5,
8.2 Hz, H-40), 5.97 (d, 1H, J = 1.3 Hz, NH); 13C NMR (CDCl3) d 25.3
(CH3–C), 25.5 (CH3–C), 26.4 (CH3–C), 27.8 (CH3–C), 28.0 (CH3–C),
28.3 (CH3–C), 33.4 (C-10), 37.8 (C-4), 58.6 (CH3O), 62.4 (CH3O),
63.0 (CH2-70), 65.7 (CH–N), 69.8, 73.3 (two signals), 76.5, 77.4,
77.5, 78.3, 80.2, 80.5, 109.5 (OCO), 109.7 (OCO), 109.9 (OCO).
Liquid NH3 was condensed into a solution of the product from
the previous step (0.122 g, 0.242 mmol) in THF (4 mL) at ꢁ78 °C,
under an atmosphere of argon. Sodium was then carefully added
to the solution until a blue color persisted for several minutes.
After slowly warming to rt, the reaction was quenched with solid
NH4Cl, and filtered. The filtrate was concentrated in vacuo. FCC
of the residue afforded the amine derivative (0.090 g, 78%) as a col-
orless oil. Rf = 0.48 (25% methanol/ethyl acetate). 1H NMR (CDCl3) d
7.11. Pseudodisaccharide oxime 14
To a solution of ketone 13 (0.312 g, 0.439 mmol) in a 1:1 mix-
ture of THF/MeOH (5.5 mL), was added a mixture of O-methyl
hydroxylamine hydrochloride (0.367 g, 4.39 mmol) and NaOAc
(0.397 g, 4.82 mmol) in water (2.75 mL), which was adjusted to
pH 4.5 with a few drops of glacial acetic acid. The reaction mixture
was stirred at rt for 4 h then diluted with EtOAc and washed with
saturated aqueous NaHCO3 and water, dried (Na2SO4), and filtered.
The solvent was removed in vacuo to give 14 (0.280 g, 86%, 1.5:1
mixture) as a colorless oil. Rf = 0.60 (20% ethyl acetate/petroleum
ether). For major isomer: 1H NMR (CDCl3) d 1.07 (s, 9H, (CH3)3C),
1.32 (s, 3H, CH3–C), 1.34 (s, 3H, CH3–C), 1.36 (s, 3H, CH3–C), 1.42
(s, 3H, CH3–C), 1.49 (s, 3H, CH3–C), 1.50 (s, 3H, CH3–C), 1.83 (dd,
1H, J = 9.5, 14.0 Hz, H-10a), 2.05 (m, 1H, H-4), 2.40 (ddd, J = 1.9,
10.6, 14.0 Hz, H-10b), 3.12 (dd, 1H, J = 7.0, 12.5 Hz, H-3), 3.45 (dt,
1H, J = 1.7, 6.4 Hz, H-60), 3.48 (s, 3H, CH3O), 3.82 (m, 2H, CH2-70),
3.93 (s, 3H, CH3O), 4.03 (t, 1H, J = 7.0 Hz, H-6), 4.15 (t, 1H,

1110
S. K. Hans et al. / Bioorg. Med. Chem. 18 (2010) 1103–1110
1,30 (s, 3H), 1.35 (3, 6H), 1.47 (s, 3H), 1.50 (s, 6H), 2.05 (m, 2H),
2.70 (t, 1H, J = 8.8 Hz), 3.05 (dd, 1H, J = 6.4, 12.3 Hz), 3.23 (t, 1H,
J = 9.2 Hz), 3.46 (s, 3H), 3.66 (m, 2H), 3.83 (m, 2H), 4.04 (t, 1H,
J = 7.0 Hz), 4.05 (m, 2H), 4.15 (t, 1H, J = 7.4 Hz), 4.23 (t, 1H,
J = 7.0 Hz); 13C NMR (CDCl3) d 25.2, 25.5, 26.5, 27.8, 28.0, 28.3,
32.9, 37.6, 56.7, 58.5, 62.9, 73.5, 76.1, 76.7, 76.8, 77.4, 77.5, 78.2,
80.0, 80.6, 80.7, 109.5, 109.7, 110.0.
was read for change in O.D. at 340 nM. Activity was expressed as %
of basal, for example, ATP concentration for 50% inhibition.
Acknowledgment
This investigation was supported by grants R01 GM57865 from
the National Institute of General Medical Sciences of the National
Institutes of Health (NIH). ‘‘Research Centers in Minority Institu-
tions” award RR-03037 from the National Center for Research
Resources of the NIH, which supports the infrastructure and instru-
mentation of the Chemistry Department at Hunter College, is also
acknowledged.
To a solution of product from the previous step (52 mg,
0.11 mmol) in dry methanol (3 mL) was added a 2 M solution of
HCl in anhydrous ether to a pH of 2–3. The reaction mixture was
stirred for 18 h. The mixture was then diluted with methanol and
evaporated under reduced pressure. The dilution–evaporation pro-
cedure was repeated three times and the residue dried in vacuo to
give 2 as the hydrochloride salt (38 mg, 98%). Rf = 0.67 (4:4:1:2 of
2-propanol/pyridine/acetic acid/water). 1H NMR (500 MHz, D2O)
d 1.77 (ddd, 1H, J = 3.0, 7.5, 14.8 Hz, H-10a), 1.85 (ddd, 1H, J = 1.8,
9.9, 14.8 Hz, H-10b), 2.05 (m, 1H, H-4), 3.05 (t, 1H, J = 10.3 Hz, H-
30), 3.52 (t, 1H, J = 3.2 Hz, H-3), 3.40 (s, 3H, CH3O), 3.57 (dd, 1H,
J = 3.9, 8.1 Hz, H-60), 3.61 (dd, 1H, J = 3.9, J = 11.8 Hz, H-70), 3.69
(dd, 1H, J = 8.1, 11.8 Hz, H-70), 3.75 (dd, 1H, J = 3.0, 10.6 Hz, H-20),
3.77–3.81 (m, 4H, H-1, 2, 5, 6), 3.87 (d, 1H, J = 3.5 Hz, H-50), 3.90
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.056.
References and notes
1. Larner, J.; Galasko, G.; Cheng, K.; DePaoli-Roach, A.; Huang, L.; Daggy, P.;
Kellogg, J. Science 1979, 206, 1408.
2. Sleight, S.; Wilson, B.; Heimark, D.; Larner, J. Biochem. Biophys. Res. Commun.
2002, 295, 561.
3. Larner, J.; Price, J. D.; Heimark, D.; Smith, L.; Rule, G.; Piccariello, T.; Fonteles, M.
C.; Pontes, C.; Vale, D.; Huang, L. J. Med. Chem. 2003, 46, 3283.
4. Brautigan, D.; Brown, M.; Grindrod, S.; Chinigo, G.; Kruszewski, A.; Lukasik, S.;
Bushweller, J.; Horal, M.; Keller, S.; Tamura, S.; Heimark, D.; Price, J.; Larner, A.;
Larner, J. Biochemistry 2005, 44, 11067.
13
(t, 1H, J = 3.5 Hz, H-40); C NMR (CDCl3) d 30.9 (C-10), 36.6 (C-4),
54.0 (CH–N), 57.5 (CH3O), 61.3 (CH2–70), 68.0 (C-50), 70.3 (C-1/2/
5/6), 70.4 (C-20), 71.2 (C-1/2/5/6), 71.9 (C-40), 72.1 (C-1/2/5/6),
75.2 (C-1/2/5/6), 78.5 (C-60), 79.8 (C-3). HRMS (ESI) m/z calcd for
C14H28NO9 (RNH3)+ 354.1759, found 354.1762.
5. Hiraga, A.; Kikuchi, K.; Tamura, S.; Tsuiki, S. Eur. J. Biochem. 1981, 119, 503.
6. Villar-Palasi, C.; Larner, J. Biochim. Biophys. Acta 1960, 39, 171.
7. Linn, T.; Pettit, F.; Reed, L. Proc. Natl. Acad. Sci. U.S.A. 1969, 62, 234.
8. For C-glycoside reviews: (a) Postema, M. H. D. C-Glycoside Synthesis; CRC Press:
Boca Raton, Fl, 1995; (b) Levy, D. E.; Tang, C. The Synthesis of C-Glycosides;
Pergamon: Oxford, 1995; (c) Beau, J.-M.; Gallagher, T. Top. Curr. Chem. 1997,
187, 1; (d) Togo, H.; He, W.; Waki, Y.; Yokoyama, M. Synlett 1998, 700; (e) Du,
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of Sugars; Levy, D. E., Fügedi, P., Eds.; CRC Press/Taylor & Francis: Boca Raton,
2005; pp 269–348.
7.14. PP2C
a assays
The mouse wild type GST-PP2C
a
fusion protein was expressed
and purified as previously described.4 Phosphatase activity was as-
sayed at rt in 0.1 M Tris-HCl (pH 8.0), 2 mM dithiothreitol, and var-
ious concentrations of added MnCl2, INS-2, C-INS-2-OH or C-INS-2,
in a 96-well plate in 50
PP2C fusion protein and 5 mM p-nitrophenyl phosphate (PNPP).
After 30 min, the reaction was stopped by addition of 200 L of
0.5% SDS and the absorbance at 410 nm recorded with an Eldex
microplate reader. Alternatively, activity of 2 g of GST-PP2C
lL reaction mixtures with 1 lg of GST-
a
l
9. Jiménez-Barbero, J.; Espinosa, J. F.; Asensio, J. L.; Cañada, F. J.; Poveda, A. Adv.
Carbohydr. Chem. Biochem. 2000, 56, 235.
10. For examples of relative activity of O-glycosides and their exact C-glycoside
analogues: (a) Acton, E. M.; Ryan, K. J.; Tracy, M.; Arora, S. K. Tetrahedron Lett.
1986, 27, 4245; (b) Wei, A.; Boy, K. M.; Kishi, Y. J. Am. Chem. Soc. 1995, 117,
9432; (c) Uchiyama, T.; Vassilev, V. P.; Kajimoto, T.; Wong, W.; Huang, H.; Lin,
C.-C.; Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 5395; (d) Xin, Y.-C.; Zhang, Y.-M.;
Mallet, J.-M.; Glaudemans, C. P. J.; Sinaÿ, P. Eur. J. Org. Chem. 1999, 471; (e) Link,
J. T.; Sorensen, B. K. Tetrahedron Lett. 2000, 41, 9213; (f) Yang, G.; Schmieg, J.;
Tsuji, M.; Franck, R. W. Angew. Chem., Int. Ed. 2004, 43, 3818; (g) Postema, M.
H.; Piper, J. L.; Betts, R. L.; Valeriote, F. A.; Pietraszkewicz, H. J. Org. Chem. 2005,
70, 829.
11. For an example where O- and C- glycosides bind in different conformations
with respect to the intersaccharide torsions: Espinosa, J. F.; Montero, E.; Vian,
A.; Garcia, J. L.; Dietrich, H.; Schmidt, R. R.; Martín-Lomas, M.; Imberty, A.;
Cañada, F. J.; Jimenéz-Barbero, J. J. Am. Chem. Soc. 1998, 120, 1309.
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13. (a) Khan, N.; Cheng, X.; Mootoo, D. R. J. Am. Chem. Soc. 1999, 121, 4918; (b)
Cheng, X.; Khan, N.; Mootoo, D. R. J. Org. Chem. 2000, 65, 2544.
14. Keck, G. E.; Enholm, E. J.; Yates, J. B.; Wiley, M. R. Tetrahedron 1985, 41, 4079.
15. Zunszain, P. A.; Varela, O. Tetrahedron: Asymmetry 1998, 9, 1269.
16. Afarinkia, K.; Bahar, A. Tetrahedron: Asymmetry 2005, 16, 1239.
17. Ueda, M.; Miyabe, H.; Namba, M.; Nakabayashi, T.; Naito, T. Tetrahedron Lett.
2002, 43, 4369.
l
a
was assayed in the same volume of the same buffer with a syn-
thetic phosphopeptide (R-R-R-R-P-pT-P-A) at a final concentration
of 5
100
l
M. After 15 min, the reaction was stopped by addition of
lL of a malachite green solution (Millipore). After color devel-
opment for 15 min, the absorbance at 650 nm was determined
with a microplate reader. Values were corrected by subtracting
the absorbance of blanks that were reactions without added
enzyme.
7.15. PDHP assay
PDHP was assayed in triplicate samples on a 96-well plate.3 An
ATP inhibition curve was first established to determine the 50%
inhibition concentration. 10
pH 7, 2 mM DTT, 10 mM MgCl2, 0.1 mM CaCl2, 105 mg/mL BSA)
was added to each well. 10 L of 0.5–5 mM ATP is added and the
volumes all adjusted with aliquots of distilled water (DW). 10
PDHP is added and the plate gently shaken. 10 L PDH was added,
the plate gently shaken and incubated at 37 °C for 30 min. 10 L of
110 mM NaF was added and the plate gently shaken. 10 L pyru-
vate-ADP (30 mM sodium pyruvate, 30 mM ADP) was added with
gentle shaking. 45 L NAD mix (83 mM imidazole pH 7, 83 mM
lL of assay buffer (1 mM imidazole
l
lL
18. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. A. J. Mol. Biol. 1996, 261, 470.
19. For definition of scoring functions see Ref. 4.
l
20. For
U W
: O5–C1–X–C40, : C1–X–C40–H40 where X the glycosidic oxygen or ‘CH2’.
l
21. Vassylyev, D.; Symersky, J. J. Mol. Biol. 2007, 370, 417.
22. Das, A.; Helps, N.; Cohen, P.; Barford, D. EMBO J. 1996, 15, 6798.
23. Mikkelsen, L. M.; Hernáiz, M. J.; Martín-Pastor, M.; Skrydstrup, T.; Jiménez-
Barbero, J. J. Am. Chem. Soc. 2002, 124, 14940.
24. García-Herrero, A.; Montero, E.; Muñoz, J. L.; Espinosa, J. F.; Vián, A.; García, J.
L.; Asensio, J. L.; Cañada, F. J.; Jiménez-Barbero, J. J. Am. Chem. Soc. 2002, 124,
4804.
l
l
b-NAD, 1.65 mM cocarboxylase, 3.3 mM DTT, 3.3 mM CoA) was
added with gentle shaking and incubated at rt for 2 min. The plate