3
290 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Larner et al.
sieves (4 Å, 2.5 g) were vacuum-dried at 150 °C for 10 h and
then cooled and suspended with stirring in anhydrous CH
Cl (15 mL) under argon. To this suspension were added
trichloroacetimidate 5 (0.50 g, 0.80 mmol) and diacetonide 6
(2) Larner, J .; Romero, G.; Kennington, A. S.; Lilley, K.; Kilgour,
E.; Zhang, C.; Heimark, D.; Gamez, G.; Houston, D. B.; Huang,
L. C. Duality in the mechanism of action of insulin. In The
Biology of Signal Transduction; Nishizuka, Y., Ed.; Raven
Press: New York, 1990; pp 290-294.
2
-
2
(0.21 g, 0.765 mmol), both azeotroped with anhydrous benzene
(
3) (a) Larner, J .; Galasko, G.; Cheng, K.; De-Paoli-Roach, A. A.;
Huang, L.; Daggy, P.; Kellogg, J . Generation by insulin of a
chemical mediator that controls protein phosphorylation and
dephosphorylation. Science 1979, 206, 1408-1410. (b) Thomp-
son, M. P.; Larner, J .; Kilpatrick, D. Purification and partial
characterization of a putative mediator of insulin action on cyclic
AMP-dependent protein kinase. Mol. Cell. Biochem. 1984, 62,
67-75. (c) Malchoff, C. D.; Huang, L.; Gillespie, N.; Villar-Pilasi,
C.; Schwartz, C. F. W.; Cheng, K.; Hewlett, E. L.; Larner, J . A
putative mediator of insulin action which inhibits adenylate
cyclase and adenosine 3′,5′-monophosphate-dependent protein
kinase: Partial purification from rat liver: Site and kinetic
mechanism of action. Endocrinology 1987, 120, 1327-1337.
4) (a) Larner, L.; Huang, L. C.; Suzuki, S.; Tang, G.; Zhang, C.;
Schwartz, C. F. W.; Romero, G.; Luttrell, L.; Kennington, A. S.
Alpha keto acid dehydrogenase complexes. Insulin mediators and
control of pyruvate dehydrogenase. Ann. N. Y. Acad. Sci. 1989,
(
2 × 25 mL). The suspension was stirred for 30 min at room
temperature, cooled to -25 °C, and treated with CF
3
SO
2
SiMe
3
(1.92 mL of a 0.42 M solution in toluene, 0.80 mmol). The
mixture turned light-purple, was allowed to warm to room
temperature, and was stirred overnight. The reaction mixture
was filtered through Celite, and the filter cake was washed
with CH
were washed with saturated aqueous NaHCO
over MgSO , and concentrated at reduced pressure. The
residue was purified by flash chromatography (EtOAc/hexane
:3, 15 g of silica gel) to afford 7 as a colorless glassy solid
2
Cl
2
(2 × 25 mL). The combined filtrate and washings
3
(100 mL), dried
4
2
(
2
3
(377 mg, 0.51 mmol, 67%): [R]
D
+13.8° (c 0.0442 g/mL,
1
CHCl
3
); H NMR (500 MHz, CDCl
3
) δ 5.35 (s, 1H), 5.27 (d, J
)
9.5 Hz, 1H), 4.88 (d, J ) 8.0 Hz, 1H), 4.73 (br s, 1H), 4.30-
.10 (m, 7H), 3.98 (m, 1H), 3.88 (t, J ) 6.5 Hz, 1H), 3.77 (dd,
J ) 10.5, 7.0 Hz, 1H), 3.58 (s, 3H), 3.22 (m, 1H), 2.16 (s, 3H),
5
73, 297-305. (b) Lilley, K.; Zhang, C.; Villar-Pilasi, C.; Larner,
4
J .; Huang, L. Insulin mediator stimulation of pyruvate dehy-
drogenase phosphatase. Arch. Biochem. Biophys. 1992, 296,
2
3
1
7
2
.03 (s, 3H), 1.94 (s, 3H), 1.52 (s, 3H), 1.51 (s, 3H), 1.36 (s,
1
70-174. (c) Abe, S.; Huang, L.; Larner, J . Dephosphorylation
H), 1.35 (s, 3H); 13C NMR (125 MHz, CDCl
) δ 170.5, 170.4,
3
of PDH by phosphoprotein phosphatases and its allosteric
regulation by inositol glycans. In Alpha-Keto Acid Dehydrogenase
Complexes; Patel, M. S., Roche, T. E.; Harris, R. A., Eds.;
Birkhauser Verlag: Basel, Switzerland, 1996; pp 187-195. (d)
Huang, L. C.; Heimark, D.; Linko, J .; Nolan, R.; Larner, J . A
model phosphatase 2C f phosphatase 1 activation cascade via
dual control of inhibitor-1 (INH-1) and DARPP-32 dephos-
phorylation by two inositol glycan putative insulin mediators
from beef liver. Biochem. Biophys. Res. Commun. 1999, 255,
70.3, 154.3, 109.8, 109.5, 100.9, 95.6, 80.0, 79.8, 79.1, 78.2,
6.1, 75.8, 74.3, 70.8, 70.7, 66.4, 61.2, 60.2, 53.0, 27.8, 27.7,
5.3 (3C), 20.6 (2C) ppm.
4
-(2-Am in o-2-d eoxy-â-D-ga la ctop yr a n osyl)-3-O-m eth yl-
D-ch ir o-in ositol (INS-2, 1). A solution of fully protected INS2
7, 4.07 g, 5.31 mmol) in 80% AcOH (50 mL) was heated at 80
C for 5 h. Solvent was removed under reduced pressure, and
the residue was purified by flash chromatography (MeOH/
CHCl 1:9, 80 g of silica gel) to afford 2.94 g of a colorless solid.
This material was dissolved in 2-propanol/H O (120 mL, 1:5),
and Amberlite 420 resin (Cl form, 45 g, washed with 10
volumes of distilled deionized (DDI) H O, 5 volumes of 2-pro-
panol, 10 volumes of 1 N NaOH, and 20 volumes of DDI H
until neutral) was added. The suspension was heated at 65
C for 3 h. The mixture was cooled, filtered, and concentrated
(
°
1
50-156. (e) Rademacher, T. W.; Caro, H.; Kunjara, S.; Wang,
D. Y.; Greenbaum, A. L.; McLean, P. Inositolphosphoglycan
3
second messengers. Braz. J . Med. Biol. Res. 1994, 27, 327-341.
2
(5) (a) Romero, G.; Luttrell, L.; Rogol, A.; Zeller, K.; Hewlett, E.;
Larner, J . Phosphatidylinositol-glycan anchors of membrane
proteins: Potential precursors of insulin mediators. Science
2
1
988, 240, 509-511. (b) M u¨ ller, G.; Wied, S.; Crecelius, A.;
2
O
Kessler, A.; Eckel, J . Phosphoinositolglycan-peptides from yeast
potently induce metabolic insulin actions in isolated rat adipo-
cytes, cardiomyocytes, and diaphragms. Endocrinology 1997,
138, 3459-3475.
°
1
under reduced pressure to /
3
of the initial volume. Filtration
through Whatman 42 paper and concentration afforded 1 as
(6) (a) Plourde, R.; d’Alarcao, M.; Saltiel, A. R. Synthesis and
characterization of an insulin-mimetic disaccharide. J . Org.
Chem. 1992, 57, 2606-2610. (b) Campbell, A. S.; Fraser-Reid,
B. First synthesis of a fully phosphorylated GPI membrane
anchor: Rat brain Thy-1. J . Am. Chem. Soc. 1995, 117, 10387-
10388. (c) Mart ´ı n-Lomas, M.; Khiar, N.; Garc ´ı a, S.; Koessler, J .-
L.; Nieto, P.; Rademacher, T. W. Inositolphosphoglycan media-
tors structurally related to glycosyl phosphatidylinositol an-
chors: Synthesis, structure and biological activity. Chem.sEur.
J . 2000, 6, 3608-3621. (d) Frick, W.; Bauer, A.; Bauer, J .; Wied,
S.; M u¨ ller, G. Structure-activity relationship of synthetic phos-
phoinositolglycans mimicking metabolic insulin action. Biochem-
istry 1998, 37, 13421-13436. (e) Morris, J . C.; Ping-Sheng, L.;
Shen, T.-Y.; Mensa-Wilmot, K. Glycan requirements of glyco-
sylphosphatidylinositol phospholipase C from Trypanosoma
brucei. J . Biol. Chem. 1995, 270, 2517-2524. (f) Gigg, R.; Gigg,
J . Synthesis of glycosylphosphatidylinositol anchors. In Glyco-
peptides and Related Compounds: Synthesis, Analysis, and
Applications; Large, D. G., Warren, C. D., Eds.; Marcel Dekker:
New York, 1997; pp 327-392.
2
3
a colorless solid (1.47 g, 4.14 mmol, 78%): [R]
D
+43.8° (c
1
0
.0295 g/mL, H O); H NMR (D O, pH 7.8, 500 MHz) δ 4.47
2
2
(
2
1
d, J ) 8.5 Hz, 1H, H-1), 3.88-3.85 (m, 2H), 3.82 (dd, J ) 10.0,
.8 Hz, 1H), 3.77-3.67 (m, 4H), 3.63 (dd, J ) 12.0, 4.5 Hz,
H), 3.53 (ddd, J ) 7.5, 4.5, 1.0 Hz, 1H, H-5), 3.47 (s, 3H,
OCH
3
), 3.44 (dd, J ) 10.0, 3.3 Hz, 1H), 3.31 (t, J ) 9.5 Hz,
H, H-3′), 2.73 (dd, J ) 10.0, 8.5 Hz, 1H, H-2); 13C NMR (D
1
1
6
2
O,
25 MHz) δ 104.2, 80.5, 80.3, 74.8, 72.7, 71.0, 70.9, 70.3, 69.2,
7.5, 60.8, 58.9, 53.5 ppm. Exact mass calculated for C13
H
26
-
+
NO10 (M + H ) 356.1557, found 356.1587. Anal. (C13
xH
N, calcd 3.84, found 3.78.
P r ep a r a tion of INS-2/Mn 2 Ch ela te. An aqueous solution
of INS-2 (36 mg, 0.10 mmol, 20 mM) was adjusted to pH 6.8
by the addition of 100 mM aqueous HOAc. The solution was
H
25NO10
‚
2
O): C, calcd 42.86, found 42.58; H, calcd 7.19, found 7.41;
+
concentrated under vacuum, and 1.00 mL of 0.40 M MnCl
0.40 mmol) was added. This solution was concentrated, and
the material was used in vitro.
2
(
7) Denton, R. M.; Midgley, P. J . W.; Rutter, G. A.; Thomas, A. P.;
McCormack, J . G. Studies into the mechanism whereby insulin
activates pyruvate dehydrogenase complex in adipose tissue.
Ann. N. Y. Acad. Sci. 1989, 573, 285-296.
(
Ack n ow led gm en t. We acknowledge with thanks
the financial support of J .S.B.
(8) (a) Huang, L. C.; Fonteles, M. C.; Houston, D. B.; Zhang, G.;
Larner, J . Chiro-inositol deficiency and insulin resistance III.
Acute glycogenic and hypoglycemic effects of two inositol phos-
phoglycan insulin mediators in normal and streptozotocin-
diabetic rats in-vivo. Endocrinology 1993, 132, 652-657. (b)
Fonteles, M. C.; Huang, L. C.; Larner, J . Infusion of pH 2.0
D-chiro-inositol glycan insulin putative mediator normalizes
plasma glucose in streptozotocin diabetic rats at a dose equiva-
lent to insulin without inducing hypoglycemia. Diabetologia
Su p p or tin g In for m a tion Ava ila ble: A figure comparing
1
the H spectra of the acidic (isolated) and neutral (synthetic)
forms of compound 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
996, 39, 731-734.
(
9) (a) Koto, S.; Inada, I.; Zen, S. The synthesis of R-D-galactopy-
ranosyl- and R-D-mannopyranosyl-2-amino-2-deoxy-R-D-glucopy-
ranosides and the conformation of their glycoside linkage. Bull.
Chem. Soc. J pn. 1981, 54, 2728-2734. (b) Garreg, P.; Kvarn-
str o¨ m, I. Synthesis of 2-O- and 5-O-(R-D-galactopyranosyl)-4-O-
methyl-D-chiro-inositol: preference for equatorial hydroxyl groups
in the imidate galactosylation procedure. Carbohydr. Res. 1981,
90, 61-69.
Refer en ces
(
1) (a) Larner, J .; Huang, L. C.; Schwartz, C. F. W.; Oswald, A. S.;
Shen, T.-Y.; Kinter, M.; Tang, G.; Zeller, K. Rat liver insulin
mediator which stimulates pyruvate dehydrogenase phosphatase
contains galactosamine and D-chiro-inositol. Biochem. Biophys.
Res. Commun. 1988, 151, 1416-1426. (b) Larner, J .; Price, J .;
Piccariello, T.; Huang, L. U.S. Patent 5,652,221, 1997.