Separate synthesis and evaluation of glucitol bis-phosphate and mannitol bis-phosphate, as competitive inhibitors of fructose bis-phosphate aldolases

Article abstract of DOI:10.1016/j.bmcl.2008.01.076

We report the first unambiguous syntheses of glucitol-1,6-bis-phosphate and mannitol-1,6-bis-phosphate and their competitive inhibition of various fructose bis-phosphate aldolases.

Full text of DOI:10.1016/j.bmcl.2008.01.076

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1735–1737
Separate synthesis and evaluation of glucitol bis-phosphate
and mannitol bis-phosphate, as competitive inhibitors
of fructose bis-phosphate aldolases
Charles-Gabin Mabiala-Bassiloua,a Magdalena Zwolinska,a Helene Therisod,b
Jurgen Syguschc and Michel Therisoda,*
aECBB, ICMMO, Univ Paris-Sud, UMR 8182, F-91405 Orsay, France
bIBBMC, Univ Paris-Sud, UMR 8619, F-91405 Orsay, France
cUniv Montreal, Biochimie, CP 6128, Stn Centre-ville, Montreal, Canada PQ H3C 3J7
Received 4 December 2007; revised 7 January 2008; accepted 8 January 2008
Available online 26 January 2008
Abstract—We report the first unambiguous syntheses of glucitol-1,6-bis-phosphate and mannitol-1,6-bis-phosphate and their com-
petitive inhibition of various fructose bis-phosphate aldolases.
Ó 2008 Elsevier Ltd. All rights reserved.
Hexitol bis-phosphate (HBP), a diastereoisomeric mix-
ture of mannitol bis-phosphate and glucitol bis-phos-
phate, is known to inhibit or activate several enzymes.
This mixture was first tested on class I muscle fructose
bis-phosphate aldolase (Fba), for which it is a competi-
tive inhibitor1,6 (Ki ꢀ 1.2 lM). Later, it was also recog-
nized as an inhibitor of yeast (class II) Fba2
(Ki ꢀ 200 lM), of pyruvate kinase3 and of fructose bis-
phosphate phosphatase.4 On an other hand, HBP is an
activator of 6-phosphofructo-kinase.5
our own observations, there may be doubt as to its iden-
tity. Nevertheless, it was reported to be a good compet-
itive inhibitor of liver8 and of muscle Fba,7 with
Ki ꢀ 4.5 lM and 12 lM, respectively.
OTr
R
OH
R
OH
R
OTr
R
2
2
1
2
1
1
2
1
R
R
R
R
a
c
b
HO
HO
BnO
BnO
OH
OH
OTr
OBn
OBn
OH
OH
OH
OH
OBn
OBn
OTr
Hexitol bis-phosphate is routinely synthesized according
to Ginsburgh by reaction of sodium borohydride on
fructose bis-phosphate.6 We determined by GC of a
resultant per-silylated mixture that it was composed of
60% mannitol bis-phosphate and 40% glucitol bis-
phosphate.
1
2
3
4
OPO(OBn)
OPO H
OPO
H
3
2
2
3
2
2
2
2
1
1
1
R
R
R
R
R
R
e
d
f
BnO
BnO
HO
OBn
OBn
OBn
OBn
OH
OH
Surprisingly, the two constituents of this mixture have
never been synthesized nor tested separately against gly-
colytic aldolases, and the kinetic constants reported
above are thus only apparent constants. Glucitol bis-
phosphate was tentatively prepared by Hartman,7 how-
ever the product was not characterized, and in view of
OPO(OBn)
OPO H
3
OPO
H
2
2
2
3
5
6
7
1
2
1
2
Glucitol 1a, 2a, 7a:
R = H, R = OH; 3-6a: R = H, R = OBn
1
2
1
2
Mannitol 1b, 2b, 7b: R = OH, R = H; 3-6b: R = OBn, R = H
Scheme 1. Synthesis of glucitol bis-phosphate 7a and mannitol bis-
phosphate 7b.11 Reagents and conditions: (a) TrCl/pyridine RT 48 h;
(b) BnBr NaH/DMF RT; (c) TFA/BuOH/CH2Cl2 RT 48 h; (d)
Keywords: Aldolase; Fructose-bis-phosphate; Enzymes inhibitors;
Glycolysis; Mannitol-bis-phosphate; Glucitol-bis-phosphate; Hexitol-
bis-phosphate.
t
iPr2NP(OBn)2/Imidazole/Triazole/AcCN RT 48 h, then BuOOH; (e)
H2 (1 bar)/Pd–C/NEt3, then Dowex 50 (H+); (f) H2 (1 bar)/Pd–C.
*
Corresponding author. E-mail: therisod@icmo.u-psud.fr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.076

1736
C.-G. Mabiala-Bassiloua et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1735–1737
The synthesis of mannitol bis-phosphate has simply
never been reported. This last compound, however, is
of special interest, especially regarding its action on
Fba: It was recently reported that by soaking crystals
of Fba with a solution of HBP, only mannitol bis-phos-
phate was retained in the active site of the enzyme.9
ily migrate to this free hydroxyl.10 In polyols, the result
is a complex mixture of regioisomers. This scrambling
can be avoided if the phosphoryl groups are deprotected
prior to the hydroxyls. Thus, we used a two-step depro-
tection protocol: the phosphoryl groups were first deb-
enzylated in presence of triethylamine. In these
conditions, the rate of hydrogenolysis of a benzyl ether
is apparently considerably reduced. After removal of
the tertiary amine on an acidic ion-exchange resin, the
benzyl protecting-groups of the hydroxyls were removed
classically from the acidic intermediate. The two com-
pounds were subsequently crystallized and characterized
as their cyclohexylammonium salts.
We report hereby the synthesis and separate testing of
glucitol and mannitol bis-phosphate on class I and class
II Fba (EC 4.1.2.13).
Although the synthesis (Scheme 1) appears straightfor-
ward, successful yield of a pure product is strongly
dependant on particular attention paid during final
deprotection steps. It is well known that a protected
phosphoryl group adjacent to a free hydroxyl can read-
Glucitol bis-phosphate and mannitol bis-phosphate
were each tested for their inhibition properties against
200
60
A
B
50
40
30
20
10
0
150
100
50
0
-0.05
0
0
0
0.05
0.1
0.15
-0.05
0
0.05
1/S (uM -1
0.1
0.15
1/S (uM -1
)
)
)
)
30
25
20
15
10
5
30
25
20
15
10
5
C
D
0
0
-5
0
5
10
15
-5
5
10
15
1/S (uM -1
)
1/S (uM -1
50
40
30
20
10
0
30
25
20
15
10
5
E
F
0
-0.05
0.05
0.1
0.15
-0.05
0
0.05
1/S (uM -1
)
0.1
0.15
1/S (uM -1
Figure 1. Inhibition of Fba by glucitol and mannitol bis-phosphate. (A) Rabbit muscle Fba, 7a 0.15 mM; (B) Rabbit muscle Fba, 7b 0.01 mM. (C)
Yeast Fba, 7a 0.3 mM; (D) Yeast Fba, 7b 0.6 mM; (E) H. pylori Fba, 7a 0.5 mM; (F) H. pylori Fba, 7b 0.1 mM.

C.-G. Mabiala-Bassiloua et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1735–1737
1737
Table 1. Enzymatic kinetics constants (lM) measured on substrates/
found, in the online version, at doi:10.1016/
j.bmcl.2008.01.076.
inhibitors13
Aldolase source
KM (FBP)
Ki (KM/Ki)
7a
7b
References and notes
Rabbit muscle
Yeast
H. pylori
20
200
20
100 (0.2)
60 (3.33)
170 (0.12)
7.3 (2.74)
400 (0.5)
73 (0.27)
1. Mehler, A. H.; Viswanatha, T. Fed. Proc. 1961, 20, 232.
2. (a) Ingram, J. M. Biochem. Biophys. Res. Commun. 1970,
38, 624; (b) Smith, G. M.; Mildvan, A. S.; Harper, E. T.
Biochemistry 1980, 19, 1248.
rabbit muscle aldolase (representative of class I aldol-
ases), yeast and Helicobacter pylori aldolases (represen-
tative of class II aldolases), using established protocols
based on use of fructose bis-phosphate (FBP) as a sub-
strate.12 The two compounds displayed purely competi-
tive inhibition patterns against the three enzymes
(Fig. 1). The measured kinetic constants are reported
in Table 1.
3. Fishbein, R.; Benkovic, P. A.; Benkovic, S. J. Biochemistry
1975, 14, 4060.
4. Marcus, C. J. J. Biol. Chem. 1976, 251, 2963.
5. Kelley, E. L.; Voll, R. J.; Voll, V. A.; Younathan, E. S.
Biochemistry 1986, 25, 1245.
6. Ginsburg, A.; Mehler, A. H. Biochemistry 1966, 5, 2623.
7. Hartman, F. C.; Barker, R. Biochemistry 1965, 4, 1068.
8. Waud, J. M.; Feldman, E.; Schray, K. J. Arch. Biochem.
Biophys. 1981, 206, 291.
9. St-Jean, M.; Lafrance-Vanasse, J.; Liotard, B.; Sygusch, J.
J. Biol. Chem. 2005, 280, 27262.
The data shown in Table 1 shows that mannitol bis-
phosphate 7b is a better inhibitor of class I aldolase than
the glucitol epimer 7a. This observation is in full accor-
dance with the structural results reported by St-Jean
et al.9
10. Brown, D. M.; Usher, D. A. J. Chem. Soc. 1965, 6558.
11. Selected analytical data: Glucitol-1,6-bis-phosphate (tet-
rakis-cyclohexylammonium salt): 1H NMR (D2O) d 1.2
(m, 10H) 1.5–1.9 (m, 10H) 3 (m, 2H) 3.6–3.9 (m, 8H).
13C NMR (BB) (D2O) d (23.95, 24.5, 30.5, 50.5: CHA)
65.58 (d, JC–P4.7 Hz, C-6 or C-1), 65.8 (d, JC–P 4.7 Hz,
C-6 or C-1), 69.7 (s, C-3 or C-4), 70.76 (s, C-3 or C-4),
70.5 (d, JC–P 6.5 Hz, C-2 or C-5), 72.4 (d, JC–P 6.5 Hz,
For class II aldolases, no trend is readily discernable
with regard to preferential inhibition of either enzyme.
Compound 7a is a better inhibitor of the yeast enzyme
while 7b is better against H. pylori aldolase. 7a and 7b
give comparable KM/Ki values on yeast and rabbit mus-
cle aldolases, respectively. In conclusion, we have re-
ported for the first time separate synthesis of glucitol-
and mannitol-1,6-bis-phosphate. The two products have
been tested separately as inhibitors of fructose bis-phos-
phate aldolases from various sources. New inhibitors of
these enzymes are of special interest. Fba is active in gly-
colysis, a major metabolic pathway of virtually all living
organisms. Inhibitors of class II Fba can be broad po-
tential drugs against microorganisms.16 Like other
inhibitors of glycolytic enzymes, and depending on their
selectivity, inhibitors of class I Fba can also be active
against parasites17 and even cancer.18 In this perspective,
compound 7b is a promising basis for further syntheses
of selective inhibitors of class I aldolases.
C-2 or C-5). 31P NMR, (BB) (D2O)
d 2.5, 3.07.
20
½aꢁD ꢂ 1:4ꢃ (c 2, H2O) Mannitol-1,6-bis-phosphate (tet-
rakis-cyclohexylammonium salt): 1H NMR (D2O) d 1–
1.25 (m, 20H) 1.5–2 (m, 20H)) 3 (m, 4H) 3.6–3.9 (m,
8H). 13C NMR (BB) (D2O) d (23.75, 24.25, 30.3, 50.2:
CHA) 65.5 (d, JC–P 3.9 Hz, C-1, C-6) 68.4 (C-3, C-4)
70.1 (d, JC–P 6.3 Hz, C-2, C-5). 31P NMR, (BB) (D2O) d
20
4.5. ½aꢁD ꢂ 3:25ꢃ (c 8, H2O).
12. Enzymes: Rabbit muscle aldolase was from Fluka. Aldol-
ase from baker yeast was partly purified according to Ref.
14 after disruption of the cells in a French press. Aldolase
from H. pylori is a recombinant enzyme expressed in
E. coli JM 109. Enzymatic assays: DHAP formed by
cleavage of FBP by aldolase was estimated by measuring
spectrophotometrically (at 340 nm) the consumption of
NADH in a coupled system employing a 300-fold excess
of glycerophosphate dehydrogenase and triose phosphate
isomerase in glycyl-glycine buffer 0.1 M, pH 7.4, contain-
ing 0.2 M potassium acetate.
13. The same buffer system was used for the three enzymes. It
should be noted that KM (Ki) values of rabbit muscle
aldolase for its substrate (inhibitor) are influenced by the
presence of salts15 and kinetic parameters can thus vary
depending on the salt composition in the assay protocol.
This statement assumes that KM and Ki are affected to the
same extent.
14. Rutter, W. J.; Hunsley, J. R. Methods Enzymol. 1966, 9,
479.
15. Mehler, A. H. J. Biol. Chem. 1963, 238, 100.
16. Lewis, D. J.; Lowe, G. J. Chem. Soc., Chem. Commun.
1973, 713.
Acknowledgments
This work was supported by scholarship to C.-G.M.-B.
´
from Government of Republique du Congo. M.Z. was
on leave from Politechnika Warszawska in an Erasmus
EC program. J.S. was supported by the Natural Science
and Engineering Research Council (Canada) and Cana-
dian Institutes for Health Research.
17. Dax, C.; Duffieux, F.; Coincon, M.; Sygusch, J.;
Michels, P. A. M.; Blonski, C. J. Med. Chem. 2006,
49, 1499.
18. Geschwind, J. F. H.; Ko, Y. H.; Torbenson, M. S.; Magee,
C.; Pedersen, P. L. Cancer Res. 2002, 62, 3909.
Supplementary data
Supporting informations: detailed synthesis of 7a, 7b.
Supplementary data associated with this article can be