Syntheses of 4-deoxy-D-fructose and enzymatic affinity study

Article abstract of DOI:10.1016/S0957-4166(98)00398-X

Enantiomerically pure 4-deoxy-D-fructose has been prepared and characterised in a protected form, acidic hydrolysis of which led to an aqueous solution of 4-deoxy-D-fructose. Activities of this compound with enzymes of the glycolysis pathway involved in glucose metabolism make possible access to 4-deoxy-D-fructose-6-phosphate, 4-deoxy-D-glucose-6- phosphate and 4-deoxy-D-gluconate-6-phosphate.

Full text of DOI:10.1016/S0957-4166(98)00398-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 3737–3739
Syntheses of 4-deoxy-D-fructose and enzymatic affinity study
Corinne André, Jean Bolte and Colette Demuynck ∗
Université Blaise-Pascal, Département de Chimie (UMR 6504-SEESIB), 63177 Aubière Cédex, France
Received 18 September 1998; accepted 29 September 1998
Abstract
Enantiomerically pure 4-deoxy-D-fructose has been prepared and characterised in a protected form, acidic
hydrolysis of which led to an aqueous solution of 4-deoxy-D-fructose. Activities of this compound with enzymes of
the glycolysis pathway involved in glucose metabolism make possible access to 4-deoxy-D-fructose-6-phosphate,
4-deoxy-D-glucose-6-phosphate and 4-deoxy-D-gluconate-6-phosphate. © 1998 Elsevier Science Ltd. All rights
reserved.
Fructose plays a crucial role in all living organisms, in the free or phosphorylated form. It acts on the
primary metabolism and cannot be considered as a therapeutic target. However, close fructose analogues
such as deoxy-D-fructoses help one to understand the mechanism of action of the glycolysis pathway
enzymes (hexokinase, phosphofructokinase, phosphoglucose isomerase, fructose-1,6-bisphosphate aldo-
lase). They are also important to elucidate specific systems such as: the fructose carrier in trypanosoma1
or the glucosamine-6 phosphate synthase involved in the biosynthesis of hexosamines required in the
construction of microbial cell walls.2
In the course of our studies on the chemo-enzymatic syntheses of sugar analogues, we were inter-
ested in obtaining 4-deoxy-D-fructose, 4-deoxy-D-fructose-6-phosphate and 4-deoxy-D-fructose-1,6-
bisphosphate, the latter being a potential competitive inhibitor of aldolase. Very few of these compounds
have been mentioned in the literature. 4-Deoxy-D-fructose was prepared in 1973 by Haylock and Slessor3
by microbial oxidation of a mixture of 3-deoxy-D-arabinohexitol and 3-deoxy-D-lyxohexitol. However,
4-deoxy-D-fructose was not fully characterised either by derivative preparation or by spectroscopic data.
Later, in 1982, Kucar et al.4 studied the isomerisation of 4-deoxy-D-glucose into 4-deoxy-D-fructose,
but this compound was characterised only by its specific rotation ([α]D=3.3 against −3.93). Recently,
Enders et al.5 published a synthesis of various protected deoxy ketoses by alkylation of protected
dihydroxyacetone by the ‘SAMP’ method.
This summary of the literature data, suggests that new methods for the synthesis of 4-deoxy-D-fructose
(and its phosphorylated derivatives) are necessary. We attempted to obtain this compound by enzymatic
∗
Corresponding author. E-mail: demuynck@chisgl.univ-bpclermont.fr
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00398-X

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C. André et al. / Tetrahedron: Asymmetry 9 (1998) 3737–3739
catalysis of transketolase. However, the activities are rather low and we were unable to carry out the
synthesis on a preparative scale.6
This paper deals with the chemical synthesis of 4-deoxy-D-fructose 1 and the activities of hexokinase,
glucose phosphate isomerase and glucose phosphate dehydrogenase, towards this compound.
The synthesis (Scheme 1) is based on the stereoselective alkylation of SAMP hydrazone of protected
dihydroxyacetone 2, (S)-(+)-1-(2,2-dimethyl-1,3-dioxan-5-ylidenamino)-2-methoxy methyl pyrrolidine
3 as described by Enders.7 We synthesized this compound in four steps with an overall yield of 37%,
according to described procedures.8 Alkylation of this compound with (R)-2,2-dimethyl-4-(iodomethyl)-
1,3-dioxolane 5 should lead to the desired protected 4-deoxy-D-fructose. To prepare this iodide, commer-
cial (S)-2,2-dimethyl-1,3-dioxolane-4-methanol 4 was converted into the (S)-tosylate9 by action of tosyl
chloride in triethylamine in 85% yield. The iodide was obtained from tosylate by treatment with sodium
iodide in acetone, in 75% yield.10 The metallation of SAMP-hydrazone 3 was achieved by addition of 1.1
equivalent of t-butyllithium (1.7 M solution in hexane) at −78°C, in THF. After 2 h at this temperature,
the mixture was cooled to −100°C and a slight excess of alkyliodide 5 (1.2 equiv.) was added. After
1.5 h, the mixture was slowly warmed at 20°C, ether was added, and the organic phase was washed and
concentrated in vacuo, leading to monoalkylated hydrazone 6 as a pale yellow oil in 84% yield.
Scheme 1.
Spectroscopic data11 are consistent with the expected structure. No trace of the other diastereoisomer
1
was detected by H or 13C NMR spectroscopy. The chiral auxiliary was removed by ozonolysis with
a 46% yield, leading to the protected 4-deoxy-D-fructose 7. Spectroscopic data for this compound
1
were recorded.12 1H NMR signals were attributed by H–1H NMR correlation. The hydrolysis of the
diacetonide was achieved in the presence of acid resin (Dowex) in aqueous solution. The reaction was
followed by TLC and was complete after 1 h. 4-Deoxy-D-fructose was not isolated and the solution was
used directly for enzymatic assays.
As the first enzyme of the glycolytic pathway, hexokinase (HK) phosphorylates glucose and fructose
into glucose-6-phosphate and fructose-6-phosphate, respectively, in the presence of adenosine triphos-
phate (ATP). We measured the activity of HK towards 4-deoxy-D-fructose according to the literature
method.13 For 1 mM concentration in substrate, the activity of HK was 3% relative to its Vmax for D-
fructose. Due to the probably high value of Km for this analogue of D-fructose, the activity should increase
with the concentration of 4-deoxy-D-fructose, and allow the enzymatic synthesis of 4-deoxy-D-fructose-
6-phosphate. We carried out the synthesis on a 50 µmol scale (50 mM in substrate) in the presence

C. André et al. / Tetrahedron: Asymmetry 9 (1998) 3737–3739
3739
of an equimolecular amount of ATP, and 50 U of HK. After 3 h, all starting materials were consumed
(Scheme 2). The formation of 4-deoxy-D-fructose-6-phosphate was controlled by coupled reactions of
phosphoglucose isomerase (PGI) and glucose-6-phosphate dehydrogenase13 (G6 PDH) (Scheme 2). The
activities of these enzymes at 1 mM concentration in substrate in this assay were 6% and 1%, respectively,
relative to their Vmax for fructose-6-phosphate.
Scheme 2.
In conclusion, enantiomerically pure 4-deoxy-D-fructose has been prepared and characterised in a
protected form. Acidic hydrolysis of the protected form led to an aqueous solution of 4-deoxy-D-fructose.
This close analogue of D-fructose is a substrate for hexokinase, allowing the synthesis of 4-deoxy-
fructose-6-phosphate. Activities of other enzymes of glucose metabolism could make possible the access
to other interesting analogues, such as 4-deoxy-D-glucose-6-phosphate.
References
1. Chevalier, N.; Callens, M.; Michels, A. M. Protein Expression and Purification 1995, 6, 39–44.
2. (a) Mengin-Lecreulx, D.; Van Heijenoort, J. J. Bacteriol. 1994, 276, 5788–5795. (b) Golinelli Pimpaneau, B.; Le Goffic,
F.; Badet, B. J. Am. Chem. Soc. 1989, 111, 3029–3034. (c) Badet-Denisot, M.-A.; Rene, L.; Badet, B. Bull. Soc. Chim. Fr.
1993, 130, 249–255.
3. Haylock, C. H.; Slessor, K. N. Can. J. Biochem. 1973, 51, 969–971.
4. Kucar, S.; Zemek, J.; Zamocky, J. Chem. Zvesti. 1982, 36(2), 241–244.
5. Enders, D.; Jegelka, U. Tetrehedron Lett. 1993, 34, 2453–2456.
6. (a) André, C.; Guérard, C.; Hecquet, L.; Demuynck, C.; Bolte, J. J. Mol. Catal. 1998, 5, 113–118. (b) André, C.;
Demuynck, C.; Geffllaut, T.; Guérard, C.; Hecquet, L.; Lemaire, M.; Bolte, J. J. Mol. Catal. 1998, in press.
7. (a) Enders, D.; Bocksteigel, B. Synthesis 1989, 493–496. (b) Enders, D.; Eichenauer, H. Chem. Ber. 1979, 112, 2933–2960.
(c) Enders, D.; Fey, P.; Kippardt, H. Org. Synth. 1987, 65, 173–183.
8. (a) Linden, G. B.; Gold, M. H. J. Org. Chem. 1956, 21, 1175–1176. (b) Jones, R. A. Y.; Katritzki, A. R.; Record, K. A. F.;
Scattergood, R.; Sullivan, J. M. J. Chem. Soc., Perkin Trans. 2 1974, 402–406.
9. Jung, M. E.; Shaw, T. J. J. Am. Chem. Soc. 1980, 102, 6304–6311.
10. Baer, E.; Basu, H. Can. J. Biochem. 1969, 47, 955–960.
11. Selected physical data of 6: 1H NMR (400 MHz, CDCl3) 1.28 (s, 3H), 1.31 (s, 3H), 1.43 (s, 3H), 1.55 (m, 1H, 55B), 1.63
00
00
(m, 1H, H6 ), 1.67–2.03 (m, 4H, 2H3 and 2H4), 2.35 (m, 1H, H6 ), 2.90–3.27 (m, 4H, H2, 2H6 and H5A), 3.35 (s, 3H),
B
A
3.58 (dd, 1H, H5 , Jgem=7.9, Jvic=7.1), 4.02 (dd, 1H, H5 , Jgem=8.1, Jvic=5.8), 4.09–4.35 (m, 3H, H4 , 2H6 , H4 ); 13C
00
00
0
0
00
B
A
NMR (100 MHz, CDCl3) 23.51, 23.63, 24.09, 24.36 (Ca, Cb, Ca0, Cb0), 25.02 and 25.91 (C3 and C4), 32.53 (C6 ), 56.23
00
0
00
00
0
0
00
(C7), 58.67 (C5), 60.32 (C2), 63.56 (C6 ), 66.71 (C4 ), 69.21 (C5 ), 70.70 (C4 ), 75.68 (C6), 100.01 (C2 ), 107.98 (C2 ),
0
100.31 (C5 ); MS, 341 (M−CH3).
25
23
12. Selected physical data of 7: [α]D −149.6 (c CHCl3); lit.6 [α]D −154.7 (neat). MS, 229 (M−CH3). 1H NMR (400 MHz,
0
0
0 0
=4.9),
6 B,4
CDCl3) 1.35 (s, 3H), 1.41 (s, 3H), 1.45 (s, 3H), 1.48 (s, 3H), 1.62 (ddd, 1H, H6 B, Jgem=14.4, Jvic 6 B,4=10.7, Jvic
0
0
0
0
0
0 0
=7.2 cis), 4.02 (d, 1H,
5 B,4
2.24 (ddd, 1H, H6 A, Jgem=14.4, Jvic
=8.3, Jvic 6 A,4=2.6), 3.57 (dd, 1H, H5 B, Jgem=8.1, Jvic
6 A,4
0
0
0
0 0 0
0 0
6H6B, Jgem=17.1), 4.04 (dd, 1H, H5 A, Jgem=8.3, Jvic
=5.8 trans) 4.22 (m, 1H, H4 , Jvic 4 ,6 A=8.2, Jvic 4 ,5 B=7.0 cis, Jvic
5 A,4
0
0
0
0
0
0
4 ,5 A=5.9 trans, Jvic 4 ,6 B=4.9), 4.28 (d, 1H, H6A, Jgem=17, J6A,4=1.4 Jw), 4.17 (dd, 1H, H4, Jvic 4,6 B=10.7, Jvic 4,6 A=2.6,
0 0
4,6A=1.4 Jw); 13C NMR (100 MHz, CDCl3) 23.7, 23.8 (Ca, Cb), 25.9, 27.1 (Ca , Cb ), 32.4 (C6 ), 66.6 (C4 ), 69.7 (C5 ),
0 0 0
J
0
72.0 (C6), 72.5 (C4), 101.1 (C2), 108.8 (C2 ), 209.1 (C5).
13. Bergmeyer, H. U. In Methods of Enzymatic Analysis, Verlag Chemie; 2nd Edition; Bergmeyer, H. U., Ed.; Academic Press,
Inc.: New York and London, 1974; pp. 1304–1307.