form of FHAP remained nearly unchanged after the first addition
of enzyme.24 These observations are in agreement with the
proposed enzymatic reaction mechanism for the natural substrate
(DHAP) with RAMA,25 since only the keto form can lead to a
covalent linked Schiff base intermediate with the enzyme. In
analogy to the stereospecificity of RAMA with DHAP as a
substrate,26 the fast proton–deuterium exchange of the pro-S
hydrogen of FHAP can be assumed.
3 (a) T. D. Machajewski and C.-H. Wong, Angew. Chem., Int. Ed.,
2000, 39, 1352; (b) L. J. Whalen and C.-H. Wong, Aldrichimica
Acta, 2006, 39, 63.
4 (a) T. Gefflaut, C. Blonski, J. Perie and M. Willson, Prog. Biophys.
Mol. Biol., 1995, 63, 301; (b) D. J. Lewis and G. Lowe, Eur. J.
Biochem., 1977, 80, 119; (c) I. A. Rose and E. L. O’Connell, J. Biol.
Chem., 1969, 244, 126; (d) C. Blonski, T. Gefflaut and J. Pe
Bioorg. Med. Chem., 1995, 3, 1247; (e) T. Gefflaut, C. Blonski and
J. Perie, Bioorg. Med. Chem., 1996, 4, 2043; (f) P. Page, C. Blonski
and J. Perie, Bioorg. Med. Chem., 1999, 7, 1403.
rie,
´ ´
´
´
´
´
5 (a) F. C. Hartman, Biochemistry, 1970, 9, 1776; (b) F. C. Hartman,
Biochemistry, 1970, 9, 1783; (c) D. W. Slater, I. L. Norton and
F. C. Hartman, Biochemistry, 1973, 12, 1.
6 4-Hydroxy-3-butanone-1-phosphonate: (a) D. Stribling, Biochem.
J., 1974, 141, 725; (b) H.-L. Arth and W.-D. Fessner, Carbohydr.
Res., 1998, 305, 313; (c) ref. 4f.
7 1,3-Dihydroxy-2-butanone-3-phosphate: (a) N. Bischofberger,
H. Waldmann, T. Saito, E. S. Simon, W. Lees, M. D. Bednarski
and G. M. Whitesides, J. Org. Chem., 1988, 53, 3457; (b) ref. 2a.
The low overall yield of the enzymatic aldol addition
requires some comment. Since product formation correlates
with the accessibility of the carbonyl moiety,17a the low
isolated yield may mainly be attributed to the high degree of
hydration (91%) of the keto functionality and to the general
difficulties in acetylation of pentuloses.27 Furthermore, the
enamine intermediates of the natural substrates DHAP and
fructose-1,6-bisphosphate are stabilized by a hydrogen bond
between the carboxyl functionality of Asp-33 of the enzyme
and the hydroxyl group at C3 of the substrates.28 The
importance of this crucial interaction was demonstrated by
mutations of Asp-33 into alanine, serine, glutamate and
asparagines, which lowered Vmax of the enzyme dramatically.29
Our entire findings point to a similar positioning of FHAP in
the active site of RAMA to that of DHAP. In contrast to
DHAP, FHAP cannot form this seminal interaction with
Asp-33, due to fluorine’s property of lacking hydrogen bond
donor capacities and functioning solely as a hydrogen bond
acceptor.
8 Thiophosphoric
acid
S-(3-hydroxy-2-oxopropyl)
ester:
(a) R. Duncan and D. G. Drueckhammer, J. Org. Chem., 1996,
61, 438; (b) W.-F. Fessner and G. Sinerius, Angew. Chem., Int. Ed.
Engl., 1994, 33, 209.
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R. Eisenthal and R. Harrison, Biochem. J., 1975, 145, 501.
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Chemistry, Wiley, NewYork, 1991; (b) J.-P. Begue and D. Bonnet-
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Delpon, Bioorganic and Medicinal Chemistry of Fluorine, Wiley,
New York, 2007.
11 J. B. Silverman, P. S. Babiarz, K. P. Mahajan, J. Buschek and
T. P. Fondy, Biochemistry, 1975, 14, 2252.
12 C.-H. Wong, D. G. Drueckhammer and H. M. Sweers, ACS Symp.
Ser., 1988, 374, 29.
13 J. Villieras and M. Rambaud, Synthesis, 1982, 924.
14 The yield was decreaseddue to partial coevaporation of allyl
alcohol 2 with CH2Cl2.
15 D. T. Fox and C. D. Poulter, J. Org. Chem., 2005, 70, 1978.
16 pH and pD measurements were accomplished using a glass elec-
trode. (pD = pH + 0.4): T. H. Fife and T. C. Bruice, J. Phys.
Chem., 1961, 65, 1079.
In summary, we have developed an efficient gram scale
synthesis of FHAP (5). In contrast to earlier reports12 FHAP
could be identified as a novel donor substrate for RAMA-
catalyzed aldol condensations. This fact was demonstrated in
the synthesis of 1,4,5-tri-O-acetyl-3-deoxy-3-fluoro-D-threo-
pent-2-ulose 8. FHAP is the first analog of DHAP with a
modification at C3 accepted as a donor substrate by RAMA.
The stereochemical configuration at C3 and C4 (D-threo) of
the aldol condensation product has proven to be identical as
for the natural substrate DHAP, which was confirmed by an
independent chemical synthesis of pentulose 8. Furthermore, a
fast proton–deuterium exchange and the different binding
preferences of the gem-diol and keto form of FHAP were
analyzed by 19F-NMR. A direct evolution of aldolases30 might
result in highly efficient catalysts for the aldol condensation of
FHAP with aldehydes, giving access towards a great variety of
fluoro containing carbohydrates with biological and medicinal
relevance.10
17 (a) S. J. Reynolds, D. W. Yates and C. I. Pogson, Biochem. J., 1971,
122, 285; (b) G. R. Gray and R. Barker, Biochemistry, 1970, 9, 2454.
18 M. Fischer, C. Schmolzer, T. Nowikow and W. Schmid, Eur. J.
¨
Org. Chem., 2011, 1645.
19 (a) M. Hudlicky, in Organic Reactions, ed. A. S. Kende, Wiley, US,
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J. DeChancie and K. N. Houk, J. Fluorine Chem., 2003, 125, 537.
20 S. Fan, C.-Y. He and X. Zhang, Tetrahedron, 2010, 66, 5218.
21 G. Lowe and R. F. Pratt, Eur. J. Biochem., 1976, 66, 95.
22 The rate of deuterium incorporation had been determined in
analogy to following procedures: (a) P. Page, C. Blonski and
´ ´
J. Perie, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol.,
1998, 1386, 59; (b) R. F. Pratt, Biochemistry, 1977, 16, 3988.
23 This observation may be explained due to enhanced acidity of the
protons at the C3 of FHAP compared to DHAP.
24 The slight increase in linewidth by the first addition of RAMA may
be attributed to changes in viscosity of the sample solution.
25 (a) E. Grazi, P. T. Rowely, T. Chang, O. Tchola and B. L. Horecker,
Biochem. Biophys. Res. Commun., 1962, 9, 38; (b) ref. 4a.
26 (a) I. A. Rose, J. Am. Chem. Soc., 1958, 80, 5835; (b) M. St-Jean
and J. Sygusch, J. Biol. Chem., 2007, 282, 31028.
27 C. Demuynck, J. Bolte, L. Hecquet and H. Samaki, Carbohydr.
Res., 1990, 206, 79. (Acetylation of xylulose yields triaceteylxylulose
in 38%).
We thank Dr Ernst Pittenauer for MS and Prof. Vladimir
Arion for X-ray diffraction measurements. We are grateful to
Prof. Robert Konrat and Prof. Fritz Pittner for valuable
discussions.
Notes and references
28 (a) M. St-Jean, J. Lafrance-Vanasse, B. Liotard and J. Sygusch,
J. Biol. Chem., 2005, 280, 27262; (b) ref. 26b.
1 C.-H. Wong, in Enzyme Catalysis in Organic Synthesis, ed. K. Drauz
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30 (a) N. J. Turner, Nat. Chem. Biol., 2009, 5, 567; (b) A. Bolt,
A. Berry and A. Nelson, Arch. Biochem. Biophys., 2008, 474, 318.
2 (a) M. D. Bednarski, E. S. Simon, N. Bischofberger, W.-D. Fessner,
M.-J. Kim, W. Lees, T. Saito, H. Waldmann and G. M. Whitesides,
J. Am. Chem. Soc., 1989, 111, 627; (b) C.-H. Wong and
G. M. Whitesides, J. Org. Chem., 1983, 48, 3199; (c) P. Page,
C. Blonski and J. Perie, Tetrahedron, 1996, 52, 1557.
´ ´
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 6647–6649 6649