A simple route for synthesis of 4-phospho-D-erythronate

Article abstract of DOI:10.1016/j.tetlet.2011.02.045

4-Phospho-d-erythronate is an intermediate in the synthesis of pyridoxal 5′-phosphate in some bacteria and an inhibitor of ribose 5-phosphate isomerase. Previous synthetic schemes for the preparation of 4-phospho-d-erythronate required expensive precursors and typically gave low yields. We report a straightforward synthesis of 4-phospho-d-erythronate from the inexpensive precursor d-erythronolactone in five steps with a preparatively useful yield of 22%.

Full text of DOI:10.1016/j.tetlet.2011.02.045

Tetrahedron Letters 52 (2011) 1913–1915
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A simple route for synthesis of 4-phospho-D-erythronate
Yehor Novikov a, Shelley D. Copley a, , Bruce E. Eaton b
⇑
a Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences,
University of Colorado at Boulder, Boulder, CO 80309, USA
b Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Phospho-
and an inhibitor of ribose 5-phosphate isomerase. Previous synthetic schemes for the preparation of 4-
phospho- -erythronate required expensive precursors and typically gave low yields. We report a
straightforward synthesis of 4-phospho- -erythronate from the inexpensive precursor -erythronolac-
tone in five steps with a preparatively useful yield of 22%.
D
-erythronate is an intermediate in the synthesis of pyridoxal 50-phosphate in some bacteria
Received 2 December 2010
Revised 7 February 2011
Accepted 9 February 2011
Available online 13 February 2011
D
D
D
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
4-Phospho-
D-erythronate
D-Erythronolactone
Phenyldiazomethane
Benzylation
4-Phospho-D-erythronate (4PE, 1) is an intermediate in the syn-
thesis of pyridoxal 50-phosphate (PLP) in some bacteria. In gamma
proteobacteria such as Escherichia coli, 4PE dehydrogenase (PdxB)
converts 4PE to 2-oxo-3-hydroxy-4-phospho-butanoate (2) with
concomitant reduction of NAD+ to NADH. Efforts to study this en-
zyme have been frustrated by the puzzling finding that multiple
turnovers of the substrate could not be achieved in vitro.1 4PE is
also oxidized to 2 by alpha proteobacteria such as Sinorhizobium
meliloti, but the reaction is catalyzed by a flavoenzyme called PdxR
that is not homologous to PdxB.2 Studies of these enzymes have
been hampered by the unavailability of synthetic 4PE. Synthesis
of 4PE by the strategy described herein enabled mechanistic stud-
ies of E. coli PdxB, leading to the discovery that the nicotinamide
cofactor is not released from the enzyme after substrate turnover.
The tightly bound NADH formed after oxidation of 4PE must be
regenerated by reduction of an a-keto acid such as a-ketoglutarate,
oxaloacetate, or pyruvate before a second turnover can occur (see
Scheme 1).3 The availability of 4PE will facilitate further studies
of the mechanism and structure of this unusual enzyme.
4PE is also of interest because it is an inhibitor of ribose 5-phos-
phate isomerase (Rpi).4 Rpi is required for synthesis of ribose and
therefore for synthesis of ribonucleotides, deoxyribonucleotides,
and histidine. In plants, Rpi is also involved in the Calvin cycle car-
bon fixation pathway. Two unrelated families of Rpi’s have been
recognized. RpiA is found in all three domains of life, while RpiB
is found only in some bacteria and protozoa, including pathogens
such as Yersinia pestis, Listeria monocytogenes, Clostridium
Scheme 1. Oxidation of 4PE by PdxB generates NADH at the active site, which must
be re-oxidized by reaction with an
ketoglutarate.
a-keto acid such as pyruvate, oxaloacetate or a-
perfringens, Clostridium botulinum, Rickettsia rickettsii and prow-
azekii, Trypanosoma cruzi and brucei, Leishmania major, Entamoeba
histolytica and Giardia lamblia. Rpis are potential targets for drugs
⇑
Corresponding author.
E-mail address: shelley@cires.colorado.edu (S.D. Copley).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.02.045

1914
Y. Novikov et al. / Tetrahedron Letters 52 (2011) 1913–1915
and herbicides, and analogs of 4PE have been synthesized and eval-
uated as potential inhibitors of these enzymes.5,6
Our synthetic strategy is shown in Scheme 2. The hydroxyl
groups of commercially available -erythronolactone (3) were
D
Most published syntheses of 4PE have involved oxidation of a
phosphorylated sugar precursor. The earliest reported synthesis
used lead tetraacetate to oxidize glucose-6-phosphate.7 However,
4PE was neither purified from by-products nor adequately
analyzed to assure the purity of 4PE. Later, traces of 4PE were
obtained from fructose 1,6-diphosphate employing a multi-step
procedure followed by a complicated purification.8 Woodruff and
Wolfenden prepared 4PE by oxidizing erythrose-4-phosphate with
bromine,4 as previously reported by Horecker.9 However, the
reaction yield and purification procedure were not described, and
characterization of the product was limited to a comparison of
its Rf with that of the product reported in Ref. 7 The reference for
the Horecker paper provided by Woodruff and Wolfenden appears
to be erroneous since we were unable to locate the original Letter.
Gupta et al. explored the oxidation of erythrose 4-phosphate by
gold(III) salts, but the product of the reaction was neither purified
nor characterized.10 Although direct oxidation of 4-erythrose
4-phosphate to 4PE is appealing, we chose not to pursue this route
protected using benzyl bromide in the presence of freshly prepared
Ag2O12 (as described by Marshall et al.).13 The yellowish oil was
purified on a silica column (ca. 50 g silica) using toluene/ethylace-
tate 8:2 mixture as the eluent. Compound 4 was obtained as a
white semi-solid (712 mg, Rf = 0.35). Recrystallization from boiling
heptanes gave white crystals (34% yield, >99% pure, melting point
87–89 °C). It should be noted that we did not attempt to optimize
the yield of this step because the starting material is inexpensive. A
variation of this procedure was reported to give a 91% yield,
although in our hands the two procedures gave similar yields.14
The lactone ring of 4 was opened using LiOH (1.1 equiv) in
water at 4 °C for 48 h with stirring. The reaction mixture was
neutralized with Dowex 50W-X12 (H+-form). After addition of
20 mL of methanol, the slurry was stirred for 20 min at 4 °C. The
suspension was filtered and the filtrate containing 5 (ca. 95%, by
1H NMR) was used for the next step without isolation.
Compound 5 was converted to the corresponding methyl ester
by treatment with diazomethane. However, this compound under-
went cyclization upon purification by silica gel chromatography.
Furthermore, hydrolysis of the methyl ester after phosphitylation
and oxidation of the phosphite (as shown in Scheme 2 for the
corresponding benzyl ester) proved problematic due to extensive
b-elimination. Consequently, we explored the possibility of gener-
ating the benzyl ester of 5 using phenyldiazomethane. Use of
phenyldiazomethane for this purpose is uncommon, but has been
reported.15 Formation of 6 using phenyldiazomethane generated
as described by Dudman and Reeze16 proved successful. Gratify-
ingly, in contrast to the corresponding methyl ester, 6 was stable
under the conditions required for silica gel chromatography and
was purified as a clear oil (84% yield).
because the starting material, D-erythrose 4-phosphate, is available
only as an impure preparation at a current cost of >$6500 per gram.
Thus, preparation of the gram quantities of 4PE needed for kinetic
and structural studies would be prohibitively expensive.
An interesting alternative strategy for synthesis of 4PE employs
selective phosphorylation of the 4-hydroxyl of a protected eryth-
ronic acid.11 The methyl ester of erythronic acid was converted
to methyl 2,3-O-dibenzoyl 4-O-trityl D-erythronic acid. After re-
moval of the trityl group, the 4-hydroxyl was phosphorylated with
diphenyl phosphorochloridate. After deprotection, 4PE was iso-
lated by fractional crystallization as the bis(dicyclohexyl ammo-
nium) salt in 19% yield from methyl 2,3-O-dibenzoyl 4-O-trityl D-
erythronic acid. The product was characterized only by elemental
analysis. We were able to reproduce the reported synthesis of
methyl erythronate. However, purification of the product by silica
gel chromatography was unsuccessful because methyl erythronate
underwent spontaneous cyclization, regenerating erythronolac-
tone. Preparation of methyl 2,3-O-dibenzoyl 4-O-trityl D-erythro-
nate directly from the impure methyl erythronate was successful,
but proceeded in less than 10% yield.
Compound 6 was converted to 7 (76% yield) by phosphitylation
with dibenzylphosphoramidite followed by oxidation of the phos-
phite with tert-butyl peroxide in the same vessel. Removal of the
benzyl protecting groups by hydrogenation over Pd/C did not pro-
ceed to completion in H2O even after 24 h at 60 psi H2. However,
complete deprotection was achieved in methanol/H2O (95:5) after
24 h using 20–60 psi H2, yielding 4PE in 22% overall yield from
D
-erythronolactone (3).17
We sought a more robust method for synthesis of 4PE from an
inexpensive precursor to supply the gram quantities required for
kinetic and structural studies of enzymes such as PdxB, PdxR and
Rpi. The strategy we report here requires five steps, most of which
are accomplished with good to high yield, beginning from the inex-
It is now possible to synthesize ample quantities of 4PE in
excellent yield in five steps from an inexpensive precursor. Utiliza-
tion of a benzyl ester as opposed to a methyl ester was the key to
the success of this synthetic strategy. There are numerous exam-
ples where synthesis of carbohydrate derivatives could benefit
from a strategy employing complete benzyl protection followed
pensive precursor D-erythronolactone ($30 per gram, TCI America).
Scheme 2. Conditions and yields: (a) i. Ag2O (4 equiv), BnBr (2.7 equiv), ether, 12 h, 25 °C; ii. silica gel chromatography (toluene/ethyl acetate, 8:2, Rf = 0.35);
iii. crystallization (boiling heptanes), 34%; (b) i. LiOH (1.1 equiv), H2O, 48 h, 4 °C; ii. neutralization with Dowex 50W-X12 (H+-form, 2.2 equiv) in 50% MeOH; iii. removal of
Dowex 50W-X12 by filtration; (c) i. slight excess benzyldiazomethane in CH2Cl2; ii. silica gel chromatography (pentane/ethyl acetate, 4:1, Rf = 0.3), clear oil, 84%;
(d) i. dibenzylphosporamidite (1.3 equiv), CH3CN, tetrazole (1.3 equiv), 1 h, 4 °C; ii. tBuOOH (1.7 equiv), 2 h, 4 °C; iii. silica gel chromatography (ethyl acetate:CH2Cl2, 1:9,
Rf = 0.40), 76%; (e) H2, 60 psi, Pd/C, MeOH, 12 h, 4 °C, quantitative yield.

Y. Novikov et al. / Tetrahedron Letters 52 (2011) 1913–1915
1915
2. Tazoe, M.; Ichikawa, K.; Hoshino, T. J. Bacteriol. 2006, 188, 4635–4645.
by hydrogenolysis. This strategy will be of particular use in the
case of C-1 carboxylates given the tendency of methyl esters of
such compounds to cyclize to the corresponding lactone.
3. Rudolph, J.; Kim, J.; Copley, S. D. Biochemistry 2010, 49, 9249–9255.
4. Woodruff, W. W.; Wolfenden, R. J. Biol. Chem. 1979, 254, 5866–5867.
5. Burgos, E.; Salmon, L. Tetrahedron Lett. 2004, 45, 753–756.
6. Burgos, E.; Salmon, L. Tetrahedron Lett. 2004, 45, 3465–3469.
7. Klybas, V.; Schramm, M.; Racker, E. Arch. Biochem. Biophys. 1959, 80, 229–
237.
Acknowledgments
8. Lee, J. B. J. Org. Chem. 1963, 2473–2475.
9. Horecker, B. L. Methods Enzymol. 1957, 172–174.
10. Gupta, K. K.; Pal, B.; Begum, B. A. Carbohydr. Res. 2001, 330, 115–123.
11. Barker, L.; Wolf, F. J. Org. Chem. 1963, 28, 1847–1851.
12. Tanabe, M.; Peters, R. H. Organic Synth. 1981, 60, 92.
This work was supported by NIH GM083285 to S.D.C. We thank
Dr. Richard K. Shoemaker for assistance with NMR spectroscopy.
Supplementary data
13. Marshall, J. A.; Seletsky, J. A.; Luke, G. P. J. Org. Chem. 1994, 59, 3413–3420.
14. Smith, A. B., 3rd; Fox, R. J.; Vanecko, J. A. Org. Lett. 2005, 7, 3099–3102.
15. Elix, J. A.; Parker, J. L. Aust. J. Chem. 1987, 40, 187–192.
16. Dudman, C. C.; Reeze, C. B. Synthesis 1982, 419–421.
Supplementary data associated with this Letter can be found, in
the online version, at doi:10.1016/j.tetlet.2011.02.045.
17. 1H NMR (500 MHz, D2O, free acid) d 3.75–3.87 (m, 2H), 3.94 (distorted dd,
J1 = 9.4 Hz, J2 = 4.6 Hz, 1H), 4.20 (d, J = 4.6 Hz, 1H). 13C APT NMR (100.6 MHz,
CDCl3) d 65.7 (2, poorly resolved d), 71.0 (1), 71.5 (1, d, JCꢀP = 8.4 Hz), 175.5(0).
References and notes
13P NMR (161.9 MHz, D2O)
d
1.1. Predicted exact mass for C4H8O8Pꢀ
214.995130; observed mass 214.996458.
1. Zhao, G.; Pease, A. J.; Bharani, N.; Winkler, M. E. J. Bacteriol. 1995, 177, 2804–
2812.