A novel strategy for the preparation of naturally occurring phosphocitrate and its partially esterified derivatives

Article abstract of DOI:10.1021/jo061709c

(Chemical Equation Presented) A novel method for the synthesis of phosphocitrate (1, PC) starting from triethyl ester of citric acid and MeOPCl₂ is described. The method is based on selective stepwise hydrolysis of ester moieties from the intermediate Me-O-P(O)(Cl)(Z) (Z = triethylcitrate), 4a, which also allows one to prepare partially esterified derivatives of PC with good yield and purity without chromatographic purifications.

Full text of DOI:10.1021/jo061709c

A Novel Strategy for the Preparation of
Naturally Occurring Phosphocitrate and Its
Partially Esterified Derivatives
form in the extracellular matrix of the cartilage and can then
be released into joint space where they can promote cartilage
2-4
5
2+
degeneration. Ca -containing crystal deposition in cartilage
4b
as a feature of osteoarthritis is widely recognized. For example,
data indicate that in degenerative joints calcium pyrophosphate
dihydrate and basic calcium phosphate crystals are more
common than in normal joints. In addition, osteoarthritis is more
,
†
‡
Petri A. Turhanen,* Konstantinos D. Demadis,
†
,†
Sirpa Per a¨ niemi, and Jouko J. Veps a¨ l a¨ inen*
2
+
Department of Chemistry, UniVersity of Kuopio, P.O. Box 1627,
FIN-70211, Kuopio, Finland, and Crystal Engineering, Growth
and Design Laboratory, Department of Chemistry, UniVersity of
Crete, Heraklion, Crete GR-71003, Greece
common and severe in patients with detected Ca -containing
4
c
crystals.
A therapeutic strategy should include chemical compounds
2+
that possess no or low toxicity and block Ca -containing crystal
formation or inhibit intracellular responses stimulated by crystal
formation and growth. Phosphocitrate (PC, Figure 1) is a
petri.turhanen@uku.fi; jouko.Vepsalainen@uku.fi
ReceiVed August 17, 2006
6
naturally occurring biomolecule found in mammalian mito-
7
chondria and crab hepatopancreas, likely produced by cytosolic
8
phosphorylation of citrate. PC does not produce any significant
toxic side effects in rats or mice when given in doses up to 150
8
µmol/kg/day. Furthermore, in vitro studies suggested that PC
concentrations up to 1.5 mM (4.5 mg/mL) do not have any effect
on normal cellular functions such as DNA and protein
9
-11
synthesis.
PC was found to inhibit crystal-induced matrix metallopro-
teinase synthesis and mitogenesis in cells, but it has no effect
on similar processes induced by growth factors or serum.1
This inhibitory action can be explained by the influence that
0,12,13
A novel method for the synthesis of phosphocitrate (1, PC)
2
+
starting from triethyl ester of citric acid and MeOPCl
2
is
PC exerts on the interaction of Ca -containing crystals with
biomembranes, as studied by computational methods.14
described. The method is based on selective stepwise
hydrolysis of ester moieties from the intermediate Me-O-
P(O)(Cl)(Z) (Z ) triethylcitrate), 4a, which also allows one
to prepare partially esterified derivatives of PC with good
yield and purity without chromatographic purifications.
PC was also found to be a powerful inhibitor of hydroxy-
1
5
apatite crystal formation in vitro. The precise mechanism of
action of PC has been an issue of intense speculation. Tew et
(
2) Rosenthal, A. K. Rheum. Dis. Clin. N. Am. 2006, 32, 401.
(3) (a) Halverson, P. B.; Derfus, B. A. Curr. Opin. Rheumatol. 2001,
Osteoarthritis and rheumatoid arthritis are two of the most
widespread diseases in the elderly population. In 2002, TIME
magazine dedicated a “cover story” on these diseases in order
to emphasize their significance for society and medicine.
According to the data presented, 20 million Americans today
suffer from arthritis-related issues, a number likely to climb to
13, 221. (b) Halverson, P. B.; Greene, A.; Cheung, H. S. Osteoarthr.
Cartilage 1998, 6, 324.
(
4) (a) Chen, L. X.; Schumacher, H. R. Curr. Opin. Rheumatol. 2006,
8, 171. (b) Dai, L.; Pessler, F.; Chen, L. X.; Clayburne, G.; Schumacher,
H. R. Rheumatology 2006, 45, 533. (c) Cheung, H. S. Curr. Opin. Biol.
1
1
2
000, 123, 223.
(5) (a) Baillon, B.; Salvia, P.; Feipel, V.; Rooze, M. ReV. Chir. Orthop.
2006, 92, 464. (b) Cheung, H. S. Front. Biosci. 2005, 10, 1336.
(6) Cheung, H. S. Curr. Opin. Rheumatol. 2005, 17, 336.
40 million in the coming years, mainly due to changes in
(
7) Moro, L.; Stagni, N.; Luxich, E.; Sallis, J. D.; DeBernard, B. Biochem.
Biophys. Res. Commun. 1990, 170, 251.
8) (a) Howard, J. E. John Hopkins Med. J. 1976, 139, 239. (b) Tew,
lifestyle of modern humans in developed societies. Some of the
well-recognized factors that result in osteoarthritis in humans
are genetic disposition, joint trauma, and joint disuse. An
inadequately investigated aspect of articular cartilage and other
(
W. P.; Malis, C. D.; Howard, J. E.; Lehninger, A. L. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 5528.
(9) (a) Shankar, R.; Crowden, S.; Sallis, J. D. Atherosclerosis 1984, 52,
2+
articular tissue is their tendency to form Ca -containing
crystals. Deposition of these crystals may be triggered by tissue
trauma or abnormal fluctuations in intracellular Ca2 concentra-
tions. Deposits may initially accumulate in a “soft” and
amorphous form, which subsequently transforms into a hard,
crystalline Ca2 salt of greatly reduced solubility. These crystals
then act to activate further events for the development of the
specific pathological disease state. This scenario prevails not
only in osteoarthritis and rheumatoid arthritis but also in a broad
range of related diseases, such as heart valve calcification, renal
calcinosis, soft tissue tumor calcification, urinary lithiasis,
1
91. (b) Krug, H. E.; Mahowald, M. L.; Halverson, P. B.; Sallis, J. D.;
Cheung, H. S. Arthritis Rheumatol. 1993, 36, 1603.
(10) Cheung, H. S.; Sallis, J. D.; Mitchell, P.; Struve, J. A. Biochem.
Biophys. Res. Commun. 1990, 171, 20.
+
(11) Cheung, H. S.; Kurup, I. V.; Sallis, J. D.; Ryan, L. M. J. Biol. Chem.
1996, 271, 28082.
+
(12) Nair, D.; Misra, R. P.; Sallis, J. D.; Cheung, H. S. J. Biol. Chem.
1997, 272, 18920.
13) (a) Reuben, P. M.; Wenger, L.; Cruz, M.; Cheung, H. S. Conn.
Tiss. Res. 2001, 42, 1. (b) Cheung, H. S.; Sallis, J. D.; Struve, J. A. Biochem.
Biophys. Acta 1996, 1315, 105.
(14) (a) Dalal, P.; Zannotti, A.; Wierzbicki, A.; Madura, J. D.; Cheung,
H. S. Biophys. J. 2005, 89, 1. (b) Wierzbicki, A.; Cheung, H. S. J. Mol.
Struct. THEOCHEM 1998, 454, 287. (c) Wierzbicki, A.; Cheung, H. S. J.
Mol. Struct. THEOCHEM 2000, 529, 73. (d) Wierzbicki, A.; Dalal, P.;
Madura, J. D.; Cheung, H. S. J. Phys. Chem. B 2003, 107, 12346.
(15) (a) Sallis, J. D.; Demadis, K. D.; Cheung, H. S. Curr. Rheumatol.
ReV. 2006, 2, 95. (b) Sallis, J. D.; Cheung, H. S. Curr. Opin. Biol. 2003,
15, 321.
(
2+
arteriosclerosis, and chondrocalcinosis. Ca -containing crystals
†
University of Kuopio.
University of Crete.
‡
(1) Gorman, C.; Park, A. Time 2002, 160 (24), 72.
10.1021/jo061709c CCC: $37.00 © 2007 American Chemical Society
1
468
J. Org. Chem. 2007, 72, 1468-1471
Published on Web 01/18/2007

after oxidation and purification with 68% yield (procedure ii in
Scheme 1). Oxidation by sulfuryl chloride yielded 4a, which
contains a P-Cl moiety. This is expected according to results
1
7
published earlier (note, the reaction mechanism in ref 17 is
incorrect). The P-bound chlorine substituent in 4a is easily
removed by addition of ethanol in the presence of NaHCO3
leading to 4c at 92% yield and g95% purity after isolation
(procedure iv in Scheme 1). Triethylamine was also tested as
HCl scavenger to prepare 4c from 4a, but the purified yield is
only ∼10%. The main reason for this low yield is the ability of
3
1
triethylamine, according the P NMR spectrum, to dealkylate
FIGURE 1. Schematic structure of PC in its protonated form.
1
8
phosphorus esters since unsymmetrical dimers and two
monomers are detected in crude product. Compound 4a is an
interesting intermediate since it allows one to prepare (1)
monomethyl ester 4b after water addition (procedure iii), (2)
mixed esters, such as 4c (procedure iv), (3) phosphates 4d, first
by treatment with trimethylsilyl bromide, followed by desilyl-
ation with methanol (procedure v), and (4) 5a by reaction with
al. proposed that PC inhibits hydroxyapatite (Ca5(PO4)3(OH))
precipitation in cells or cellular compartments by maintaining
8b
high hydroxyapatite supersaturation. Sequestration/complex-
ation of serum Ca2+ resulting in formation of Ca-PC complexes
at pH 7.4 was disproven.8b As proposed before, PC acts as a
powerful hydroxyapatite growth inhibitor by strongly binding
to its surface, thus poisoning its growth.
2
M HCl at 38-40 °C (above 42 °C the PO-Z bond also starts
to hydrolyze) for 20 h (procedure vii). The high selectivity (94%)
of demethylation versus removal of the phosphate ethyl group
is observed when KI in acetone is used as the dealkylation agent
when compound 4e is prepared from 4c (procedure vi).
Compound 4a seems to be quite stable since no degradation
was observed during a 2-month storage period (without N2) at
The available literature synthetic procedures for PC report
7
-61% overall yields, while laborious chromatographic puri-
1
6
fications are needed for isolation of pure final product. Also,
the methods reported thus far do not allow access to preparation
of partial ester PC derivatives. Our approach involves a synthetic
strategy to improve overall yields for PC preparation, as well
as to give access to several partial ester derivatives of PC. These
modified PC molecules are needed for more complete in vivo
and in vitro testing and detailed structure/function anticalcifi-
cation relationships.
-
18 °C. Compound 5a, which is a new type of partially
esterified phosphocitrate, is a challenge to obtain in pure form
since it is typically obtained in ∼66% yield with 83-90% purity
1
and contains 5c (R ) H), diethyl monomethyl PC, and a trace
1
31
of 1 as impurities (according to H and P NMR spectra).
Alkaline hydrolysis was also tested with poor success since
the PO-Z bond tends to hydrolyze as well under the conditions
used. However, when 5a is treated with acetone and KI, as
described elsewhere for the demethylation of bisphosphonate
The difficulty in efficient synthesis of PC is phosphorylation of
the sterically hindered tertiary alcohol group of citric acid ester
(Scheme 1). Previously, PC had been prepared by several meth-
2
ods starting from trialkyl esters of citric acid and powerful phos-
1
6a
phorylating agents such as o-phenylene phosphorochloridate,
1
9
-cyanoethyl phosphate,1 and diphenyl chlorophosphate.
6b
16c
and phosphonate compounds, 5b is obtained in quantitative
yield and g95% purity (procedure viii).
2
Also, partially substituted 1,3-dibenzyl-2-phosphonooxy citrate
16d
For the last step (from 5b to 1, procedure ix), we resorted to
a modification of a previously published method, in which an
excess of NaOH and CaCl2 was used at 4 °C for 4 h. New
modification was used to avoid the aforementioned chromato-
graphic purification, with precipitation of the final product.16
Thus, in our approach, exactly 4.0 equiv of NaOH and 1.2 equiv
CaCl2 were added to 5b at 65 °C and reacted for 2 h. A white
had been synthesized starting from tribenzyl citrate and PCl5.
Our strategy to prepare PC and its derivatives is based on
utilization of MeOPCl2, which is a readily available, inexpen-
sive, very reactive, and sterically nonhindered phosphitylating
agent of trivalent P. Our key intermediate 3 (produced when
1
.5 equiv of MeOPCl2 is used, but not isolated) is proposed to
31
form almost in quantitative yield according to P NMR data
1
procedure i in Scheme 1; 31P spectrum was measured after
precipitate formed almost instantaneously. On the basis of H
(
3
1
and P NMR spectra, the organic portion of the precipitate
addition of methanol to 3 and was 132.1 ppm). The next step
procedure ii in Scheme 1) leads to formation of 4a after addition
+
2+
contains exclusively 1 as the mixed K /Ca salt (C6H4O10-
PKCa2‚2.5 H2O) at 92% yield according to elemental analysis.
We also tried to use 2.0 equiv of CaCl2, which in theory allows
preparation of 1 in quantitative yield, but the inorganic phosphate
impurity formed co-precipitated as insoluble calcium phosphate.
Similar results were obtained when 1.5 equiv of CaCl2 was used.
As mentioned above, the best results were obtained upon
addition of 1.2 equiv of CaCl2, yielding the final compound 1,
with no co-precipitation of calcium phosphate. Compound 1 is
insoluble in water but can be easily converted to a water-soluble
(
of methanol and oxidation. 4a is purified on dry silica, thus
further chromatographic purifications are avoided. The absence
of triethyl citrate starting material (2) in the reaction mixture is
crucial for an efficient purification step. This is because of
simultaneous elution of 4a and 2 under these chromatographic
conditions. Use of 1.5 equiv of MeOPCl2 in our procedure
caused none of the above-mentioned issues, ensuring depletion
of all triethyl citrate.
We also attempted to oxidize 3 directly to the five-coordinated
phosphorus derivative 4a, but complicated mixtures resulted.
However, after treatment of 3 with methanol, 4a can be obtained
+
Na salt of PC in quantitative yield by treatment with the Dowex
(
(
17) Poshkus, A. C.; Herweh, J. E. J. Am. Chem. Soc. 1957, 79, 6127.
18) Ahlmark, M. J.; Veps a¨ l a¨ inen, J. J. Tetrahedron 1997, 53, 16153.
(
16) (a) Tew, W. P.; Mahle, C. D.; Benavides, J.; Howard, J. E.;
(19) (a) Niemi, R.; Turhanen, P.; Veps a¨ l a¨ inen, J.; Taipale, H.; J a¨ rvinen,
T. Eur. J. Pharm. Sci. 2000, 11, 173. (b) Turhanen, P. A.; Ahlgren, M. J.;
J a¨ rvinen, T.; Veps a¨ l a¨ inen, J. J. Synthesis 2001, 4, 633. (c) Turhanen, P. A.;
Veps a¨ l a¨ inen, J. J. Synthesis 2004, 7, 992. (d) He, H. W.; Wang, T.; Yuan,
J. L. J. Organomet. Chem. 2005, 690, 2608.
Lehninger, A. L. Biochemistry 1980, 19, 1983. (b) Williams, G.; Sallis, J.
D. Anal. Biochem. 1980, 102, 365. (c) Meyer, J.; Bolen, R. J.; Stakelum, I.
J. J. Am. Chem. Soc. 1959, 81, 2094. (d) Pankowski, A. E.; Meehan, J. D.;
Sallis, J. D. Tetrahedron Lett. 1994, 35, 927.
J. Org. Chem, Vol. 72, No. 4, 2007 1469

SCHEME 1. Preparation of PC (1) and Its Derivatives (3-5)a
a
Conditions: (i) 1.5 equiv of MeOPCl2, triethylamine, 24 h, rt, ca. 100%; (ii) MeOH, triethylamine, 3 h, rt then SO2Cl2, -5-0 °C and 1 h, rt, 68%; (iii)
H2O, NaHCO3, 1 h, rt, 100%; (iv) abs. EtOH, NaHCO3, 2 h, reflux, 92%; (v) (CH3)3SiBr, CH3CN, NaHCO3, 2 h, rt, then MeOH, 1 h, rt, 100%; (vi) KI,
acetone, 48 h, 60 °C, 63%; (vii) 2 M HCl, 20 h, 38-40 °C, 66%; (viii) KI, acetone, 24 h, 60 °C, 100%; (ix) 4 equiv of 1 M NaOH, 1.2 equiv of CaCl2, H2O,
2
h, 65 °C, 92%.
5
0W cation-exchange resin (H+ form) followed by partial
confirmed the aforementioned formula. 1 was converted to a water-
soluble Na salt by the following method: 1 (30 mg, 0.071 mmol)
+
neutralization with NaOH (see Experimental Section).
All compounds are identified by H, C, and 31P NMR
spectroscopy. Elemental analyses were determined for the
compounds 1 and 5b; ESI-MS spectra were recorded for the
rest of the compounds. Assignment of the 31P NMR spectra of
the compounds described herein is straightforward since only
1
13
was suspended in distilled H O (3 mL), Dowex 50W × 8 cation-
2
+
exchange resin (H -form) was added (∼300 mg), and the mixture
was stirred until no white solids were observed in the reaction
mixture (this takes a few minutes). The resin was filtered off, 1 M
NaOH (142 µL, 2 equiv) was added to the filtrate, and the reaction
mixture was further stirred for a few minutes. Then, the solution
1
3
one signal for each compound is observed. In the C NMR
spectra, signals were assigned based on 2,3JCP couplings.
Symmetrical -CH2CO2 moieties in 4c show different chemical
+
was taken to dryness in vacuo. The PC Na salt was obtained as a
white solid in quantitative yield. H, C, and P NMR spectra for
the PC Na salt were comparable to those of 1.
1
13
31
+
1
shifts due to prochirality. Due to the same reason, H NMR
Typical Preparation of 5a. 4a (600 mg, 1.54 mmol) was
dissolved in 2 M HCl (5 mL), and the reaction mixture was stirred
for 20 h at 38-40 °C (above 42 °C, the P-O-C bridge tends to
hydrolyze!) followed by evaporation to dryness in vacuo. Diethyl
ether (∼10 mL) was added to the residue, and the mixture was
stirred until a fluffy white suspension was obtained. The precipitate
spectra of all PC compounds contain a typical AB quartet.
Furthermore, in the case of 4a and 4c, the ester O-CH2 protons
are found at different chemical shifts.
In conclusion, the novel method described herein opens a
new route to prepare PC and several new partial ester derivatives
not reported earlier. The overall yield to prepare 1 (calculated
based on the composition C6H4O10PKCa2‚2.5H2O) starting from
triethyl citrate 2 is 41.3%, which is quite reasonable for a four-
step reaction procedure and is over 5 times higher than that
published earlier. Final product 1 is easily separated and
collected from the reaction mixture without further chromato-
graphic purifications in contrast to the methods reported earlier
and can be converted quantitatively to water-soluble Na salt
of PC. Availability of PC and its derivatives will allow further
and more systematic testing of these anticalcification agents in
vivo and in vitro.20 PC derivatives of various solubilities may
allow control of bioavailability of “active” PC, as has been
reported for CaNa(PC)2(H2O).20
(
5a) was isolated by filtration, dried in vacuo, and was finally
obtained as a white powder in 66% yield and 83-90% purity
containing diethyl monomethyl PC, monoethyl PC, and traces of
(
PC as impurities).
1
6b
Preparation of 5b. 5a (330 mg, 1.05 mmol) was dissolved in
acetone (10 mL), KI (oven dried, 178 mg, 1.07 mmol) was added,
and the reaction mixture was stirred for 24 h at 60 °C. The
precipitate that formed was separated by centrifugation, washed
with acetone, and dried in vacuo. 5b was obtained quantitatively
as white powder.
16
+
Preparation of 4a. Triethyl citrate (6.3 mL, 25.9 mmol) and
dry triethylamine (5.4 mL, 38.7 mmol) were dissolved in diethyl
ether (220 mL). MeOPCl
slowly to the reaction mixture, which was then stirred under nitrogen
in a glovebox) at room temperature for 24 h. The precipitate that
formed was filtered off under nitrogen, and excess MeOPCl was
removed under vacuum. The residue was redissolved in diethyl ether
∼50 mL), and a mixture of dry MeOH (1.1 mL, 27.0 mmol) and
2
(3.8 mL, 40.0 mmol) was then added
(
Experimental Section
2
Synthetic Procedures. Preparation of 1. Compound 5b (200
(
mg, 0.52 mmol) was dissolved in distilled H
NaOH (2370 µL, ∼4 equiv) was added, and reaction mixture was
stirred until a clear solution was obtained. CaCl (79 mg, 1.2 equiv)
was then added, and the reaction mixture was stirred for 2 h at
5 °C until a white precipitate appeared. The solid was filtered by
2
O (2.5 mL), 1 M
dry triethylamine (4.0 mL, 28.7 mmol) in diethyl ether (∼25 mL)
was added in portions to the reaction mixture under stirring in a
nitrogen glovebox for 3 h at room temperature. Stirring was
continued, and diethyl ether was added to dilute the reaction mixture
if it was too viscous. A precipitate formed and was removed under
a nitrogen atmosphere, and the volume was reduced to ∼70-80
mL and cooled to -5-0 °C. Then, distilled sulfuryl chloride (2.08
mL, 25.9 mmol) was added slowly and carefully. The reaction
mixture was then stirred for 1 h at room temperature. After filtration
to remove any formed solids, diethyl ether was removed from the
filtrate by evaporation. The crude product was purified by silica
column chromatography using ethyl acetate/hexane (1:1) as eluent
2
6
suction, washed with very small portions of ice-cold water, and
finally washed with a small volume of acetone. The precipitate was
dried in vacuo, and 1 was obtained as white “felting like” solid in
92% yield based on the composition C
6 4
H O10PKCa
2 2
‚2.5 H O. K
and Ca contents were quantitatively analyzed by flame AAS and
CHN and were quantified by elemental analysis. These results
(
20) (a) Demadis, K. D.; Sallis, J. D.; Raptis, R. G.; Baran, P. J. Am.
Chem. Soc. 2001, 123, 10129. (b) Demadis, K. D. Inorg. Chem. Commun.
003, 6, 527. (c) Sun, Y.; Reuben, P.; Wenger, L.; Sallis, J. D.; Demadis,
(
silica was oven-dried at 120 °C and ethyl acetate over MgSO
before use). The final product was obtained as a colorless oil in
8% yield. Compound 4a seems to be quite stable since no
degradation was observed during a 2-month storage period (without
2
N ) at -18 °C.
4
2
6
K. D.; Cheung, H. S. Front. Biosci. 2005, 10, 803. (d) Cheung, H. S.;
Sallis, J. D.; Demadis, K. D.; Wierzbicki, A. Arthritis Rheumatol. 2006,
5
4, 2452.
1
470 J. Org. Chem., Vol. 72, No. 4, 2007

Preparation of 4b. 4a (200 mg, 0.51 mmol) was dissolved in
O (1 mL), and NaHCO (43 mg, 0.51 mmol) was added to it.
The reaction mixture was then stirred for 1 h at room temperature,
and the solvent was removed in vacuo. The residue was dissolved
3-Ethoxycarbonyl-3-(ethoxymethoxyphosphoryloxy) Pen-
1
H
2
3
tanedioic Acid Diethyl Ester (4c): H NMR (CDCl
2H, J ) 7.2 Hz), 4.09-4.04 (m, 2H), 4.07 (q, 4H, J ) 7.1 Hz),
3
) δ 4.21 (q,
3
2
3.68 (d, 3H, JHP ) 11.5 Hz), 3.258 (d, 1H, JHH ) -16.21 Hz),
2
2
in chloroform (∼5 mL), dried over MgSO
4
, and subsequently
evaporated in vacuo. 4b was obtained as a colorless oil in
quantitative yield.
3.258 (d, 1H, JHH ) -16.15 Hz), 3.242 (d, 1H, JHH ) -16.15
2 4
Hz), 3.238 (d, 1H, JHH ) -16.21 Hz), 1.26 (td, 3H, JHP ) 1.0
Hz, J ) 7.1 Hz), 1.25 (t, 3H, J ) 7.2 Hz), 1.19 (t, 6H, J ) 7.1
Hz); 13C NMR (CDCl
) δ 168.2 (d, JCP ) 6.1 Hz), 167.7 (s, 2C),
3
Preparation of 4c. 4a (200 mg, 0.51 mmol) was dissolved in
3
2
2
absolute EtOH (1 mL), NaHCO
3
(44 mg, 0.52 mmol) was added
79.4 (d, JCP ) 6.5 Hz), 63.3 (td, JCP ) 6.3 Hz), 61.3 (t), 59.8 (t,
3
3
to it, and the reaction mixture was refluxed for 2 h. A residue was
obtained after evaporation of EtOH in vacuo and was then
suspended in diethyl ether. The solid was removed by filtration,
and the filtrate was evaporated to dryness in vacuo. 4c was obtained
as slightly yellow oil in 92% yield.
2C), 53.5 (qd, JCP ) 6.5 Hz), 39.96 (td, JCP ) 1.1 Hz), 39.93 (td,
3
3
3
JCP ) 1.2 Hz), 14.9 (qd, JCP ) 7.1 Hz), 13.1 (q, 2C), 12.9 (q);
1
P NMR (CDCl
3
) δ -7.89; ESI-MS 399.1 (M + 1; 100%).
3-Ethoxycarbonyl-3-phosphonooxy Pentanedioic Acid Diethyl
1
Ester (4d): H NMR (CD OD) δ 4.23 (q, 2H, J ) 7.0 Hz), 4.12
3
Preparation of 4d. 4b (177 mg, 0.48 mmol) was dissolved in
CH CN (3 mL), NaHCO (41 mg, 0.49 mmol) and trimethylsilyl
3 3
(q, 4H, J ) 7.0 Hz), 3.35 (s, 2H), 3.28 (s, 2H), 1.29 (t, 3H, J ) 7.0
Hz), 1.24 (t, 6H, J ) 7.0 Hz); 13C NMR (CD OD) δ 171.2 (d, J
3
3
CP
2
bromide (140 µL, 1.06 mmol) were added to it, and the reaction
mixture was stirred for 2 h at room temperature. A solid formed
that was filtered off, and the filtrate was evaporated to dryness in
vacuo. The residue was redissolved in MeOH (1 mL) and stirred
for 1 h at room temperature and then evaporated to dryness in vacuo.
) 6.5 Hz), 170.5 (s, 2C), 80.7 (d, J ) 5.0 Hz), 63.3 (t), 61.9 (t,
3 31
CP
2C), 42.0 (td, 2C, J ) 2.5 Hz), 14.4 (q, 2C), 14.2 (q); P NMR
CP
(CD OD) δ -4.19; ESI-MS 357.1 (M + 1; 75%).
3
3
-Ethoxycarbonyl-3-(ethoxyhydroxyphosphoryloxy) Pen-
1
tanedioic Acid Diethyl Ester Monopotassium Salt (4e): H NMR
4
d was obtained quantitatively as an orange oil.
Preparation of 4e. 4c (186 mg, 0.47 mmol) was dissolved in
(
2
D
2
O) δ 4.22 (q, 2H, J ) 7.0 Hz), 4.16-4.07 (m, 4H), 3.89 (qv,
2
2
H, J ) 7.0 Hz), 3.40 (d, 2H, JHH ) 16.5 Hz), 3.17 (d, 2H, JHH
acetone (4 mL), KI (oven-dried, 76 mg, 0.46 mmol) was added to
it, and the reaction mixture was stirred for 48 h at 60 °C. It was
then evaporated to dryness in vacuo, diethyl ether was added, and
the resulting suspension was stirred for 15 min at room temperature.
After centrifugation, the solids were separated and dried in vacuo.
e was obtained as a slightly yellow amorphous solid in 63% yield
and 94% purity (contained triethyl monomethyl PC monopotassium
salt as impurity).
13
)
16.5 Hz), 1.27 (t, 3H, J ) 7.0 Hz); C NMR (D
2
O) δ 172.8 (d,
3
2
J
CP ) 9.7 Hz), 171.3 (s, 2C), 78.7 (d, JCP ) 6.3 Hz), 63.0 (t),
2
3
6
1
2.6 (td, JCP ) 5.9 Hz), 61.8 (t, 2C), 42.3 (td, 2C, JCP ) 2.3 Hz),
3
3
1
3
6.8 (qd, JCP ) 7.9 Hz), 14.4 (q, 2C), 14.3 (q); P NMR (CD -
OD) δ -3.51; ESI-MS 383.4 (M; 18%).
4
3
-Ethoxycarbonyl-3-(hydroxymethoxyphosphoryloxy) Pen-
1
tanedioic Acid (5a): H NMR (D O) δ 4.27 (q, 2H, J ) 7.2 Hz),
2
3
2
3
(
.55 (d, 3H, JHP ) 11.1 Hz), 3.33 (d, 2H, JHH ) 16.4 Hz), 3.20
Characterization of Compounds. 3-Carboxy-3-phosphonooxy
2
1
3
d, 2H, JHH ) 16.4 Hz), 1.28 (t, 3H, J ) 7.2 Hz); C NMR (D
2
O)
1
Pentanedioic Acid, Phosphocitrate (1): H NMR (D
2
O + 1 drop
3
2
δ 175.65 (d, JCP ) 10.9 Hz), 175.61 (s, 2C), 81.0 (d, JCP ) 6.1
2
2
of 6 M DCl) δ 3.33 (d, 2H, JHH ) 16.5 Hz), 3.18 (d, 2H, JHH
)
3
3
Hz), 66.3 (t), 56.1 (qd, JCP ) 6.1 Hz), 44.8 (td, 2C, JCP ) 1.7
3
1
6.5 Hz); 13C NMR (D
11.2 Hz), 175.6 (s, 2C), 81.2 (d, JCP ) 6.3 Hz), 44.9 (t, 2C);
1
2
O + 1 drop of 6 M DCl) δ 178.0 (d, JCP
31
Hz), 16.0 (q); P NMR (D O) δ -3.53; mp 146-148 °C; ESI-
2
2
)
MS 315.2 (M + 1; 22%).
3
P NMR (D
10PKCa
.10. Mp > 310 °C (did not melt at 310 °C, so the exact mp could
not be reasonably identified).
-(Chloromethoxyphosphoryloxy)-3-ethoxycarbonyl Pen-
2
O + 1 drop of 6 M DCl) δ -4.91. Anal. Calcd for
3-Ethoxycarbonyl-3-phosphonooxy Pentanedioic Acid Mo-
C
6
H
4
O
2
‚2.5 H O: C, 16.71; H, 2.10. Found: C, 17.04; H,
2
nopotassium Salt, Phosphocitrate Monoethyl Ester Monopo-
2
1
tassium Salt (5b): H NMR (D O) δ 4.26 (q, 2H, J ) 7.2 Hz),
2
2
2
3
.35 (d, 2H, JHH ) 16.2 Hz), 3.17 (d, 2H, JHH ) 16.2 Hz), 1.28
13
3
(
t, 3H, J ) 7.2 Hz); C NMR (D
2
O) δ 176.1 (s, 2C), 175.8 (d,
1
tanedioic Acid Diethyl Ester (4a): H NMR (CDCl
3
) δ 4.31 (q,
3
2
J
J
CP ) 10.6 Hz), 80.7 (d, JCP ) 5.9 Hz), 66.3 (t), 45.0 (td, 2C,
2
H, J ) 7.0 Hz), 4.17 (q, 2H, J ) 7.0 Hz), 4.16 (q, 2H, J ) 7.0
3
31
CP ) 1.9 Hz), 16.0 (q); P NMR (D
2
O) δ -4.21. Anal. Calcd
3
2
Hz), 3.90 (d, 3H, JHP ) 14.0 Hz), 3.37 (d, 1H, JHH ) -16.1 Hz),
8 12
for C H O
3 2
10PK‚0.5(CH )
CO: C, 31.07; H, 4.12. Found: C, 30.98;
2
3
.33 (s, 2H), 3.30 (d, 1H, JHH ) -16.1 Hz), 1.33 (t, 3H, J ) 7.0
H, 4.15. Mp 242-244 °C (at 147 °C, a gas boiled off, most likely
13
Hz), 1.273 (t, 3H, J ) 7.0 Hz), 1.268 (t, 3H, J ) 7.0 Hz);
C
acetone).
3
NMR (CDCl
3
) δ 168.4 (d, JCP ) 5.2 Hz), 168.2 (s), 168.1 (s),
2
3
8
7
2
1
4.0 (d, JCP ) 8.8 Hz), 62.9 (t), 61.2 (t), 61.1 (t), 56.0 (qd, JCP )
Acknowledgment. We would like to thank Mrs. Maritta
Salminkoski for expert technical assistance, Mrs. Tiina Koivunen
for performing the elemental analyses, Mrs. Helena Veps a¨ l a¨ inen
for the AAS analyses, and Mrs. Miia Reponen for the ESI-MS
analyses.
3
3
.8 Hz), 41.3 (td, JCP ) 5.0 Hz), 41.1 (td, JCP ) 3.9 Hz), 14.1 (q,
C), 13.9 (q); 31P NMR (D
00%).
O) δ -1.16; ESI-MS 389.0 (M + 1;
2
3
-Ethoxycarbonyl-3-(hydroxymethoxyphosphoryloxy) Pen-
1
tanedioic Acid Diethyl Ester (4b): H NMR (CDCl
3
) δ 4.27 (q,
3
2
1
H, J ) 7.0 Hz), 4.15 (q, 4H, J ) 7.0 Hz), 3.75 (d, 3H, JHP )
Supporting Information Available: 1H, 13C, and P NMR
31
2
2
1.5 Hz), 3.30 (d, 2H, JHH ) -15.9 Hz), 3.29 (d, 2H, JHH ) -15.9
1
13
spectra of key intermediate 4a and final compound 1, H NMR
Hz), 1.31 (t, 3H, J ) 7.0 Hz), 1.25 (t, 6H, J ) 7.0 Hz); C NMR
3
2
spectra of compounds 4b-e and 5a,b. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
6
2
CDCl
3
) δ 169.3 (d, JCP ) 5.8 Hz), 168.9 (s, 2C), 80.5 (d, JCP
)
3
.9 Hz), 62.5 (t), 61.0 (t, 2C), 54.4 (qd, JCP ) 6.2 Hz), 40.9 (td,
C, JCP ) 3.8 Hz), 14.1 (q, 2C), 13.9 (q); P NMR (CDCl
5.86; ESI-MS 371.0 (M + 1; 100%).
3
31
3
) δ
-
JO061709C
J. Org. Chem, Vol. 72, No. 4, 2007 1471