Asymmetric synthesis of a 3-acyltetronic acid derivative, RK-682, and formation of its calcium salt during silica gel column chromatography

Article abstract of DOI:10.1248/cpb.49.206

RK-682 was reported to be a potent protein tyrosine phosphatase inhibitor. We found that (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (I) was easily converted to its calcium salt during column chromatography on Silica gel 60, and this calcium salt was identical to RK-682 originally isolated from a natural source. Here we report details of the asymmetric synthesis of (R)-I and its conversion to the calcium salt. Fast atom bombardment mass spectrometric (FAB-MS) analysis of the free and calcium salt forms of RK-682 is also reported.

Full text of DOI:10.1248/cpb.49.206

206
Chem. Pharm. Bull. 49(2) 206—212 (2001)
Vol. 49, No. 2
Asymmetric Synthesis of a 3-Acyltetronic Acid Derivative, RK-682, and
Formation of Its Calcium Salt during Silica Gel Column Chromatography
Mikiko SODEOKA, ,a,b,c Ruriko SAMPE,b Sachiko KOJIMA,b Yoshiyasu BABA,a,b Naoko MORISAKI,c and
*
c
Yuichi HASHIMOTO
Institute for Chemical Reaction Science, Tohoku University,a Sendai, Miyagi 980–8577, Japan, Sagami Chemical Research
Center,b Nishi-Ohnuma, Sagamihara, Kanagawa 229–0012, Japan, and Institute of Molecular & Cellular Biosciences,
University of Tokyo,c Bunkyo-ku, Tokyo 113–0032, Japan. Received September 20, 2000; accepted December 4, 2000
RK-682 was reported to be a potent protein tyrosine phosphatase inhibitor. We found that (R)-3-hexade-
canoyl-5-hydroxymethyltetronic acid (1) was easily converted to its calcium salt during column chromatography
on Silica gel 60, and this calcium salt was identical to RK-682 originally isolated from a natural source. Here we
report details of the asymmetric synthesis of (R)-1 and its conversion to the calcium salt. Fast atom bombard-
ment mass spectrometric (FAB-MS) analysis of the free and calcium salt forms of RK-682 is also reported.
Key words RK-682; tetronic acid derivative; silica gel; calcium salt; FAB-MS
RK-682 was isolated by a RIKEN group from Strepto- paper, we report structural analysis of this less-soluble com-
myces sp. 88-682 and found to have interesting biological ac- pound, as well as details of the asymmetric synthesis of (R)-
tivities.1) These activities may be due to blocking of protein 1.
phosphorylation, since RK-682 inhibits protein tyrosine
phosphatases (CD45, VHR). The chemical structure of RK- Results and Discussion
682 was assigned as 3-hexadecanoyl-5-hydroxymethyl-
Asymmetric Synthesis of (R)-1 For efficient synthesis
tetronic acid (1) at that stage.1a,b) The same structure had al- of optically pure (R)-1, the commercially available optically
ready been assigned by a Takeda group to a compound iso- active glyceric acid derivative (R)-2 was chosen as a starting
lated from cultures of Streptomyces sp. AL-462 that inhibits material,6) and synthesis was achieved as shown in Fig. 3.
the activity of phospholipase A2.2) The Takeda group also re- Specifically, (R)-2 was first converted to the hydroxyester
ported a synthesis of (ϩ)-1 from D-ribose, and the structure (R)-3 via deprotection of the acetonide group and selective
of their compound, including the absolute stereochemistry, protection of the primary alcohol with a trityl group. The b-
was confirmed to be (R)-1. However, the reported spectral ketoester group was introduced into (R)-3 by silver salt-pro-
data, including optical rotation and melting point, for RK- moted condensation with the thioester 6 to give (R)-7 in 74%
682 were not identical with those of Takeda’s compound. To
clarify this structural problem of RK-682, we have developed
a versatile synthetic route to optically active 1, and in our
preliminary communication, we briefly reported the asym-
metric synthesis of (R)- and (S)-1.3,4)
Since 3-acyltetronic acid is known to exist in two sets of
tautomeric forms (i/ii and iii/iv in Fig. 2),5) we first suspected
Fig. 1
that such tautomeric properties may be the origin of the dif-
ference of the spectral data between RK-682 and (R)-1. How-
ever, this was not the case, and we found that RK-682 was
distinct from (R)-1. Acid treatment of RK-682 gave (R)-1,
and, interestingly, Silica gel column chromatography of (R)-1
afforded an unknown less-soluble compound whose spectral
data were identical to those reported for RK-682. In this
Fig. 2
(a) 1 N aq. HCl, MeOH, 23 °C; (b) Ph3CCl, NEt3, cat. DMAP, 23 °C, 69% (2 steps); (c) CDI, THF, 23 °C, then 5, 93%; (d) CF3COOAg, 6, THF, 23 °C, 74%; (e) Bu4NF, THF,
23 °C, 95%; (f) 1 N aq. HCl, MeOH, 75%.
Fig. 3
To whom correspondence should be addressed. e-mail: sodeoka@icrs.tohoku.ac.jp
© 2001 Pharmaceutical Society of Japan

February 2001
207
Fig. 4
yield. The thioester 6 was synthesized from hexadecanoic
acid (4) by using the magnesium salt 5.7) Construction of the
key 3-acyltetronic acid skeleton without racemization was
achieved under the conditions reported by Ley and co-work-
ers.8) Cyclization of the b-ketoester (R)-7 proceeded
smoothly with tetrabutylammonium fluoride to give the de-
sired 3-acyltetronic acid (R)-8 in 95% yield. Deprotection of
the trityl group using 1 N HCl afforded (R)-1 in 75% yield.
Fig. 5
The overall yield of the last three steps was improved to 75% trum, IR spectrum and melting point of (R)-9 were similar to
by omitting the chromatographic purification of 7 and 8. All those of RK-682, but those of (R)-10 were distinct. Sec-
spectral data, including 1H-NMR in dimethyl sulfoxide ondary ion mass spectrometry (SI-MS) of (R)-9, however,
(DMSO)-d6, the optical rotation, and the melting point of clearly eliminated the possibility of RK-682 being a sodium
synthetic (R)-1, were identical to those reported by the salt. SI-MS of (R)-9 showed molecular-related ion peaks at
Takeda group.2)
m/z 391 (MϩHϩ) and 413 (MϩNaϩ), but RK-682 showed
Structural Analysis of RK-682 As shown in Fig. 2, 3- only a low intensity peak at m/z 377. This clearly indicated
acyltetronic acid is known to exist in two sets of tautomeric that RK-682 is not a Na salt. It appeared that RK-682 might
1
forms (i/ii, iii/iv).5) In fact, the H-NMR spectrum of 3- be some other metal salt.
acetyltetronic acid in CDCl3 was reported to show two sets of
Conversion of (R)-1 to Its Calcium Salt 11 by Silica Gel
peaks corresponding to the two tautomers (i/ii, iii/iv). Saito Chromatography In the course of these studies, we found
and Yamaguchi have suggested that 3-acetyltetronic acid ex- that (R)-1 was converted to a new compound 11, which is
ists as the i/ii form in methanol-d4, but as the iii/iv form in less soluble in organic solvent, during column chromatogra-
1
DMSO-d6.5b) Since the H-NMR spectrum reported by the phy on Silica gel 60 (Merck) (MeOH–CHCl3). The spectral
Takeda group was measured in DMSO-d6, and that reported data and melting point of this less-soluble compound 11 were
by the RIKEN group was measured in methanol-d4, we first found to be identical to those of RK-682. In addition, (R)-1
1
measured the H-NMR spectrum of the synthetic (R)-1 in was recovered by the treatment of 11 with 0.5 N aqueous hy-
1
methanol-d4 for comparison. But the H-NMR spectrum of drochloric acid as well as the case of RK-682. Since Silica
this synthetic (R)-1 in methanol-d4 was not identical to that gel column chromatography (MeOH–CHCl3) was employed
previously reported for RK-682,1a) indicating that the two in the isolation of RK-682, we concluded that RK-682 was
samples are distinct compounds.
identical to this less-soluble compound 11.13)
Next, we found that treatment of the original RIKEN sam-
To identify the unknown element in 11, we next carried
ple of RK-6829) with 0.5 N aqueous hydrochloric acid af- out X-ray fluorescence analysis of 11. Since 11 was obtained
forded a compound whose spectral data, including optical ro- upon Silica gel column chromatography, we first suspected
tation, were identical with those of synthetic (R)-1. This sug- that RK-682 might be a silicon complex; however, the con-
gested that RK-682 was a metal salt. Indeed, 3-acyltetronic tent of silicon was less than 0.5 wt%. The data indicated that
acids are known to be extremely acidic (pKa values of 3- a significant amount of calcium (ca. 3.8 wt%) was contained
acetyltetronic acid and 3-acetyl-5-methyltetronic acid were in 11, in addition to carbon (ca. 65 wt%) and oxygen (ca.
reported to be 0.8 and 0.5, respectively),5a) and a Ciba-Geigy 29 wt%). No potassium was detected, and the contents of
group reported isolation of the sodium salt of 1 from Actino- other atoms (Na, Mg, Al, S, Cl) were less than 1%. Quantita-
mycete strain DSM7357.10) It is also known that the antibiotic tive analysis of 11 by ICP-AES (inductively coupled plasma
magnesidin, a structurally related tetramic acid derivative, is atomic emission spectroscopy) gave a calcium content of
a magnesium salt.11) Another tetramic acid derivative, tenua- 3.4 wt%. Thus, we presumed that 11 is mainly a calcium salt
zoic acid, was reported to form complexes with several metal of (R)-1. Analysis data supplied by Merck suggested that Sil-
ions.12) Therefore, to examine the possibility that 1 could be ica gel 60 contains 0.11 wt% of calcium. It is surprising that
isolated as a salt, the sodium salt (R)-9 and magnesium salt 1 can react efficiently with such a small amount of calcium
(R)-10 were prepared by the treatment of (R)-1 with 1 eq of contained in the Silica gel 60 to give 11.14) The contents of
sodium methoxide in methanol or with 1/2 mol eq of magne- ions are variable from sample to sample, depending on the
1
sium ethoxide in tetrahydrofuran (THF). The H-NMR spec- amount and lot of the Silica gel used, and no salt formation

208
Vol. 49, No. 2
Fig. 6. a, b Positive and Negative Ion FAB-MS of 1 [Matrix: Glycerol, m-Nitrobenzyl Alcohol (2 : 1)]
c, d Positive and Negative Ion MS of 11 [Matrix: (c) Dithiothreitol, a-Thioglycerol (1 : 1),15) (d) Triethanolamine].
was observed when spherical Silica gel 60 was used.
formed from the cation B by elimination of formaldehyde.
Finally, the structure of 11 was confirmed by detailed Since no significant peak at m/z 339, corresponding to the
analysis of fast atom bombardment mass spectra (FAB-MS). elimination of formaldehyde from protonated 1, was ob-
Figure 6 shows positive and negative ion FAB-MS of 1 and served in the spectrum of 1 (Fig. 6a), this elimination is pre-
11. A strong (MϩH)ϩ peak at m/z 369 and (MϪH)Ϫ peak at sumably enhanced by the calcium salt formation. The struc-
367 were observed in the spectra of 1 (Figs. 6a, b). In con- ture of the cation D (Calcd for C18H31O2Ca: 319.1950,
trast, no significant peak at m/z 369 was observed in the Found: 319.1960) is unclear, but a possible candidate is ex-
spectrum of 11 (Fig. 6c). FAB-mass spectral patterns of 11 pected to be formed by elimination of the C2H2O2 fragment
(Figs. 6c, d) were completely different from those of 1. Peaks from C. The cation F (Calcd for C40H65O8Ca2: 753.3931,
observed in the FAB-MS of 11 were assigned on the basis of Found: 753.3991) would be formed by double elimination of
HR-FAB-MS data and collisionally activated dissociation formaldehyde from A and further association of Ca2ϩ. Figure
(CAD) spectra as shown in Fig. 7.16) Dissociation of proto- 8 shows CAD spectra of the ions A (m/z 775) and C (m/z
nated 11 (ion A, Calcd for C42H71O10Ca: 775.4673, Found: 377). The cation A gave the ions E, B and C, and the cation
775.4681) would give the cation B at m/z 407. Association of C gave the ion D. Fragmentation of the ion B to C was
cation B to 11 affords the cation G (Calcd for C63H105O15Ca2: shown by the metastable ion at m/z 349.2 in the spectrum of
1181.6705, Found: 1181.6709). It is likely that the cation C Fig. 6c. Anions at m/z 367, 337, 279, 713 and 1141 are for-
(Calcd for C20H33O4Ca: 377.2005, Found: 377.2029) is mally corresponding to the cations B, C, D, F and G minus

February 2001
209
Fig. 7. Assignment of the Peaks Observed in FAB-MS
Only one structure is shown for each ion, although several tautomeric forms are possible.
Ca2ϩ, respectively. Although no large molecular-related ion
peak, which would be helpful to identify the counter cation,
was observed in the FAB-MS of 11, the characteristic 40
atom mass unit difference observed in the positive and nega-
tive mass spectra (m/z 1181 vs. 1141, 753 vs. 713, 407 vs.
367, 377 vs. 337, and 319 vs. 279) suggested involvement of
Ca2ϩ in the structure. Thus, comparison of the positive and
negative ion mass spectra is useful for analyzing the structure
of divalent cation salts.
of 1 and 11. Peaks were assigned using the heteronuclear
multipe quantum coherence (HMQC) and heteronuclear mul-
tiple bond correlation (HMBC) techniques. Two types of
chelate, 11a and 11b, are expected to exist for the calcium
salt. Since large chemical shift changes were observed for
the carbonyl carbons at positions 2 and 7, 11a might be the
predominant form in methanol-d4.
In summary we have efficiently synthesized (R)-1, and
found that it is easily converted to its calcium salt 11 during
Silica gel column chromatography. RK-682 originally iso-
lated from a natural source was found to be identical to this
calcium salt 11. This unusual property of 1 suggests that care
must be taken in handling a wide variety of 3-acyltetronic
acid derivatives, which are frequently found as components
of biologically active natural products.17) It is noteworthy that
the acid-treated form, (R)-1, is now supplied as “RK-682”
1
The H-NMR spectra of 1 and 11 in methanol-d4 show
only one set of peaks. The proton at the C5 position of 11
(4.40 ppm) shows an upfield shift compared to that of 1
(4.73 ppm). The 1H-NMR spectrum of a mixture of 1 and 11
shows only one set of peaks with intermediate chemical shift,
suggesting a rapid equilibrium between 1 and 11 in
methanol. Table 1 shows the 13C-NMR chemical shift values

210
Vol. 49, No. 2
Fig. 8. CAD Spectra of a) Ion A at m/z 775 and b) Ion C at m/z 377 from the Spectrum of 11 in Fig. 3c
residue was neutralized with saturated aqueous NaHCO3. The mixture was
successively extracted with AcOEt containing 10% iso-PrOH. The organic
layers were combined, dried over Na2SO4, and concentrated to give crude
methyl (R)-2,3-dihydroxypropionate (1.39 g, 72%) as a colorless oil. 1H-
NMR (CDCl3, 200 MHz) d: 2.67 (2H, br s), 3.85 (3H, s), 3.87 (2H, m), 4.30
(1H, t, 3.5 Hz).
To a solution of the crude methyl (R)-2,3-dihydroxypropionate (1.39 g,
11.6 mmol) in CH2Cl2 (60 ml) were added triphenylchloromethane (4.20 g,
15.1 mmol), triethylamine (2.3 ml, 16.5 mmol), and N,N-dimethylaminopyri-
dine (DMAP) (99.4 mg, 0.814 mmol), and the mixture was stirred at 23 °C
for 24 h. The reaction mixture was poured into ice-water and extracted with
CH2Cl2. The organic layer was washed with brine, dried (Na2SO4), and con-
centrated. The residue was purified by Silica gel column chromatography
(CH2Cl2) to give (R)-3 (3.99 g, 95%) as a colorless solid.
Table 1. 13C-NMR Chemical Shifts of 1 and 11
1
11
2-C
3-C
4-C
5-C
6-C
7-C
174.59
100.79
196.10
83.69
61.63
196.48
178.63
99.19
196.69
83.18
62.65
198.96
1H-NMR (CDCl3, 200 MHz) d: 3.16 (1H, d, Jϭ7.7 Hz), 3.35 (1H, dd, Jϭ
9.5, 3.4 Hz), 3.48 (1H, dd, Jϭ9.5, 3.0 Hz), 3.77 (3H, s), 4.25 (1H, ddd, Jϭ
7.7, 3.4, 3.0 Hz), 7.10—7.50 (15H, m). IR (neat) cmϪ1: 3520, 1745, 1450,
1230, 1125. EI-MS m/z: 362 (Mϩ), 285 (MϩϪOH, COOCH3), 243 (Ph3Cϩ).
HR-MS (Mϩ) Calcd for C23H22O4: 362.1516, Found 362.1515. [a]2D0 Ϫ6.93°
(cϭ1.36, CH3OH : CHCl3ϭ1 : 4); mp 88—90 °C.
S-tert-Butyl 3-Oxo-octadecanethioate (6) To a solution of [(tert-
butylthio)carbonyl]acetic acid18) (2.76 g, 15.6 mmol) in THF (45 ml) was
added magnesium ethoxide (897 mg, 7.84 mmol), and the mixture was
stirred at 23 °C for 24 h. Evaporation of the solvent afforded its magnesium
salt 5 (3.16 g, quant.) as a colorless solid. To a solution of palmitic acid (4)
(100 mg, 0.39 mmol) in THF (4 ml) was added 1,1Ј-carbonyldiimidazole
(CDI) (103 mg, 0.78 mmol), and the mixture was stirred at 23 °C for 6 h.
After addition of the magnesium salt (267 mg, 0.78 mmol), the mixture was
Fig. 9
for biological research use.13)
Experimental
General Methods Infrared (IR) spectra were measured on an FT/IR- stirred at 23 °C for 1.5 h, then the reaction mixture was quenched by the ad-
5300 spectrometer. 1H- and 13C-NMR spectra were recorded with a Brucker dition of saturated aqueous NH4Cl and extracted with AcOEt. The organic
AM-400, AC-200P, or AVANCE 500 NMR spectrometer with tetramethylsi- layer was washed with saturated aqueous NH4Cl and brine, dried (Na2SO4),
lane used as an internal standard. Electron ionization mass spectra (EI-MS) and concentrated. The residue was purified by Silica gel column chromatog-
and chemical ionization mass spectra (CI-MS) were obtained with a Hitachi raphy (hexane : AcOEt, 50 : 1) to give 6 (134 mg, 93%) as a colorless oil
M-80B mass spectrometer. SI-MS were obtained with a Hitachi M-80A
1H-NMR (CDCl3, 200 MHz) d: 0.88 (3H, m), 1.25 (24H, m), 1.48 (9H, s),
mass spectrometer. FAB-MS were measured on a JEOL JMS-HX110 dou- 1.61 (2H, m), 2.53 (2H, t, Jϭ7.2 Hz), 3.56 (2H, s). IR (neat) cmϪ1: 2930,
ble-focusing mass spectrometer of EBE arrangement. The ion acceleration 1725, 1680, 1620. EIMS m/z: 314 (MϩϪtert BuϩH), 281 (MϩϪStBu), 239
voltage was 10 kV, and xenon gas was accelerated at a voltage of 6 kV. CAD (MϩϪCH2COS tert Bu). CI-MS (isobutane) m/z: 371 (MϩϩH). HRMS
spectra were obtained with helium as the collision gas. Ultraviolet (UV) (Mϩ) Calcd for C22H42O2S: 370.2902, Found 370.2879.
spectra were measured on a Hitachi U-3210 spectrophotometer. Optical rota-
Methyl (R)-2-(3-Oxo-octadecanoyl)oxy-3-triphenylmethyloxypropio-
tion was measured on a Horiba SEPA-200 polarimeter. In general, reactions nate (7) To a solution of 6 (570 mg, 1.54 mmol) and (R)-3 (557 mg,
carried out under anhydrous conditions utilized dry solvents under an argon 1.54 mmol) in THF (12 ml) was added silver trifluoroacetate (0.41 g,
atmosphere.
1.84 mmol), and the mixture was stirred overnight at 23 °C while shielded
Methyl (R)-2-Hydroxy-3-triphenylmethyloxypropionate (3) To a so- from light. The mixture was diluted with ether, passed through a short Silica
lution of methyl (R)-2,2-dimethyl-1,3-dioxolane-4-carboxylate 2 (2.57 g, gel column, and concentrated. The residue was purified by Silica gel column
16.0 mmol) in methanol (40 ml) was added 1 N aqueous HCl (20 ml), and the
chromatography (hexane : AcOEt, 10 : 1) to give 7 (728 mg, 74%, a 6 : 1 mix-
mixture was stirred at 23 °C for 4 h. After the evaporation of methanol, the ture of keto and enol forms) as a colorless oil.

February 2001
211
1H-NMR (CDCl3, 400 MHz) d: 0.88 (3H, t, Jϭ6.7 Hz), 1.25 (24H, m),
1.52 (2H, m), 2.24 (2/7H, t, Jϭ7.5 Hz), 2.62 (6/7H, dt, Jϭ17.5, 7.3 Hz),
2.63 (6/7H, dt, Jϭ17.5, 7.3 Hz), 3.46—3.56 (2H, m), 3.55 (6/7H, d, Jϭ
15.4 Hz), 3.59 (6/7H, d, Jϭ15.4 Hz), 3.74 (18/7H, s), 3.75 (3/7H, s), 5.20
(1/7H, s), 5.26 (6/7H, dd, Jϭ4.6, 3.5 Hz), 5.30 (1/7H, t, Jϭ4.1 Hz), 7.22—
7.33 (9H, m), 7.38—7.44 (6H, m), 11.76 (1/7H, s). IR (neat) cmϪ1: 2930,
1750, 1720, 1220, 1100. EI-MS m/z: 362 (MϩϪCOCH2CO(CH2)14CH3ϩH),
285, 259 (Ph3COϩ), 243 (Ph3Cϩ). CI-MS (isobutane) m/z: 642 (Mϩ), 643
(MϩϩH). HR-MS (MϩϪC18H32O2) Calcd for C23H22O4: 362.1516, Found
362.1543.
(R)-3-Hexadecanoyl-5-(triphenylmethyloxymethyl)tetronic Acid (8)
To a solution of 7 (436 mg, 0.68 mmol) in THF (2.2 ml) was added tetrabutyl-
ammonium fluoride (1 M THF solution, 0.88 ml, 0.88 mmol), and the mixture
was stirred at 23 °C for 2 h. After the addition of tetrabutylammonium fluo-
ride (0.14 ml, 0.14 mmol), the mixture was further stirred overnight at 23 °C.
The reaction was quenched by the addition of 6 N aqueous HCl (0.17 ml,
1.03 mmol) and the mixture was poured into ice-water; the whole was then
extracted with ether. The organic layer was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by Silica gel column
chromatography (CHCl3 : MeOH, 1 : 0—20 : 1). The combined and concen-
trated fractions containing the desired product were redissolved in AcOEt
(50 ml), and the solution was washed with 0.5 N aqueous HCl and water,
dried (Na2SO4), and concentrated to give 8 (395 mg, 95%) as a pale yellow
oil.
centrated to give (R)-1 (967 mg, 75% in 3 steps) as a colorless solid.
(R)-3-Hexadecanoyl-5-hydroxymethyltetronic Acid Sodium Salt (9)
To a solution of (R)-1 (26.3 mg, 0.071 mmol) in methanol (2.5 ml) was
added sodium methoxide (1 M MeOH solution, 71.3 ml, 0.071 mmol) at 0 °C.
The mixture was stirred at 23 °C for 1 h, and the solvent was removed in
vacuo to give the sodium salt (R)-9 (27.5 mg, quant) as a colorless solid.
1H-NMR (methanol-d4, 400 MHz) d: 0.90 (3H, t, Jϭ6.9 Hz), 1.28 (24H,
m), 1.56 (2H, m), 2.74 (2H, m), 3.73 (1H, dd, Jϭ12.3, 5.0 Hz), 3.88 (1H, dd,
Jϭ12.3, 2.8 Hz), 4.30 (1H, dd, Jϭ5.0, 2.8 Hz). IR (neat) cmϪ1: 3400, 2920,
2850, 1720, 1645, 1570, 1460, 1030. SI-MS (m-nitrobenzyl alcohol) m/z:
413 (MϩNaϩ), 391 (MϩHϩ), 176, 136, 73, 43, 41 23 (Naϩ, bp). UV lmax
(MeOH) nm (e): 233 (13821), 265 (17343). [a]2D0 ϩ55.7° (cϭ0.42, CHCl3);
mp 220—230 °C (dec.).
(R)-3-Hexadecanoyl-5-hydroxymethyltetronic Acid Magnesium Salt
(10) To a solution of (R)-1 (17.4 mg, 0.047 mmol) in THF (2.0 ml) was
added magnesium ethoxide (2.7 mg 0.024 mmol) at 0 °C. The mixture was
stirred at 23°C for 4 h, and the solvent was removed in vacuo to give the
magnesium salt (R)-10 (19.9 mg, quant) as a colorless solid. 1H-NMR
(methanol-d4, 400 MHz) d: 0.90 (6H, t, Jϭ6.9 Hz), 1.29 (48H, m), 1.64 (4H,
m), 2.86 (4H, m), 3.87 (2H, dd, Jϭ12.6, 3.3 Hz), 3.94 (2H, dd, Jϭ12.6,
2.7 Hz), 4.72 (2H, m). IR (neat) cmϪ1: 3395, 2925, 2855, 1735, 1635, 1565,
1530, 1500, 1475, 1340, 1080, 1040. SI-MS (m-nitrobenzyl alcoholϩNaCl)
m/z: 781 (MϩNaϩ), 759 (MϩHϩ). [a]2D0 ϩ161.3° (cϭ0.19, CHCl3); mp
245—260 °C (dec.).
1H-NMR (DMSO-d6, 400 MHz) d: 0.84 (3H, t, Jϭ6.7 Hz), 1.21 (24H, m),
1.54 (2H, br tt, Jϭ7.3, 7.3 Hz), 2.78 (2H, m), 3.20 (1H, dd, Jϭ10.3, 3.7 Hz),
3.34 (1H, dd, Jϭ10.3, 2.4 Hz), 4.73 (1H, m), 7.24—7.34 (15H, m). IR (neat)
cmϪ1: 3450, 2925, 2860, 1775, 1700, 1610, 710. EI-MS m/z: 350 (MϩϪ
Ph3COH), 243 (Ph3Cϩ). CI-MS (isobutane) m/z: 610 (Mϩ), 611 (MϩϩH).
HR-MS (MϩϪC19H16O) Calcd for C21H34O4: 350.2455, Found 350.2454.
[a]2D0 ϩ48.27° (cϭ1.02, CHCl3).
(R)-3-Hexadecanoyl-5-hydroxymethyltetronic Acid Calcium Salt (11)
A solution of (R)-1 (50.4 mg, 0.137 mmol) in CHCl3 : MeOH (5 : 1) was
loaded onto a Silica gel column (Merck, Silica gel 60, particle size 0.040—
0.063 mm, cat. # 109385, 5.0 g), and eluted with the same solvent. Removal
of the solvent of the combined fractions afforded the calcium salt 11
(52.8 mg) as a colorless solid. No salt formation was observed when spheri-
cal Silica gel 60 (Kanto Chemical, particle size 0.04—0.050 mm, cat. #
37562) was used.
(R)-3-Hexadecanoyl-5-hydroxymethyltetronic Acid (1) To a solution
of 8 (315 mg, 0.52 mmol) in methanol (30 ml) was added 1 N aqueous HCl
(0.52 ml, 0.52 mmol), and the mixture was stirred at 23 °C for 48 h. After re-
moval of the solvent in vacuo, the residue was purified by Silica gel column
chromatography (CHCl3 : MeOH, 1 : 0—20 : 1—10 : 1). The combined and
concentrated fractions containing the desired product were redissolved in
AcOEt (100 ml), washed with 0.5 N aqueous HCl and water, dried (Na2SO4),
and concentrated to give 1 (143 mg, 75%) as a colorless solid.
1H-NMR (methanol-d4, 400 MHz) d: 0.90 (3H, t, Jϭ6.9 Hz), 1.25—1.50
(24H, m), 1.60 (2H, br tt, Jϭ7.6, 7.6 Hz), 2.79 (2H, t, Jϭ7.6 Hz), 3.81 (1H,
dd, Jϭ12.3, 4.2 Hz), 3.90 (1H, dd, Jϭ12.3, 2.7 Hz), 4.40 (1H, dd, Jϭ4.2,
2.7 Hz). 13C-NMR (methanol-d4, 125 MHz, 30 °C) d: 14.46 (22-CH3), 23.75
(21-CH2), 26.25 (9-CH2), 30.50, 30.77, 30.80, 30.84, 30.86 (10—19-CH2),
33.10 (20-CH2), 40.68 (8-CH2), 62.65 (6-CH2O), 83.18 (5-CH), 99.19 (3-C),
178.63 (2-CϭO), 196.69 (4-CϭO), 198.96 (7-CϭO). IR (neat) cmϪ1: 3400,
2925, 2855, 1730, 1635, 1560, 1470, 1040—1100 (broad). Elemental Analy-
sis Found C 62.51, H 9.32 (residual material 7.61%).19) mp 180—200 °C
(dec). The [a]D value was unstable due to the low solubility of 11.
1H-NMR (DMSO-d6, 400 MHz) d: 0.85 (3H, t, Jϭ6.8 Hz), 1.23 (24H, m),
1.49 (2H, m), 2.73 (2H, t, Jϭ7.4 Hz), 3.66 (1H, dd, Jϭ12.3, 3.6 Hz), 3.74
1
(1H, dd, Jϭ12.3, 2.6 Hz), 4.66 (1H, m). H-NMR (methanol-d4, 500 MHz)
d: 0.90 (3H, t, Jϭ6.9 Hz), 1.22—1.46 (24H, m), 1.65 (2H, br tt, Jϭ7.5, 7.5
Hz), 2.85 (2H, t, Jϭ7.5 Hz), 3.86 (1H, dd, Jϭ12.6, 3.5 Hz), 3.94 (1H, dd,
Jϭ12.6, 2.7 Hz), 4.68 (1H, br t, Jϭ2.9 Hz). 13C-NMR (methanol-d4,
125 MHz, 33 °C) d: 14.45 (22-CH3), 23.74 (21-CH2), 26.49 (9-CH2), 30.46,
30.60, 30.73, 30.76, 30.78 (10—19-CH2), 33.09 (20-CH2), 37.51 (8-CH2),
61.63 (6-CH2O), 83.69 (5-CH), 100.79 (3-C), 174.59 (2-CϭO), 196.10 (4-
CϭO), 196.48 (7-CϭO); IR (KBr) cmϪ1: 3350, 2925, 2850, 1750, 1665,
1610, 1470, 1050. EI-MS m/z: 368 (Mϩ), 350 (MϩϪH2O), 337
(MϩϪCH2OH), 319, 185, 172, 154. HR-MS (Mϩ) Calcd for C21H36O5:
368.2560, Found 368.2557. UV lmax (MeOH) nm (e): 232 (12319), 267
(15083). [a]2D0 ϩ58.06° (cϭ0.47, CHCl3); mp 105—108 °C.
Improved Synthesis of (R)-1 from (R)-3 To a solution of 6 (1.32 g,
3.50 mmol) and (R)-3 (1.27 g, 3.51 mmol) in THF (25 ml) was added silver
trifluoroacetate (930 mg, 4.21 mmol), and the mixture was stirred for 23 h at
23 °C while shielded from light. Silver trifluoroacetate (231 mg after 5 h and
387 mg after 22 h) was further added to complete the reaction. The mixture
was diluted with ether, passed through a short Silica gel column, and con-
centrated to give crude 7.
To a solution of this crude 7 in THF (12 ml) was added tetrabutylammo-
nium fluoride (1 M THF solution, 4.6 ml, 4.6 mmol), and the mixture was
stirred at 23 °C for 41 h. Tetrabutylammonium fluoride (0.7 ml after 2 h,
1.75 ml after 17 h, and 1.75 ml after 25 h) was further added to complete the
reaction. The reaction was quenched by the addition of 1 N aqueous HCl
(2 ml) and poured into ice-water; the mixture was then extracted with ether.
The organic layer was washed with brine, dried (Na2SO4), and concentrated
to give crude 8 (2.916 g).
Acknowledgments We thank Nissan Chemical Industries, Ltd. for ICP-
AES and X-ray fluorescence analysis. We are grateful to Dr. Hiroyuki Osada
of RIKEN for the generous gift of their RK-682 sample. We also thank Dr.
Akio Fujii and Mr. Ichiro Kudaka for their measurement of mass spectra.
This work was partially supported by the Hayashi Memorial Foundation for
Female Natural Scientists and the Kato Memorial Bioscience Foundation.
References and Notes
1) a) Osada H., Kohinata K., Hamaguchi T., Jpn. Kokai Tokkyo Koho,
1995, JP 07-242545; b) Hamaguchi T., Sudo T., Osada H., FEBS Lett.,
372, 54—58 (1995); c) Fujii S., Kato H., Furuse H., Ito K., Osada H.,
Hamaguchi T., Kuroda Y., Neurosci. Lett., 187, 130—132 (1995), d)
Fujii S., Ito K., Osada H., Hamaguchi T., Kuroda Y., Kato H., ibid.,
187, 133—136 (1995).
2) Shinagawa S., Muroi M., Itoh T., Tobita T., Jpn. Kokai Tokkyo Koho,
1993, JP 05-43568.
3) Sodeoka M., Sampe R., Kagamizono T., Osada H., Tetrahedron Lett.,
37, 8775—8778 (1996).
4) For a synthesis of racemic 1, see also: Mittra A., Yamashita M.,
Kawasaki I., Murai H., Yoshioka T., Ohta S., SYNLETT 1997, 909—
910.
5) a) Yamaguchi T., Saito K., Tsujimoto T., Yuki H., J. Heterocyclic
Chem., 13, 533—537 (1976); b) Saito K., Yamaguchi T., Bull. Chem.
Soc. Jpn., 51, 651—652 (1978); c) Gelin S., Pollet P., Tetrahedron
Lett., 21, 4491—4494 (1980).
6) Both enantiomers of 2 are commercially available, or can be easily
prepared from inexpensive D-mannitol and L-ascorbic acid, respec-
tively. See, a) Hebert N., Beck A., Lenox R. B., Just G., J. Org. Chem.,
57, 1777—1783 (1992); b) Dumont R., Pfander H., Helv. Chim. Acta.,
55, 814—823 (1983); c) Emons C. H. H., Kuster B. F. M., Vekemans
J. A. J. M., Sheldon R. A., Tetrahedron: Asymmetry, 2, 359—362
To a solution of the crude 8 (2.907 g) in methanol (70 ml) was added 1 N
aqueous HCl (4 ml), and the mixture was stirred at 23 °C for 66 h. After re-
moval of the solvent in vacuo, the residue was purified by Silica gel column
chromatography (CHCl3 : MeOH, 1 : 0—10 : 1—3 : 1). The combined and
concentrated fractions containing the desired product were redissolved in
AcOEt, washed with 0.5 N aqueous HCl and water, dried (Na2SO4), and con-

212
Vol. 49, No. 2
(ϩϩϩ), Ti (0.02%), Zr (0.006%). We used ca. 50—100 fold excess
(by weight) of silica gel for the conversion of 1 to 11. This means that
the compound 1 extracted calcium from silica gel quite efficiently. It is
also noteworthy that the Mg content in the same sample of 11 quanti-
fied by ICP-AES was 0.7 wt%, which was significantly higher than
those of other metals such as sodium (sodium content of 11 quantified
by atomic absorption spectrum was 1600 ppm) indicating that 1
prefers divalent cations.
(1991).
7) Brooks D. W., Lu L. D.-L., Masamune S., Angew. Chem. Int. Ed.
Engl., 18, 72—74 (1979).
8) Booth P., M. Fox C. M. J., Ley S. V., J. Chem. Soc. Perkin Trans. I,
1987, 121—129.
9) The RK-682 sample isolated from Streptomyces sp. 88-682 was gener-
ously provided by Dr. H. Osada (RIKEN).
10) a) Roggo B. E., Petersen F., Delmendo R., Jenny H.-B., Peter H. H.,
Roesel J., J. Antibiot., 47, 136—142 (1994); b) Roggo B. E., Hug P.,
Moss S., Raschdorf F., Peter H. H., ibid., 47, 143—147 (1994).
11) Kohl H., Bhat S. V., Patell J. R., Gandhi N. M., Nazareth J., Divekar P.
V., de Souza N. J., Bersheid H. G., Fehlhaber H.-W., Tetrahedron Lett.
1974, 983—986.
15) DTT/TG11 (Tokyo Kasei Kogyo Co. Ltd.). See, Takayama, M., J.
Mass Spectrom. Soc. Jpn., 42, 11—23 (1994). The FAB-MS of 11
using glycerol-m-nitrobenzylalcohol as a matrix showed similar peaks,
but with weaker intensity.
16) Morisaki N., Inoue K., Iwasaki S., J. Mass Spectrom. Soc. Jpn., 45,
471—477 (1997).
17) a) Ley S. V., Clase J. A., Mansfield D. J., Osborn H. M. I., J. Hetero-
cyclic Chem., 33, 1533—1544 (1996); b) Yoshii E., Yakugaku Zasshi,
112, 358—374 (1992).
18) Imamoto T., Kodera M., Yokoyama, M., Bull. Chem. Soc. Jpn., 55,
2303—2304 (1982).
12) Lebrun, M.-H., Duvert, P., Gaudemer, F., Gaudemer, A., Deballon, C.,
Boucly, P., J. Inorg. Biochem., 1985, 167—181.
13) (R)-1 and 11 showed similar inhibitory activity to a dual-specificity
phosphatase, VHR, suggesting that 11 is hydrolyzed in the assay
buffer (pH 6.0).3) RK-682 (acid-treated free form isolated from a nat-
ural sourceϭ(R)-1) is commercially available from Wako Pure Chemi-
cals Industries Inc.
14) According to the ICP-AES analysis data supplied by Merck, the fol-
lowing elements were present in Silica gel 60: Al (0.035%), Ca
(0.11%), Fe (0.01%), Mg (0.018%), Na (0.06%), S as SO4 (0.1%), Si
19) Quantification by ICP-AES and atomic absorption suggested that the
molar ratio of Ca : Mg : Na is about 72 : 25 : 6. The elemental analysis
data are in good agreement with the calculated values for
(C21H35O5)2Ca0.72Mg0.25Na0.06·2H2O: C 62.48, H 9.24.