Stereochemical assignment of four diastereoisomers of 3,4- dimethylpyroglutamic acid, A moiety of callipeltin B

Article abstract of DOI:10.3987/COM-14-13003

The synthesis and stereochemical assignment of four diastereoisomers of 3,4-dimethylpyroglutamic acid (pDME) (1) are described. Stereo-divergent synthesis of four pDMEs (1) was achieved starting from Thottathil's synthon and Garner's aldehyde for comparison of synthetic pDMEs (1) by ¹H NMR analysis and CD spectra. The stereochemistry of pDME (1) in cyclic depsipeptide callipeltin B (2) with cytotoxic and anti-HIV activities was confirmed to be 2S,3S,4R. Coupling constant between H2 and H3 of synthetic (2S,3S,4R)-pDME (1a) showed a smaller value than those of other isomers. In addition, synthetic pDMEs (1) hardly showed cytotoxicity against HeLa cells.

Full text of DOI:10.3987/COM-14-13003

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HETEROCYCLES, Vol. 89, No. 7, 2014
HETEROCYCLES, Vol. 89, No. 7, 2014, pp. 1620ꢀ- 1631. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 9th April, 2014, Accepted, 2nd May, 2014, Published online, 27th May, 2014
DOI: 10.3987/COM-14-13003
STEREOCHEMICAL ASSIGNMENT OF FOUR DIASTEREOISOMERS
OF 3,4-DIMETHYLPYROGLUTAMIC ACID, A MOIETY OF
CALLIPELTIN B
Mari Kikuchi and Hiroyuki Konno*
Department of Biochemical Engineering, Graduate School of Science and
Technology, Yamagata University, Yonezawa, Yamagata 992!8510, Japan
*E-mail address: konno@yz.yamagata-u.ac.jp
Abstract – The synthesis and stereochemical assignment of four diastereoisomers
of 3,4-dimethylpyroglutamic acid (pDME) (1) are described. Stereo-divergent
synthesis of four pDMEs (1) was achieved starting from Thottathil’s synthon and
1
Garner’s aldehyde for comparison of synthetic pDMEs (1) by H NMR analysis
and CD spectra. The stereochemistry of pDME (1) in cyclic depsipeptide
callipeltin B (2) with cytotoxic and anti-HIV activities was confirmed to be
2S,3S,4R. Coupling constant between H2 and H3 of synthetic (2S,3S,4R)-pDME
(1a) showed a smaller value than those of other isomers. In addition, synthetic
pDMEs (1) hardly showed cytotoxicity against HeLa cells.
INTRODUCTION
3,4-Dimethylpyroglutamic acid (pDME) (1) is an unusual amino acid, including callipeltin B (2) isolated
from marine sponge Callipelta sp.1 3,4-Dimethylglutamine (DMQ) (3), an acyclic unusual amino acid as a
precursor of pDME (1), is a common component of callipeltin A (4),2 papuamide A,3 neamphamides,4
theopapuamides,5 mirabamides,6 homophymines7 and pipecolidepsin A.8 The stereochemistry of DMQ (3)
was determined to be 2S,3S,4R on the basis of a positive Cotton effect on the CD spectrum and NMR
analysis of the hydrolysate, which was pDME (1) derived from callipeltin A (4). Therefore, the
stereochemistry of pDME (1) in callipeltin B (2) was also concluded to be 2S,3S,4R. Synthetic studies to
construct unusual amino acids in the above-mentioned cyclic depsipeptides have been reported by several
groups.9-14 In particular, synthesis of pDME (1a) is important research to achieve the total synthesis of
complexed cyclic depsipeptides since DMQ (3) could be converted by a ring-opening reaction for pDME
(1a).12 Preparation of DMQ and pDME derivatives has been reported independently by Lipton’s and
Hamada’s groups to date. Lipton et al. obtained pDME (1a) in 9 steps via cuprate addition and enolate

HETEROCYCLES, Vol. 89, No. 7, 2014
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alkylation, followed by kinetic epimerization of the methyl substituent starting from L-pyroglutamic acid
(L-pGlu) (5).12 On the other hand, Hamada’s group chose a Thottathil’s synthon15 as the starting material
for pDME (1a) and produced in 6 steps.13 For our research program of the synthesis of callipeltins and
homophymines, we reported the preparation of L-N-methylalanine, L-N-methylglutamine, (2S,3R)-D-!-
methoxytyrosine (!MOY), D-allothreonine and pDME (1a) for the solid phase synthesis of callipeltin B
1
(2) in our previous works.16-19 However, H NMR spectral data of pDME (1), which was obtained on
large-scale synthesis based on previous synthetic route starting from Garner’s aldehyde, and those of
natural product1 were slightly different. Synthetic pDME (1a) derived from Garner’s aldehyde was
simultaneously recognized as the equilibrium mixture. Because of the formal synthesis of pDME (1a)
derived from Garner’s aldehyde in our previous report,19 the stereochemistry of pDME (1a) has been
hardly investigated in detail. In addition, spectral data of 3,4-dimethylpyroglutaminol (7a) reported by
us,19,20 which was the substrate of the final oxidation reaction for pDME (1a), were similar to those of
Hamada’s report.13 This result might suggest that the stereochemistry of pDME (1) needed reassignment
by comparison of all diastereoisomers since Lipton’s and Hamada’s groups synthesized only the natural
form and spectral data for comparison of other diastereoisomers were not indicated. In contrast, Lipton’s
group reported that callipeltin B (2) showed cytotoxicity against HeLa cells with IC50 = 98 "M and
desmethoxycallipeltin B, in which substitution of D-Tyr for !MOY indicated IC50 = 128 "M.21 The results
showed that a methoxy group of !MOY was not needed to show cytotoxicity. Furthermore, we supposed
that dimethyl groups of pDME (1a) were essential for cytotoxicity in our previous study22 using
callipeltin B analogues; however, evaluation of pDMEs (1) against the cell lines has never been reported.
For this reason, we herein disclose the preparation of four possible diastereoisomers, reconfirmation of
Figure 1. Dimethylpyroglutamic acid (pDME) (1), natural form of pDME (1a) and DMQ (3) and
callipeltins B (2) and A (4).

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the stereochemistry of the natural form, and evaluation of cytotoxicity against HeLa cells for synthetic
pDMEs (1) (Figure 1).
RESULTS AND DISCUSSION
At first, we synthesized pDMEs (1a,b) using Thottathil’s synthon.15 Preparation of the requisite dimethyl
compound 6 was already described in 7 steps starting from L-pGlu (5) by Hamada and co-workers and
therefore verification of the stereochemistry of the starting material (6) was performed correctly. After the
spectral data of the obtained 6 were found to be identical to those of Hamada’s group, cleavage of N,
O-acetal of 6 using 9% TFA/CH2Cl2 proceeded smoothly to afford alcohol (7b) in quantitative yield.
Direct epimerization of H4-proton and/or oxidation of the hydroxy group of 7b afforded the expected
products in extremely low yield. Thereby, after the protection of 7b with TBS and Boc groups, treatment
with NaHMDS at -80 °C gave a mixture of the desired isomerized product (8a) and substrate (8b) in a
ratio of 3:1 in satisfactory yield. Separation of the mixture of 8a and 8b afforded pure 8a by column
chromatography in 67% yield, which is a known product reported by Lipton’s group.12 Boc carboxylic
acid (9a) derived from 8a was obtained in the Jones condition with CrO3/H2SO4 in acetone at 0 °C
(deprotection of TBS group and oxidation) and subsequently, deprotection of the Boc group with 50%
TFA/CH2Cl2 proceeded to give the desired (2S,3S,4R)-pDME (1a) according to Lipton’s procedures
(Scheme 1).12 It was noted that treatment of 9a with neat TFA afforded an undesired product (1b) by
epimerization of H4-proton. Similarly, (2S,3S,4S)-pDME (1b) as a single diastereomer was given from
8b using the Jones condition followed by cleavage of the Boc group in 68% yield over 2 steps. As a result,
1H, C NMR, CD, IR, mp, [!]D and MS spectral data of synthetic 1a resembled those of the natural
13
product1 and Hamada’s group,13 and furthermore, an equilibrium mixture of synthetic 1a could hardly be
observed. On the contrary, pDME (1a) described in our previous report was not identified. Therefore, we
Scheme 1. Synthesis of (2S,3S,4R)- and (2S,3S,4S)-pDME (1a,b).

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synthesized pDME (1a) according to the previous route to reassign the spectral data of all compounds
obtained from previous study.19 As a result, we focused on the reaction of diastereoselective
hydrogenation of 10. Thereby, hydrogenation of olefin (10), which was obtained starting from serine
derivatives in 7 steps, by H2/Pd-C in MeOH/AcOEt afforded a single product in quantitative yield. In
contrast, Hanessian has demonstrated that treatment of olefin (11) with Gilman reagent gave ethyl ester
(12) in 98% yield at the high diastereoselectivity by internal 1,2-asymmetric induction.23-26 1H NMR
spectral data of 12 derived from 11 by Hanessian’s protocol were clearly identical to the product obtained
by hydrogenation of 10 in the present study (Scheme 2). Hanessian’s 1,4-addition reaction with
Me2CuLi/TMSCl for 12 was proposed via Felkin-Ahn type conformation.27 We conjectured that 13 would
be obtained due to the addition of hydrogen from the bottom at hydrogenation via similar conformation.
However, both spectral data of 12 by Hanessian and our synthetic 13 in the previous literature19
conformed completely. In other words, two plausible reactions in Hanessian’s and our groups proceeded
via distinct mechanisms to give the same compound (12). Kim et al. reported stereoselective
dihydroxylation reaction of "-amino-!,#-unsaturated esters via N-outside or N-inside conformers.28,29
According to these results, we assumed that the hydrogenation of E-olefin (10) proceeded via N-inside
conformer to give 12 (Scheme 2).
Scheme 2. Diastereoselection by internal 1,2-asymmetric induction.
The improved synthesis of other pDME isomers (1c,d) was carried out for comparison of spectral data. 12
was treated with NaHMDS and MeI in the presence of HMPA to provide the dimethylated product (14) in
92% yield as a single isomer. In the process of the purification of 14, pyrrolidinone (7c), transformed via
deprotection of N, O-acetal, was afforded in trace amount. Therefore, treatment of 14 with SiO2 in toluene
at reflux proceeded pyrrolidinone formation to afford 7c and its isomer (7d) in 54% yield at a ratio of 5:1
with amino lactone (15) as a byproduct. In an attempt to separate the mixture of 7c and 7d, we found that

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these isomers (7c,d) easily isomerized under neutral conditions at room temperature for a few hours.
Spectral data of the mixture of 7c and 7d resembled those of 7a20 and 7b, thus determination of the
natural form was extremely difficult. In our previous study, we concluded that alcohol (7) existed as a
rotamer in the 1H NMR spectrum. Consequently, it was not easy to determine the natural form using 7a-d,
although we have reported the synthesis of pDME (1a). Since TEMPO mediated oxidation using the
mixture of 7c and 7d gave 1c and 1d in 77% yield at a ratio of 3:1, their coupling constants between H2
and H3-proton were indicated as JH2, H3 = 7.8~8.7 Hz, which did not conform with those of the natural
compound and synthetic 1a or 1b. These results propose that the stereochemistry of 1c and 1d is
2S,3R,4R and 2S,3R,4S (Scheme 3).30 By measurement of the CD spectrum, 1a and the mixture of 1c and
1d showed a positive Cotton effect at 211 and 217 nm, respectively. These CD spectra were similar to
those of the natural product having a positive Cotton effect at 208 nm. Because of the negative Cotton
effect at 219 nm, 1b could be rejected completely.
Scheme 3. Synthesis of (2S,3R,4R)- and (2S,3R,4S)-pDME (1c,d).
Next, the molecular force field for four diastereoisomers of pDME (1) was calculated by SPARTAN.
Four stable conformers were consequently indicated for each diastereoisomer and the energies of most
stable conformations were calculated as -29.16, -30.09, -21.13 and -20.44 kJ/mol for 1a-d, respectively.
On account of these results, the dihedrals of H3C3C2H2 of 1a-d were 93.5°, 153.6°, 30.8°and 35.7°. These
coupling constants calculated by the Karplus equation confirmed that the observed value of the H2-proton
of 1a was much smaller than other forms (Figure 2). We concluded that the stereochemistry of natural
pDME (1) was 2S,3S,4R through the synthesis of four diastereoisomers and its simulation. (2S,3S,4R)-1a
only indicated a small coupling constant between H2 and H3-protons at JH2, H3 = 3.2 Hz, and the other
three isomers (1b-d) had similar coupling constants of JH2, H3 = 7.8~8.7 Hz. These results were confirmed
1
by the comparison of H NMR spectral data of natural callipeltin B (2) (JH2, H3 = 3.0 Hz). In addition, 1a
and 1b have never been observed in the equilibrium state in spite of the isomerized mixture of 1c and 1d
with H4-proton in neutral conditions.

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Figure 2. Stable conformation of pDMEs (1a-d) by SPARTAN.
In our previous works on the cytotoxicity of callipeltin B analogues, we suggested that dimethyl groups
of pDME (1) might be important for potent activities.22 A cytotoxicity assay was performed against HeLa
cells using dimethyl and normal pyroglutamic acids, 1a-d, L-pGlu (5), D-pGlu (ent-5) for cytotoxicity
alongside rotenone, which served as the control. The cytotoxicities of these compounds were determined
by measuring live-cell succinate-tetrazolium reductase activity (MTT assay). The normal L- and D-pGlus
(5, ent-5), pDMEs (1a, b), and the mixture of 1c and 1d showed practically no cytotoxicity. The results
suggested that dimethyl groups of pDME (1) needed to be incorporated into the peptide, callipeltin B (2),
for cytotoxicity against HeLa cells.
In conclusion, we prepared four diastereoisomers of pDMEs (1a-d) for the first time, and reconfirmation
the stereochemistry of natural pDME (1) in callipeltin B (2) to be 2S,3S,4R. We supposed that 10 gave 12
by hydrogenation in high diastereoselectivity via N-inside conformer. Furthermore, 1H NMR spectral data
of 7a-d were similar and consequently it was difficult to identify the stereochemistry of natural form
pDME (1). However the comparison of all diastereomers of pDME (1) was efficient, especially using 1H
NMR analysis and CD spectra. We revise the assignment of the stereochemistry of synthetic compounds
in our previous research.19 In addition, cytotoxicity against HeLa cells was shown by the peptide with
pDME (1). A synthetic study of callipeltin B (2) containing synthetic (2S,3S,4R)-pDME (1a) on a solid
support is now underway.
EXPERIMENTAL
General. All solvents were reagent grade. CH2Cl2 was distilled from CaH2. All commercial reagents were
of the highest purity available. Analytical TLC was performed on silica gel (Merck Silica gel 60 F254).
Column chromatography was carried out on Silica Gel 60 N [Kanto, particle size, (spherical, neutral)
40-100 $m or 63-210 $m]. 1H (400 MHz) and 13C NMR (100, 75 MHz) spectra were recorded on either a
JEOL JNM-ECX400 or a BRUKER AM-300. Chemical shifts are expressed in ppm relative to TMS (0

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ppm), CHCl3 (7.26 ppm for 1H and 77.1 ppm for 13C) or H2O (4.79 ppm for 1H). IR spectra were obtained
on a HORIBA FT-720 spectrometer. Optical rotations were recorded on a JASCO DIP-371 polarimeter at
the sodium D line. High-resolution mass spectra (HRMS) were obtained using a JEOL AccuTOF
JMS-T100LC (ESIMS) or a JMS-T100GC-EI (EIMS). Melting points were determined on an AS ONE
ATM-02. Conformational searches and stable energy calculations were performed with Spartan’14
Parallel Suite for Windows (Wavefunction, Inc). Absorbances for cytotoxicity assay were measured with
the CORONA MTP-310Lab. The CD spectra were recorded on a JASCO J-820.
(3S,4S,5S)-5-Hydroxymethyl-3,4-dimethylpyrrolidin-2-one (7b). To a solution of 6 (235 mg, 1.02
mmol) in CH2Cl2 (6 mL) was added TFA (600 $L). After the stirring for 1.5 h at room temperature, the
mixture was evaporated under reduced pressure. The product was purified with silica gel column
chromatography (CHCl3/MeOH=95:5) to give 7b (146 mg, 1.02 mmol, quant.) as a brown oil. [!]27D 45.8
(c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz) %: 1.13 (3H, d, J=6.4 Hz), 1.14 (3H, d, J=7.2 Hz), 1.63 (1H,
ddq, J=10.4, 8.4, 6.8 Hz), 2.08 (1H, dq, J=10.4, 7.2 Hz), 3.31 (1H, td, J=7.6, 2.4 Hz), 3.46 (1H, dd,
13
J=11.6, 7.6 Hz), 3.76 (1H, d, J=11.6 Hz); C NMR (CDCl3, 100 MHz) %: 13.9, 16.5, 40.0, 44.6, 62.4,
64.2, 180.6; IR &max (film) cm-1: 3338, 2962, 2931, 2875, 1685; EIHRMS m/z [M]+: 143.0909, Calcd. for
C7H13NO2: 143.0946.
(3S,4S,5S)-N-(tert-Butoxycarbonyl)-5-(tert-butyldimethylsilyloxy)methyl-3,4-dimethylpyrrolidin-2-
one (8b). To a solution of 7b (288 mg, 2.01 mmol) in DMF (6 mL) was added TBSCl (603 mg, 4.02
mmol) and imidazole (545 mg, 8.04 mmol) and then the mixture was stirred at ~50 °C. After being stirred
for 3.5 h, H2O and AcOEt were added to the solution. The organic layer was washed with brine, dried
over MgSO4, and evaporated under reduced pressure. The residue was purified with silica gel column
chromatography (CHCl3/MeOH=99:1) to give TBS ether (453 mg, 1.76 mmol, 88%) as a pale yellow oil.
1
[!]28 31.8(c 0.4, CHCl3); H NMR (CDCl3, 400 MHz) %: 0.05 (5H, s), 0.08 (1H, s), 0.87 (8H, s), 0.90
D
(1H, s), 1.13 (3H, d, J=7.2 Hz), 1.16 (3H, d, J=6.8 Hz), 1.55 (1H, m), 2.05 (1H, m), 3.27 (1H, td, J=8.2,
13
2.8 Hz), 3.38 (1H, dd, J=9.6, 8.7 Hz), 3.74 (1H, dd, J=10.1, 2.8 Hz), 5.87 (1H, brs); C NMR (CDCl3,
100 MHz) %: -5.4, 14.2, 16.9, 18.2, 25.8, 40.6, 44.4, 61.4, 66.0, 179.2; IR &max (film) cm-1: 3218, 3106,
2956, 2929, 2857, 1704, 1122, 839; ESIHRMS m/z [M+Na]+: 280.1708, Calcd. for C13H27NNaO2Si:
280.1709. To a solution of TBS ether (43.8 mg, 170 $mol) in MeCN (1 mL) was added (Boc)2O (371 mg,
1.70 mmol) and DMAP (cat.). After the stirring for 1.5 h at room temperature, H2O and AcOEt were
added to the solution. The organic layer was washed with brine, dried over MgSO4, and evaporated under
reduced pressure. The residue was purified with silica gel column chromatography (hexane/AcOEt=6:1)
to give 8b (58.9 mg, 165 $mol, 97%) as a pale yellow solid. mp 50-52 °C; [!]26D -55.5(c 2.7, CHCl3); 1H

HETEROCYCLES, Vol. 89, No. 7, 2014
1627
NMR (CDCl3, 400 MHz) %: 0.03 (3H, s), 0.04 (3H, s), 0.87 (9H, s), 1.15 (3H, d, J=6.9 Hz), 1.22 (3H, d,
J=7.4 Hz), 1.53 (9H, s), 1.96 (1H, m), 2.09 (1H, m), 3.54 (1H, td, J=4.1, 2.3 Hz), 3.69 (1H, dd, J=10.5,
1.8 Hz), 4.03 (1H, dd, J=10.6, 4.1 Hz); 13C NMR (CDCl3, 100 MHz) %: -5.4, 14.8, 18.3, 18.6, 25.8, 28.1,
35.0, 45.4, 61.3, 64.4, 82.6, 150.5, 176.6; IR &max (film) cm-1: 2958, 2931, 2857, 1789, 1754, 1716, 1340,
1201, 837; ESIHRMS m/z [M+H]+: 358.2451, Calcd. for C18H36NO4Si: 358.2414.
(3R,4S,5S)-N-(tert-Butoxycarbonyl)-5-(tert-butyldimethylsilyloxy)methyl-3,4-dimethylpyrrolidin-2-
one (8a). To a solution of 8b (1.17 g, 3.28 mmol) in THF (15 mL) at -80 °C was added NaHMDS in
THF (2.30 mL, 1.9 mol/L, 4.26 mmol) and then the mixture was stirred at -80 °C. After being stirred for
1 h, AcOH (377 $L) was added. The mixture was quenched by saturated NH4Cl aq. and diluted with Et2O.
The solution was allowed to warm to room temperature and then extracted with Et2O. The organic layer
was washed with brine, dried over MgSO4, and evaporated under reduced pressure. The residue was
purified with silica gel column chromatography (hexane/AcOEt=8:1) to give 8a (786 mg, 2.20 mmol,
1
67%) as a pale yellow oil. [!]28 -43.5(c 1.7, CHCl3); H NMR (CDCl3, 400 MHz) %: 0.03 (3H, s), 0.05
D
(3H, s), 0.87 (9H, s), 0.99 (3H, d, J=7.2 Hz), 1.08 (3H, d, J=8.0 Hz), 1.54 (9H, s), 2.44 (1H, m), 2.97 (1H,
13
m), 3.67 (1H, td, J=5.2, 2.8 Hz), 3.75 (1H, dd, J=10.8, 2.8 Hz), 3.85 (1H, dd, J=10.8, 5.2 Hz); C NMR
(CDCl3, 100 MHz) %: -5.6, 10.1, 15.7, 18.1, 25.8, 28.0, 32.9, 40.6, 63.0, 64.7, 82.6, 150.5, 176.7; IR &max
(film) cm-1: 2956, 2931, 2857, 1789, 1754, 1712, 1309, 1159, 837; ESIHRMS m/z [M+Na]+: 380.2212,
Calcd. for C18H35NNaO4Si: 380.2233.
(2S,3S,4R)-N-(tert-Butoxycarbonyl)-3,4-dimethylpyroglutamic acid (9a). To a solution of 8a (317
mg, 887 $mol) in acetone (5 mL) at 0 °C was added Jones reagent (30% CrO3/H2SO4, 225 $L). After
being stirred for 6.5 h at 0 °C, the reaction was quenched by addition of 2-propanol (225 $L) at 0 °C and
saturated NaHCO3 aq. (125 $L) was added. The mixture was diluted with H2O and extracted with AcOEt.
The aqueous layers were combined and acidified with 0.1 M HCl. The solution was extracted with AcOEt.
The combined organic layers were washed with brine, dried over MgSO4, and evaporated under reduced
pressure. The residue was purified with silica gel column chromatography (CHCl3/MeOH=98:2 to 95:5)
to give 9a (199 mg, 773 $mol, 87%) as a colorless amorphous. mp 112-113 °C; [!]29D -8.0(c 0.6, CHCl3);
1H NMR (CDCl3, 400 MHz) %: 1.12 (3H, d, J=7.3 Hz), 1.13 (3H, d, J=7.3 Hz), 1.52 (9H, s), 2.58 (1H, m),
13
2.82 (1H, m), 4.24 (1H, d, J=1.8 Hz); C NMR (CDCl3, 100 MHz) %: 9.9, 15.3, 27.9, 34.3, 40.7, 64.2,
83.9, 149.9, 175.7; IR &max (film) cm-1: 3218, 2979, 2937, 2879, 1781, 1751, 1720, 1311, 1155; ESIHRMS
m/z [M+Na]+: 280.1137, Calcd. for C12H19NNaO5: 280.1161.
(2S,3S,4R)-3,4-Dimethylpyroglutamic acid (1a). To a solution of 9a (49.5 mg, 192 $mol) in CH2Cl2

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(2 mL) was added TFA (2 mL). After the stirring for 1.5 h at room temperature, the mixture was
evaporated under reduced pressure to give 1a (30.2 mg, 192 $mol, quant.) as a white powder. mp
177-180 °C; [!]29D 48.3(c 0.3, MeOH); CD [']211 nm +29728 (c 0.001 M, H2O); 1H NMR (D2O, 400 MHz)
%: 1.05 (3H, d, J=6.8 Hz), 1.13 (3H, d, J=6.8 Hz), 2.68 (2H, m), 3.96 (1H, d, J=3.2 Hz); 13C NMR (D2O,
100 MHz) %: 9.5, 13.7, 38.0, 39.2, 61.4, 176.3, 183.8; IR &max (film) cm-1: 3259, 2971, 1735, 1720, 1155;
EIHRMS m/z [M]+: 157.0749, Calcd. for C7H11NO3: 157.0739.
(2S,3S,4S)-N-(tert-Butoxycarbonyl)-3,4-dimethylpyroglutamic acid (9b). To a solution of 8b (137
mg, 530 $mol) in acetone (2.5 mL) at 0 °C was added Jones reagent (30% CrO3/H2SO4, 100 $L). After
being stirred for 3.5 h at 0 °C, the reaction was quenched by addition of 2-propanol (100 $L) at 0 °C and
saturated NaHCO3 aq. (50 $L) was added. The mixture was diluted with H2O and extracted with AcOEt.
The aqueous layers were combined and acidified with 0.1 M HCl. The solution was extracted with AcOEt.
The combined organic layers were washed with brine, dried over MgSO4, and evaporated under reduced
pressure. The residue was purified with silica gel column chromatography (CHCl3/MeOH=99:1 to 80:20)
1
to give 9b (73.9 mg, 287 $mol, 76%) as a pale yellow oil. [!]23 -29.9(c 0.7, CHCl3); H NMR (CDCl3,
D
400 MHz) %: 1.24 (3H, d, J=7.3 Hz), 1.30 (3H, d, J=6.9 Hz), 1.50 (9H, s), 1.94 (1H, m), 2.21 (1H, m),
13
4.07 (1H, d, J=8.2 Hz); C NMR (CDCl3, 100 MHz) %: 13.9, 17.2, 27.9, 38.8, 45.6, 64.4, 84.1, 149.4,
175.1; IR &max (film) cm-1: 3219, 2978, 2937, 1778, 1753, 1724, 1155; ESIHRMS m/z [M+Na]+: 280.1175,
Calcd. for C12H19NNaO5: 280.1161.
(2S,3S,4S)-3,4-Dimethylpyroglutamic acid (1b). To a solution of 9b (65.9 mg, 256 $mol) in CH2Cl2
(800 $L) was added TFA (800 $L). After the stirring for 2 h at room temperature, the mixture was
evaporated under reduced pressure to give 1b (36.1 mg, 230 $mol, 90%) as a colorless amorphous. mp
129-130 °C; [!]24D -13.3(c 0.4, MeOH); CD [']219 nm -26424 (c 0.001 M, H2O); 1H NMR (D2O, 400 MHz)
%: 1.06 (3H, d, J=7.3 Hz), 1.22 (3H, d, J=6.9 Hz), 2.03 (1H, m), 2.19 (1H, m), 3.88 (1H, d, J=8.2 Hz); 13C
NMR (D2O, 100 MHz) %: 13.5, 16.6, 42.7, 43.9, 61.2, 175.9, 182.9; IR &max (film) cm-1: 3276, 2969, 2929,
1739, 1722, 1660; EIHRMS m/z [M]+: 157.0752, Calcd. for C7H11NO3: 157.0739.
(3R,4R,5S)-5-Hydroxymethyl-3,4-dimethylpyrrolidin-2-one (7c) and (3S,4R,5S)-5-hydroxymethyl-
3,4-dimethylpyrrolidin-2-one (7d). To a solution of 14 (195 mg, 647 $mol) in toluene (10 mL) was
added SiO2 (2.0 g) and heated to reflux for 8 h. The mixture was filtrated, washed with 30%
MeOH/CHCl3 and evaporated under reduced pressure. The residue was purified with silica gel column
chromatography (CHCl3/MeOH=97:3 to 95:5) to give mixture of 7c and 7d (5:1, 50.4 mg, 352 $mol,
13
54%) as a brown oil. C NMR (CDCl3, 75 MHz) %: 13.1, 13.8, 13.9, 16.4, 39.8, 40.0, 42.6, 44.4, 57.3,

HETEROCYCLES, Vol. 89, No. 7, 2014
1629
58.4, 62.0, 62.1, 63.9, 180.4, 181.6; IR &max (film) cm-1: 3385, 2962, 2931, 2877, 1685; EIHRMS m/z
1
[M]+: 143.0944, Calcd. for C7H13NO2: 143.0946; 7c: H NMR (CDCl3, 400 MHz) %: 1.11-1.14 (6H, m),
1
2.16 (2H, m), 3.55-3.76 (3H, m). 7d: H NMR (CDCl3, 400 MHz) %: 1.13-1.15 (6H, m), 1.69-2.04 (2H,
m), 3.31 (1H, m), 3.45 (1H, m), 3.79 (1H, m).
(2S,3R,4R)-3,4-Dimethylpyroglutamic acid (1c) and (2S,3R,4S)-3,4-dimethylpyroglutamic acid (1d).
To a solution of mixture 7c and 7d (105 mg, 732 $mol) in MeCN (500 $L) and 0.5 M phosphate buffer
(pH 6.6) (500 $L) was added NaClO (2 mL), NaClO2 (132 mg, 1.46 mmol) and TEMPO (16.3 mg, 105
$mol). After the stirring for 4.5 h at room temperature, 1 M HCl and AcOEt were added to the solution.
The organic layer was washed with brine, dried over MgSO4, and evaporated under reduced pressure. The
residue was purified with silica gel column chromatography (CHCl3/MeOH=95:5) to give mixture of 1c
and 1d (3:1, 88.8 mg, 565 $mol, 77%) as a colorless oil. CD [']217 nm +21097 (c 0.001 M, H2O); 13C NMR
(D2O, 100 MHz) %: 14.3, 14.4, 14.6, 15.0, 41.2, 41.6, 43.0, 43.2, 69.2, 71.0, 173.9, 179.8, 180.0, 181.3;
IR &max (film) cm-1: 3417, 2972, 2939, 1732, 1716, 1213; EIHRMS m/z [M]+: 157.0730, Calcd. for
1
C7H11NO3: 157.0739; 1c: H NMR (D2O, 400 MHz) %: 1.07 (3H, d, J=7.7 Hz), 1.11 (3H, d, J=7.2 Hz),
2.27 (1H, m), 2.47 (1H, m), 4.37 (1H, d, J=8.7 Hz). 1d: 1H NMR (D2O, 400 MHz) %: 1.14 (3H, d, J=6.9
Hz), 1.22 (3H, d, J=6.9 Hz), 2.11 (1H, m), 2.28 (1H, m), 4.04 (1H, d, J=7.8 Hz).
Chemical shift calculations. Synthetic pDMEs (1a-d) were submitted to a conformational search
employing Molecular Mechanics to afford four stable conformers, respectively. The most stable
conformer about each diastereomer was calculated the energies, dihedrals of H3C3C2H2 and chemical
shifts.
Evaluation of the cytotoxicity by MTT assay. HeLa cells were maintained in a suspension culture of
DMEM supplemented with 5% FBS (Fetal Bovine Serum) containing 1% of a penicillin-streptomycin
mixture. A 100 $L aliquot of HeLa cells (5000 cells/mL) was added to a 96 well plate and incubated for
24 h at 37 °C in a humidified incubator containing 5% CO2 in air. After 24 h, a 10 $L aliquot of
compound 1a, 1b, mixture of 1c and 1d, L-pGlu (5), D-pGlu (ent-5) (concentrations varying in the range
of 5, 4, 3, 2, 1, 0.1 mM) and rotenone as a control was added to each of the 96 wells and incubated for
24 h. A 10 $L WST-8 solution (mixture of WST-8 and 1-Methoxy PMS) was added to each well and the
incubation continued for 2 h. The visible absorbance at 450 nm and 630 nm as the reference wavelength
of each well was quantified using a microplate reader.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant-in aid from the Japan Society for the Promotion of the

1630
HETEROCYCLES, Vol. 89, No. 7, 2014
Science (25450145) to H. K. and M. K. is grateful to the Japan Society for the Promotion of Science
Fellowships for young scientists (8696).
SUPPORTING INFORMATION AVAILABLE
1H, C NMR, IR and MS spectra of 1a-d, and 7a-d and CD spectral data for 1a-d. This material
13
associated with this article can be found in the online version.
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1
other isomers. [!]23 76.2 (c 0.2, CHCl3); H NMR (CDCl3, 400 MHz) %: 1.01 (3H, d, J=6.9 Hz),
D
1.07 (3H, d, J=7.8 Hz), 2.25 (1H, m), 2.55 (1H, m), 3.33 (1H, brs), 3.49 (1H, td, J=10.1, 6.9 Hz),
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