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Z. Zhang et al.
non-deuterated (non-racemized) L-DLA derivative of the
hydrolysate gave a peak only at 13.5 min (Fig. S9).
(400 μl) were added (R)-3-hydroxy-3-phenylpropanoic
acid (3.5 mg, 21 μmol) and N, N-diisopropylethylamine
(3.7 μl, 21 μmol). After stirring for 12 h at room tem-
perature, the reaction mixture was poured into ice-water
and extracted with EtOAc. The organic layer was washed
with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by preparative HPLC (Cosmosil
5C18 AR-II, 10 × 250 mm, 4 ml min−1, UV detection at
210 nm) with 30% MeCN in 0.1% HCO2H solution to
give (R,S)-2 (2.9 mg, tR 11.3 min) in 52% yield: 1H NMR
(500 MHz, CDCl3) δ1.42 (3H, d, J = 7.2 Hz, Nma-3),
2.67 (1H, m, βHpp-2), 2.71 (1H, m, βHpp-2), 2.89 (3H,
s, N-CH3), 3.73 (3H, s, OCH3), 5.15 (1H, dd, J = 4.2,
8.0 Hz, βHpp-3), 5.26 (1H, q, J = 7.3 Hz, Nma-2), 7.28
(1H, m, βHpp-7), 7.35 (2H, m, βHpp-6/8), 7.41 (2H, m,
βHpp-5/9); HR-ESITOFMS m/z 288.1211 [M + Na]+
(calcd for C14H19NNaO4, 288.1206).
Chiral HPLC analysis
Compound 1 (0.5 mg) was hydrolyzed at 110 °C in 6 M
HCl (200 μl) for 12 h, and the reaction mixture was dried in
a stream of N2. The residue was dissolved in 2 mM CuSO4
aqueous solution (200 μl) and subjected to HPLC analysis
(Sumichiral OA-5000, 4.6 × 150 mm; 15% 2-propanol in
2 mM CuSO4 aqueous solution; 1 ml min−1; UV 254 nm).
Retention times for the standards were 6.2 min for (S)-
αHpp, 10.1 min for (R)-αHpp, and 28.2 min for (S)-Hiv, and
36.6 min for (R)-Hiv, while the hydrolysate of 1 gave peaks
at 6.2 min and 28.2 min (Fig. S10).
Methanolysis of 1 to yield 2
Compound 1 (5 mg, 10 μmol) was dissolved in 28%
NaOMe in MeOH (750 μl) and stirred for 30 min with ice
cooling. The reaction mixture was neutralized with 1 M HCl
solution and the aqueous phase was extracted with EtOAc.
After removing the solvent, the residue was purified by
preparative HPLC (Cosmosil 5C18 AR-II, 10 × 250 mm,
4 ml min−1, UV detection at 210 nm) with 30% MeCN in
Synthesis of (S,S)-2
In the same manner as described for (R,S)-2, (S)-3-hydroxy-
3-phenylpropanoic acid gave (S,S)-2 in 60% yield: 1H NMR
(500 MHz, CDCl3) δ1.40 (3H, d, J = 7.3 Hz), 2.66 (1H, m),
2.75 (1H, m), 2.87 (3H, s), 3.73 (3H, s), 5.18 (1H, m), 5.26
(1H, q, J = 7.2 Hz), 7.28 (1H, m), 7.36 (2H, m), 7.40 (2H,
m); HR-ESITOFMS m/z 288.1203 [M + Na]+ (calcd for
C14H19NNaO4, 288.1206).
1
0.1% HCO2H solution to yield 2 (1.0 mg, tR 11.1 min): H
NMR (500 MHz, CDCl3) δ1.40 (3H, d, J = 7.3 Hz, Nma-3),
2.68 (1H, m, βHpp-2), 2.73 (1H, m, βHpp-2), 2.88 (3H, s,
N-CH3), 3.73 (3H, s, OCH3), 5.18 (1H, m, βHpp-3), 5.26
(1H, q, J = 7.1 Hz, Nma-2), 7.29 (1H, m, βHpp-7), 7.36
(2H, m, βHpp-6/8), 7.40 (2H, m, βHpp-5/9); HR-
ESITOFMS m/z 288.1219 [M + Na]+ (calcd for
C14H19NNaO4, 288.1206).
Bioassay
Antimicrobial activity and cytotoxicity were evaluated
according to the previously reported procedures [12].
Acknowledgements We thank Prof. Daisuke Urabe at Toyama Pre-
fectural University for his technical support on the synthesis of 2.
Synthesis of methyl N-methyl-L-alaninate
A stirred solution of N-methyl-L-alanine (50 mg, 0.48 mol)
in 5–10% HCl/MeOH (5 ml) was heated at 70 °C for 3 h.
The reaction mixture was concentrated and again heated in
5–10% HCl/MeOH (1 ml) and MeOH (5 ml) for 3 h. The
reaction mixture was concentrated in vacuo to give semi-
pure methyl N-methyl-L-alaninate (90 mg). This was
employed for the next reaction without further purification:
1H NMR (500 MHz, CD3OD) δ1.51 (3H, d, J = 6.9 Hz,
Nma-3), 2.68 (3H, s, N-CH3), 3.78 (3H, s, OCH3), 4.05
(1H, brd, J = 6.6 Hz, Nma-2); HR-ESITOFMS m/z
118.0869 [M + H]+ (calcd for C5H12NO2, 118.0863).
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
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JD. Chemical repertoire and biosynthetic machinery of the
Aspergillus flavus secondary metabolome: a review. Compr Rev
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Synthesis of (R,S)-2
To a solution of methyl N-methyl-L-alaninate (2.5 mg,
21 μmol), BOP reagent (9.3 mg, 21 μmol), and 1-
hydroxybenzotriazole (2.9 mg, 21 μmol) in dry DMF