Pithohirolide, an antimicrobial tetradepsipeptide from a fungus Pithomyces chartarum

Article abstract of DOI:10.1038/s41429-021-00423-4

Pithohirolide (1), a new depsipeptide, was isolated from an ascomycetous fungus Pithomyces chartarum TAMA 581. The planar structure of 1 was elucidated on the basis of NMR and MS analyses and the absolute configuration was determined by the advanced Marfey’s analysis, chiral-phase HPLC analysis, and synthesis of degradation product. Compound 1 possesses a cyclic structure comprising (S)-2-hydroxy-3-phenylpropanoic acid, (S)-3-hydroxy-3-phenylpropanoic acid, (S)-2-hydroxyisovaleric acid, and N-methyl-l-alanine, connected via three ester and one amide linkages. Compound 1 exhibited antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae at MIC 3.1 μg ml^?1.

Full text of DOI:10.1038/s41429-021-00423-4

The Journal of Antibiotics (2021) 74:458–463
https://doi.org/10.1038/s41429-021-00423-4
BRIEF COMMUNICATION
Pithohirolide, an antimicrobial tetradepsipeptide from a fungus
Pithomyces chartarum
1
Zhiwei Zhang1 Tao Zhou1 Tian Xing1 Takayuki Ishizaki2 Toru Okuda2 Naoya Oku
Yasuhiro Igarashi1
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Received: 2 March 2021 / Revised: 1 April 2021 / Accepted: 5 April 2021 / Published online: 7 May 2021
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2021
Abstract
Pithohirolide (1), a new depsipeptide, was isolated from an ascomycetous fungus Pithomyces chartarum TAMA 581. The
planar structure of 1 was elucidated on the basis of NMR and MS analyses and the absolute configuration was determined by
the advanced Marfey’s analysis, chiral-phase HPLC analysis, and synthesis of degradation product. Compound 1 possesses a
cyclic structure comprising (S)-2-hydroxy-3-phenylpropanoic acid, (S)-3-hydroxy-3-phenylpropanoic acid, (S)-2-hydro-
xyisovaleric acid, and N-methyl-L-alanine, connected via three ester and one amide linkages. Compound 1 exhibited
antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae at MIC 3.1 μg ml−1
.
Introduction
mostly saprophytic and commonly found in decayed plant
materials, but some are pathogenic and are occasionally
isolated from mammals [9]. For instance, Pithomyces
chartarum causes facial eczema in grazing ruminants
through the production of a mycotoxin sporidesmin [10]. At
present, only 24 compounds are known from Pithomyces
[5], but further discovery of new bioactive compounds are
expected in consideration of their pathogenic characteristics
analogous to the sibling genera.
In the course of our metabolite analysis of underexplored
microbial genera [11–13], a Pithomyces strain, isolated from a
decaying bark sample, was examined, which resulted in the
isolation of a new tetradepsipeptide, pithohirolide (1), com-
prising one each of (S)-2-hydroxy-3-phenylpropanoic acid
(αHpp), (S)-3-hydroxy-3-phenylpropanoic acid (βHpp), (S)-2-
hydroxyisovaleric acid (Hiv), and N-methyl-L-alanine (Nma),
along with two known compounds, sporidesmin and bos-
trycoidin (Fig. 1).
Fungi have served as a prolific source of bioactive natural
products since the discovery of penicillin in the last century [1].
They are capable of synthesizing a wide range of secondary
metabolites including polyketides, terpenes, and peptides
owing to their diverse biosynthetic pathways [2–4]. While
representative genera such as Penicillium and Aspergillus have
provided almost 6000 bioactive compounds [5], contributions
from less studied families/genera cannot be underestimated
because structurally unique compounds have continuously
been discovered from other minor fungi groups [6, 7].
The genus Pithomyces belongs to the family Pleospor-
aceae, which includes Alternaria and Cochliobolus, known
as plant pathogens and producers of 500 bioactive meta-
bolites [5]. These pathogenic fungi utilize secondary
metabolites for establishing infection, and thus the patho-
genicity is considered as a trait of fungi with high ability of
secondary metabolism [8]. Members of Pithomyces are
Results and discussion
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1038/s41429-
021-00423-4.
Compound 1 was obtained as a pale yellow amorphous
solid. HR-ESITOFMS analysis gave a sodium adduct ion
[M + Na]+ at m/z 504.1990 (Δ −0.3 mmu, calcd for
C27H31NNaO7, 504.1993), corresponding to the molecular
formula of C27H31NO7 with 13 degrees of unsaturation.
Spectroscopic analyses of 1H, 13C, and HSQC spectra
presented 27 carbons assignable to four carbonyl carbons
(δC 169.3, 169.4, 171.7, 173.3), two sp2 nonprotonated
* Yasuhiro Igarashi
yas@pu-toyama.ac.jp
1
Biotechnology Research Center and Department of
Biotechnology, Toyama Prefectural University, Toyama, Japan
2
HyphaGenesis, Inc., Machida, Tokyo, Japan

Pithohirolide, an antimicrobial tetradepsipeptide from a fungus Pithomyces chartarum
Table 1 1H and 13C NMR data for pithohirolide (1) in CD3OD
459
Residue Position δCa, type
δH mult
HMBCb,c
(J in Hz)b
Nma
1
2
171.7, C
57.8, CH
4.85e q (7.1)
1, 3, 4 (Nma);
1 (βHpp)
3
4
16.1, CH3 1.43 d (7.1)
29.3, CH3 3.10 s
1, 2 (Nma)
2 (Nma);
1 (βHpp)
αHpp
1
2
169.3, C
76.4, CH
5.41 dd
(8.6, 5.7)
1, 3, 4 (αHpp);
1 (Nma)
3
38.4, CH2 3.25 dd
(8.6, 14.0)
1, 2, 4, 5 (αHpp)
Fig. 1 Structures of secondary metabolites isolated from Pithomyces
chartarum TAMA 581
3.29 dd
(5.7, 14.0)
1, 2, 4, 5 (αHpp)
carbons (δC 136.8, 140.6), 10 sp2 methine (δC 127.6 × 2,
128.6, 129.6, 129.8 × 2, 129.9 × 2, 130.7 × 2), five sp3
methine (δC 32.0, 57.8, 76.4, 77.2, 78.1; four are nitrogen/
oxygen-bonded), two sp3 methylene (δC 38.4, 40.9), and
four methyl groups (δC 16.1, 17.2, 19.4, 29.3) (Table 1).
COSY analysis proved the connectivity of a doublet methyl
proton (δH 1.43/δC 16.1, Nma-H3) and an α-methine proton (δH
4.85/δC 57.8, Nma-H2). The latter was embedded in a huge
“water” (HDO) peak in the 1H NMR spectrum at 25 °C, but its
7.1-Hz quartet splitting was read by shifting the solvent peak
by warming the sample solution to 35 °C (Figs. S3 and S4).
Both Nma-H2 and Nma-H3 showed HMBC correlations to a
carbonyl carbon (δC 171.7, Nma-C1), providing an alanine
residue. N-Methylation in this residue was evidenced by a
HMBC correlation from a deshielded singlet methyl proton (δH
3.10/δC 29.3, Nma-4) to Nma-C2 (Fig. 2).
Two equivalent doublet methyl protons (δH 0.92/δC 17.2,
Hiv-H4; 0.93/19.4, Hiv-H5), accounting for six-proton
integration, were mutually correlated in the HMBC spec-
trum and showed COSY correlations to a multiplet methine
(δH 2.34/δC 32.0, Hiv-H3), yielding an isopropyl group.
COSY analysis also proved the connectivity of Hiv-H3 with
an oxymethine (δH 5.24/δC 77.2, Hiv-2) which was in turn
long-range coupled to a carbonyl carbon (δC 169.4, Hiv-
C1), thus establishing a 2-hydroxyisovaleric acid residue.
A bundle of overlapped protons resonating between δH
7.28 and 7.34 in the 1H NMR spectrum accounted for a total
integration area of 10 protons and was assigned to two
monosubstituted benzene rings based on the HSQC and
HMBC correlations. One of the benzene rings was con-
nected to a methylene group (δH 3.25/3.29, δC 38.4, αHpp-
3) by HMBC correlations. The methylene protons αHpp-H3
were further COSY-correlated to an oxymethine αHpp-H2
(δH 5.41/δC 76.4) and both αHpp-H2 and αHpp-H3 had
HMBC correlations to a carbonyl carbon (δC 169.3, αHpp-
C1). Thus, a 2-hydroxy-3-phenylpropanoic acid residue was
established (αHpp).
4
136.8, C
5, 9
6, 8
7
130.7, CH 7.34f
129.9,d CH 7.34f
128.6, CH 7.28f
169.4, C
3, 5, 7, 9 (αHpp)
4, 6, 8 (αHpp)
5, 9 (αHpp)
Hiv
1
2
77.2, CH
5.24 d (2.4)
1, 3, 4, 5 (Hiv);
1 (αHpp)
2, 4, 5 (Hiv)
2, 3, 4 (Hiv)
2, 3, 5 (Hiv)
3
4
5
1
2
32.0, CH
2.34 m
17.2, CH3 0.92 d (6.7)
19.4, CH3 0.93 d (6.7)
173.3, C
βHpp
40.9, CH2 2.47 dd
(3.4, 14.3)
1 (βHpp)
3.40 dd
(11.7, 14.3)
1, 3, 4 (βHpp)
3
78.1, CH
140.6, C
127.6, CH 7.33f
129.8,d CH 7.34f
129.6, CH 7.28f
5.98 dd
(3.4, 11.7)
2, 4, 5 (βHpp);
1 (Hiv)
4
5, 9
6, 8
7
3, 5, 7, 9 (βHpp)
4, 6, 8 (βHpp)
5, 9 (βHpp)
aRecorded at 125 MHz (reference δC 49.2)
bRecorded at 500 MHz (reference δH 3.31)
cHMBC correlations are from proton stated to the indicated carbons
dInterchangeable
eOverlapped by HDO peak at 25 °C
fOverlapped
The other benzene ring, on the other hand, was bonded to an
oxymethine (δH 5.98/δC 78.1, βHpp-3) by mutual HMBC
correlations between βHpp-3 and the aromatic protons βHpp-5,
and a proton of the same oxymethine was connected to
methylene protons (δH 2.47/3.40, βHpp-H2) by COSY corre-
lations. Because both of these protons showed HMBC corre-
lations to a carbonyl carbon (δC 173.3, βHpp-C1), a 3-hydroxy-
3-phenylpropanoic acid residue was identified (βHpp).

460
Z. Zhang et al.
1
2
2
2
2
2
2
R S
S S
Fig. 2 COSY and key HMBC correlations for pithohirolide (1)
2
2
2
These four residues were assembled into a cyclic struc-
ture based on a sequence of inter-residue HMBC correla-
tions. While correlations from Nma-H2 and Nma-H4 to
βHpp-C1 confirmed an amide linkage between Nma and
βHpp residues, correlations from βHpp-H3 to Hiv-C1, Hiv-
H2 to αHpp-C1, and αHpp-H2 to Nma-C1 proved ester
linkages between these residue pairs, thereby establishing a
cyclo(Nma-αHpp-Hiv-βHpp) structure for 1.
The absolute configuration of Nma was determined by the
advanced Marfey’s method [14]. Compound 1 was hydrolyzed
in 6 M HCl and the hydrolysate was derivatized with 1-fluoro-
2,4-dinitrophenyl-5-L-leucinamide (L-FDLA). The resulting
DLA derivative of Nma, detected by ion extraction at m/z 396
in an LC/MS analysis, contained both of the enantiomers
(Fig. S9), implying that racemization of this moiety took place
during hydrolysis. To verify this, 1 was hydrolyzed in 6 M
DCl/D2O, which gave peaks for α-deuterated L-Nma and D-
Nma at m/z 397 generated by H/D exchange but a single peak
for L-Nma at m/z 396 (Fig. S9). Therefore, Nma was concluded
to have an L-chilarity. The absolute configurations of αHpp and
Hiv units were determined by analyzing the hydrolysate of 1 on
a ligand-exchange type chiral-phase HPLC column (Sumichiral
OA-5000) in comparison with commercially available standard
α-hydroxy acids. αHpp and Hiv in the hydrolysate eluted at 6.2
and 28.2 min, respectively, which were consistent with the
standard (S)-αHpp (tR = 6.2 min) and (S)-Hiv (tR = 28.2 min),
respectively (Fig. S10).
The remaining stereochemistry in βHpp was determined
by synthesis of a degradation product containing this resi-
due. Methanolysis of 1 in methanolic NaOMe gave amide 2
(Fig. 3). Since the L (= S) configuration was already
assigned to Nma, two possible diastereomers of 2 were
prepared. Esterification of L-Nma in HCl/MeOH yielded
methyl ester of L-Nma, which was condensed with (R)- and
(S)-3-hydroxy-3-phenylpropanoic acid by using BOP
reagent and HOBt to give (R,S)- and (S,S)-2 (Fig. 3). The 1H
NMR spectrum of 2 derived from 1 was completely iden-
tical to that of (S,S)-2 but not of (R,S)-2. Thus, an S-con-
figuration was assigned to βHpp (Figs. S19 and S20).
Antimicrobial activity of 1 was tested against four bac-
teria, Bacillus subtilis ATCC6633, Kocuria rhizophila
Fig. 3 Partial degradation of 1 and synthesis of (R,S)- and (S,S)-2
ATCC9341, Staphylococcus aureus FDA209P JC-1, and
Escherichia coli NIHJ JC-2, two yeasts, Candida albicans
NBRC0197 and Saccharomyces cerevisiae S100, and two
filamentous fungi, Glomerella cingulata NBRC5907 and
Trichophyton rubrum NBRC5467. Compound 1 inhibited
the growth of S. aureus and S. cerevisiae at MIC of
3.1 μg ml−1. Cytotoxicity against P388 murine leukemia
cells was not observed at 50 μM.
In summary, a chemical investigation of an under-
explored fungus, Pithomyces chartarum TAMA 581, led to
the discovery of a new tetradepsipeptide, pithohirolide (1).
Compound 1 is composed of two α-hydroxy acids (αHpp,
Hiv), one β-hydroxy acid (βHpp), and one N-methylated
amino acid (Nma), which are connected into a 13-
membered ring by one amide and three ester linkages.
Contiguous triester structures in cyclic compounds are very
rare in nature. The most similar compound is pithomycolide
from Pithomyces, which is composed of five units, two
βHpps and one each of Hiv, Ala, and Nma, connected
through three contiguous ester linkages and two amide
bonds (Fig. 4) [15]. Angolide from Pithomyces is the tet-
radepsipeptide similar to 1, but two ester and two amide
linkages are aligned alternatively [16]. Pimaydolide, a
hexadepsipeptide also isolated from Pithomyces, possesses
three ester linkages, which are not contiguously connected
[17]. Discovery of 1 offers an additional proof regarding the
biosynthetic potential of Pithomyces and assures the
importance of exploiting underexplored/unexplored micro-
organisms to obtain new chemical entities.
Experimental section
General experimental procedures
The optical rotation was measured using a JASCO P-1030
polarimeter. UV and IR spectra were recorded on a

Pithohirolide, an antimicrobial tetradepsipeptide from a fungus Pithomyces chartarum
461
FeSO4·7H2O 0.0005%, and ZnSO4·7H2O 0.0005% in
deionized water. The flasks were incubated at 25 °C for
21 days statically in dark.
Extraction and Isolation
At the end of the fermentation period, 20 ml of 1-butanol
was added to each flask for extraction. Evaporation of the
solvent gave 15.8 g of extract from 1 l of culture. The
extract was subjected to silica gel column chromatography
with a step gradient of CHCl3–MeOH (1:0, 20:1, 10:1, 4:1,
2:1, 1:1, and 0:1 v/v). Fraction 3 (10:1) was concentrated to
give 11.0 g of a dark green viscous oil. A part of fraction 3
(0.50 g) was then fractionated by ODS column chromato-
graphy with a gradient of MeCN-0.1% HCO2H aqueous
solution (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 v/v). Fraction 6
(7:3) was concentrated in vacuo to give a semipure material
(18 mg), which was subjected to preparative HPLC (Cos-
mosil Cholester 5 µm, 10 × 250 mm, 4 ml min−1, UV
detection at 254 nm) with MeCN and 0.1% HCO2H aqu-
eous solution (65:35) to yield 1 (12 mg, tR 9.4 min).
Fig. 4 Natural products structurally related to 1
Shimadzu UV-1800 and a Shimadzu FT-IR-300 spectro-
photometer, respectively. NMR spectra were obtained on a
Bruker AVANCE 500 spectrometer by referring to signals
of the residual solvent protons (CD3OD: δH 3.31; CDCl3: δH
7.27) and carbons (CD3OD: δC 49.2; CDCl3: δH 77.0) as
internal standards. HR-ESITOFMS were measured on a
Bruker micrOTOF spectrometer. HPLC analysis and pur-
ification were performed on an Agilent HP1200 system
equipped with a diode array detector.
Pithohirolide (1)
Pale yellow amorphous; [α]22 -92 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 203 (4.30), 263 (2.39) nm; IR νmax
D
(ATR) 3438, 2929, 1702, 1674, 1466, 1415, 1348, 1204,
1039, 739 cm−1; H and 13C NMR data, see Table 1; HR-
1
ESITOFMS m/z 504.1990 [M + Na]+ (calcd for
C27H31NNaO7, 504.1993).
Microorganism
Marfey’s analysis
Strain TAMA 581 was isolated from rotten bark, which was
collected near Rainbow Falls & Boiling Pots, Hiro, Hawaii,
USA in 2005. Zonate colonies grew rapidly. The conidial
area showed dark red and brown, and powdery morphology.
Conidia born from short conidiogenous cells in broadly
ellipsoidal shape, with 3-4 transverse and mostly two
longitudinal septa. They were often constricted at the septa
with brown and verruculose appearence. These distinct
morphological characteristics led us to identify the strain as
Pithomyces chartarum (Berk. & M. A. Curtis) M. B. Ellis.
Compound 1 (0.5 mg) was hydrolyzed at 110 °C in 6 M
DCl-D2O (200 μl) for 12 h, and the reaction mixture was
dried under a stream of N2. The resulting residue and
the standard L-Nma (0.1 mg) were placed in vials, to which
0.1 M NaHCO3 (100 μl) and 1-fluoro-2,4-dinitrophenyl-5-L-
leucinamide (L-FDLA) or D-FDLA in acetone (0.05 mg in
50 μl) were added and sealed. The vials were warmed at
50 °C for 30 min, and then quenched by the addition of
0.2 M HCl (50 μl), followed by dilution with MeCN-0.05%
TFA (100 μl, 50:50). The DLA derivatives of the hydro-
lysate and standard amino acids were analyzed by LC/MS
on a Cosmosil 5C18-AR-II column (Nacalai Tesque Inc.,
2.0 × 150 mm) eluted with MeCN-0.1% HCO2H aqueous
solution (35:65) at a flow rate of 0.2 ml min−1, in the
negative mode, monitoring at m/z 396 (C16H22N5O7) or 397
(C16H21DN5O7). Retention times for the amino acid stan-
dards were 13.5 min for L-DLA-L-Nma and 14.3 min for D-
DLA-L-Nma (chromatographically equivalent to L-DLA-D-
Nma) and the deuterated (racemized) L-DLA derivative of
the hydrolysate gave peaks at 13.5 min 14.3 min, while the
Fermentation
Fifty 150-ml flasks each containing 20 ml buckwheat
medium were inoculated with mycelia from well-grown
slant of TAMA 581. The buckwheat medium was prepared
from buckwheat (purchased from a food vendor in Gunma,
Japan) and SB solution (1:1). SB solution was prepared
from yeast extract (Oriental Yeast) 0.01%, sodium tartrate
0.005%, K2HPO4 0.005%, MgSO4·7H2O 0.005%,

462
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.
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