Asperterrestide A, a cytotoxic cyclic tetrapeptide from the marine-derived fungus Aspergillus terreus SCSGAF0162

Article abstract of DOI:10.1021/np300897v

A new cytotoxic and antiviral cyclic tetrapeptide, asperterrestide A (1), a new alkaloid, terremide C (2), and a new aromatic butenolide, aspernolide E (3), together with 10 known compounds were isolated from the fermentation broth of the marine-derived fungus Aspergillus terreus SCSGAF0162. Their structures were elucidated by spectroscopic analysis, and the absolute configuration of 1 was determined by the Mosher ester technique and analysis of the acid hydrolysates using a chiral-phase HPLC column. Compound 1 contains a rare 3-OH-N-CH₃-Phe residue and showed cytotoxicity against U937 and MOLT4 human carcinoma cell lines and inhibitory effects on influenza virus strains H1N1 and H3N2.

Full text of DOI:10.1021/np300897v

Note
pubs.acs.org/jnp
Asperterrestide A, a Cytotoxic Cyclic Tetrapeptide from the Marine-
Derived Fungus Aspergillus terreus SCSGAF0162
Fei He,† Jie Bao,† Xiao-Yong Zhang,† Zheng-Chao Tu,‡ Yi-Ming Shi,§ and Shu-Hua Qi*,†
†Key Laboratory of Marine Bio-resources Sustainable Utilization/RNAM Center for Marine Microbiology/Guangdong Key
Laboratory of Marine Material Medical, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang
Road, Guangzhou 510301, People’s Republic of China
‡Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Road, Guangzhou 510530, People’s
Republic of China
§State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, People’s Republic of China
S
* Supporting Information
ABSTRACT: A new cytotoxic and antiviral cyclic tetrapeptide, asperterrestide A (1), a new alkaloid, terremide C (2), and a new
aromatic butenolide, aspernolide E (3), together with 10 known compounds were isolated from the fermentation broth of the
marine-derived fungus Aspergillus terreus SCSGAF0162. Their structures were elucidated by spectroscopic analysis, and the
absolute configuration of 1 was determined by the Mosher ester technique and analysis of the acid hydrolysates using a chiral-
phase HPLC column. Compound 1 contains a rare 3-OH-N-CH3-Phe residue and showed cytotoxicity against U937 and
MOLT4 human carcinoma cell lines and inhibitory effects on influenza virus strains H1N1 and H3N2.
arine microorganisms have proved to be an important
source of pharmacologically active metabolites, and a
C,13 arisugasin H,14 arisugasin D,14 territrem B,13 and territrem
A,13 were isolated. Compound 1 was tested for its cytotoxicity
toward human carcinoma U937, K562, BGC-823, MOLT-4,
MCF-7, and A549 cell lines and antiviral activity toward two
influenza virus strains.
M
growing number of marine-derived fungi have been reported to
produce metabolites with unique structures and interesting
biological activities.1 The genus Aspergillus (Moniliaceae), with
over 180 species, has attracted considerable attention as a rich
source of alkaloids, terpenoids, xanthones, and polyketides,
some of which showed antifungal, antibacterial, antifouling, and
cytotoxic activities.2−4 Aspergillus terreus is commonly isolated
from soil with worldwide distribution and has been found to
produce a number of bioactive compounds, such as terreineol,5
terrain,6 aspulvinone,7 butyrolactone I,8 and terreic acid.9 With
the aim of searching for novel bioactive natural compounds
from marine fungi, we investigated the chemical constituents of
a fermentation broth of the marine-derived fungal strain A.
terreus SCSGAF0162. Three new compounds, the cyclic
tetrapeptide asperterrestide A (1), the alkaloid terremide C
(2), and the aromatic butenolide aspernolide E (3), together
with 10 known compounds, methyl 3,4,5-trimethoxy-2-(2-
(nicotinamido)benzamido)benzoate (4),10 butyrolactone I,8
butyrolactone III,11 butyrolactone II,8 4-(4-hydroxyphenyl)-5-
(4-hydroxyphenylmethyl)-2-hydroxyfuran-2-one,12 territrem
Asperterrestide A (1) was obtained as a yellowish powder. It
was assigned a molecular formula of C26H32N4O5 on the basis
of its HRESIMS data. Analysis of the 1H and 13C NMR spectra
(Table 1) revealed the presence of three amide N-H protons
(δH 6.48, 7.18, and 9.17), four methyl groups (including one N-
methyl), one methylene group, two phenyl rings, five methines
(four of which were bound to heteroatoms), and four carbonyl
1
carbons. Interpretation of the H, 13C, and 2D NMR data of 1
suggested a peptidic nature for the molecule. By analysis of the
COSY and HMBC spectra of 1, the aromatic protons at δH 7.36
(1H, d, J = 8.0 Hz), 7.15 (1H, dd, J = 7.5, 8.0 Hz), 7.48 (1H,
dd, J = 7.5, 8.0 Hz), and 8.17 (1H, d, J = 8.0 Hz) represented
an ortho-disubstituted benzene ring. HMBC correlations from
Received: December 22, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np300897v | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
and alanine (Ala) were completely assigned. Specifically, the
loss of Ala (m/z 71) and Ile (m/z 113) fragments was deduced
from the ESIMS2 spectrum (Supporting Information). Upon
extensive analysis of these data, asperterrestide A (1) was
assigned as a cyclic tetrapeptide containing ABA, 3-OH-N-Me-
Phe, Ile, and Ala.
The amino acid sequence of 1 was deduced from NOESY
correlations between α-protons and neighboring residue NH
groups, with support from HMBC correlations. The N-H
proton of ABA (δH 9.17) showed an HMBC correlation with
the carbonyl carbon of C-8, indicating that it was acylated by 3-
OH-N-Me-Phe, and this connection was also confirmed by the
NOESY correlation between the N-H of ABA and H-9 (Figure
1). An HMBC correlation from H3-17 to C-18 supported the
Figure 1. Key NOESY correlations of compound 1.
Table 1. NMR Data for 1 (500 MHz for 1H and125 MHz for
13C NMR in CDCl3)
connection of Ile and 3-OH-N-Me-Phe. HMBC correlations
from the N-H proton (δH 7.18) and H-19 of Ile to C-24,
together with the NOESY correlation between H-25 and the N-
H proton of Ile, supported the connection of the Ile and Ala
residues. The HMBC correlation from the N-H proton (δH
6.48) of Ala to C-1 supported the connection of Ala and ABA
residues. Therefore, the complete sequence of 1 was deduced.
The large coupling constant (J = 10.0 Hz) between H-9 and
H-10 of the β-hydroxy-N-Me-phenylalanine residue suggested a
syn relative configuration for the two protons.15 A molecular
modeling experiment and the observation of NOE correlations
of H3-17 with H-10 and of H-9 with N-H (ABA) in the
NOESY spectrum of 1 (Figure 1) further confirmed this
deduction. The geometries for modeling were obtained in
Chem3D 11.0 and then optimized at the AM1 level using
Gaussian 09.16 The absolute configurations of the Ala and Ile
residues were determined by analysis of acid hydrolysates on a
chiral-phase HPLC column17,18 and Marfey’s methods.19,20
HPLC analyses of the mixture of hydrolysates and authentic
samples (co-injection) confirmed D-Ala in 1. Through a chiral-
phase HPLC analysis, the D-Ile and D-allo-Ile residues could not
be differentiated because the retention times of these two
compounds were identical. D-Ile and D-allo-Ile were not
resolved even using Marfey’s method.21 The absolute
configuration of C-10 in 1 was determined using the modified
Mosher’s ester method in an NMR tube.22 Compound 1 was
treated separately with (R)-(−)- and (S)-(+)-α-methoxy-α-
(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) in pyridine-
d5 in an NMR tube to yield the (S)- and (R)-MTPA ester
derivatives 1a and 1b, respectively. The neighboring signals of
H-10 were clearly different in the diastereomeric MTPA esters
(1a and 1b), and the observed chemical shift differences
(ΔδS−R, Figure 2) unambiguously indicated the absolute
configuration of C-10 of 1 to be S. Consequently, the structure
of 1 was elucidated as cyclo-[ABA-D-Ala-Ile-3(S)-OH-N-Me-
Phe].
b
unit
position
δC, type
δH, mult (J in Hz)
HMBC
ABA
1
170.3, C
2
126.7, C
3
125.6, CH
124.5, CH
131.5, CH
123.5, CH
135.1, C
7.36, d (8.0)
1, 5, 7
4
7.15, dd (7.5, 8.0) 2, 6
5
7.48, dd (7.5, 8.0) 3, 6, 7
6
8.17, d (8.0)
2, 4, 7
2, 6
7
NH
8
9.17, s
3-OH-N-Me-
Phe
169.1, C
9
60.9, CH
71.4, CH
138.7, C
5.76, d (10.0)
5.15, d (10.0)
8, 10, 11, 17
8, 9, 11, 12
10
11
12/16
13/15
14
17
18
19
a
127.5, CH
128.5, CH
127.6, CH
7.39
10, 13
a
7.33
a
a
7.26
31.4, CH3 2.75, s
172.5, C
9, 18
Ile
53.9, CH
4.31, dd (10.0,
10.5)
20, 21, 23
20
21
36.1, CH
1.75, m
24.7, CH2 0.30, m
19, 20, 22,
23
22
23
NH
24
25
26
10.8, CH3 0.54, dd (7.0, 7.5) 20, 21
14.5, CH3 0.79, d (6.5)
7.18, d (9.5)
19, 20, 21
Ala
171.3, C
50.1, CH
4.52, m
24, 26
24, 25
25, 26
14.8, CH3 1.43, d (7.0)
6.48, d (8.0)
NH
a
b
Overlapped signals. HMBC correlations are from proton(s) started
to the indicated carbon.
H-3 (δH 7.36) to C-1 (δC 170.3) and from N-H (δH 9.17) to C-
7 (δC 135.1) led to the identification of an anthranilic acid
(ABA) unit. Similarly, three other amino acid units including 3-
OH-N-Me-phenylalanine (3-OH-N-Me-Phe), isoleucine (Ile),
Terremide C (2) gave an HRESIMS ion peak at m/z
448.1510 [M + H]+, corresponding to the molecular formula
B
dx.doi.org/10.1021/np300897v | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
further confirmed by HMBC correlations between H-2′ (δH
8.67)/H-6′ (δH7.79) and C-3 (δC 131.5). Furthermore, the
carbon signal of C-1 shifted upfield from δC 168.1 (in 4) to δC
162.5 (in 2), revealing the existence of a quinazoline fragment
as in terremide B.3 The structure of asperterrestide B (2) was
determined as 3,4,5-trimethoxyl-2-(4-oxo-2-(pyridine-3-yl)-
quinazolin-3(4H)-yl) benzoate.
Aspernolide E (3) was assigned the molecular formula
C24H22O7 through an analysis of its HRESIMS data, requiring
14 degrees of unsaturation. From the 1H and 13C NMR spectra
(Table 2), signals for 1,2-disubstituted benzene protons at δH
7.62 (2H, d, J = 8.5 Hz) and 6.93 (2H, d, J = 8.5 Hz) and 1,2,4-
trisubstituted benzene protons at δH 6.46 (1H, d, J = 2.0 Hz),
6.52 (d, J = 8.0 Hz), and 6.56 (dd, J = 8.0, 2.0 Hz) were
recognized. Comparison of the 1H NMR data of 3 with those of
aspernolide A (5) revealed one additional cis-double bond in
3.23 In the HMBC spectrum, correlations from δH 6.12 (1H, d,
J = 10.0 Hz) to C-4″ and C-9″ and from δH 5.54 (1H, d, J =
10.0 Hz) to C-3″ and C-9″ indicated the position of the double
bond between C-7″ and C-8″. The similar specific rotation
values of 3 ([α]3D0 +100) and aspernolide A ([α]2D8 +88.7)
suggested the same R configuration at C-4.
Figure 2. Δδ (δS − δR) values in ppm for MTPA esters of 1.
C24H21N3O6, which required 16 degrees of unsaturation. The
UV spectrum displayed absorptions at λmax 225 and 268 nm.
1
The H and 13C NMR spectra (Table 2) were quite similar to
1
Table 2. H (500 MHz) and 13C (125 MHz) NMR Data for
Compounds 2 and 3 in CDCl3
b
c
2
3
δH, mult (J in
Hz)
δH, mult (J in
Hz)
position
δC, type
δC, type
d
Compound 1 showed cytotoxicity toward human carcinoma
U937 and MOLT4 cell lines with IC50 values of 6.4 and 6.2
μM, respectively. In addition, compound 1 showed inhibitory
effects on the influenza virus strains A/WSN/33 (H1N1) and
A/Hong Kong/8/68 (H3N2) with IC50 values of 15 and 8.1
μM, respectively.
1
2
3
4
5
6
162.5, C
d
131.5, C
148.0, C
127.8, C
86.7, C
169.7, C
a
a
128.2, CH 7.84
38.6, CH2 3.45, d (15)
3.55, d (15)
7
134.8, CH 7.84
EXPERIMENTAL SECTION
■
8
127.3, CH 7.56, m
127.8, CH 8.35, d (8.0)
121.0, C
General Experimental Procedures. Optical rotations were
determined with an MCP 300 (Anton Paar) polarimeter at 25 °C.
UV spectra were recorded on a U-2910 spectrometer (Hitachi). NMR
spectra were recorded with an Avance 500 spectrometer (Bruker) at
500 MHz for the 1H nucleus and 125 MHz for the 13C nucleus.
Chemical shifts (δ) are given with reference to TMS. ESIMS spectra
were obtained with an Esquire 3000 Plus spectrometer (Bruker).
HRESIMS data were acquired on a micro TOF-QII mass spectrometer
(Bruker). Column chromatography was performed using silica gel
(100−200 mesh; Qingdao Marine Chemicals) and Sephadex LH-20
(Amersham Pharmacia). HPLC was carried out on an ODS column
(250 × 4.6 mm, 5 μm; Phenomenex) with a photodiode array detector
(Shimadzu).
9
10
1′
2′
3′
4′
5′
122.8, C
149.0, CH 8.67, s
129.6, CH
115.8, CH
156.7, CH
115.8, CH
7.62, d (8.5)
6.93, d (8.5)
150.2, CH 8.49, d (4.0)
122.5, CH 7.16, dd (5.0,
5.0)
6.93, d (8.5)
7.62, d (8.5)
6′
135.7, CH 7.79, d (8.0)
123.3, C
129.6, CH
124.5, C
1″
2″
3″
4″
5″
6″
125.1, C
128.3, CH
121.0, C
6.46, d (2.0)
149.0, C
Fungal Materials. The fungal strain of Aspergillus terreus
SCSGAF0162 was isolated from the tissue of the gorgonian
Echinogorgia aurantiaca collected from Sanya, Hainan Province,
China. The strain was identified by one of the authors (X.-Y.Z.),
and a voucher specimen (A. terreus SCSGAF0162) has been deposited
in the RNAM Center for Marine Microbiology, South China Sea
Institute of Oceanology, Chinese Academy of Sciences.
Fermentation and Isolation. The fungal strain A. terreus
SCSGAF0162 was cultivated in 10 L of liquid medium (20 g D-
sorbitol, 3.0 g yeast extract, 0.3 g MgSO4·7H2O, 10 g L-lysine, 0.5 g
KH2PO4, 100 g maltose, 100 g NaCl in 1 L of water after adjusting its
pH to 7.0, in 500 mL Erlenmeyer flasks each contain 200 mL of
culture broth) at 28 °C without shaking for one month. The
fermented whole broth (10 L) was filtered through cheesecloth to
separate the supernatant from the mycelia, and the mycelia were
extracted three times with acetone. The acetone solution was
evaporated under reduced pressure to afford an aqueous solution,
which was then extracted three times with EtOAc to afford the EtOAc
extract (10 g). The EtOAc-soluble part (10 g) was chromatographed
on silica gel using gradient elution from 100% (v/v) CHCl3 to 100%
MeOH (v/v), to give five fractions (Fr. A−E). Fr. A (100% CHCl3)
contained most of the nonpolar constituents such as fatty acids and
sterols and was not further investigated. Fraction B (CHCl3/MeOH,
146.5, C
152.2, C
153.5, C
116.0, CH
131.5, CH
6.52, d (8.0)
109.5, CH 7.29, s
6.56, dd (8.0,
2.0)
7″
8″
9″
165.0, C
122.1, CH
130.6, CH
76.8, C
6.12, d (10.0)
5.54, d (10.0)
10″
11″
26.8, CH3 1.27, s
27.9, CH3 1.37, s
a
b
Overlapped signals. Chemical shifts of methoxy groups in 2: 3″-
OMe δH 3.79 (s), δC 60.9; 4″-OMe δH 3.82 (s), δC 61.5; 5″-OMe δH
c
3.87 (s), δC 56.2; 7″-OMe δH 3.72 (s), δC 52.5. Chemical shift of
d
methoxy groups in 3: 5-OMe δH 3.78 (s), δC 53.5. Not observed.
those of 4 and differed only in the absence of one amide
carbonyl signal (δC 163.2) in 4 and the appearance of a
quaternary carbon signal (δC 131.5) in 2. Comparison of the
molecular formulas of 2 and 4 also suggested that 2 might be
produced by dehydration of 4. Heating 4 in pyridine at 100 °C
for 8 h formed 2 in 80% yield, proving this hypothesis. This was
C
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Journal of Natural Products
Note
98:2−95:5 v/v elution, 1.2 g) was further purified by semipreparative
reversed-phase HPLC (MeOH/H2O, 70% v/v, 3 mL/min, UV
detector 230 nm) to yield 3 (2 mg), butyrolactone I (10 mg),
butyrolactone III (8 mg), butyrolactone II (5 mg), and 4-(4-
hydroxyphenyl)-5-(4-hydroxyphenylmethyl)-2-hydroxyfuran-2-one (6
mg). Fraction C (CHCl3/MeOH, 95:5−9:1 v/v elution, 2 g) was
chromatographed on a C18 silica gel column to afford five subfractions,
C1−C5. Fr. C2 was subjected to semipreparative reversed-phase
HPLC (MeOH/H2O, 60% v/v, 3 mL/min, UV detector 250 nm), and
compounds territrem C (3 mg), territrem B (5 mg), and territrem A
(3 mg) were isolated. Then Fr. C3 was purified by semipreparative
reversed-phase HPLC (MeOH/H2O, 50% v/v, 3 mL/min, UV
detector 250 nm), resulting in the isolation of compounds arisugasin
H (2 mg) and arisugasin D (6 mg). Fr. C4 followed by semipreparative
reversed-phase HPLC (ACN/H2O, 50% v/v, 3 mL/min, UV detector
230 nm) afforded compounds 1 (10 mg). Finally, Fr. C (CHCl3/
MeOH, 9:1−7:3 v/v elution, 800 mg) was further purified on a
Sephedex LH-20 column (MeOH) and by semipreparative reversed-
phase HPLC (CH3CN/H2O, 40% v/v, 3 mL/min, UV detector 230
nm) to yield 2 (2 mg) and 4 (5 mg).
were the same in this method, the configuration of the Ile residue was
not further determined.
Cytotoxicity Assay. Due to the small amount of the new
compounds, only compound 1 was tested for its cytotoxicity.
Cytotoxic activity was evaluated using human leukemic monocyte
lymphoma U937, erythroid leukemic K562, gastric carcinoma BGC-
823, acute lymphoblastic leukemia MOLT-4, breast adenocarcinoma
MCF-7, and lung carcinoma A549 cell lines by the MTT method as
described previously.24 Taxol was used as positive control against the
U937, K562, BGC-823, MOLT-4, Mcf7, and A549 cell lines with IC50
values of 1.9, 4.9, 3.5, 1.8, 5.0, and 3.6 nM, respectively.
Antiviral Assay. Due to the small amount of the new compounds,
only compound 1 was tested for its antiviral activity. Compound 1 was
tested against influenza virus strain A/WSN/33 (H1N1) (an M2-
resistant strain) and strain A/Hong Kong/8/68(H3N2) (an M2-
sensitive strain) by CPE assay to determine its inhibitory effect against
virus replication in the cell MDCK.25 RIBA was used as positive
control against H1N1 and H3N2 with IC50 values of 20.2 and 0.41
μM, respectively.
Asperterrestide A (1): yellowish, amorphous powder; [α]3D0 −13
ASSOCIATED CONTENT
1
■
(c 0.03, MeOH); UV (MeOH) λmax (log ε) 220 (3.57) nm; H and
13C NMR data, see Table 1; ESIMS m/z 503 [M + Na]+; HRESIMS
m/z 503.2274 [M + Na]+ (calcd for C26H32N4O5Na, 503.2265).
Terremide C (2): yellowish, amorphous powder; [α]3D0 +40 (c
0.005, MeOH); UV (MeOH) λmax (log ε) 225 (4.41), 268 (3.22) nm;
1H and 13C NMR data, see Table 2; ESIMS m/z 448 [M + H]+;
HRESIMS m/z 448.1510 [M + H]+ (calcd for C24H22N3O6,
448.1503).
S
* Supporting Information
This material (1H, 13C NMR, DEPT, HSQC, HMBC, COSY,
NOESY, ESI, and HRESIMS spectroscopic data for compounds
1−3, 1H NMR data for compounds 1a and 1b, HMBC
spectroscopic data for 1a, and Marfey’s results) is available free
of charge via the Internet at http://pubs.acs.org.
Aspernolide E (3): yellowish, amorphous powder; colorless gum;
[α]3D0 +100 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 228 (3.47),
AUTHOR INFORMATION
■
1
325 (4.13) nm; H and 13C NMR data see Table 2; ESIMS m/z 423
Corresponding Author
*E-mail: shuhuaqi@scsio.ac.cn. Tel: (86) 20-89022112.
[M + H]+; HRESIMS m/z 423.1432 [M + H]+ (calcd for C24H23O7,
423.1438).
Notes
Preparation of the (R)- and (S)-MTPA Ester Derivatives of 1.
Compound 1 (0.6 mg) was transferred to a clean NMR tube and dried
completely. Deuterated pyridine (0.5 mL), R-(−)-MTPA-Cl (12 μL),
and 0.2 mg of DMAP (4-dimethylaminopyridine) were added to the
NMR tube under a N2 gas stream. The reaction mixture was shaken
for 5 h at room temperature to yield the (S)-MTPA ester derivative of
1 (1a). The 1H NMR data of 1a were directly obtained from the NMR
tube, and the data were assigned on the basis of HMBC correlations.
In the same fashion, the (R)-MTPA ester derivative of 1 (1b) was
obtained from the reaction mixture of 1 (0.6 mg) in deuterated
pyridine (0.5 mL), S-(+)-MTPA-Cl (12 μL), and 0.2 mg of DMAP
(0.2 mg). Key 1H NMR shifts (ppm) for 1R (500 M Hz, pyridine-d5):
8.290 (H-6), 9.945 (NH-ABA), 6.473 (H-9), 6.933 (H-10), 7.529 (H-
12/H-16), 3.047 (H-17), and for 1S (500 M Hz, pyridine-d5): 8.183
(H-6), 9.854 (NH-ABA), 6.486 (H-9), 7.043 (H-10), 7.723 (H-12/H-
16), 3.030 (H-17).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the National Basic Research
Program of China (973 Program, grant 2010CB833803),
National Natural Science Foundation of China (grant
41206130), National High Technology Research and Develop-
ment Program of China (863 Program, grant 2012AA092104),
National Marine Public Welfare Research Project of China
(grant 201305017), and Chinese Academy of Sciences (grant
5060267) for financial support. The calculation sections were
supported by HPC Center of Kunming Institute of Botany,
CAS, China.
Chiral-Phase HPLC Analysis of the Acid Hydrolysates of 1.
To determine the absolute configurations of the Ala and Ile in 1,
chiral-phase HPLC analyses of the acid hydrolysates were conducted.
Compound 1 (0.50 mg) was hydrolyzed as described. Two different
analytical conditions were used to analyze the Ala and Ile isomers,
respectively. First, the dried hydrolysate was dissolved in 100 μL of 2
mM CuSO4/H2O solution. Ten microliters of this sample was
analyzed by HPLC with a chiral-phase column (MCIGELCRS10W,
4.6 × 50 mm, Mitsubishi Chemical Corporation) using a 2 mM
CuSO4/H2O solution as the mobile phase at a flow rate of 0.5 mL/min
with UV detection at 254 nm. L-Ile, L-allo-Ile, D-Ile, and D-allo-Ile were
detected as references. The retention times of D-Ile, D-allo-Ile, L-Ile,
and L-allo-Ile were 13.6, 13.6, 31.2, and 22.4 min, respectively. Second,
D-Ala and L-Ala were detected as references by HPLC with a chiral
column (MCIGELCRS10W, 4.6 × 50 mm) using a 0.5 mM CuSO4/
H2O solution as the mobile phase at a flow rate of 0.5 mL/min with
UV detection at 254 nm. The retention times of D-Ala and L-Ala were
9.09 and 7.79 min, respectively. Hence, the Ala and Ile residues in 1
were determined to be D-Ala (8.71 min) and D-Ile or D-allo-Ile (13.1
min), respectively. Because the retention times of D-Ile and D-allo-Ile
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