Trichoderins, novel aminolipopeptides from a marine sponge-derived Trichoderma sp., are active against dormant mycobacteria

Article abstract of DOI:10.1016/j.bmcl.2010.04.100

Three new aminolipopeptides, designated trichoderins A (1), A1 (2), and B (3), were isolated from a culture of marine sponge-derived fungus of Trichoderma sp. as anti-mycobacterial substances with activity against active and dormant bacilli. The chemical structures of trichoderins were determined on the basis of spectroscopic study. Trichoderins showed potent anti-mycobacterial activity against Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis H37Rv under standard aerobic growth conditions as well as dormancy-inducing hypoxic conditions, with MIC values in the range of 0.02-2.0 μg/mL.

Full text of DOI:10.1016/j.bmcl.2010.04.100

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3658–3663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Trichoderins, novel aminolipopeptides from a marine sponge-derived
Trichoderma sp., are active against dormant mycobacteria
Patamaporn Pruksakorn a, , Masayoshi Arai a, , Naoyuki Kotoku a, Catherine Vilchèze b, Anthony D. Baughn b,
Prashini Moodley c, William R. Jacobs Jr. b, Motomasa Kobayashi a,
*
a Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan
b Howard Hughes Medical Institute, Price Translational Research Center, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx, NY 10461, USA
c Nelson R. Mandela School of Medicine, University of KwaZulu Natal, 719 Umbilo Road, Congella Durban 4013, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new aminolipopeptides, designated trichoderins A (1), A1 (2), and B (3), were isolated from a cul-
ture of marine sponge-derived fungus of Trichoderma sp. as anti-mycobacterial substances with activity
against active and dormant bacilli. The chemical structures of trichoderins were determined on the basis
of spectroscopic study. Trichoderins showed potent anti-mycobacterial activity against Mycobacterium
smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis H37Rv under standard aerobic
growth conditions as well as dormancy-inducing hypoxic conditions, with MIC values in the range of
Received 2 March 2010
Revised 17 April 2010
Accepted 21 April 2010
Available online 26 April 2010
Keywords:
Trichoderin
Trichoderma sp.
Aminolipopeptide
Anti-mycobacterial activity
Dormant
0.02–2.0 lg/mL.
Ó 2010 Elsevier Ltd. All rights reserved.
Tuberculosis
Tuberculosis is one of the most common causes of morbidity
and mortality in HIV-positive adults living in poverty.1 Aggravation
of tuberculosis is evaded by host immune systems, even though
infection is concluded completely. However, a small population
of bacilli enter into a dormant state in the granuloma which is
formed by immune cells and fibroblasts in an attempt to contain
the infection. Then, the bacilli maintain their ability to resume
growth and aggravate disease as a result of deterioration of
immune system. This unique property also relates to tolerance to
conventional anti-tuberculosis drugs such as isoniazid.2 Therefore,
new lead compounds, which are effective against Mycobacterium
tuberculosis in both active and dormant states, are urgently needed.
Although physiology of the latent M. tuberculosis infection is still
unclear, hypoxic incubation was found to induce the dormant state
of Mycobacterium sp., which has a drug susceptibility profile resem-
bling that of the latent M. tuberculosis.3
three new aminolipopeptides named trichoderins A (1),5 A1 (2),6
and B (3)7 from a culture of marine sponge-derived fungus of Trich-
oderma sp. 05FI48 as anti-dormant mycobacterial substances. In
this paper, the structure elucidation of trichoderins and their
anti-mycobacterial activities are presented.
The fungal strain 05FI48 of Trichoderma sp. was isolated from
unidentified marine sponge in 2005. The strain was cultured in
the rice medium containing artificial sea water (totally 2.3 kg of
unpolished rice and 4.5 L of artificial sea water) under static condi-
tion at 30 °C for 2 weeks. The culture was extracted by acetone and
mixed organic solvent of acetone/methanol/ethyl acetate (4:2:1),
and the crude extract was partitioned into a water/2-butanone mix-
ture. The 2-butanone soluble portion (51.2 g) was further parti-
tioned with an n-hexane and 90% methanol mixture. On the
guidance of bioassay, the 90% methanol soluble portion (37.7 g,
MIC 12.5 lg/mL against Mycobacterium bovis BCG under hypoxic
Recently, we established a screening system to search for sub-
stances that have activity against dormant mycobateria, and iso-
condition) was fractionated by silica gel column chromatography
(eluted ethyl acetate/methanol) to give eight fractions (Fr. A1-Fr.
A8). The active Fr. A4 (ethyl acetate/methanol = 5:1 eluted) was dis-
solved in methanol. Then, the MeOH soluble portion (2.5 g, MIC
lated macrocyclic alkaloids, halicyclamines, from
a marine
sponge of Haliclona sp. on the basis of bioassay-guided separation.4
In the continuous screening from marine organisms, we isolated
2.5
(chloroform/methanol/water = 30:3:1, lower phase) to afford four
fractions (Fr. B1-Fr. B4). The active Fr. B2 (390 mg, MIC 0.15 g/
mL) was subjected to Sephadex LH-20 column chromatography
and eluted with methanol. The active fraction (243 mg, MIC
lg/mL) was fractionated by silica gel column chromatography
l
* Corresponding author. Tel.: +81 66879 8215; fax: +81 66879 8219.
E-mail address: kobayasi@phs.osaka-u.ac.jp (M. Kobayashi).
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.100

P. Pruksakorn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3658–3663
3659
0.04
l
g/mL) was purified by reversed-phase HPLC [CAPCELL PAK C8
ysis of the dabsylated amino acids revealed that compound 1 con-
tained Pro, Aib ( -aminoisobutyric acid), Ile and Val in the
DD (10 mm id ꢀ 250 mm); eluted with acetonitrile/water contain-
ing 0.1% TFA = 7:3] to furnish trichoderin A (1) (89 mg, 0.174% yield
from the 2-butanone extract), trichoderin A1 (2) (18 mg, 0.035%),
and trichoderin B (3) (8 mg, 0.016%) as TFA salts, respectively
(Fig. 1).
a
structure. The order of amino acid sequence was determined
through the observation of a series of the fragment ions by
ESI-TOF-MSMS (Table 1 and Fig. 2). The fragment ions from
ESI-TOF-MSMS experiments indicated cleavage of the amide bond
leading the predominant B-type fragmentation.9 In addition, the
difference of 213 amu. as shown in Figure 2 suggested the presence
of an unusual amino acid, 2-amino-6-hydroxy-4-methyl-8-oxo-
decanoic acid (AHMOD), from the reported data of MSMS analysis
in the structure-related aminolipopeptides, leucinostatins,10
trichopolyns,11 helioferins,12 and roseoferins.13
The chemical structure of 1 was also confirmed by NMR analy-
sis. The 13C NMR, DEPT, and HMQC spectra of 1 showed 60 carbon-
signals that were classified as 18 methyls, 18 methylenes, 10
methines, four quaternary carbons, nine amide carbonyls and one
ketone carbonyl. Since the last step for purification of 1 was carried
out under acidic condition, 1 was isolated as the protonated form
of the amino group in the C-terminal residue as observing of the
Trichoderin A (1) was obtained as a colorless amorphous solid.
The ESI-TOF MS of 1 showed a pseudomolecular ion peak at m/z
1163 [M]+. The molecular formula was determined as C60H111
-
N10O12 by high-resolution (HR-) ESI-TOF MS. The IR absorption at
3314 cmꢁ1and 1669 cmꢁ1 suggested the presence of hydroxyl
and/or amide groups, respectively. The observation of the signals
at 175–178 ppm in the 13C NMR spectrum and the downfield sig-
nals of the 1H NMR spectrum indicated peptide structure of 1. The
upfield signals of the 1H and 13C NMR spectra implied the presence
of aliphatic hydrocarbons in 1. These finding suggested that com-
pound 1 is an aminolipopeptide.
To confirm the amino acid composition of 1, compound 1 was
treated by dabsyl chloride following to acid hydrolysis.8 HPLC anal-
Figure 1. Chemical structures of trichoderins (1–3).
Table 1
ESI-TOF-MSMS analysis of trichoderins A (1), A1 (2) and B (3)
1
B1
B2
B3
B4
B5
B6
B7
B8
[M]+
1
2
3
169
169
169
266
266
266
479
461
479
564
546
564
649
631
649
762
744
748
861
843
847
946
928
932
1031
1013
1017
1163
1145
1149
1
2
3
MDA
MDA
MDA
Pro
Pro
Pro
AHMOD
AMOD
AHMOD
Aib 1
Aib 1
Aib 1
Aib 2
Aib 2
Aib 2
Ile
Ile
Val 1
Val 2
Val 2
Val 2
Aib 3
Aib 3
Aib 3
Aib 4
Aib 4
Aib 4
AMAE
AMAE
AMAE
MDA; 2-methyl decanoic acid. Pro; proline. AHMOD; 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid, AMOD; 2-amino-4-methy1-8-oxodec-6-enoic acid, Aib;
isobutyric acid, Ile; isoleucine, Val; valine. AMAE; 2-[(20-aminopropyl) methylamino] ethanol.
a-amino-

3660
P. Pruksakorn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3658–3663
Figure 2. ESI-TOF-MSMS spectra for 1.
proton signal at dH 8.23. The presence of this quaternary amine
clarified the connection in the C-terminal residue by 1H-1H connec-
tivities (2J(H,H) coupling) of the quaternary amino proton at dH 8.23
and the adjacent protons at dH 3.06 (N-CH3), dH 3.09 (H-10) and dH
2.92 (H-2). In addition, the heteronuclear long range coupling
(3J(C,H)) between the N-CH3 at dH 3.06 and the methylene carbons
at dC 54.5 (C-2) and dC 62.4 (C-10) completely proved the connec-
tion of this part (Fig. 3 and Table 2). Thus, the C-terminus of 1
was deduced to be 2-[(20-aminopropyl) methylamino] ethanol
(AMAE).
The existing of AHMOD was confirmed by COSY and HMBC
analysis as the correlations between the ketone carbonyl at dC
212.3 (C-8) and the two adjacent methylene protons at dH 2.52
(H-9) and dH 2.59, 2.43 (H-7). (Fig. 3 and Table 2)
Figure 3. COSY and HMBC correlations for 1.

P. Pruksakorn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3658–3663
3661
Table 2
1H and 13C NMR data for trichoderins A (1), A1 (2) and B (3)
Position
1
2
3
a
b,c
a
b,c
a
b,c
dc
dH
dc
dH
dc
dH
MDAd
1
2
3
4
5
6
7
8
177.1
38.0
34.2
27.3
29.5
29.9
29.9
32.1
29.7
14.3
15.7
177.4
37.9
34.2
27.2
29.4
29.9
29.7
32.1
29.6
14.3
15.9
177.9
38.3
34.2
27.4
29.5
30.0
30.0
32.1
29.7
14.3
16.4
2.56
1.25, 1.49
1.20
1.21
1.25
1.25
1.24
1.23
0.87
2.65
1.28, 1.50
1.23
1.21
1.25
1.25
1.24
1.24
0.88
2.81
1.34, 1.50
1.28
1.25
1.27
1.27
1.24
1.26
0.87
9
10
11
1.12 (d, 6.4)
1.12 (d, 6.3)
1.21
Pro
CO
a
b
c
d
175.2
62.7
30.4
25.3
47.6
175.1
62.7
29.9
25.3
47.6
174.6
62.7
30.4
25.3
47.8
4.17
4.18
4.18
1.96, 2.16
1.92, 2.16
3.44, 3.99
1.99, 2.24
1.92, 2.23
3.48, 4.02
1.97, 2.17
1.94, 2.14
3.48, 4.18
AHMOD/NH
AMOD
9.07 (br)
9.13 (br)
9.01 (br)
1
2
3
4
5
6
7
8
175.5
55.4
34.7
25.9
45.3
64.5
50.9
212.3
37.0
7.7
175.3
55.4
36.7
28.7
39.6
145.8
131.9
201.7
33.0
8.4
175.1
55.0
35.8
25.7
45.5
64.1
50.8
212.5
37.1
7.8
4.14
1.25, 2.16
1.99
1.26, 1.46
4.06
2.43, 2.59
4.12
1.62, 1.83
2.23
1.99, 2.34
6.75
6.02 (d, 15.7)
4.14
1.95
2.14
1.24, 1.50
4.23
2.41, 2.61
9
10
11
2.52
1.03
0.88
2.55 (q, 7.2)
1.07 (t, 7.2)
0.85
2.54
1.01
0.85
20.2
19.2
20.0
Aib 1
CO
a
b
176.6
56.5
22.6
26.8
176.4
56.3
22.4
26.7
175.9
56.7
22.6
26.9
1.38
1.49
1.42
1.51
1.48
1.54
Aib 2
CO
a
b
177.3
56.4
22.1
27.2
177.2
56.4
22.1
27.3
177.2
56.5
22.6
27.1
1.41
1.51
1.39
1.52
1.42
1.52
Ile/Val 1
NH
CO
a
b
c
7.58 (d, 5.6)
7.64 (d, 5.5)
7.48 (d, 5.6)
175.3
62.1
35.8
27.1
15.6
11.5
175.2
62.0
35.8
26.7
15.6
11.4
175.0
63.5
29.5
20.5
19.5
3.63
1.99
1.23, 1.82
0.94 (d, 6.7)
0.88
3.68
1.99
1.24, 1.50
0.96 (d, 6.7)
0.88
3.58
2.21
1.13 (d, 6.7)
0.99 (d, 7.0)
d
Val 2
NH
CO
a
b
c
7.93
7.96 (d, 4.9)
7.83 (d, 5.6)
175.7
65.5
28.7
21.2
19.2
175.8
65.5
30.2
21.2
18.9
175.5
65.2
28.8
21.1
19.2
3.36 (dd, 11.4, 5.3)
3.37 (dd, 11.6, 5.5)
2.23
1.01 (d, 6.3)
0.88
3.38
2.20
1.02
0.87
2.22
1.02
0.87
Aib 3
Aib 4
AMAE
CO
a
b
177.4
56.8
22.9
27.4
177.3
56.8
22.9
27.4
177.0
57.0
22.9
27.4
1.41
1.52
1.41
1.54
1.41
1.52
CO
a
b
175.9
57.1
22.5
26.6
176.0
57.1
22.5
27.1
176.5
56.6
22.9
27.1
1.49
1.54
1.42
1.52
1.38
1.50
NH (20-aminopropyl)
1
7.71 (d, 10.0)
56.3
54.5
62.4
39.8
7.74 (d, 9.6)
56.2
54.4
62.5
39.8
7.69 (d, 10.0)
56.3
54.2
62.5
40.0
3.88, 4.11
2.92, 3.77
3.09, 3.58
4.60
3.87, 4.11
2.84, 3.80
3.04, 3.60
4.61
3.90, 4.12
2.92, 3.81
3.17, 3.58
4.62
2
10
20
30
18.1
1.25
18.1
1.24
18.0
1.24
N-CH3
NH (quaternary amine)
42.4
3.06 (d, 4.4)
8.23 (br)
42.3
3.05 (d, 4.2)
8.25 (br)
42.5
3.08 (d, 5.0)
8.24 (br)
a
b
c
13C NMR: dC (ppm), (150 MHz, CDCl3).
1H NMR: dH (ppm, J in Hz), (600 MHz, CDCl3).
Multiplicity was not reported because of overlapping resonances.

3662
P. Pruksakorn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3658–3663
The observation of heteronuclear long range coupling between
[between the pro-R d-proton of proline (dH 3.99) and the
a-proton
the amide carbonyl at dC 177.1 (C-1) and the d-protons of proline
at dH 3.99, 3.44 confirmed that the N-terminus of 1 was associated
with 2-methyl decanoic acid (MDA) (Fig. 3 and Table 2). Moreover,
of proline (dH 4.17) and the -methyl protons of MDA (dH 1.12); the
b-proton of MDA (H-3, dH 1.25) and the pro-S d-proton of proline
(dH 3.44)] were observed as shown in Figure 4. The observation
a
the strong NOE correlation between the
a-methyl protons of MDA
of the NOE correlations [between the
a-proton (H-2, dH 4.14) and
(dH 1.12) and the d-proton of proline (dH 3.99) also completely
proved the connection of N-terminal residue (Fig. 4). All the pro-
ton- and carbon-signals were assigned as shown in Table 2.
Stereostructure of chiral amino acids were deduced by HPLC
analysis of the derivatized amino acids by 1-fluoro-2,4-dinitro-
the -methyl protons of AHMOD (dH 0.88); the H-5 proton (dH
c
1.26) and the adjacent protons (H-4 proton (dH 1.99) and the H-6
proton (dH 4.06)); the H-3 proton (dH 2.16) and the H-6 proton]
suggested that AHMOD might take a conformation as shown in Fig-
ure 4. Furthermore, the NOE correlations [between the 20-proton
(H-20, dH 4.60) and the adjacent proton (H-10, dH 3.09), the 2-meth-
ylene protons (H-2, dH 2.92 and 3.77); the 10-proton (H-10, dH 3.58)
and the N-methyl protons of AMAE (dH 3.06)] revealed a conforma-
tion of AMAE residue as shown in Figure 4. From these evidences
and the comparison with stereochemistry of trichopolyn I (4), the
relative stereostructure of 1 was tentatively assigned as depicted in
Figure 4.
phenyl-5-
analysis indicated that all the constituting chiral amino acid of 1
were found to be -amino acids. Stereostructure of N-terminal,
L
-alanine-amide (Marfey’s reagent).14 The result of HPLC
L
AHMOD and C-terminal were tentatively assigned based on the
NOE correlations and the comparison with the structurally related
compounds, trichopolyns.11 Thus, the N-terminal region was
presumed to take a conformation where the NOE correlations
Figure 4. NOE correlations and plausible stereochemistry of 1.

P. Pruksakorn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3658–3663
3663
the more potent anti-mycobacterial activity against the pathogenic
strain M. tuberculosis H37Rv with MIC values of 0.12 and 0.13 g/
l
mL, respectively. This observation suggested that the AHMOD moi-
ety is important for anti-mycobacterial activity of trichoderins. Ac-
tion-mechanism of trichoderins is currently under study.
Acknowledgments
The authors thank Ms. Salona Moodley, Mr. Yushir R. Maharaj,
Ms. Karnishree Govender (University of KwaZulu Natal) for excel-
lent technical assistance. A.D.B. was supported by a Grant from
the Helen Hay Whitney Foundation. This study was financially sup-
ported by the Hoh-ansha Foundation, the Uehara Memorial Foun-
dation, JSPS-South Africa (NRF) Joint Research Program, and
Grant-in-Aid for scientific research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Figure 5. COSY and HMBC correlations for AMOD moiety in 2.
Table 3
MIC values of trichoderins (1–3) and isoniazid against M. smegmatis, M. bovis BCG and
M. tuberculosis H37Rv under both aerobic and hypoxic conditions
References and notes
Compounds
MIC (
lg/mL)
M. smegmatis
M. bovis BCG
M. tuberculosis
1. Idemyor, V. J. Natl. Med. Assoc. 2007, 99, 1414.
H37Rv
2. Wayne, L. G.; Sohaskey, C. D. Annu. Rev. Microbiol. 2001, 55, 139.
3. (a) Wayne, L. G.; Hayes, L. G. Infect. Immun. 1996, 64, 2062; (b) Lim, A.;
Eleuterio, M.; Hutter, B.; Murugasu-Oei, B.; Dick, T. J. Bacteriol. 1999, 181, 2252;
(c) Dick, T.; Lee, B. H.; Murugasu-Oei, B. FEMS Microbiol. Lett. 1998, 163, 159.
4. (a) Arai, M.; Sobou, M.; Vilchèze, C.; Baughn, A.; Hashizume, H.; Pruksakorn, P.;
Ishida, S.; Matsumoto, M.; Jacobs, W. R., Jr.; Kobayashi, M. Bioorg. Med. Chem.
2008, 16, 6732; (b) Arai, M.; Ishida, S.; Setiawan, A.; Kobayashi, M. Chem.
Pharm. Bull. 2009, 57, 1136.
Aerobic Hypoxic Aerobic Hypoxic Aerobic
Hypoxic
1
2
3
0.1
0.1
0.02
0.16
0.02
0.03
0.02
0.16
0.02
>100
0.12
2.0
0.13
0.05
0.12
2.0
0.13
>100
1.56
0.63
2.5
1.56
0.63
25
Isoniazid
5. Trichoderin A (1): colorless amorphous solid. ½a D20
ꢁ17 (c 0.7, MeOH). IR mmax
ꢂ
(KBr) cmꢁ1: 3314, 2928, 1669, 1535, 1200. ESI-MS: m/z 1163 [M]+. High
resolution ESI-MS: calcd for C60H111N10O12: m/z 1163.8383. Found 1163.8851.
1H NMR (600 MHz, CDCl3, dH), 13C NMR (150 MHz, CDCl3, dC) spectra: as shown
in Table 2.
Trichoderin A1 (2) was obtained as a colorless amorphous solid.
The ESI-TOF-MS of 2 showed a pseudomolecular ion peak at m/z
1145 [M]+, which was smaller than that of 1 by 18 mass units,
and the molecular formula was determined as C60H109N10O11 by
HR-ESI-TOF-MS. The comparison of the fragmentation patterns be-
tween 1 and 2 by ESI-TOF-MSMS analysis suggested that com-
pound 2 has AMOD moiety instead of AHMOD as shown in Table
1. This moiety was confirmed by the heteronuclear long range cou-
pling (3J(C,H)) between the olefinic proton at H-6 (dH 6.75) and the
ketone carbonyl at C-8 (dC 201.7) (Fig. 5). All the proton- and car-
bon-signals were assigned as shown in Table 2.
Trichoderin B (3) was obtained as a colorless amorphous solid.
The ESI-TOF-MS of 3 showed a pseudomolecular ion peak at m/z
1149 [M]+, and the molecular formula was determined as
C59H109N10O12 by HR-ESI-TOF-MS. The ESI-TOF-MSMS analysis of
3 confirmed that the B5 fragment of 3 was assigned to Val instead
of Ile in 1 (Table 1). The presence of valine in 3 was also supported
by the detailed analysis of 2D NMR spectra. All the proton- and car-
bon-signals were assigned as shown in Table 2.
MIC values of trichoderins (1–3) and isoniazid against Mycobac-
terium smegmatis, M. bovis BCG and M. tuberculosis H37Rv under
the both aerobic condition and hypoxic condition of nitrogen
atmosphere containing 0.2% oxygen inducing dormant state were
determined by the established method previously.4 The dormant
M. tuberculosis was highly resistant against isoniazid, which inhib-
its inhA of the type II fatty acid synthase.3a As shown in Table 3, the
MIC values of isoniazid against M. smegmatis, M. bovis BCG, and M.
6. Trichoderin A1 (2): colorless amorphous solid. ½a D20
ꢂ
ꢁ23 (c 0.3, MeOH). IR mmax
(KBr) cmꢁ1: 3322, 2932, 2361, 1667, 1534, 1200. UV kmax (MeOH) nm (
e
): 232
(sh) (14100). ESI-MS: m/z 1145 [M]+. High resolution ESI-MS: calcd for
C60H109N10O11: m/z 1145.8277. Found 1145.8325. 1H NMR (600 MHz, CDCl3,
dH), 13C NMR (150 MHz, CDCl3, dC) spectra: as shown in Table 2.
7. Trichoderin B (3): colorless amorphous solid. ½a D20
ꢁ59 (c 0.1, MeOH). IR mmax
ꢂ
(KBr) cmꢁ1: 3318, 2930, 1667, 1535, 1200. ESI-MS: m/z 1149 [M]+. High
resolution ESI-MS: calcd for C59H109N10O12: m/z 1149.8226. Found 1149.8474.
1H NMR (600 MHz, CDCl3, dH), 13C NMR (150 MHz, CDCl3, dC) spectra: as shown
in Table 2.
8. Trichoderins (5 mg each) were treated with 5 mL of 6 N HCl aq, and heated at
110 °C for 20 h. The reaction mixture was partitioned with ether. The aqueous
portion was dried under reduced pressure to obtain crude amino acids. The crude
amino acids from each trichoderins and authentic amino acids were dissolved in
40
l
L of 50 mM sodium bicarbonate (pH 8.1) in 3 mL screw-cap glass tubes,
respectively. Then, 80
l
L of freshly prepared 4-dimethylaminoazobenzene-40-
sulfonyl chloride (DABS-Cl) solution (4 mM in acetonitrile) was added to each
sample. The samples were sealed with caps and parafilm. The samples were then
heated at 70 °C for 10 min. After dabsylation, the samples were analyzed by
reversed-phase HPLC under the following condition; Cosmosil 5C18 AR, (4.6 mm
id ꢀ 250 mm),
a 100 min linear gradient from acetonitrile/25 mM sodium
acetate buffer (pH 6.5) = 15:85–70:30, 37 °C, 1 mL/min, and detection at
436 nm. Authentic dabsylated Pro, Val, Aib and Ile were eluted with retention
times of 44, 46, 48 and 51 min, respectively.
9. Roepstorff, P.; Hojrup, P.; Moller, J. Biomed. Mass Spectrom. 1985, 12, 181.
10. Fukushima, K.; Arai, T.; Mori, Y.; Tsuboi, M.; Suzuki, M. J. Antibiot. 1983, 36,
1613.
11. (a) Fujita, T.; Takaishi, Y.; Okamura, A.; Fujita, E.; Fuji, K.; Hiratsuka, N.;
Komatsu, M.; Arita, I. J. Chem. Soc., Chem. Commun. 1981, 585; (b) Iida, A.;
Mihara, T.; Fujita, T.; Takaishi, Y. Bioorg. Med. Chem. Lett. 1999, 9, 3393.
12. Grafe, U.; Ihn, W.; Ritzau, M.; Schade, W.; Stengel, C.; Schlegel, B.; Fleck, W. F.;
Kunkel, W.; Hartl, A.; Gutsche, W. J. Antibiot. 1995, 48, 126.
13. Degenkolb, T.; Heinze, S.; Schlegel, B.; Dornberger, K.; Mollmann, U.; Dahse, H.
M.; Grafe, U. J. Antibiot. 2000, 53, 184.
tuberculosis H37Rv were 2.5
under aerobic condition, respectively. While, the MIC values of iso-
niazid against these strains were more than 25 g/mL under nitro-
lg/mL, 0.03 lg/mL, and 0.05 lg/mL
14. The crude amino acids from each trichoderins and
acids were dissolved in 100 L of 0.5 M sodium bicarbonate in 3 mL screw-cap
glass tubes, respectively. Then, 200 of 1-fluoro-2,4-dinitrophenyl-5-
alanine amide ( -FDLA, Marfey’s reagent, 10 mg/mL in acetone) was added to
each sample. The samples were sealed with caps and incubated at 40 °C for
90 min. After addition of 25 L of 2 M hydrochloric acid, the reaction mixture
was diluted with methanol to suitable volumes (15–20-fold dilution). An
aliquot of the -FDLA derivatives was analyzed by reversed-phase HPLC under
L- and D-authentic amino
l
l
lL
L-
gen atmosphere containing 0.2% oxygen. On the other hand,
trichoderins (1–3) showed potent anti-mycobacterial activity
against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv un-
der both aerobic conditions and dormancy-inducing hypoxic con-
L
l
L
the following condition; Cosmosil 5C18 AR, (4.6 mm id ꢀ 250 mm), a 60 min
linear gradient from acetonitrile/50 mM triethylamine phosphate (TEAP)
buffer (pH 3.0) = 1:9–1:1, 1.5 mL/min, and detection at 340 nm. Authentic
ditions, with MIC values in the range of 0.02–2.0 lg/mL. These
results indicated that trichoderins were effective against Mycobac-
terium sp. in both actively growing and dormant states. Among
them, 1 and 3 having AHMOD moiety in the structures showed
derivatized
L-Pro, D-Pro, L-Val, L-Ile, D-Val and D-Ile were eluted with retention
times of 26, 28, 34, 40, 41 and 45 min, respectively.