Minutissamides A-D, antiproliferative cyclic decapeptides from the cultured cyanobacterium Anabaena minutissima

Article abstract of DOI:10.1021/np2002226

Four cyclic decapeptides, minutissamides A-D (1-4), were isolated from the cultured cyanobacterium Anabaena minutissima (UTEX 1613). The planar structures were determined using various spectroscopic techniques including HRESIMS and 1D and 2D NMR experiments. The absolute configurations of the α-amino acid residues were assigned using Marfey's method after acid hydrolysis. The absolute configuration of a β-amino acid residue was assigned by a combination of the advanced Marfey's method, J-based configurational analysis, and ROE spectroscopic analysis. The structures of minutissamides A-D (1-4) were characterized by the presence of three nonstandard α-amino acid residues (two α,β-dehydro-α-aminobutyric acids and one N-methylated Asn) and one β-amino acid residue (2-hydroxy-3-amino-4-methyldodecanoic acid or 2-hydroxy-3-amino-4-methylhexadecanoic acid). Minutissamides A-D (1-4) exhibited antiproliferative activity against the HT-29 human colon cancer cell line with IC₅₀ values of 2.0, 20.0, 11.8, and 22.7 μM, respectively.

Full text of DOI:10.1021/np2002226

ARTICLE
pubs.acs.org/jnp
Minutissamides AꢀD, Antiproliferative Cyclic Decapeptides from the
Cultured Cyanobacterium Anabaena minutissima
Hahk-Soo Kang, Aleksej Krunic, Qi Shen, Steven M. Swanson, and Jimmy Orjala*
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612,
United States
S Supporting Information
b
ABSTRACT:
Four cyclic decapeptides, minutissamides AꢀD (1ꢀ4), were isolated from the cultured cyanobacterium Anabaena minutissima
(UTEX 1613). The planar structures were determined using various spectroscopic techniques including HRESIMS and 1D and 2D
NMR experiments. The absolute configurations of the R-amino acid residues were assigned using Marfey’s method after acid
hydrolysis. The absolute configuration of a β-amino acid residue was assigned by a combination of the advanced Marfey’s method,
J-based configurational analysis, and ROE spectroscopic analysis. The structures of minutissamides AꢀD (1ꢀ4) were characterized
by the presence of three nonstandard R-amino acid residues (two R,β-dehydro-R-aminobutyric acids and one N-methylated Asn)
and one β-amino acid residue (2-hydroxy-3-amino-4-methyldodecanoic acid or 2-hydroxy-3-amino-4-methylhexadecanoic acid).
Minutissamides AꢀD (1ꢀ4) exhibited antiproliferative activity against the HT-29 human colon cancer cell line with IC50 values of
2.0, 20.0, 11.8, and 22.7 μM, respectively.
yanobacteria (blue-green algae) have been shown to be
R-hydroxy or β-hydroxy amino acids, and Dhb (R,β-dehydro-R-
aminobutyric acid).
C
prolific producers of bioactive secondary metabolites.1ꢀ3
A major class of secondary metabolites from cyanobacteria is
polyketide-conjugated nonribosomal peptides, commonly known
as lipopeptides.4,5 Numerous cyclic lipopeptides have been iso-
lated from both freshwater and marine cyanobacteria with ring
sizes of up to 12 amino acid residues. Cyclic deca- and undeca-
peptides, containing 10 and 11 residues, have been reported to
exhibit a wide spectrum of biological activities, including
antifungal,6,7 cardiotonic,8,9 and cytotoxic.10,11 A common struc-
tural feature of cyclic deca- and undecapeptides is the occurrence of
one lipophilic β-amino acid residue with a structurally unique side
chain.12 Amination of fatty acids at C-3 forming β-amino acids
provides the branching point via an amide bond with other R-
amino acids, whereas C-2 is usually a methylene, methylated or
hydroxylated. Further fatty acid modifications include methyla-
tion, oxidation, chlorination, and/or hydroxylation of the side
chain, resulting in the high diversity of modified β-amino fatty
acids. Most of the cyclic lipopeptides in this class also possess
nonstandard amino acids, such as N-methylated R-amino acids,
In our continuing search for secondary metabolites from
laboratory-cultured cyanobacteria, we evaluated the cell extract
of cultured Anabaena minutissima (UTEX 1613). Herein we
report the isolation, structure elucidation, and biological activity
of four new cyclic decapeptides, named minutissamides AꢀD,
each possessing two Dhbs, one N-methylated Asn, and one
β-amino acid, Hamd (2-hydroxy-3-amino-4-methyldodecanoic
acid) or modified Hamh (2-hydroxy-3-amino-4-methylhexade-
canoic acid). The planar structures were determined using a
combination of spectroscopic techniques including HRESIMS,
tandem MS, and 1D and 2D NMR (COSY, TOCSY, HSQC,
HMBC, and ROESY) experiments. The absolute configurations
of the R-amino acid residues were determined by Marfey’s
method, whereas the stereoconfiguration of the Hamd residue
Received: March 10, 2011
Published: June 23, 2011
r
Copyright
2011 American Chemical Society and
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|

Journal of Natural Products
ARTICLE
in 1 was solved by a combination of the advanced Marfey’s
method, J-based configurational analysis, and ROE correlations.
Figure 1. Key 2D correlations of minutissamide A (1) that were used
for the determination of the planar structure of 1.
(δH 3.93) and 3-NH (δH 6.85) and between H-4 (δH 1.65) and
4-Me (δH 0.58). Further COSY correlations from H-4 (δH 1.65)
to H2-5 (δH 1.17 and 1.61), H2-5 to H2-6 (δH 1.18 and 1.24),
and H2-6 to the region of highly overlapped methylene signals
(H2-7ꢀ11, δH 1.26ꢀ1.27), corresponding to five carbons in the
HSQC spectrum (δC 30.1, 29.2, 29.7, 31.8, and 22.6), and finally
to the methyl triplet protons H3-12 (δH 0.87), completed the
structure of Hamd.
’ RESULTS AND DISCUSSION
A. minutissima (UTEX 1613) was grown in Z media.13 The
freeze-dried cells were extracted with a mixture of CH2Cl2 and
MeOH (1:1) and dried in vacuo. The cell extract was subse-
quently fractionated by column chromatography using Diaion
HP-20 resin and an increasing amount of isopropyl alcohol (IPA)
in H2O. LC-MS analysis of the third fraction (40% IPA)
indicated the presence of peptides with molecular weights of
1118, 1152, 1188, and 1190 Da. Subsequent Sephadex LH-20
column chromatography followed by reversed-phased HPLC
yielded minutissamides AꢀD (1ꢀ4).
The sequence of the 10 residues was established by analysis of
the ROESY and selective HMBC spectra (Figure 1 and Table 1).
A selective HMBC spectrum for the carbonyl region was used to
resolve the overlapped amide carbonyl signals.14 HMBC correla-
tions from Dhb2-NH (δH 9.17) to Val C-1 (δC 169.2), from Val
H-2 (δH 4.29) to Hamd C-1 (δC 170.1), and from Hamd H-3
(δH 3.93) to Pro C-1 (δC 171.9) suggested a partial sequence of
Dhb2-Val-Hamd-Pro. ROE correlations between Dhb2-NH (δH
9.17) and Val H-2 (δH 4.29), between Val-NH (δH 6.85) and
Hamd H-2 (δH 4.18), and between Hamd-NH (δH 6.85) and
Pro H-2 (δH 4.19) confirmed this partial sequence. HMBC
correlations from Pro H-2 (δH 4.19) to NMeAsn C-1 (δC 168.3)
and from NMeAsn N-Me (δH 2.96) to Thr C-1 (δC 170.4),
together with ROE correlations between Pro H2-5 (δH 4.10) and
NMeAsn H-2 (δH 5.53) and between NMeAsn N-Me (δH 2.96)
and Thr H-2 (δH 4.63), further expanded the sequence to Dhb2-
Val-Hamd-Pro-NMeAsn-Thr. HMBC correlations from Thr NH
(δH 7.30) to Ala C-1 (δC 172.5) and from Ala NH (δH 8.27) to
Asn1 C-1 (δC 171.2), together with ROE correlations between
Thr-NH (δH 7.30) and Ala H-2 (δH 4.29) and between Ala-NH
(δH 8.27) and Asn1 H-2 (δH 4.40), established the sequence of
Dhb2-Val-Hamd-Pro-NMeAsn-Thr-Ala-Asn1, leaving two resi-
dues (Dhb1 and Asn2) unassigned. The complete sequence of
Dhb2-Val-Hamd-Pro-NMeAsn-Thr-Ala-Asn1-Dhb1-Asn2 was
deduced by an HMBC correlation from Asn1-NH (δH 8.45) to
Dhb1 C-1 (δC 163.4) and a ROE correlation between Dhb1-NH
(δH 10.18) and Asn2 H-2 (δH 4.92). Lastly, HMBC correlations
from Asn2 H-2 (δH 4.92) and NH (δH 8.73) to Dhb2 C-1 (δC
163.6) closed the ring, completing the planar structure of 1.
Mass fragmentation analysis was performed to further verify
the structure of 1.15 The ESIMS/MS analysis, using a quadru-
pole-time-of-flight (Q-TOF) tandem mass spectrometer, sup-
ported the proposed planar structure of 1. The parent peak was
observed at m/z [M + H]+ 1118.6. The ring-opening occurred
Minutissamide A (1) was obtained as a colorless, amorphous
powder. The molecular formula of 1was determined as C51H83N13O15
by HRESIMS analysis (m/z 1140.6049 [M + Na]+). The H
1
NMR spectrum in DMSO-d6 showed a signal distribution typical
for lipopeptides, including several exchangeable amide NH
signals (6.5ꢀ10.5 ppm), signals in the R-proton region (4ꢀ6 ppm),
methylene signals (1.2ꢀ1.3 ppm), and numerous aliphatic
doublet and triplet methyl signals (0.5ꢀ2.0 ppm). The structures
of the amino acid residues were elucidated by interpretation of
the COSY and TOCSY spectra (Figure 1 and Table 1) and
indicated the presence of 10 residues, including nine R-amino
acid residues and one β-amino acid residue. Six of the R-amino
acid residues were identified as the standard amino acids proline
(Pro), threonine (Thr), alanine (Ala), valine (Val), and two
asparagines (Asn). In addition, the structures of three nonstan-
dard amino acid residues were determined by analysis of COSY,
HSQC, and HMBC data (Figure 1). An HMBC correlation from
the N-methyl singlet (δH 2.96) to the C-2NMeAsn (δC 49.4) in
combination with a COSY correlation between NMeAsn H-2
(δH 5.53) and NMeAsn H2-3 (δH 2.00 and 3.02) identified the
structure of N-methylasparagine (NMeAsn). The presence of
two Dhb residues was deduced by COSY correlations observed
between the methyl protons (H3-4Dhb1, δH 1.90 and H3-4Dhb2
,
δH 1.66) and the corresponding olefinic proton (H-3Dhb1, δH
5.71 and H-3Dhb2, δH 5.30), and HMBC correlations from the
methyl protons (H3-4Dhb1 and H3-4Dhb2) to the corresponding
olefinic carbon (C-2Dhb1, δC 130.1 and C-2Dhb2, δC 133.0). The
2-hydroxy-3-amino-4-methyl portion in Hamd was identified by
sequential COSY correlations between H-2 (δH 4.18)/H-3 (δH
3.93)/H-4 (δH 1.65), as well as COSY correlations between H-3
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Table 1. NMR Spectroscopic Data for Minutissamides A and B (1 and 2) in DMSO-d6
minutissamide A (1)
minutissamide B (2)a
b
c
f
c
δC
δH
mult. (J in Hz)
COSY
HMBC
ROESY
δC
δH
mult.(J in Hz)
Hamd or Hamcd
1
2
3
4
5
170.1
70.0
56.6
32.6
33.8
170.0
70.0
56.6
32.6
33.7
4.18 d (4.8)
3
3
4.18 overlapped
1,e 2, 1Pro
3.92
1.65
1.17
1.61
1.16
1.23
1.23
1.23
1.26
1.37
1.70
M
m
m
m
m
m
m
m
m
m
m
e
3.93
1.65
1.17
1.61
1.18
1.24
1.26
1.26
1.26
1.26
1.27
m
m
m
m
m
m
m
m
m
m
m
2, 4
3, 5, 4-Me
4, 6
6
25.9
5, 6
25.9
7
30.1
29.2
29.7
31.8
22.6
14.5
6, 8
7, 9
8, 10
10, 11
11, 12
11
30.1
29.7
29.1
26.6
32.4
45.8
8
9
10
11
12
0.87 t (7.2)
10, 11
3.62 t (6.6)
ndd
2-OH
3-NH
4-Me
1
5.51 br s
2
e
6.85 partly overlapped
0.58 d (6.6)
3
1Pro
3, 2Pro
3
6.88 partly overlapped
0.57 d (6.6)
16.3
171.9
60.5
4
3, 4, 5
16.4
171.9
60.4
Pro
2
4.19
1.91
1.85
1.92
3.26
4.10
m
m
m
m
m
m
3
3, 4, 1NMeAsn
1
4.18
1.92
1.83
1.92
3.25
4.09
m
m
m
m
m
m
3
30.7
2, 4
3, 5
30.7
4
24.2
24.2
5
47.3
4
2
2NMeAsn
47.3
NMeAsn
1
2
3
168.3
49.4
34.4
168.3
49.3
34.4
5.53 dd (12.0, 3.0)
2.00
3.02 dd (15.6, 12.0)
3
1,e 3, 4e
1e, 2e, 4e
5Pro, 3, N-Me
2Thr, 3Thr
N-Me, 3, 4
2, 3
5.53 br d (12.0)
m
2, 4
2.00
3.01
m
m
4
172.1
30.7
172.1
30.7
e
N-Me
NH2
2.96
5.92
7.52
s
s
s
2, 1Thr
2.95
6.03
7.52
s
3
m
m
Thr
1
170.4
55.3
66.8
20.0
170.4
55.3
66.8
19.9
1,e 3, 1Ala
4.62
3.92
m
m
e
2
4.63 dd (8.4, 3.6)
3.92
2-NH, 3
3
m
2, 4
3
4
1.00 d (6.0)
5.13 br s
2, 3
1Ala
0.99 d (6.6)
ndd
3-OH
NH
1
7.30 d (7.8)
2
7.30 d (8.4)
Ala
172.5
48.9
17.3
172.5
48.7
17.3
1,e 1Asn1
4.28
1.22 d (7.2)
m
e
2
4.29 q (7.2)
1.23 d (7.2)
8.27 d (8.4)
2-NH, 3
3
2
2
1,e 2
NH
1
2, 1Asn1
2, 2Asn1, NHThr, NHAsn1
3
Asn1
171.2
50.5
36.2
171.2
50.3
36.2
2
4.40
2.56
m
m
2-NH, 3
2
3
4.39
2.53
m
m
3
1,e 2, 4e
2.87 dd (18.6, 7.2)
2.88 dd (16.8, 7.2)
4
173.2
173.2
NH
NH2
8.45 d (7.2)
2
1Dhb1
3
2
8.45 d (7.2)
7.04
7.58
s
s
7.04
7.59
s
s
Dhb1
1
2
163.4
130.1
163.4
130.1
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Table 1. Continued
minutissamide A (1)
COSY HMBC
minutissamide B (2)a
b
c
f
c
δC
δH
mult. (J in Hz)
ROESY
δC
δH
mult.(J in Hz)
3
125.6
13.7
5.71 q (7.2)
1.90 d (7.2)
4
3
1, 2, 4
2, 3
NH
3, 2Asn2
3
125.6
13.6
5.71 q (7.2)
1.89 d (7.2)
4
e
NH
1
10.18
s
1, 1Asn2
10.20
s
Asn2
172.4
49.4
39.1
172.4
49.4
39.2
1,e 3, 1Dhb2
1,e 2, 4e
4.91 q (7.8)
e
2
4.92 q (7.2)
2-NH, 3
2
3
2.67 dd (15.0, 7.2)
2.77 dd (15.0, 7.8)
2.67 dd (15.0, 7.2)
2.77 dd (15.0, 7.8)
4
172.6
172.7
NH
NH2
8.73 d (7.8)
2
1Dhb2
3
8.74 d (7.8)
7.11
7.58
s
s
7.12
7.59
s
s
Dhb2
Val
1
163.6
133.0
116.5
13.5
163.6
133.0
116.4
13.3
2
3
5.30 q (7.2)
1.66 d (7.2)
4
3
1, 2, 4
2, 3
1,e 3, 1Val
NH
5.29 q (7.2)
1.65 d (7.2)
4
e
NH
1
9.17
s
3, 2Val
9.21
s
169.2
56.2
33.1
19.4
19.1
169.2
56.2
33.1
19.3
19.0
1,e 1Hamd
1e
2, 3, 40
4.28
1.74
m
m
e
2
4.29 d (10.8)
1.75
2-NH, 3
3
m
2, 4
3
4
40
0.83 d (6.6)
0.82 d (6.6)
0.90 d (6.6)
3
2, 3, 4
0.89 d (6.6)
NH
6.85 partly overlapped
2
2Hamd
6.88 partly overlapped
a Complete NMR table for minutissamide B (2) including 2D NMR data can be found in the Supporting Information. b Carbon chemical shifts were
assigned from DEPT-Q spectrum measured at 226 MHz. c Measured at 600 MHz. d nd: not detected. e Assigned from the selective HMBC spectrum.
f Assigned from the HSQC and HMBC spectra.
between Pro and NMeAsn, forming an acylium ion. The con-
tinuous fragmentation yielded the fragment ions at m/z 990.6
[M + H ꢀ NMeAsn]+, 889.5 [M + H ꢀ NMeAsn ꢀ Thr]+, 704.4
[M + H ꢀ NMeAsn ꢀ Thr ꢀ Ala ꢀ Asn1]+, 621.4 [M + H ꢀ
NMeAsn ꢀ Thr ꢀ Ala ꢀ Asn1 ꢀ Dhb1]+, 507.4 [M + H ꢀ
NMeAsn ꢀ Thr ꢀ Ala ꢀ Asn1 ꢀ Dhb1 ꢀ Asn2]+, and 396.3
[M + H ꢀ NMeAsn ꢀ Thr ꢀ Ala ꢀ Asn1 ꢀ Dhb1 ꢀ Asn2 ꢀ
Dhb2]+ (Figure 2). This fragmentation pattern was in complete
agreement with the structure determined by 2D NMR analysis.
Minutissamide A (1) contained three different types of
stereogenic elements, the geometric configuration of Dhbs, the
configurations of the R-amino acid residues, and the configura-
tions of the three consecutive asymmetric centers in the Hamd
residue. A number of techniques were used for the assignment of
stereoconfiguration of 1, including Marfey’s method and the
advanced Marfey’s method, J-based configurational analysis, and
ROE correlations. A strong ROE correlation between the
respective NH (δH 10.18 or δH 9.17) and the corresponding
olefinic proton (H-3, δH 5.71 or δH 5.30) was observed for both
Dhb1 and Dhb2, assigning the geometric configuration of both
double bonds as E (Figure 1). The absolute configurations of the
common amino acids and NMeAsn were assigned as L-Pro,
L-Thr, L-Asn1, L-Asn2, L-Val, D-Ala, and L-NMeAsn by chromato-
graphic comparison of Marfey’s derivatives of the acid hydro-
lysate of 1 and appropriate amino acid standards.16 The relative
configurations of the three asymmetric centers (C-2, C-3, and
C-4) in the Hamd residue were established by a combination of
J-based configurational analysis and ROE correlations (Figure 3).
Homonuclear (3JHꢀH) coupling constants were acquired from
the DQF-COSY spectrum, and the phase-sensitive HMBC
spectrum was used to obtain the heteronuclear (2JCH and 3JCH
)
coupling constants.17 The small 3JHH (5.6 Hz) between H-2 and
H-3, the large 3JCH (5.4 Hz) between H-3 and C-1, and the large
2JCH (ꢀ5.1 Hz) between H-3 and C-2 allowed for two possible
conformations (A2 and A6) out of the six conformations shown
in Figure 3a. The conformation A6 was found to be a correct
conformation based on a ROE correlation observed between H-2
and H-4, assigning the relative configuration between C-2 and
C-3 as “erythro”. The same method was applied for the assign-
ment of the relative configuration between C-3 and C-4. On the
basis of the large 3JHH (11.0 Hz) measured between H-3 and H-4,
only two conformations (B3 and B4) were possible, as shown in
Figure 3b. A ROE correlation observed between H-2 and H2-5
confirmed B3 as the correct confirmation, assigning the relative
configuration between C-3 and C-4 as “threo”. The nearly
identical carbon chemical shifts of C-1, C-2, C-3, and C-4 of
Hamd (δC 170.1, 70.0, 56.6, and 32.6) to those of Hamh
(δC 169.8, 69.9, 56.2, and 32.4) in puwainaphycin E provided
further evidence for this relative configuration.10 Because the
relative configurations of C2ꢀC3 and C3ꢀC4 were erythro and
threo, respectively, the absolute configurations of the three
stereogenic centers (C-2, C-3, and C-4) in the Hamd residue
could be either “RRS” or “SSR”. The advanced Marfey’s method
was applied to assign the absolute configuration of the Hamd
residue.18,19 The acid hydrolysate of 1 was derivatized with L- and
DL-FDLA, respectively. Subsequent LC-MS analysis of DL-FDLA
derivatives identified two peaks of the FDLA derivatives of Hamd
(m/z 538 [M ꢀ H]ꢀ) at 43.3 and 48.8 min, while the L-FDLA
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Figure 2. ESIMS/MS fragmentation of minutissamide A (1).
Figure 3. Newman projections for (a) C-2/C-3 and (b) C-3/C-4. All possible relative conformations are shown. The DQF-COSY and phase-sensitive
HMBC spectra were used for the calculation of homo- and heteronuclear coupling constants. Labels below projections denote the predicted size of
coupling constants. The predicted values that are consistent with observed values are highlighted by a box. Observed ROE correlations are presented as
double-headed arrows: (a) H-2/H-4 and (b) H-2/H2-5.
derivative of Hamd resulted in one peak at 48.8 min. Previous
application of this method for the determination of the absolute
configuration of Ahda (amino-2-hydroxydecanoic acid) identi-
fied that the R-L and S-D derivatives are more hydrophobic than
the R-D and S-L derivatives.20 In our experiment, the L-FDLA
derivative of 1 corresponded to the later peak of DL-FDLA
derivatives, suggesting the presence of R-L. As a result, the
absolute configuration of C-3 in Hamd was assigned as R, which
allowed for the assignment of the absolute configurations at C-2,
C-3, and C-4 in Hamd as “RRS”.
Minutissamide B (2) was obtained as a colorless, amorphous
powder. The molecular formula of 2was deduced as C51H82ClN13O15
on the basis of HRESIMS analysis (m/z 1174.5680 [M + Na]+). A M
+ 2 isotope peak verified the presence of chlorine in2. The 1Hand13C
NMR spectra of 2 were almost identical to those of 1 except for the
Hamd residue, where the terminal methyl proton signal had been
replaced by a new triplet methylene resonance at 3.62 ppm. This
indicated that chlorine was positioned on the terminal carbon of
Hamd, thus renaming it Hamcd (2-hydroxy-3-amino-4-methyl-12-
chlorododecanoic acid). The sequential COSY correlation network of
1601
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1
Table 2. H and 13C NMR Data for Minutissamides C and D (3 and 4) in DMSO-d6
minutissamide C (3)a
minutissamide D (4)a
b
c
b
c
δC
δH
mult. (J in Hz)
δC
δH
mult. (J in Hz)
Hamoh or Hamhh
1
2
3
4
5
170.0
70.0
56.5
32.6
33.8
170.1
70.0
56.6
32.6
33.9
4.17
3.92
1.65
1.17
1.62
1.16
1.23
1.23
1.23
1.23
1.23
1.23
1.44
d (4.8)
4.18
3.92
1.65
1.17
1.61
1.16
1.23
1.25
1.25
1.25
1.25
1.25
1.16
1.35
1.26
1.32
3.28
1.16
1.35
0.83
d (4.2)
m
m
m
m
m
m
m
m
6
25.9
m
25.9
m
m
m
7
30.1
29.1
29.7
29.4
29.4
23.6
m
30.1
29.1
29.7
29.4
29.4
25.8
m
8
m
m
9
m
m
10
11
12
m
m
m
m
q (7.2)
m
m
13
41.9
2.38
t (7.2)
q (7.8)
35.4
m
m
14
15
211.5
35.4
71.5
30.1
m
2.40
0.90
m
m
16
8.2
t (7.8)
ndd
10.6
t (7.2)
ndd
2-OH
3-NH
6.84
0.57
partly overlapped
d (6.6)
6.87
0.57
partly overlapped
d (6.6)
4-CH3
16.4
171.9
60.6
16.5
172.0
60.6
Pro
1
2
3
4
4.18
1.92
1.84
1.91
3.26
4.10
m
m
m
m
m
m
4.18
1.92
1.83
1.91
3.25
4.09
m
m
m
m
m
m
30.8
30.8
24.2
24.3
5
47.3
47.3
NMeAsn
1
2
3
168.2
49.4
34.4
168.3
49.1
34.4
5.52
2.00
3.01
dd (12.0, 3.0)
m
5.53
1.99
3.01
dd (12.0, 3.0)
m
dd (16.2, 12.6)
dd (15.6, 12.6)
4
172.1
30.8
172.1
30.8
N-Me
NH2
2.95
5.95
7.52
s
s
s
2.95
6.01
7.52
s
s
s
Thr
1
170.4
55.3
66.8
20.0
170.4
55.3
66.8
20.0
2
4.62
3.93
0.99
dd (9.0, 3.6)
m
4.62
3.91
0.98
dd (8.4, 3.6)
m
3
4
d (6.0)
ndd
d (7.8)
ndd
3-OH
NH
1
7.29
d (9.0)
7.30
d (7.8)
Ala
172.6
48.9
17.3
172.6
49.0
17.4
2
4.28
1.22
8.26
m
4.28
1.22
8.26
m
3
d (7.2)
d (8.4)
d (7.2)
d (8.4)
NH
1
Asn1
171.2
50.5
36.2
171.2
50.5
36.2
2
4.39
2.53
td (7.2, 3.0)
m
4.39
2.52
td (7.2, 3.0)
m
3
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Journal of Natural Products
ARTICLE
Table 2. Continued
minutissamide C (3)a
minutissamide D (4)a
b
c
b
c
δC
δH
mult. (J in Hz)
δC
δH
mult. (J in Hz)
2.87
dd (16.8, 7.2)
2.87
dd (16.8, 7.2)
4
173.2
173.2
NH
NH2
8.45
7.04
7.58
d (7.2)
8.44
7.04
7.59
d (7.2)
s
s
s
s
Dhb1
Asn2
1
163.4
130.1
125.6
13.7
163.4
130.1
125.8
13.7
2
3
5.70
1.89
10.18
q (7.2)
d (7.2)
s
5.70
1.89
10.19
q (7.8)
d (7.8)
s
4
NH
1
172.4
49.4
39.0
172.4
49.5
39.2
2
4.90
2.66
2.76
q (7.2)
4.91
2.67
2.76
q (7.2)
3
dd (15.0, 7.2)
dd (15.0, 7.8)
dd (15.0, 7.2)
dd (15.0, 7.8)
4
172.6
172.7
NH
NH2
8.73
7.12
7.59
d (7.8)
8.73
7.12
7.59
d (7.8)
s
s
s
s
Dhb2
Val
1
163.6
133.0
116.4
13.5
163.7
133.0
116.5
13.5
2
3
5.29
1.65
9.18
q (7.2)
d (7.2)
s
5.29
1.64
9.19
q (7.2)
d (7.2)
s
4
NH
1
169.2
56.2
33.1
19.4
19.1
169.3
56.3
33.1
19.4
19.1
2
4.28
1.74
0.82
0.89
6.84
m
4.28
1.74
0.82
0.89
6.86
m
3
m
m
4
40
d (6.6)
d (6.6)
d (6.6)
d (6.6)
NH
partly overlapped
partly overlapped
a Complete NMR tables for minutissamides C (3) and D (4) including 2D NMR data can be found in the Supporting Information. b Carbon chemical
shifts were assigned from the DEPT-Q spectrum measured at 226 MHz. c Measured at 600 MHz. d nd: not detected.
H2-12/H2-11/H2-10 and the slightly downfield chemical shifts of H2-
11 (δH 1.70) and H2-10 (δH 1.37) further supported the structure of 2.
The stereoconfiguration of 2was determined by comparison of proton
and carbon chemical shifts as well as specific rotation to those of 1. The
carbon chemical shifts of all of the stereogenic centers in 2 were similar
to those observed for 1 (deviation <0.2 ppm), suggesting the same
relative configurations. The specific rotations of both 1 and 2 displayed
positive values (1,[R]D +4; 2,[R]D +5).Onthebasisofthesedataand
a shared biosynthesis, we propose that the absolute configurations of all
of the stereogenic centers in 2 are the same as those found in 1.
Minutissamide C (3) was obtained as a colorless, amorphous
powder. The HRESIMS analysis (m/z 1210.6440 [M + Na]+)
suggested the molecular formula of 3 to be C55H89N13O16.
Detailed comparison of the NMR spectra indicated that com-
pounds 1 and 3 share the same cyclic decapeptide core structure.
The difference was the replacement of the dodecanoic acid
residue found in 1 with an oxidized hexadecanoic acid residue
in 3 (Table 2). The 1H NMR spectrum of 3 displayed three addi-
tional methylene protons, H2-12 (δH 1.44), H2-13 (δH 2.38),
and H2-15 (δH 2.40), as compared to 1. In addition to three
methylene carbon signals (δC 23.6, 41.9, and 35.4), a signal
NMR spectrum. The molecular weight difference of 70 between
3 and 1 corresponded to three methylenes and one ketone, indi-
cating the presence of a Hamoh (2-hydroxy-3-amino-4-methyl-
14-oxohexadecanoic acid) moiety in 3. The location of the
ketone group at C-14 in Hamoh was determined by the proton
chemical shifts of H2-13 (δH 2.38) and H2-15 (δH 2.40) and a
TOCSY correlation observed between H2-15 (δH 2.40) and H3-
16 (δH 0.90). An HMBC correlation from the terminal methyl
protons (H3-16, δH 0.90) to the ketone carbon (C-14, δC 211.5)
further confirmed this location. The stereoconfiguration of 3 was
assigned by comparison of spectroscopic data to those of 1. No
significant difference (deviation <0.1 ppm) was found for the 13C
chemical shifts of all of the stereogenic centers in 3 when com-
pared to 1. Also, compound 3 displayed a similarly small positive
specific rotation ([R]D +3) to 1. Together, these data suggested
the absolute configurations of the stereogenic centers in 3 to be
identical to those of 1.
Minutissamide D (4) was also obtained as a colorless, amor-
phous powder. The HRESIMS spectrum of 4 displayed a major
ion peak at 1212.6632 [M + Na]+, suggesting a molecular
formula of C55H91N13O16. The 1H and 13C NMR spectra of 4
were almost identical to those of 3, except that the ketone in the
characteristic of a ketone (δC 211.5) was also apparent in the 13
C
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Journal of Natural Products
ARTICLE
Hamoh moiety had been replaced by resonances for a carbinol
moiety (δH 3.28 and δC 71.5). This, together with the molecular
weight difference of 2 between 4 and 3, suggested that the ketone
in Hamoh of 3 had been reduced to a hydroxy group in 4, forming
a Hamhh residue (2-hydroxy-3-amino-4-methyl-14-hydroxyhex-
adecanoic acid). The structure of Hamhh was further supported
by analysis of the COSY and HMBC spectra. Sequential COSY
correlations between H3-16 (δH 0.83)/H2-15 (δH 1.16 and
1.35)/H-14 (δH 3.28) and an HMBC correlation from H3-16
(δH 0.83) to C-14 (δC 71.5) placed the hydroxy group at C-14.
The absolute configurations of the amino acid residues and the
three asymmetric centers in the Hamhh residue appeared to be
the same as those found for 1 by comparison of 13C chemical
shifts and specific rotation ([R]D +2). Compound 4 possessed
one more stereogenic hydroxy-bearing carbinol carbon (C-14) in
the Hamhh residue. Mosher’s ester analysis was attempted in an
effort to assign the absolute configuration of this carbinol center.
However, the presence of three hydroxy groups (one in Thr and
two in Hamhh) resulted in a mixture of products and a very
complex 1H NMR spectrum of the Mosher’s ester products of 4.
Hydrolysis of 4 followed by isolation of the Hamhh residue
would be necessary for Mosher’s ester analysis, but was not
attempted due to the limited amount of sample available. Thus,
the absolute configuration of the C-13 carbinol carbon in Hamhh
could not be assigned conclusively.
The structures of minutissamides AꢀD (1ꢀ4) were charac-
terized by the presence of a β-amino acid residue, a 2-hydroxy-3-
amino-4-methyldodecanoic or -hexadecanoic acid, and three
nonstandard amino acid residues (NMeAsn and two Dhbs).
The β-amino acid residues were further modified by chlorination
in 2, by oxidation to ketone in 3, or by hydroxylation in 4. Inter-
estingly, the structures of minutissamides AꢀD (1ꢀ4) revealed
some similarities to the cyclic decapeptides puwainaphycins AꢀE,
isolated from a Hawaiian terrestrial Anabaena sp.9 The sequence
of the five residues in the puwainaphycins, including Dhb, Val,
β-amino acid unit, Pro, and NMeAsn, was conserved in the
minutissamides. The other five amino acid residues, OMT
(O-methyl-Thr), Gly, Gln, Thr-2 (or Val-2), and Thr-1, found
in the puwainaphycins were replaced by Thr, Ala, Asn1, Dhb1,
and Asn2 in the minutissamides. The core structure of the
lipophilic β-amino acid residues, characterized by 2-hydroxy-3-
amino-4-methyl substitution, and the chlorination and oxidation
patterns were also similar to those observed in the puwainaphycins.
Minutissamides AꢀD (1ꢀ4) were evaluated for their anti-
proliferative activity against the HT-29 human colon cancer cell
line. Minutissamide A (1) displayed antiproliferative activity with
an IC50 value of 2.0 μM, whereas lower activity was observed for
minutissamides BꢀD (2ꢀ4) with IC50 values of 20.0, 11.8, and
22.7 μM, respectively. All four minutissamides have identical
cyclic peptide cores and only differ in the lipophilic β-amino acid
residue. For example, the only structural difference between
minutissamides A (1) and B (2) was the presence of a chlorine
atom at C-12 of this residue, but minutissamide A (1) was found
to be 10-fold more active in the HT-29 assay. This suggests that
the β-amino acid residue plays an important role in the anti-
proliferative activity of these compounds.
190 to 360 nm. 1D and 2D NMR spectra were obtained on a Bruker
Avance DRX 600 MHz NMR spectrometer with a 5 mm CPTXI
Z-gradient probe and a Bruker Avance II 900 MHz NMR spectrometer
with a 5 mm ATM CPTCI Z-gradient probe. 1H and 13C NMR chemical
shifts were referenced to the DMSO-d6 solvent signals (δH 2.50 and δC
39.51, respectively). A mixing time of 60 ms was set for the TOCSY
experiments and 200 ms for the T-ROESY experiment. The HMBC
spectra were recorded with the 3JCꢀH set to 8 Hz, and the HSQC spectra
were collected with the 1JCꢀH set to 140 Hz. High-resolution ESI mass
spectra were obtained using a Shimadzu IT-TOF LC mass spectrometer.
Tandem mass analysis was performed using a Micromass Q-TOF LC
mass spectrometer.
Biological Material. Anabaena minutissima was acquired from the
Culture Collection of Algae at the University of Texas at Austin (UTEX
1613). The cyanobacterium was grown in 20 L flasks containing 18 L of
inorganic media (Z media).13 Cultures were illuminated with fluores-
cent lamps at 1.03 klx. The temperature of the culture room was
maintained at 22 °C. After 6ꢀ8 weeks, the biomass of cyanobacteria
was harvested by centrifugation and then freeze-dried.
Extraction and Isolation. The freeze-dried biomass (5.2 g) from
the total 54 L (3 ꢁ 18 L) culture was harvested and extracted with
CH2Cl2ꢀMeOH (1:1) and concentrated in vacuo to yield 1.1 g of the
extract. The extract was fractionated using Diaion HP-20 with increasing
amounts of IPA in H2O to generate eight subfractions (0, 20, 40, 60, 70,
80, 90, 100% aqueous IPA). Fraction 3 (40% IPA) indicated the pres-
ence of four peptides by LC-MS. This fraction was further purified by
Sephadex LH-20 column chromatography in MeOH. Fractions 7ꢀ9
containing the peptides were subjected to reversed-phased HPLC
(Varian C8 semipreparative column, 10 mm ꢁ 250 mm, 3 mL/min)
eluting with a linear gradient from 60 to 80% aqueous MeOH for 45 min.
Minutissamaides AꢀD (1ꢀ4) were eluted at 37.9 min (1, 1.4 mg), 31.6 min
(2, 0.4 mg), 30.0 min (3, 1.0 mg), and 33.5 min (4, 0.7 mg), respectively.
Minutissamide A (1): colorless, amorphous powder; [R]25 +4
D
(c 0.06, MeOH); UV (MeOH) λmax (log ε) 243 (3.45) nm; IR (neat)
3317 (br), 2925, 2861, 1681, 1635 (br), 1561, 1541 cmꢀ1; 1H and 13
C
NMR (see Table 1); HRESIMS m/z 1140.6049 [M + Na]+ (calcd for
C51H83N13O15Na, 1140.6029).
Minutissamide B (2): colorless, amorphous powder; [R]25 +5
D
(c 0.02, MeOH); UV (MeOH) λmax (log ε) 230 (4.24) nm; IR (neat)
3315 (br), 2926, 2852, 1670, 1647 (br), 1545, 1516, 1459, 1402 cmꢀ1
;
1H and 13C NMR (see Table 1); HRESIMS m/z 1174.5680 [M + Na]+
(calcd for C51H83ClN13O15Na, 1174.5640).
Minutissamide C (3): colorless, amorphous powder; [R]25 +3
D
(c 0.07, MeOH); UV (MeOH) λmax (log ε) 242 (2.62) nm; IR (neat)
3315 (br), 2926, 2857, 1664, 1630 (br), 1533, 1447, 1402 cmꢀ1; 1H and
13C NMR (see Table 2); HRESIMS m/z 1210.6440 [M + Na]+ (calcd
for C55H89N13O16Na, 1210.6448).
Minutissamide D (4): colorless, amorphous powder; [R]25 +2
D
(c 0.05, MeOH); UV (MeOH) λmax (log ε) 240 (3.94) nm; IR (neat)
3320 (br), 2926, 2852, 1664, 1636 (br), 1539, 1453, 1379 cmꢀ1; 1H and
13C NMR (see Table 2); HRESIMS m/z 1212.6632 [M + Na]+ (calcd
for C55H91N13O16Na, 1212.6604).
Determination of Amino Acid Configurations. Approxi-
mately 0.3 mg of minutissamide A was hydrolyzed using 6 N HCl
(500 μL) in a high-pressure tube for 16 h at 110 °C. The hydrolysate was
dried under vacuum and redissolved in H2O. This procedure was
repeated three times to completely remove the remaining HCl. For
the derivatization with Marfey’s reagent (FDAA), the hydrolysate (0.1
mg) or amino acid standard was dissolved in 50 μL of H2O, and 20 μL of
1 N NaHCO3 and 110 μL of acetone were added. The reaction was
initiated by adding 20 μL of FDAA solution (10 mg/mL w/v in
acetone), proceeded for 1 h at 40 °C, and was quenched by adding 20
μL of 1 N HCl. The reactant was dried under vacuum and redissolved in
CH3CN for HPLC analysis. Chromatographic analysis of the FDAA
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured using a Perkin-Elmer 241 polarimeter. UV spectra were
recorded on a Shimadzu UV spectrometer UV2401 and scanned from
1604
dx.doi.org/10.1021/np2002226 |J. Nat. Prod. 2011, 74, 1597–1605

Journal of Natural Products
ARTICLE
derivatives was performed on an Alltech C18 reversed-phased column
(5 μm, 250 ꢁ 4.6 mm) with the flow rate of 1.0 mL/min. Aqueous
CH3CN containing 0.1% formic acid (FA) was used as a mobile phase
eluting with a linear gradient from 10 to 100% (A: H2O containing 0.1%
FA; B: CH3CN containing 0.1% FA; gradient conditions: 0 min 10% B,
5 min 10% B, 35 min 20% B, 65 min 30% B, 70 min 60% B, 72 min 100%
B, and 80 min 100% B). The absolute configurations of the amino acids
were assigned by comparing the retention times with those of the
corresponding amino acid standards. The retention times of the FDAA
derivatives of the authentic amino acid standards were identified at 30.70
(L-Thr and N-Me-D-Asp), 32.30 (L-Asp), 36.17 (N-Me-L-Asp), 36.94
(D-Asp), 41.06 (L-Ala), 42.54 (D-Thr), 44.57 (L-Pro), 49.84 (D-Pro), 51.76
(D-Ala), 59.09 (L-Val), and 71.45 (D-Val) min. The FDAA derivatives of
the amino acids from the hydrolysate of 1 showed the peaks at 32.30,
36.17, 44.57, 51.76, and 59.09 min, corresponding to L-Asp, N-Me-L-
Asp, L-Pro, and L-Val, respectively. The peak at 30.70 min was
identified as L-Thr by LC-MS analysis (m/z 372, [M + H]+).
Advanced Marfey’s Analysis. L- and D-FDLA (1-fluoro-2,4-
dinitrophenyl-5-leucinamide) was synthesized using the protocol pub-
lished for Marfey’s analysis.21 The acid hydrolysate (0.2 mg) was
separated into two equal portions for derivatization with either L-FDLA
or DL-FDLA. Each portion was dissolved in 50 μL of H2O and then
mixed with 20 μL of 1 N NaHCO3 and 110 μL of acetone. Finally, 20 μL
of L-FDLA or DL-FDLA (10 mg/mL w/v in acetone) was added, and the
mixtures were heated to 40 °C for 1 h. The reaction mixtures were cooled to
room temperature, and 20 μL of 1 N HCl was added to quench the reaction.
After drying, the FDLA derivatives were dissolved in CH3CN for LC-MS
analysis. The chromatograms of L-FDLA and DL-FDLA derivatives were
compared for the assignment of the absolute configuration of Hamd in 1.
LC-MS analysis was performed using a Varian reverse-phased C18 column
(250 ꢁ 2.0 mm) at the flow rate of 0.4 mL/min. The linear gradient eluting
from 20 to 75% aqueous CH3CN (0.1% FA) for 50 min was used as a
mobile phase, and ESI was used as the ionization method. Two peaks
corresponding to the L- and D-FDLA derivatives of Hamd were observed at
43.33 and 48.84 min (m/z 538, [M ꢀ H]ꢀ), respectively. The L-FDLA
derivative of Hamd gave one peak at 48.84 min.
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(21) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
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HT-29 Antiproliferative Assay. Antiproliferative activity
against the HT-29 cancer cell line was performed according to
established protocols.22
’ ASSOCIATED CONTENT
S
Supporting Information. Complete NMR data tables of
b
2ꢀ4; HPLC chromatograms of Marfey’s and the advanced
1
Marfey’s derivatives of 1; H, COSY, TOCSY, HSQC, and
HMBC spectra of 1ꢀ4; DEPTQ spectra of 1, 3, and 4; ROESY
spectrum of 1; Q-TOF Tandem MS spectrum of 1. This informa-
tion is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +1-312-996-5583. Fax: +1-312-996-7107. E-mail: orjala@
uic.edu.
’ ACKNOWLEDGMENT
This research was supported by PO1 CA125066 from NCI/
NIH. We thank L. Dong of the Mass Spectrometry Laboratory at
UIC for acquiring Q-TOF tandem MS data.
’ REFERENCES
(1) Tan, L. T. Phytochemistry 2007, 68, 954–979.
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dx.doi.org/10.1021/np2002226 |J. Nat. Prod. 2011, 74, 1597–1605