sp. UIC 10035 was isolated from a sample collected near the town of Homestead, South Florida, 2007, and cultured in an inorganic Z medium.15
The freeze-dried cells (7.5 g from 20 L) were extracted with a mixture of CH2
and MeOH (1:1, v/v), and dried in vacuo. The resulting extract (0.5 g) was fractionated using Diaion HP-20 resin with an increasing amount of i
PrOH in H2
O. Fractions eluting at 40 and 60 % aqueous i
PrOH (v/v) exhibited antiproliferative activity against MD-MB-435 cells. LC-MS analysis of these fractions indicated the presence of a series of nitrogen-containing compounds with molecular weights ranging between 1150 and 1250 Da. These fractions were combined and subjected to HPLC purification using reversed-phase columns to yield eight cyclic lipodecapeptides, named minutissamides E - L (1
Minutissamide E (1) was obtained as a colorless, amorphous powder. The molecular formula was determined as C59H100N12O17 by HRESIMS analysis. The signal distribution pattern observed in the 1H NMR spectrum (amide NH, H 6.0 – 10.0; amino acid H, H 3.5 – 5.5; largely overlapped aliphatic CH2, H 1.25; aliphatic doublet CH3, H 0.5 – 2.0) suggested this compound to be a lipopeptide. The presence of ten amino acid residues were identified by 2D NMR analysis, including one lipophilic -amino acid, six standard amino acids and three non-standard amino acids. Analysis of the COSY and TOCSY spectra established the structures of six standard amino acids as Pro, Ala, Gln, Thr (2×) and Val. The structures of three non-standard amino acids were determined as NMeAsn, OMeThr and Dhb by interpretation of the COSY, HSQC and HMBC spectra (). The structure of the -amino acid residue was also determined by analysis of 2D NMR data as described below. COSY correlations between NH (H 6.77) and H-3 (H 3.92), and between a doublet methyl (H 0.57) and H-4 (H 1.68), as well as sequential COSY correlations of H-2/H-3/H-4/H2-5, which in turn coupled with the overlapped methylene signals at H 1.25, indicated the presence of a 3-amino-2-hydroxy-4-methyl functionality coupled to a lipid portion (). The isolated TOCSY correlation fragment of H2-16/H2-17/H3-18 and HMBC correlations from H2-17 (H 1.47) to a ketone carbon (C 210.6) indicated that a ketone was positioned at C-15, determining the structure of the -amino acid residue as Ahmoo (3-amino-2-hydroxy-4-methyl- 15-oxooctadecanoic acid).
Key 2D correlations of minutissamide E (1) used for the determination of the planar structure of 1.
The established ten amino acid residues were connected by combined analysis of the HMBC and ROESY spectra (). Starting from the Ahmoo residue, HMBC correlations from Ahmoo NH to Pro C-1 (C 171.2) and from N-Me (H 2.93) to NMeAsn C-2 (C 49.7) and OMeThr C-1 (C 169.6), combined with NOE correlations between Ahmoo NH (H 6.77) and Pro H-2 (H 4.25), between Pro H2-5 (H 3.11 and 4.21) and NMeAsn H-2 (H 5.52), established a partial sequence of Ahmoo-Pro-NMeAsn-OMeThr. This sequence was further expanded into Ahmoo-Pro-NMeAsn-OMeThr-Ala-Gln-Thr1 by HMBC correlations from OMeThr NH (H 6.74) to Ala C-1 (C 171.9), from Ala NH (H 7.58) to Gln C-1 (C 171.1) and from Gln NH (H 7.25) to Thr1 C-1 (C 170.4), as well as NOE correlations between OMeThr NH (H 6.74) and Ala H-2 (H 4.19), between Ala NH (H 7.58) and Gln H-2 (H 4.08), and between Gln NH (H 7.25) and Thr1 H-2 (H 3.90). The complete sequence of Ahmoo-Pro-NMeAsn-OMeThr-Ala-Gln-Thr1-Thr2-Dhb-Val was established by HMBC correlations from Thr2 NH (H 8.37) to Dhb C-1 (H 163.9) and from Dhb NH to Val C-1 (C 168.8), together with NOE correlations between Thr1 NH (H 8.84) and Thr2 H-2 (H 5.02), and between Dhb NH (H 9.08) and Val H-2 (H 4.31). Lastly, HMBC correlations from Val NH (H 6.85) and Val H-2 (H 4.31) to Ahmoo C-1 (C 169.6), and a NOE correlation observed between Val NH and Ahmoo H-2, closed the ring, completing the cyclic lipodecapeptide structure of 1.
To confirm the amino acid sequence of 1
, MS fragmentation analysis was carried out using quadrupole ion trap CID (collision-induced dissociation) MS/MS data (). In the MS1
analysis, the most intense peak corresponded to the sodium adduct ion of 1
1271.7). CID fragmentation of this sodiated precursor ion generated a set of y-type fragment ions instead of b-type, which are commonly observed in CID fragmentation of protonated precursor ions. This predominant formation of y-type fragments by CID of sodiated precursor ions has been previously reported. The two most intense ions at m/z
1227.7 and 1183.7, corresponding to [M + Na - CONH2
and [M + Na - 2CONH2
, respectively, resulted from the sequential loss of the side chain amides from N
MeAsn and Gln. Ring-opening at two different sites yielded two sets of y-type fragment ions (). Ring-opening (A) between Dhb and Thr yielded ions at m/z
755.5, 870.5, 941.6 and 1069.6, corresponding to y5
fragments, respectively (). The second set of ions, resulted from ring-opening) between N
MeAsn and Pro, included m/z
465.2, 667.3, 750.3, 849.4 and 1174.7, corresponding to y4
fragments, respectively (). This fragmentation pattern agreed well with the sequence determined by 2D NMR analysis, thus further confirming the amino acid sequence of 1
ESI-MS/MS fragmentation of minutissamides E and I (1 and 5).
The stereoconfiguration of 1
, including the geometric configuration of Dhb and the absolute configurations of - and -amino acids, was determined on the basis of NOE correlations and the advanced Marfey's method as well as comparison of 1
H and 13
C NMR chemical shifts. A strong NOE correlation, observed between Dhb NH and Dhb H-3, determined E
-geometry for the Dhb residue. For the assignment of amino acid configurations, advance Marfey's analysis was carried out as previously described.17-19
LC-MS comparison between l
-FDLA and dl
-FDLA derivatives of the acid hydrolysate of 1
-configuration to Pro, Gln, and Val, and d
-configuration to Ala. The l
-FDLA derivative of the acid hydrolysate of 1
was further compared with those of authentic standards of Thr (l
-Thr and d
-Asn and N
-Asn) and O
-Thr and O
-Thr), assigning l
-configuration to these amino acids. Minutissamide E (1
) contained the same asymmetric centers (3-amino2-hydroxy-4-methyl) in the -amino acid residue as the previously described puwainaphycins and minutissamides A - D.9,14
These three consecutive stereocenters in Ahmoo showed nearly identical 1
H (< 0.1 ppm) and 13
C (< 1 ppm) NMR chemical shifts to those of the puwainaphycins and minutissamides A - D. In addition, a small 3JHH
between H-2 and H-3 (4.8 Hz) and a large 3JHH
between H-3 and H-4 (10.8 Hz) observed in Ahmoo of 1
were similar to those of the puwainaphycins (5.5 Hz and 11.3 Hz) and minutissamides A - D (5.6 Hz and 11.0 Hz). This suggested the relative configuration of Ahmoo in 1
to be the same as those found in the puwainaphycins and minutissamides A - D. The absolute configuration of Ahmoo was then determined using the advanced Marfey's method as previously described.14, 19
In LC-MS analysis, the dl
-FDLA derivative of the acid hydrolysate of 1
exhibited two peaks at 67.0 and 72.2 min, corresponding to the molecular weight of the FDLA derivative of Ahmoo (m/z
638.3 [M + H]+
), while the l
-FDLA derivative resulted in one peak at 72.2 min. This result was consistent with those reported for the -amino acids Ahda (3-amino-2-hydroxydecanoic acid) in microginin19
and Hamd (3-amino-2-hydroxy-4-methyldodecanoic acid) in minutissamide A,14
suggesting the same absolute configuration (3R
) in Ahmoo. Taken together, the absolute configuration of the three consecutive stereocenters (C-2, C-3 and C-4) in Ahmoo was assigned as 2R
, completing the stereoconfiguration of 1
Minutissamide F (2) was obtained as a colorless, amorphous powder. The molecular formula was determined as C55H93ClN12O16 by HRESIMS, which exhibited a molecular ion peak at m/z 1213.6645 ([M + H]+) and a M + 2 isotope peak. Comparison of the 1D and 2D NMR spectra of 2 and 1 revealed that compound 2 shared the same amino acid sequence as 1 and differed only in the -amino acid side chain. The four carbon differences in the molecular formula between 2 and 1 suggested that the -amino acid residue of 2 was composed of 14 carbons (tetradecanoic acid) instead of 18 carbons. The down-field proton signal of H-12 (H 3.99), as well as sequential COSY correlations from the terminal methyl H3-14 (H 0.96) to H-12 via one diastereotopic methylene H2-13 (H 1.64 and 1.78) and an HMBC correlation from H3-14 to C-12 (C 66.4), positioned the chlorine at C-12, establishing the structure of the -amino acid residue as Achmt (3-amino-12-chloro-2-hydroxy-4-methyltetradecanoic acid). Compound 2 showed nearly identical 1H and 13C NMR chemical shifts to those of 1 at all of the stereocenters, and the negative specific rotation ([ ] D -19) was also similar to that of 1 ([ ] D -29), thus indicating the stereoconfiguration of the stereocenters of 2 to be the same as those observed for 1. The absolute configuration of the additional chlorine-bearing stereocenter (C-12), located distant from other stereocenters in Achmt, was not assigned.
Minutissamide G (3) was obtained as a colorless, amorphous powder. The HRESIMS spectrum of 3 displayed a molecular ion at m/z 1251.7564 ([M + H]+), suggesting the molecular formula as C59H102N12O17, which was two mass units higher than that of 1. The 1H NMR spectrum of 3 also closely resembled that of 1, except for in the -amino acid residue. The difference was the up-field shift of signals for H2-14 (H 1.26 and 1.30) and H2-16 (H 1.28) in the -amino acid residue as compared to those of 1. These two methylene signals were connected by the oxygenated methine proton H-15 (H 3.35) in the COSY spectrum, indicating the structure of the -amino acid in 3 to be Adhmo (3-amino-2,15-dihydroxy4-methyloctadecanoic acid). The position of a hydroxy group at C-15 was further supported by an HMBC correlation from the terminal methyl proton signal of H3-18 (H 0.86) to the down-fielded carbon signal of C-16 (C 39.9, the -carbon of a hydroxy group). The stereoconfiguration of 3 was suggested to be the same as that of 1, based on the nearly identical NMR chemical shifts, observed for all the stereocenters. In addition, compound 3 showed the negative specific rotation ([ ]D -22) similar to that of 1 ([ ] D -29). The assignment of the absolute configuration for the additional hydroxy-bearing carbinol stereocenter (C-15) in Adhmo was not attempted due to the limited amount of sample available.
Minutissamide H (4) was obtained as a colorless, amorphous powder. The molecular formula was deduced as C60H102N12O16 by HRESIMS analysis (m/z 1247.7622 [M + H]+). Detailed analysis of the COSY and TOCSY spectra suggested that the structure of 4 was similar to that of 1, but differed by one amino acid in the cyclic peptide core. In the TOCSY spectra, a new spin system, corresponding to the amino acid Val, replaced the spin system of Thr1, indicating the presence of Val1 instead of Thr1. The amino acid configurations of 4 were determined by advanced Marfey's analysis as described for 1. LC-MS comparison between the l- and dl-FDLA derivatives of the acid hydrolysate of 4 assigned the l-configuration to Val. The result of advanced Marfey's analysis also suggested that the absolute configurations of the rest of the other amino acids in 4 are the same as those found for 1.
Minutissamide I (5), a colorless, amorphous powder, displayed a molecular ion peak at m/z 1235.7328 ([M + H]+) in HRESIMS analysis, indicating a molecular formula of C58H98N12O17. The molecular weight of 5 was smaller than that of 1 by 14 mass units and identical to that of the previously reported cyclic lipodecapeptide, puwainaphycin A.9 Detailed analysis of the 2D NMR spectra revealed that the structure of 5 differs from that of 1 by the presence of Gly instead of Ala, thus sharing the same cyclicpeptide core as puwainaphycin A. The only difference found between 5 and puwainaphycin A was in the position of the ketone in the -amino acid residue Ahmoo. An isolated spin system, composed of the proton signals of H3-18 (H 0.83), H2-17 (H 1.46) and H2-16 (H 2.36), was found in the TOCSY spectrum. This, together with HMBC correlations from H3-18 and H2-17 to C-15 (C 210.9), indicated the ketone to be positioned at C-15 in 5 instead of at C-14 as found for puwainaphycin A. The amino acid sequence of 5 was further confirmed by analysis of tandem MS data, which showed a nearly identical fragmentation pattern to that of 1 (). Lastly, advanced Marfey's analysis of the acid hydrolysate of 5, as well as 1H and 13C NMR data, suggested the absolute configurations of all of the amino acids to be the same as those reported for puwainaphycin A.
Minutissamide J (6) was also obtained as a colorless, amorphous powder. The HRESIMS spectrum of 6 displayed a molecular ion peak at m/z 1199.6477 ([M + H]+) as well as a M + 2 isotope peak, indicating the presence of a chlorine, thus the molecular formula of 6 was deduced as C54H91ClN12O16. Analysis of 1D and 2D NMR data of 6 revealed that the structure of 6 is similar to that of 5 except for the -amino acid residue. The 36 Da mass difference between 6 and 5 and the presence of a chlorine instead of an oxygen indicated the structure of the -amino acid residue in 6 to be Achmt, the same as found in 2. The chlorine was placed at C-12 by an HMBC correlation from the terminal methyl protons H3-14 (H 0.96) to the chlorinated carbon C-12 (C 66.4). The stereoconfiguration of 6 was considered to be the same as that found for 5 based on the nearly identical 1H and 13C NMR chemical shifts at all of the stereocenters and the negative specific rotation ([ ]D -19) similar to that of 5 ([ ]D -26). The absolute configuration of the additional chlorine-bearing stereocenter (C-12) in Achmt of 6 was not assigned.
The last two compounds, minutissamides K (7) and L (8), were obtained as a mixture in a ratio of 3:5 as determined by 1H NMR analysis. The HRESIMS spectrum of the mixture showed the two molecular ion peaks at m/z 1237.7381 (7, [M + H]+) and m/z 1233.7482 (8, [M + H]+), suggesting the molecular formulas as C58H100N12O17 and C59H100N12O16, respectively. Attempts to separate 7 and 8 using reversed-phase HPLC failed due to complete overlap of the two peaks, thus structure determination was carried out using the mixture. The majority of signals in the 1H NMR spectrum appeared to be identical between 7 and 8, except for one amino acid and the -amino acid residue. In the TOCSY spectrum, two doubled NH (H 9.03 and H 8.67) and -H (H 3.80 and H 3.82) signals, the sum of which showed the same integration as other signals, indicating thatthese signals belong to different spin systems, Thr1 and Val1. In addition, analysis of the COSY and HMBC spectra identified the presence of the two -amino acids, Adhmo and Ahmoo. The molecular weight and the integration ratio suggested the presence of Thr1 and Adhmo in 7, and Val1 and Ahmoo in 8. Advanced Marfey's analysis of the acid hydrolysate of the mixture assigned the l-configuration to all of the amino acids present in 7 and 8. Nearly identical 1H and 13C NMR data and of stereogenic centers (C2, C3 and C4) in the -amino acid residue to those of other compounds (1 - 6) as well as minutissamides A - D and the puwainaphycins and the negative specific rotation ([ ]D -26) suggested the absolute configuration of the -amino acid residue for both 7 and 8 to be the same (2R,3R,4S). The determination of the absolute configuration at C-15 in Adhmo was not attempted in this study due to the limited amount of sample available.
sp. UIC 10035 produced several cyclic lipodecapeptides in a laboratory culture that showed close structural relationships with the previously reported cyclic lipodecapeptides, puwainaphycins A - E and minutissamides A - D isolated from Anabaena
sp. BQ-16-1 and Anabaena minutissima
UTEX 1613, respectively.9,14
However, minutissamides E - L showed higher structural diversity than the puwainaphycins and minutissamides A - D. This high structural diversity of cylic lipodecapeptides produced by the strain UIC 10035 is likely derived from its biosynthetic flexibility to incorporate two different amino acids (Ala/Gly and Thr1/Val1) by adenylation domains in two NRPS modules, and two different fatty acids (probably hexadecanoic acid/dodecanoic acid) by an acyl ligase in a PKS loading module followed by variable modifications including oxidation, chlorination and hydroxylation. As observed for the adenylation domain in anabaenapeptin biosynthesis,20
minutissamides E - L (1
) produced by the strain UIC 10035 might also represent a good example of nature's strategy to diversify their structures by substrate promiscuity.
Minutissamides E - L (1 - 8) were evaluated for their antiproliferative activity against the MDA-MB-435 human melanoma cancer cell line. All of the compounds exhibited similar levels of activity with IC50 values ranging between 1 and 10 μM (1, 2.9 μM; 2, 1.2 μM; 3, 9.9 μM; 4, 1.1 μM; 5, 2.9 μM; 6, 2.6 μM; the mixture of 7 and 8, 2.9 μg/ml). Compounds 1 - 8 were also evaluated for antibacterial activity against Mycobacterium (M. smegmatis), Gram-positive (S. aureus, E. faecalis and S. pneumoniae) and Gram-negative (A. baumannii, E. coli and P. aeruginosa) bacteria, but showed no activity at the highest concentrations tested (10 μg/ml).
Taxonomic identification of UIC 10035 was established on the basis of morphological observation and phylogenetic analysis using a partial 16S rRNA gene sequence (1.2 Kb). The strain UIC 10035 was filamentous and developed heterocysts in nitrogen-deficient media, indicating it belonging to the order Nostocales. Long straight and yellow-green colored trichomes and the lack of mucilage indicated UIC 10035 potentially being of the genus Anabaena. However, the partial 16S rRNA gene sequence of UIC 10035 showed the highest sequence homology to those of Nostoc sp. PCC8112 (99.9 %), Nostoc sp. PCC8976 (98.5 %) and Nostoc elgonense TH3S05 (97.8 %). In the phylogenetic tree constructed using cyanobacterial 16S rRNA gene sequences (), the monophyletic clade, containing the UIC 10035 strain and three Nostoc spp., was not clustered with any of the genus reference strains for either Nostoc or Anabaena, complicating the taxonomic assignment of the UIC 10035 strain. Pairwise distance analysis between clades indicated that this clade is the most closely related with the clade containing the strain Anabaena minutissima UTEX 1613 (0.03), the producer of minutissamides A - D, and the clade of the genus Anabaenopsis (0.03). Based on the morphological similarity and close phylogenetic relationship to the strain Anabaena minutissima UTEX 1613, the UIC 10035 strain was designated as a cf. Anabaena sp.
Figure 3 Phylogenetic relationships of 16S rRNA genes from cyanobacteria. Evolutionary distances were determined using the minimum evolution method with 1,000 replicate bootstrap re-samplings to construct the phylogenetic tree. Strains denoted with an asterisk (more ...)