column, gel filtration on a Sephadex LH-20 column, and
repeated purification on a reversed phase HPLC column to
produce a yellow glassy solid. Its positive HR ESI TOFMS
quasi-molecular ion at m/z 1156.4196 matched the molecular
formula C53H66N13O11S3 with a 2.9 mDa error. The lower field
1H NMR spectrum of 1 in DMSO-d6 revealed three sharp
singlets between 8.20 and 8.10 ppm, suggesting that 1 contains
three thiazole moieties. Additional signals in this region were
two exchangeable broad singlet protons, six exchangeable broad
doublet protons, and three exchangeable broad triplet protons,
as well as signals of a monosubstituted phenyl and a para-
substituted phenol moiety. In the middle region of the 1H NMR
spectrum of 1, 12 protons were observed, and in the higher
field of the spectrum, six doublet methyls were present. The
13C NMR spectrum exposed 49 absorption lines, suggesting that
four of the signals are doubled as a result of symmetry. Of the
49 carbon signals, 13 resonate between 174 and 159 ppm,
suggesting that they belong to carboxamide carbons. Three of
these signals, resonating around 160 ppm, were of conjugated
carboxamide groups characteristic of carboxazole moieties.8 The
1D (one dimensional) NMR data thus suggest that 1 is a peptide
containing three thiazole moieties.
Figure 1. Substructures assigned for 1.
The structure elucidation of the amino acids that compose 1
was initiated with the analysis of the COSY and TOCSY
correlation maps measured in DMSO-d6. This resulted in the
assignment of the R-amide and side chains as three glycines,
two valines, a leucine, three ABMX spin systems where X is
an amide proton, a primary amide (δH 7.47 brs and 7.00 brs
ppm), a monosubstituted phenyl ring, and a para-substituted
phenol moiety, accounting for 62 out of the 65 protons of 1.
fragments could be assembled to the complete structure by
HMBC correlations of the carboxamide of a subunit with amide
(2J) or R-proton (3J) of the adjacent subunit, namely, the
carboxamide of Tyr with the 5-amide proton of Tzl-Asn, the
carboxamide of Tzl-Leu with Tyr-R-amide proton, the car-
boxamide of Tzl-Val with the 5-proton and 5-amide proton of
Tzl-Leu, the carboxamide of Phe with the 5-amide proton of
Tzl-Val, the carboxamide of Val with Phe-R-amide proton, the
carboxamide of Gly(1) with Val-R-amide proton, the carboxa-
mide of Gly(2) with Gly(1)-R-amide proton, the carboxamide
of Gly(3) with Gly(2)-R-amide proton, and the carboxamide
of Tzl-Asn with Gly(3)-R-amide proton that assigned the closure
of the macrocyclic ring (see Figure 2). The NOE correlations
1
Data from the H-13C HMQC experiment (see Table 1)
assigned all of the protonated carbons to the fragments suggested
by the COSY and TOCSY experiments, as well as the thiazoles
singlet protons (δH 8.20, 8.16, and 8.14 ppm) to the corre-
sponding sp2 carbons (δC 124.5, 124.4, and 124.8 ppm,
1
respectively). The H-15N HMQC experiment (see Table 1)
assigned the nine secondary nitrogens and the primary amide
to the amino acid fragments (see Table 1). The structure
elucidation of the thiazole moieties was based on HMBC
correlations. Each one of the thiazole singlet protons (δH 8.20,
8.16, and 8.14 ppm) presented, in the 1H-13C HMBC experi-
ment, a 2J correlation with C-2 of the thiazole moiety (resonating
3
at δC 148.5, 149.0, and 148.8 ppm, respectively) and J
correlations with C-1 (resonating at δC 160.1, 160.9, and 159.7
ppm, respectively) and C-4 (resonating at δC 171.2, 173.5, and
171.2 ppm, respectively). The latter thiazole protons presented
1
3J correlations in the H-15N HMBC experiment with the
quaternary nitrogen atoms resonating at δN 309.2, 307.7, and
307.3 ppm, respectively, thus establishing the structure of the
three thiazole rings. The structure elucidation of the thiazole
moieties was completed with the assignment of the 2J correlation
of the R-protons of the amino acid with C-4 of the thiazole
system (the carboxyl of the precursor amino acid) (see Table 1
and Figure 1). For the other six amino acids, a 2J (and in some
cases J H-C correlation) correlation of the R-proton(s) and
the corresponding carboxamide assigned the latter to the
complete substructures (see Table 1 and Figure 1). The nine
Figure 2. HMBC and NOE correlations between the subunits of
aeruginazole A (1).
3
from the ROESY experiment, on the other hand, could assign
only small fragments because the thiazole protons did not show
correlations to the neighboring amino acids (see Table 1 and
Figure 2).
(8) Banker, R.; Carmeli, S. J. Nat. Prod. 1999, 61, 1248–1251.
3538
Org. Lett., Vol. 12, No. 15, 2010
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