Mannopeptimycins from Streptomyces hygroscopicus
A R T I C L E S
Aiha-A, the HMBC correlations from HR at δ 4.24 to CdO at
173.2, to Câ at 71.5, and to C-4′ at 57.8, and from H-4′ at 2.26
and H2-5′ at 3.46 and 3.11 to a guanidino carbon, C-2′, at 161.7,
were observed. For Aiha-B, the HMBC correlations from Hâ
at δ 4.25 to CR at 57.5 and to C-4′ at 63.5, and from H-4′ at
4.23 and H2-5′ at 3.78 and 3.76 to C-2′ at 161.6, were observed.
glycine, â-methylphenylalanine, tyrosine, Ahia-A, and Ahia-
B, and the R-proton signals of Ahia-B, serine, glycine, â-me-
thylphenylalanine, tyrosine, and Ahia-A in a ROESY spectrum
also supported the sequence (Figure 5).
The molecular formula of mannopeptimycin γ (3) was
determined by high-resolution FTICR mass spectrometry to be
1
C59H86N12O26. Compared to 1, the H and 13C NMR spectral
Furthermore, a careful analysis of the TOCSY and COSY
spectral data led to the identification for a third mannosyl
moiety. The seven-proton spin system from H-1 to H2-6 and
the correlation from its anomeric proton signal (H-1) at δ 5.13
to C-5 at 82.7 in the HMBC spectrum were indicative of a
pyranose moiety. The large 3JH1-H2 (8.0 Hz), and small 3JH2-H3
data of 3 showed the presence of an isovaleryl group in addition
to the cyclic peptide core and three mannosyl moieties. In a
COSY spectrum, the signal of two methyl doublet signals at δ
0.96 (6H, d, 6.6 Hz) was coupled to a methine signal at 2.09
(m) which was in turn coupled to methylene protons at 2.40
(2H, m). The H-2 signal of Man-B at δ 5.18 (dd, 4.0, 1.8 Hz)
was coupled to H-1 of the same sugar at 5.33 in the COSY
spectrum and to C-1 of the isovaleryl group at 177.3 in an
HMBC spectrum. The isovaleryl group was thus determined to
be attached to C-2 of Man-B. Mannopeptimycin γ (3) was
hydrolyzed with 5% sodium carbonate at ambient temperature
to afford 1.
Mannopeptimycins δ (4) and ꢀ (5) were also assigned to be
isovaleryl esters, differing from 3 only in the positions of the
ester group, where the isovaleryl was connected to C-3 and C-4
of Man-B in 4 and 5, respectively. The same hydrolysis
condition was respectively applied to 4 and 5 to obtain 1. For
13C NMR data of 3, 4, and 5, see Table 3.
Absolute Stereochemistry. Mannopeptimycin R (1) was
subjected to standard peptide hydrolysis (6 N HCl, 100 °C) to
yield the component amino acids. The absolute configurations
of the tyrosine and serine were respectively established to be D
and L by LC/MS analysis of the Marfey’s derivatives.8 The
â-methylphenylalanine isolated from the hydrolysate was found
to have a (2S, 3S) configuration by comparing its chemical shift
data, 1H-1H coupling constants, and optical rotation value with
literature data for the four isomers obtained by synthesis.9 The
configurations of the remaining chiral centers in Aiha-A and
-B and the conformation of the cyclic hexapeptide core were
determined by derivatization and further NMR experiments.
The mixture of the polar amino acids of the hydrolysate was
reacted with benzyl chloroformate (CBZCl) to yield a benzyl
carbamate mixture of diastereoisomeric Aiha-A and -B, not
separable by HPLC. The mixture was treated with methyl iodide
and the resulting methyl esters were carefully purified by reverse
phase HPLC to afford 9 and 10. The coupling constants,
3JHR-Hâ’s, were measured as 5.9 and 1.5 Hz respectively for 9
and 10 (Figure 4). This was indicative of an erythro configu-
ration for the former and threo for the latter, compared with
literature data for R-amide-â-hydroxycarboxylic acid deriva-
tives.10 It was interesting to note that the presence of a pair of
L- and D-R-amino-â-[4′-(2′-iminoimidazolidinyl)]propionic acid
residues, designated enduracididine and alloenduracididine, was
reported in the cyclic peptide enduracidin.11
(3.0 Hz), JH3-H4 (4.5 Hz), and 3JH4-H5 (3.0 Hz), together with
3
a ROESY cross-peak between H-1 at δ 5.13 and H-6 at 4.11,
suggested a mannose moiety with an axial C-6 hydroxymeth-
ylene. A similar coupling pattern and corresponding ROESY
cross-peak were observed in the spectra of compound 2.
The attachment of C-1 (δ 82.8) of this sugar to N-3′ in Aiha-B
was required by three-bond HMBC correlations from H-1 at δ
5.13 to C-4′ at 63.5 and to the guanidino carbon C-2′ at 161.6.
In the electrospray ionization mass spectrum, the doubly charged
molecular ion (M + 2H)2+ was displayed as a prominent signal,
supporting the presence of two basic groups in the molecule.
Evidence for the cyclic nature of the peptide core of 1 came
from the molecular formula, which required an additional degree
of unsaturation beyond those already accounted for in the
substructures. The sequence of the peptide core was primarily
determined by analysis of 1H-13C HMBC data. The respective
three-bond correlations in an HMBC spectrum from R-protons
of serine, glycine, â-methylphenylalanine, tyrosine, Aiha-A, and
Aiha-B to carbonyls of Aiha-B, serine, glycine, â-methylphe-
nylalanine, tyrosine, and Aiha-A established the amino acid
sequence for the cyclic hexapeptide to be c-[Ser-Gly-Mephe-
Tyr-Aiha-A-Aiha-B] (Table 1). In the HMBC spectrum, the 1H-
13C correlation from H-1 in Man-A at δ 5.47 to C-4′ in Tyr at
157.5 required that the mannosyl disaccharide moiety be
attached to the tyrosine. Thus, the elucidation of the planar
structure for compound 1 was completed.
The molecular formula of mannopeptimycin â (2) was
determined by high-resolution FTICR mass spectrometry to be
C42H58N12O15, which was C12H20O10 less than compound 1.
Compound 2 showed 1H and 13C NMR spectral data very similar
to those of 1, except that signals for the mannosyldisaccharide
moiety on the tyrosine were lacking (for 13C NMR data of 2,
see Table 3). In addition, compound 2 was obtained by
hydrolysis of 1 with 5% aqueous HCl at 60 °C (Figure 3);
therefore, the structure was confirmed.
Compound 2 was oxidized with potassium periodate to afford
a dialdehyde 6, which was reduced to the corresponding alcohol
7 by sodium borohydride. The hydrolysis of 7 with 5% aqueous
hydrochloric acid at 80 °C afforded the aglycone (Figure 3).
The TFA salt of the aglycone (8), obtained by HPLC with TFA-
containing mobile phase, was subjected to NMR studies (spectral
data acquired in DMSO-d6). The respective two-bond 1H-13C
correlations from NH proton signals of serine, glycine, â-me-
thylphenylalanine, tyrosine, Ahia-A, and Ahia-B to carbonyls
of Ahia-B, serine, glycine, â-methylphenylalanine, tyrosine, and
Ahia-A in an HMBC spectrum confirmed the assigned sequence
for the cyclic hexapeptide (Table 2). In addition, the respective
strong ROESY cross-peaks between NH proton signals of serine,
As indicated, the strong NOE cross-peaks between the R-H’s
of the amino acid residues and the NH’s of their adjacent
(8) Fuji, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.-i. Anal. Chem. 1997,
69, 5146-51.
(9) Kataoka, Y.; Seto, Y.; Yamamoto, M.; Yamada, T.; Kuwata, S.; Watanabe,
H. Bull. Chem. Soc. Jpn. 1976, 49 (4), 1081-4.
(10) (a) Rassu, G.; Zanardi, F.; Cornia, M.; Casiraghi, G. J. Chem. Soc., Perkin
Trans. 1 1994, 17, 2431-7. (b) Monache, G. D.; Di Giovanni, M. C.; Misiti,
D.; Zappia, G. Tetrahedron: Asymmetry 1997, 8 (2), 231-43.
(11) (a) Horii, S.; Kameda, Y. J. Antibiot. 1968, 665-7. (b) Hatano, K.; Nogami,
I.; Higashide, E.; Kishi, T. Agric. Biol. Chem. 1984, 48 (6), 1503-8.
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J. AM. CHEM. SOC. VOL. 124, NO. 33, 2002 9731