1448
D. L. Jakeman et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1447–1449
Scheme 1. Synthesis of non-natural amino acid analogues (1) and (2).
Table 1. Enhanced product ion scan (m/z), parent ions observed for
ESI-MS of ethyl acetate culture extracts of Streptomyces venezuelae
ISP5230
structural analogues with potentially interesting bioac-
tivity. Altering growth conditions by using different ami-
no acids was initially reported by Doull et al.3 Herein,
initial mass spectrometry data are presented on the
incorporation of two non-natural amino acid analogues,
O-methoxy-L-threonine (1) and O-methoxy-methylene-
L-threonine (2), D-val, D-ile and all 20 naturally occur-
ring L-amino acids.
Amino
acid
[M+H]+ Amino [M+H]+ Amino [M+H]+
acid
acid
1
2
552.0
582.1
L-Alaa 507.3
L-Cysa 540.2
L-Meta 568.3
L-Gly
L-Leu
L-Val
L-Trp
L-Tyr
L-Asn
L-Gln
494.5
550.2
536.4
623.2
600.5
551.4
565.3
Jadomycin B 550.3
L-Asp
L-Glua 566.2
L-Arg 593.3
552.3
D-Ile
550.3
536.5
524.3
538.3
583.9
D-Val
L-Ser
L-Thr
L-Phe
The syntheses of the isosteric isoleucine analogue 1 and
novel analogue 2, starting from commercially available
N-carbobenzyloxy-L-threonine methyl ester, are pre-
L-Hisa 574.4
L-Lysa 565.4
L-Proa 536.2
1
sented in Scheme 1 (1D and 2D H and 13C NMR data
a Parent ion fragmentation to the phenanthroviridin [M+H]+ 306 was
not observed.
are included in the Supplementary data for compounds
1 and 2).
Streptomyces venezuelae ISP5230 cultures were grown as
described previously using one of the amino acid ana-
logues as a sole nitrogen source.2 The cultures grew,
based on doubling of optical density (OD) 600 measure-
ments, and were shocked with ethanol after the standard
6.5 h. In comparison to growths to produce jadomycin
B, the cultures were then left for an additional 24 h be-
fore harvesting and extracting due to a less intense col-
our build-up post ethanol shock. The delay in colour
build-up may potentially arise from difficulties in meta-
bolizing these amino acid analogues. Electrospray ioni-
zation mass spectrometry (ESI-MS) data of the infused
culture extracts is presented in Table 1 and the corres-
ponding enhanced product ion (EPI) scan data in Figure
2 (EPI mass spectra for all extracts are included in the
Supplementary data). These data provide definitive evi-
dence for the incorporation of the non-natural amino
acids into the jadomycin aglycone by the observation
of the parent ion ([M+H]+) and the unequivocal break-
down of this ion to the non-glycosylated aglycone ([agly-
cone+H]+) and subsequently to phenanthroviridin.
10 h, however, over the remaining 48 h the intensity of
the culture colour was comparable to growths on L-ami-
no acids. From the ESI-MS data in Table 1 it was ob-
served that the two amino acids were incorporated into
a corresponding jadomycin B analogue.à As expected,
the enhanced product ion spectra of these amino acid
substituted jadomycins also broke down into the agly-
cone and ultimately the phenanthroviridin. Table 1 also
summarizes electrospray mass spectral (ESI-MS) data of
all ethyl acetate culture extracts from the naturally
occurring L-amino acids. These data were also acquired
in enhanced product ion mode providing definitive evi-
dence of parent ions [M+H]+ that are unequivocally
the results of incorporating naturally occurring L-amino
acids into the jadomcyin aglycone. The ESI-MS data
intensities of parent ion and fragmentation peaks for
the [aglycone+H]+ and [phenanthroviridin+H]+ ob-
served in enhanced product ion mode were highly depen-
dent on the collision energy between Q0 and Q2; these
fragments were not observed unambigiously for all acidic
or all basic amino acid extracts. The importance of the
oxazolone ring upon pharmacological activity and sub-
sequently pharmacokinetic, pharmacodynamic parame-
ters will be reported in due course.
When D-Val and D-Ile were used as sole nitrogen sources,
the cultures initially grew at a reduced rate on the mini-
mal media and ethanol shock was delayed from 6.5 to
The incorporation of novel non-natural amino acid ana-
logues by Streptomyces venezuelae ISP5230 into jadomy-
cin B analogues significantly broadens the potential
range of secondary metabolites that can be produced
The molar concentration of amino acid was 70 mM.3 Strain VS1099
was used to obtain a twofold increase in production levels of the
jadomycin analogue.14 Extracted masses varied between 1 and 30 mg
of material. Extracts were standardized to an Abs313 = 0.01 in 50%
methanol (0.5% formic acid) prior to ESI analysis on an Applied
Biosystems-MDS SCIEX triple quadrupole linear ion trap with an
ambient source temperature, N2 as collision gas for enhanced product
ion scans, and with a turbospray ion source loaded by infusion at
à For D-val and D-ile extracts, the stereochemical assignment at the a-
(and b-) carbons remains to be determined. The structure of the
isolated product resulting from growth on L-pro remains to be
determined and will be reported in due course.
10 lL minÀ1
.