4-methoxyphenylboronic acid (6) with ethyl 13C2-bromoacetate. In
a four step one-pot process, the resulting ester was saponified,
converted to the corresponding acyl chloride and reacted with
triethyl phosphite, resulting in the a-keto phosphonate (8). This
unstable compound was converted without isolation to the oxime
with hydroxylamine, which was immediately reduced in situ in the
presence of zinc–formic acid, resulting in the respective amine.
Global deprotection was achieved with hydrobromic acid and the
final product rac-AHEP (2) was obtained in a 22% overall yield
(based on ethyl bromoacetate-13C2) after purification by ion
exchange filtration. AHEP was obtained in this two-pot process
with only one chromatographic step required and . 95% purity
likely that tyrosine is a more direct metabolic precursor than
tyramine in the biosynthetic pathway leading to AHEP. If tyrosine
is indeed the direct precursor, this also suggests the possibility that
decarboxylation is coupled to the C–P bond forming reaction in a
single enzymatic reaction. In any event, these data demonstrate
that C–P bond forming biochemistry other than PEP mutase is
operative in the biosynthetic pathway of K-26.
A priori, it is expected that the K-26 gene cluster will encode an
N-acetyltransferase enzyme, which may acetylate isoleucine prior
or subsequent to peptide coupling. In the biosynthesis of
phosphinothricin, the c-phosphonyl amino acid is acetylated prior
to loading onto a nonribosomal peptide synthetase (NRPS) and is
subsequently deacetylated at a later stage to produce the bioactive
product.13 To determine if acetylation of isoleucine occurs prior to
peptide coupling, as it does in the case of phosphinothricin, feeding
studies with 13C1-labeled isoleucine and N-acetyl isoleucine were
designed (Scheme 3). N-acetyl isoleucine was synthesized by
reacting 13C2-labeled acetyl chloride and 13C1-isoleucine according
to literature precedent.14 The 13C-labeled isoleucine and N-acetyl
isoleucine were separately introduced to growing cultures of
Astrosporangium hypotensionis (0.4 mM/day for 4 days). Samples
were prepared as described above and incorporation levels were
calculated in the same manner. Incorporation of 13C1-isoleucine
and 13C2-N-acetyl 13C1-isoleucine into K-26 was calculated to be
24% and 43%, respectively (Fig. 1). Interestingly, 13C2-N-acetyl
13C1-isoleucine was not incorporated intact. That N-acetyl
isoleucine was incorporated only after being deacetylated was
indicated by the enhancement of only the M + 1 peak and not of
the M + 3. The higher level of enrichment of the M + 1 peak in the
case of 13C2-N-acetyl 13C1-isoleucine relative to isoleucine may be
due to more favorable intracellular transport properties of this less
charged compound. The requisite acetyltransferase may be a
component of an NRPS, in which the acetyltransferase would be
the first domain on the megasynthetase. Our results are not
inconsistent with this scenario. Alternatively the acetyltransferase
enzyme may be discrete from the NRPS, in which case our data
suggest that the acetylation would occur subsequent to tripeptide
elaboration (in contrast to phosphinothricin).13 A less typical
possibility is that the tripeptide is formed by the consecutive action
of two free synthetases, as in the biosynthesis of glutathione.15 In
this instance, our results would suggest that acetylation occurs
subsequent to the formation of the first peptide bond.
1
was confirmed by 31P, 13C, H NMR and mass spectrometry.
Liquid cultures of Astrosporangium hypotensionis were sepa-
rately supplemented with labeled and unlabeled synthetic racemic
AHEP (0.3 mM/day for 4 days). After six days of incubation,
K-26 samples were isolated from fermentation supernatant by
solid phase extraction with Diaion HP-20 polystyrene resin at
pH = 3 and fractionated by 5 K centrifugal molecular weight
filtration. K-26 was further separated from co-metabolites by
reverse phase HPLC/MS. Cultures that were supplemented with
unlabeled AHEP were used as a reference and grew equally well as
unsupplemented control cultures. The specific incorporation of
13C2-AHEP into K-26 amino acids was determined using the SRM
method previously described.9 The isotopic mass distribution of
K-26 from 13C2-AHEP fed cultures indicated the major isoto-
pomer at m/z = 536, a two Th shift relative to the unlabeled
standard (Fig. 1). Deconvolution of mass isotopomer data
indicates that 13C2-AHEP was incorporated into K-26 at a level
of 85% natural abundance.
The high level of incorporation of labeled AHEP in combina-
tion with previous data suggests that indeed AHEP is a discrete
precursor in the biosynthetic pathway of K-26 and the substrate of
the C–P bond forming enzyme is most likely tyrosine or a closely
related metabolite. Since decarboxylation of tyrosine is formally
required for its conversion to AHEP we also synthesized labeled
tyramine via decarboxylation of d4-tyrosine with tyrosine dec-
arboxylase from Streptococcus faecalis (Scheme 2).12 Four separate
incorporation studies (at 1.0 mM/day for 4 days) failed to
demonstrate any detectable incorporation of d4-tyramine into
K-26. While this result does not absolutely rule out the
intermediacy of tyramine in the biosynthesis of K-26, it is most
Fig. 1
Scheme 3
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 4518–4520 | 4519
Ntai, Ioanna
