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afford vinylphosphonate 5 and its constitutional isomer 6
(Scheme 2). Both compounds were separated, deprotected,
and coupled to the protected dipeptide CbzNH-Gly-Leu by
The structure of synthetic 2 was confirmed unambiguously by
X-ray crystallography (see the Supporting Information), and
therefore the previously reported structure of A53868 must
necessarily be reassigned.
Given the identical masses of the synthetic and natural
products, one possibility that was explored involved changing
the order of the Gly and Leu residues. This peptide was
prepared analogously to the route described for 2, but NMR
spectroscopy clearly showed it was not A53868 (not shown).
Based on the previously reported NMR spectroscopy data,[11]
two candidate isomeric structures 10 and 11 were envisioned.
Since crystallization attempts with the natural product to
confirm its structure were unsuccessful, the producing organ-
ism was grown on minimal media with (15NH4)2SO4 as the sole
nitrogen source to probe whether 10 or 11 could be the
structure of A53868.
The compound produced was purified, and its 31P NMR
spectrum was recorded. A clear doublet was observed with a
coupling constant of 8.6 Hz. However, no coupling was
Scheme 2. Synthetic route to peptide 2 and isomer 9. Fmoc=9fluor-
enylmethoxycarbonyl, cod=1,5-cyclooctadiene, Bn=benzyl,
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, DIC=1,3-diisopropylcarbo-
diimide, HOBt=1-hydroxybenzotriazole, Cbz=benzyloxycarbonyl.
1
observed in the H NMR spectrum between the 15N nucleus
and the doublet at d = 3.3 ppm corresponding to the putative
methylene protons, thus ruling out structure 11. Furthermore,
the coupling constant of 8.6 Hz between the 31P and 15N nuclei
appeared small for compound 10. Since we could not find any
literature values for the coupling expected for a structure like
10, the producer organism was subsequently grown on
minimal media with (15NH4)2SO4 as the only exogenous
source of nitrogen and with [13C6]glucose as the carbon source
to allow characterization by 13C NMR spectroscopy. The
NMR spectrum of the purified and uniformly 15N,13C-labeled
A53868 displayed the expected splitting patterns for the vinyl
carbons due to interaction with each other and couplings to
the phosphorus and nitrogen atoms. Unexpectedly, however,
the putative allylic methylene carbon atom was still present as
a doublet, showing only the splitting from the phosphorus
atom that is seen in unlabeled A53868, while no splitting from
13C or 15N nuclei was detected. These observations are
incompatible with either structures 10 or 11 as candidates
for A53868. Our data require that the putative methylene
carbon atom cannot be directly attached to nitrogen, phos-
phorus, or carbon atoms.
The assignment of a methylene group to the signal at d =
3.3 ppm for the proton and d = 52 ppm for the 13C NMR
spectra in the original work was based on the multiplicity of
the signal in the 13C NMR spectrum (reported as a triplet).[11]
However, our 13C NMR spectroscopy data indicate that this
signal is in fact a methyl group.[14] On the basis of this
assignment, data from 13C,13C COSY, HMBC, and HMQC
NMR spectroscopy experiments, and the splitting patterns
observed in the 13C NMR spectrum of uniformly 15N,13C-
solution-phase peptide synthesis. After global deprotection
with boron tribromide,[13] peptide 2 and its isomer 9 were
obtained.
Surprisingly, both synthetic phosphonates displayed dif-
ferent 31P NMR spectra than the material produced by
S. luridus (see Figure 1a for a mixture of the natural product
and synthetic 2). The natural product was therefore purified
from the spent medium of S. luridus by HPLC, and its
1H NMR spectra was recorded (Figure 1c), again showing
different resonances compared to the NMR spectra of the
synthetic compounds 2 (Figure 1b) and 9. Whereas a trans-
vinylphosphonate structure such as in 9 can be readily ruled
out for A53868 from the NMR data, the spectra of the natural
product and 2 are remarkably similar, both exhibiting two
vinyl protons with clear splittings owing to interaction with
the phosphorus atom as well as a doublet signal that displays a
smaller coupling constant to the phosphorus atom. The only
obvious differences are the reversed relative chemical shifts
of the protons cis and trans to the phosphorus atom (arrows)
as well as reversed positions of the aforementioned doublet
and the resonance from the glycine a protons (asterisks).
Furthermore, the synthetic and natural products were found
to have identical masses as determined by high-resolution
Fourier-transform mass spectrometry, indicating identical
atomic compositions, and both compounds showed fragmen-
tation patterns consistent with a Gly-Leu dipeptide fragment.
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 9089 –9092
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