The new metabolite (S)-cinnamoylphosphoramide from Streptomyces sp. and its total synthesis

Article abstract of DOI:10.1002/ejoc.200800654

The tunicate-associated strain Streptomyces sp. JP90 produces the unprecedented metabolite cinnamoylphosphoramide (1) among several other compounds. Structure elucidation was accomplished by NMR spectroscopic studies and efficient total synthesis. The absolute configuration at phosphorus was determined by synthesis of both enantiomers of 1 performing a resolution of the corresponding diastereomeric phosphoramides of L-phenylalanine ethyl ester. Unusual cinnamic acid derivative 1 represents the first bacterial organophosphoramide. As it matches the Schrader's formula for insecticidal organophosphates, its biological activity was investigated. A weak inhibition of acetylcholinesterase was observed in in vitro tests, and water-soluble analogues of 1 were prepared. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800654

FULL PAPER
DOI: 10.1002/ejoc.200800654
The New Metabolite (S)-Cinnamoylphosphoramide from Streptomyces sp. and
Its Total Synthesis
Melanie Quitschau,[a] Tim Schuhmann,[a] Jörn Piel,[b] Paultheo von Zezschwitz,*[a] and
Stephanie Grond*[a]
Keywords: Configuration determination / Natural products / Phosphorus / Streptomyces / Total synthesis
The tunicate-associated strain Streptomyces sp. JP90 pro-
duces the unprecedented metabolite cinnamoylphosphor-
amide (1) among several other compounds. Structure eluci-
dation was accomplished by NMR spectroscopic studies and
efficient total synthesis. The absolute configuration at phos-
phorus was determined by synthesis of both enantiomers of 1
performing a resolution of the corresponding diastereomeric
namic acid derivative 1 represents the first bacterial organo-
phosphoramide. As it matches the Schrader’s formula for in-
secticidal organophosphates, its biological activity was inves-
tigated. A weak inhibition of acetylcholinesterase was ob-
served in in vitro tests, and water-soluble analogues of 1
were prepared.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
phosphoramides of L-phenylalanine ethyl ester. Unusual cin-
methyl cinnamate moiety para-substituted with an unusual
phosphoramide methyl ester, which is unique among natu-
ral products. However, phosphoramides are known as syn-
thetic anti-HIV pharmaceuticals [e.g., -alaninyl-d4T-MP
(2)] and antihepatitis C virus (HCV) drug candidates (e.g.,
azidoadenosin phosphoramides from the current promising
“ProTide technology” approach).[6] Also, they are structur-
ally related to organophosphate poisons, for example, the
famous synthetic insecticide methyl paraoxon (3), an inhibi-
tor of acetylcholinesterase (AChE).[7] Moreover, tetracoor-
dinate organophosphates show high configurational sta-
bility and, therefore, are of great importance for asymmetric
synthesis in modern organic chemistry.[8] On the contrary,
Introduction
Marine organisms are a rich source of secondary metab-
olites many of which show unique structures and possess a
wide range of pharmacological properties.[1] Natural prod-
ucts isolated from marine – and also terrestrial – macroor-
ganisms are frequently assumed to actually be of bacterial
origin. Occurring structural similarities to known metabo-
lites from microorganisms, bacteria or non-related eukary-
otes, underline this hypothesis.[2] For example, several bac-
teria were cultivated from tunicates and yielded structurally
diverse metabolites; however, systematic studies of such
bacterial communities and their metabolite patterns, as well
as compound localizations, are rare.[3,4]
One of us (J. P.) and his coworkers identified a large com-
munity of Actinomycetes bacteria associated with the ma-
rine ascidian Aplidium lenticulum found in the Great Barrier
Reef (Australia), which is a prolific source of new natural
products.[5] Cultivations of one of the isolated strains Strep-
tomyces sp. JP90 yielded several metabolites of low molecu-
lar weight, such as the new organophosphate cinnamoyl-
phosphoramide (1; Figure 1). Its structure comprises a
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: +49-551-39-3228
E-mail: zezschwitz@chemie.uni-marburg.de
sgrond@gwdg.de
[b] Kekulé-Institut für Organische Chemie und Biochemie, Rheini-
sche Friedrich-Wilhelms-Universität Bonn,
Gerhard-Domagk Str. 1, 53121 Bonn, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Figure 1. Structures of phosphorus-containing bioactive com-
pounds.
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M. Quitschau, T. Schuhmann, J. Piel, P. von Zezschwitz, S. Grond
FULL PAPER
only very few such compounds originating from microbial Total Synthesis
secondary metabolism are described in the literature. The
To unambiguously confirm the constitution of cinnam-
well-known herbicide bialaphos, the potent antimalarial
fosmidomycin (4), and the strongly insecticidal cyclophostin
(5) all originate from Streptomyces sp.[9] In addition, the
indole alkaloid psilocybin (6) and its dephosphorylated an-
alogue psilocin are ingredients of “magic mushrooms” and
responsible for their hallucinogenic effect.[10]
Herein, we present a comprehensive study of new cinna-
moylphosphoramide (1) comprising isolation, structure elu-
cidation, total synthesis, and biological activity.
oylphosphoramide a total synthesis of rac-1 was per-
formed by starting from methyl 4-hydroxycinnamate (7),
which was readily prepared by Heck reaction of 4-bro-
mophenol with methyl acrylate (Scheme 1).[17] A one-pot
procedure was envisioned for the attachment of the phos-
phoramide moiety involving consecutive phosphorylation,
methoxylation, and amidation with aqueous ammonia, and
phenol was used as a model substrate.[18] Different methods
for its selective reaction with phosphoryl chloride were
tested, yet all yielded complex mixtures of mono- and di-
phenoxylated species, which decomposed upon attempted
separation by distillation.[19] Therefore, the reaction se-
quence was altered and methoxy phosphoryl dichloride (8)
was used as starting material.[20] Slow addition of a
prestirred mixture of sodium hydride and phenol 7 to this
reagent yielded 9,[21] which was immediately treated with
gaseous ammonia to afford rac-cinnamoylphosphoramide
(1) in 55% yield over the two steps from 7 (Scheme 1).[22]
Results and Discussion
Isolation and Chemical Characterization
Fermentations of Streptomyces sp. (strain JP90) were ini-
tially performed in 300-mL shaking flasks for 96 h by using
a complex medium (1% starch, 1% glucose, 1% glycerol,
0.25% cornsteep powder, 0.5% peptone). The culture fil-
trate was adsorbed on XAD-16 resin and elution with
methanol yielded the crude extract. The metabolite pattern
was analyzed,[11] and several substances were isolated by
column chromatography on Sephadex LH-20/MeOH and
on silica gel.[5] This led to pure compounds such as the im-
munosuppressive siderophore desferrioxamine E, the
monoterpene rosiridol, the new compound 6-hydroxy-6-
methylheptanoic acid, cyclo(tyrosylprolyl), N-acetyl tyr-
amine, and 3-indolecarboxylic acid, which were identified by
NMR spectroscopy, MS analysis, and database search.[12–16]
However, an apparently unknown secondary metabolite
aroused our interest. Pure colorless solid 1 was isolated
from the crude extract in a yield of 1.2 mgL–1.
The ESI mass spectrum of 1 showed an ion peak at m/z
= 294 [M + Na]+, and the molecular formula was deter-
mined by ESI HRMS to be C11H14NO5P. Characteristic IR
absorption peaks suggested an α,β-unsaturated ester
(1716 cm–1), an alkene (1602, 1508 cm–1), and a P=O moi-
ety (1250–1300 cm–1). The UV spectra showed an absorp-
tion maximum at 283 nm with a significant bathochromic
Scheme 1. Total synthesis of rac-1.
The aminomethoxyphosphoryloxy moiety proved to be
highly sensible towards acidic and basic conditions, and de-
composition to phenol 7 was observed during chromatog-
raphy on silica gel. However, 1 could be successfully puri-
fied on reverse-phase (RP) silica gel. Comparison of the
spectroscopic data confirmed the assigned structure of the
first microbial organophosphoramide 1.
Absolute Configuration
shift upon addition of HCl (316 nm) or NaOH (359 nm).
For the determination of the absolute configuration at
The constitution of 1 was deduced from H, 13C, 31P, and phosphorus, the single enantiomers were both prepared fol-
2D NMR spectroscopic experiments (CD2Cl2). The 1H lowing a protocol of Koizumi et al.[23] Treatment of racemic
NMR spectrum exhibited signals of 14 protons with two phosphoryl monochloride 9 with the hydrochloride of -
signals pointing at methoxy groups [δH = 3.76 (s), 3.79 (d, phenylalanine ethyl ester afforded a diastereomeric mixture
3JH,P = 11.8 Hz) ppm]. The presence of a para-substituted of phosphoramides 10a,b in a 13:7 dr, as judged from the
1
1
methyl cinnamate was confirmed by two methine proton integrals of the P–OMe signals in the H NMR spectrum
signals [δH = 6.37, 7.62 ppm, (E)-3JH,H = 16.0 Hz] and four (Scheme 2).
aromatic protons (δH = 7.23, 7.50 ppm). The 13C NMR
Unfortunately, the diastereomers could not be separated
spectrum showed nine resonances, three of which appeared by crystallization (e.g., from n-hexane) or various chroma-
as doublets at δ = 53.9 (2JC,P = 5.8 Hz), 121.0 (3JC,P
=
tographic methods including chromatography on silica gel
4.5 Hz), 152.7 (2JC,P = 6.8 Hz) ppm and thus indicated (CHCl3/MeOH; n-hexane/EtOAc/MeOH), reverse-phase
13C,31P coupling. In conclusion, compound 1 could be iden- silica gel (MeOH/water), chiral plates (MeOH/water; aceto-
tified as methyl 4-(aminomethoxyphosphoryloxy)cinnam- nitrile/water; MeOH/water/acetonitrile), or HPLC (Nucleo-
ate, although an imido ester could not be finally excluded sil C8 column, acetonitrile/water). Therefore, the mixture of
on the basis of all spectroscopic data.[17]
10a,b was subjected to N-chlorination with tert-butyl hypo-
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The New Metabolite (S)-Cinnamoylphosphoramide from Streptomyces sp.
This configurational assignment to the synthetic enantio-
mers of cinnamoylphosphoramide (1) was performed by
comparison of the physicochemical properties of com-
pounds 11a,b with those of the analogous diastereomeric
phosphoramides 14a,b, which have previously been pre-
pared by Koizumi et al. (Figure 2).[23] A characteristic fea-
ture of both pairs of diastereomers is the significant differ-
ence between the chemical shifts of the P–OMe signals in
1
the H NMR spectra (∆δ = 0.8–0.9 ppm, Table 1).
Figure 2. Analogous N-chlorophosphoramides 14a,b.[23]
Table 1. Comparison of physicochemical properties of N-chloro-
phosphoramides 14a,b and 11a,b.
14a[a]
14b[a]
11a
11b
Configuration
S,S
2.98
R,S
3.80
X,S
3.03
Y,S
3.93
1H NMR, δ [ppm] P–OMe
Rf values on silica gel
0.67[b]
0.58[b] 0.54[c] 0.36[c]
Optical rotation values of
respective hydrolysis product [°]
Deduced configurations X
and Y at the phosphorus atom
–13.6
+14.0
–6.6
+6.6
S
R
S
R
[a] Data taken from refs.[23,27] [b] Benzene/EtOAc, 5:1. [c] Hexane/
EtOAc, 2:1.
Moreover, in both pairs the diastereomers with the high-
field shifted methoxy signal also have higher Rf values on
silica gel. Another analogy arises from the optical rotation
values of the cleaved phosphoramides, that is, of enantio-
mers 1 in the case of diastereomers 11a,b and their respec-
tive phenoxy analogues from 14a,b: the enantiomers ob-
tained from the diastereomers with the high-field shifted
methoxy signal exhibit negative rotation values. On the ba-
sis of X-ray crystal structure analysis of the dechlorinated
derivative of 14b, Koizumi et al. had assigned the (R,S) con-
figuration to this diastereomer.[27] By analogy, the (S,S) and
(R,S) configuration were assigned to diastereomers 11a and
11b, respectively. Thus, pure enantiomers 1 are (S)-(–)-cin-
namoylphosphoramide and (R)-(+)-cinnamoylphosphor-
amide. Isolated metabolite 1 was compared with the syn-
thetically prepared enantiomers by chiral HPLC analysis
(Daicel Chiralpak IA column, n-hexane/2-propanol). Single
chromatography runs with the three single compounds and
with mixed samples represent experimental sets,[17] which
unambiguously proved the identity of the natural product
with the (S)-(–)–enantiomer 1.
Scheme 2. Resolution of racemic phosphoryl chloride 9 and prepa-
ration of (S)-1 and (R)-1.
chlorite in 4% methanolic borax yielding chloro derivatives
11a,b.[24] Both diastereomers were readily distinguishable by
1
the H NMR signals of the P–OMe group (δ = 3.03 and
3.93 ppm, dr 13:10) and by their Rf values on silica-gel
plates (hexane/EtOAc, 2:1).
A
rapid flash column
chromatography at 4 °C furnished pure 11a and 11b in
yields of 31 and 21%, respectively.[25] This ratio does not
reflect the original dr, as partial dechlorination of com-
pounds 11a,b occurred during chromatography leading to
the formation of some starting material 10a,b. Separated
diastereomers 11a,b were then treated with sodium meth-
oxide to give N,O-acetals 12 from 11a, and 13 from 11b
(Scheme 2).[26] These reactions destroyed the configura-
tional integrity of the former amino acid, as they proceeded
by initial elimination of HCl and thus formation of an N-
phosphorylimine, which then undergoes addition of
MeOH. Finally, cleavage of the C,N bonds in crude prod-
ucts 12 and 13 was effected by acid-catalyzed hydrolysis by
employing 5% methanolic H2SO4, and purification by RP
Biosynthesis
The carbon skeleton of cinnamoylphosphoramide (1) in-
silica gel chromatography yielded (S)-1 in 54% yield and dicates biosynthesis by the shikimate pathway.[17] Within
(R)-1 in 30% yield from 11a and 11b, respectively.
the scope of biosynthetic studies on other metabolites,
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M. Quitschau, T. Schuhmann, J. Piel, P. von Zezschwitz, S. Grond
FULL PAPER
tracer experiments were carried out by adding the sodium phosphoryl chloride 8 was added to the sodium salt of 15
salts of [1,2-13C2]acetate and [U-13C3]glycerol to the grow- at –78 °C (Scheme 3). After subsequent treatment with gas-
ing cultures of the strain Streptomyces sp. JP90 (12 h of eous ammonia, desired acid 17 was obtained in 43% overall
continuous feeding starting 48 h after inoculation). The iso- yield from 15.
lated metabolites were purified and analyzed by 13C NMR
spectroscopy. Whereas other compounds such as polyket-
ides, amino acid derived metabolites, and terpenoids were
intensively labeled,[5] no specific enrichments were detected
for 1. This might indicate that cinnamate 1 or precursors
thereof were possibly produced within the first 48 h of fer-
mentation.[17]
Biological Activity
Scheme 3. Synthesis of water-soluble rac-17.
Cinnamoylphosphoramide (1) comprises an unusual nat-
Ellmann tests performed with 17 again showed only very
ural phosphoramide methyl ester, which is very rare among
weak inhibition of AChE (IC50 Ͼ 350 µ), whereas control
natural products, and it entirely matches Schrader’s formula
experiments with the aminoacridine tacrine gave the ex-
describing the chemical structure of highly active organo-
pected results (IC50 = 1.5 nM).[30] Moreover, the inhibition
phosphate poisons and the resulting mode of action as
potential of 1 and 17 on butyryl cholinesterase (BChE) was
strong electrophiles in acetylcholinesterase (AChE) inhibi-
investigated in collaboration with C. Meier et al. and re-
tion (Figure 3).[28] The important structural motifs are the
vealed an IC50 = 250 µ for 1, but no significant activity
P,O double bound, basic substituents R1 and R2 such as
for 17.
alkoxy, alkyl, or amino groups, and a leaving group X such
Cinnamoylphosphoramide (1) showed no antibacterial
as a halide or phenoxy substituent, which provides the elec-
and antifungal activity in plate diffusions assays with E.
trophilicity at the phosphorus atom as a prerequisite for the
coli, B. subtilis, S. aureus, and C. albicans. Phosphates and
biological activity as an AChE inhibitor. On the basis of
their derivatives are known as ubiquitous energy-rich inter-
this analogy, we investigated the biological activity of 1.
mediates in metabolic pathways and mediators for the acti-
vation of organic substances. Thus, it was assumed that me-
tabolite 1 with its unusual phosphoramide moiety could be
an activated biosynthetic precursor. To evaluate the bio-
logical function of 1 for the producer strain JP90, feeding
experiments were performed by adding synthetic 1 and re-
lated cinnamic acid derivatives (e.g., methyl 4-hydroxycin-
namate, cinnamic acid, methyl cinnamate) to the growing
cultures (12 and 16 h after inoculation). The metabolite
Figure 3. Schrader’s formula.
pattern was analyzed by methods of chemical screening
In a cooperation with U. Holzgrabe et al., Ellmann tests
(HPLC–MS–UV and TLC), yet revealed no detectable
were performed but revealed only very weak inhibition of
amounts of new metabolites, but production of already
AChE by methyl ester 1 (IC50 Ͼ 350 µ).[29] This was as-
known compounds. Therefore, the biological function of
cribed to its poor solubility in the used buffer solution, and
cinnamoylphosphoramide (1) for the producer strain re-
thus, we decided to prepare free carboxylic acid 17 as a
mains unclear, as for most other interesting Streptomycetes
water-soluble analogue of 1. This turned out to be quite
products, but might be of future interest in terms of investi-
challenging, as compound 1 undergoes fast dephosphoryla-
gations of chemical biology.
tions under acidic and basic conditions, and decomposition
to phenol 7 was observed upon attempted ester hydrolysis
with lithium hydroxide.[17] Enzymatic ester hydrolysis with
Conclusions
different enzymes was investigated next, but the desired
compound was not obtained.[17] A reduction–oxidation se-
The new cinnamoylphosphoramide (1) is a structurally
quence was also examined for the transformation of 1 into unusual microbial natural product from the tunicate-associ-
the free acid, but treatment with LiAlH4 just led to decom- ated Streptomyces sp. strain JP90 due to its phosphoramide
position, whereas reaction of 1 with DIBAL-H at –78 °C moiety. Although its constitution could be deduced from
merely furnished a mixture of the desired allylic alcohol of analysis of the isolated material, the elucidation of its con-
1 and the dephosphorylated 4-[(E)-3-hydroxyprop-1-enyl] figuration required a total synthesis of both enantiomers
phenol.[17] Finally, synthesis of water-soluble analogue 17 (S)-1 and (R)-1. The evaluation of the biological activity of
was successfully accomplished by starting from TBS-ester 1 was of special interest, as its organophosphate structure
15 (TBS = tert-butyldimethylsilyl). In contrast to the syn- complies with the criteria of Schrader’s formula for insecti-
thesis of 1, the addition order had to be reversed in that cides and nerve poisons of extraordinary potency. Whereas
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The New Metabolite (S)-Cinnamoylphosphoramide from Streptomyces sp.
phoric acid solution; 5% molybdatophosphoric acid in ethanol.
strong AChE and BChE inhibition was not observed in vi-
tro, preliminary studies indicate biological activity against
arthropods. Similar apparently opposed results have been
described before and additional inhibition of cell membrane
functions are presently discussed for organophos-
phates,[28b,31] which invites us to more future investigations
of 1.
Fermentation was carried out in a Braun Incubator BS4, Braun
Certomat RM, 250-mL or 1-L Erlenmeyer flasks with three spo-
ilers; Nutrient solutions: Medium S: starch (10 gL–1), glycerol
(4 gL–1), casein peptone (4 gL–1), yeast extract (0.5 gL–1), meat ex-
tract (0.5 gL–1), liver extract (0.5 gL–1), NaCl (1 gL–1) adjusted to
pH 7.0 prior to sterilization; complex medium (SGG): glucose
(10 gL–1), glycerol (10 gL–1), starch (10 gL–1), cornsteep powder
(2.5 gL–1), casein peptone (5 gL–1), yeast extract (2.0 gL–1), NaCl
(1 gL–1), CaCO3 (3 gL–1) adjusted to pH 7.3 prior to sterilization;
The physiological function of such unique microbial or-
ganophosphates like 1 for the tunicate-associated Strepto-
myces strain is yet fully unknown. We tackled this question medium M2+: malt extract (10 gL–1), yeast extract (4 gL–1), glucose
(4 gL–1), CaCO3 (0.3 gL–1) adjusted to pH 7.0 prior to steriliza-
with a first experimental series of feeding experiments of
synthetic 1 and derivatives to growing cultures of the pro-
ducer strain, and have not yet detected altered metabolite
tion. Labeled precursors: sodium [1,2-13C2]acetate (99% 13C; Cam-
bridge Isotope Lab.), [U-13C3]glycerol (99% 13C; Chemotrade).
patterns. Our finding of the first microbial organophos-
phate amide emphasizes the value of natural product dis-
coveries in raising questions and enriching research in both
chemical synthesis and chemical biology.
Fermentation: Strain JP90 (Streptomyces sp.) was incubated for 6 d
at 28 °C and maintained on agar plates (medium M2+). A 1-cm2
piece of incubated agar plates was used to inoculate 100 mL of
medium SGG in 250-mL flasks (180 rpm, 28 °C, 48 h). Production
in medium SGG was carried out in 250-mL Erlenmeyer flasks
(with three spoilers, 180 rpm, 28 °C, 96 h) by using 1% of the pre-
culture as inoculum. Under optimized conditions, a 1-cm2 piece of
incubated agar plates was used to inoculate 100 mL of medium S
in 300-mL flasks (180 rpm, 28 °C, 48 h). Production in medium S
was carried out in 1-L Erlenmeyer flasks (180 rpm, 28 °C, 96 h).
Experimental Section
1
General: H, 13C, 31P, and 2D NMR spectra were recorded with a
Varian Inova-600, Varian Mercury-300, Varian Unity-300, or
Bruker AM 250 spectrometer. Chemicals shifts are reported as δ
values (ppm) with the residual protonated solvent as the internal
reference; the multiplicity of the carbon signals was determined by
the DEPT or APT technique and quoted as follows: (+) for CH3
and CH, (–) for CH2, and (Cquat) for quaternary carbon atoms.
Electron impact (EI) mass spectra were recorded with a Finnigan
MAT 95 spectrometer (70 eV), electrospray ionization (ESI) mass
spectra with a Finnigan LC-Q spectrometer (70 eV), high-resolu-
tion (HR) mass spectra (ESI) with a Bruker APEX-Q 7T IV spec-
trometer; preselected ion-peak matching at R ϾϾ 10000 were
within Ϯ2 ppm of the exact masses. Elemental analysis was per-
formed at the Mikroanalytisches Labor der Universität Göttingen
(Germany). IR spectra were recorded with a Perkin-Elmer Model
1600 spectrometer. UV spectra were recorded with a Varian Model
Cary 3E spectrophotometer. Optical rotations were recorded with
a Perkin-Elmer 241 polarimeter. Melting points are uncorrected.
The solvents used for extraction and chromatography were of tech-
nical grade and distilled prior to use. All moisture-sensitive reac-
tions were carried out under nitrogen or argon in oven- and/or
flame-dried glassware. THF was distilled from sodium benzophe-
none ketyl; dichloromethane and triethylamine were distilled from
CaH2; methanol was distilled from magnesium. Column
chromatography was carried out on silica gel (Merck; grade 60, 70–
230 mesh or Machery & Nagel; grade 60, 230–400 mesh), Sephadex
LH-20 (Pharmacia), Li-Chroprep ®RP-18 (Merck) and Lobar RP-
18 (Merck). TLC analysis was performed on silica plates (Merck
60 F254, 0.25 mm, RP-18 F/UV254 and Machery Nagel chiral
plates). HPLC analyses were performed by using a Grom Supher-
sphere-100 RP-18, 4 µm (100ϫ2 mm) column, a Knauer Nucleo-
sil 100 C8, 5 µm (250ϫ3 mm) column or a Daicel Chiralpak IA
(0.46ϫ25 cm) column, a Jasco pump PU-2080 Plus, a Kontron
pump Model 322, a UV detector MD-2010 Plus and a Kontron
diode Array Detector 440. Staining reagents were anisaldehyde/sul-
furic acid [anisaldehyde (1.0 mL) in methanol (85 mL) with conc.
sulfuric acid (5 mL) and acetic acid (10 mL)], 4-dimethylami-
nobenzaldehyde/hydrochloric acid [4-dimethylaminobenzaldehyde
(1 g) in methanol (75 mL) with conc. hydrochloric acid (25 mL)],
orcin/sulfuric acid; iron(III)chloride (1 g) in sulfuric acid (100 mL)
with 6% alcoholic solution of orcin (ratio 1:1), molybdatophos-
Isolation and Purification: The culture broth was adjusted to pH
5.0 and separated by filtration from the mycelia, which was dis-
carded. The culture filtrate was adsorbed on XAD-16 resin and
elution with methanol yielded the crude extract. The solvent was
removed by evaporation. Purification was achieved by flash column
chromatography on silica gel (CHCl3/MeOH, 9:1), gel permeation
chromatography on Sephadex LH-20 (MeOH) and column
chromatography on reverse-phase silica gel (MeOH/H2O, 7:3), thus
yielding 1.2 mgL–1 of 1. Optimized conditions: The culture broth
was adjusted to pH 5.6 and separated by filtration from the myce-
lia. The culture filtrate was extracted with ethyl acetate
(2ϫ500 mL), and the solvent was removed by evaporation. Purifi-
cation was achieved by column chromatography on silica gel
(CHCl3/MeOH, 95:5), twofold chromatography on reverse-phase
silica gel (acetone/H2O, 3:1), thus yielding 2.0 mgL–1 of 1.
Methyl (S)-(2E)-3-[4-(Aminomethoxyphosphoryloxy)phenyl]acrylate
(1): The title compound was isolated as described above as a pure
colorless solid. C11H14NO5P: Mr = 272.21. Rf (silica gel) = 0.35
(CHCl3/MeOH, 9:1), Rf = 0.52 (MeOH/H2O, 7:3). M.p. 130 °C.
[α]2D0 = –5.2 (c = 0.25, MeOH). IR (KBr): ν = 3417 (N–H), 2953
˜
(C–H), 1716 (C=O), 1637 (N–H), 1602 (C=C), 1508 (C=C), 1437,
1326, 1226 (P=O), 1171, 1053, 1014, 982, 925, 836, 796 cm–1. UV
(MeOH): λmax (log ε) = 283 (4.86) nm. 1H NMR (600 MHz,
3
CD2Cl2, 25 °C): δ = 3.76 (s, 3 H, 12-H3), 3.79 (d, JH,P = 11.8 Hz,
3
3
3 H, 11-H3), 6.37 (d, JH,H = 16.0 Hz, 1 H, 2-H), 7.23 [d, JH,H
=
8.5 Hz, 2 H, 6(8)-H], 7.50 [d, 3JH,H = 8.4 Hz, 5(9)-H], 7.62 (d, 3JH,H
= 16.0 Hz, 1 H, 3-H) ppm. 13C NMR (75.5 MHz, CD2Cl2, 25 °C):
2
δ = 51.8 (+, C-12), 53.9 (+, d, JC,P = 5.8 Hz, C-11), 117.9 (+, C-
2), 121.0 [+, d, JC,P = 4.5 Hz, C-6(8)], 129.8 [+, C-5(9)], 131.4
3
2
(Cquat, C-4), 143.7 (+, C-3), 152.7 (d, JC,P = 6.8 Hz, C-7, Cquat),
167.4 (Cquat, C-1) ppm. 31P NMR (121.5 MHz, CD2Cl2, 25 °C): δ
= 6.1 (br.) ppm. MS (ESI): m/z (%) = 294 (32) [M + Na]+, 565
(100) [2M
+
Na]+. HRMS (ESI): calcd. for C11H15NO5P
[M + H]+ 272.0681; found 272.0684.
Methyl
(2E)-3-[4-(Chloromethoxyphosphoryloxy)phenyl]acrylate
(9): Methyl 4-hydroxycinnamate (7; 301 mg, 1.68 mmol) was dis-
solved in dichloromethane (22 mL) and cooled to 0 °C, and sodium
hydride (60% in mineral oil, 72.9 mg, 1.82 mmol), was added. After
Eur. J. Org. Chem. 2008, 5117–5124
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5121

M. Quitschau, T. Schuhmann, J. Piel, P. von Zezschwitz, S. Grond
FULL PAPER
stirring for 0.5 h at 0 °C, the suspension was warmed to room tem-
perature and stirred for another 2 h. The resulting suspension was
added dropwise to a vigorously stirred solution of methoxy phos-
phoryl dichloride (8; 503 mg, 3.38 mmol)[20] in dichloromethane
(6 mL) over a period of 9 h at 0 °C. The reaction mixture was
warmed to room temperature and stirred for 16 h. The mixture was
filtered under a nitrogen atmosphere through a 2-cm pad of Celite.
The solids were washed with dichloromethane (3ϫ50 mL), and the
filtrate was concentrated in vacuo, thus yielding 586 mg of 9 as a
yellow oil, which without purification was transformed in the fol-
[2M + Na]+, 470 (12) [M + Na]+. HRMS (ESI): calcd. for
C22H27NO7P [M + H]+ 448.1520; found 448.1520.
Methyl (S,S)- and (R,S)-(2E)-3-{4-[N-Chloro-N-(1-ethoxycarbonyl-
2-phenylethyl)aminomethoxyphosphoryloxy]phenyl}acrylate (11a,b):
A solution of tert-butyl hypochlorite (1.28 mL, 11.3 mmol) in 4%
methanolic borax (34 mL) was added to a vigorously stirred solu-
tion of diastereomers 10a,b (dr 13:7, 2.96 g, 6.62 mmol) in 4%
methanolic borax (20 mL). Due to incomplete conversion, an ad-
ditional amount of tert-butyl hypochlorite (1.02 mL, 9.02 mmol) in
4% methanolic borax (31 mL) was added after 50 min, and the
mixture was stirred for another 45 min. The resulting mixture was
diluted with chloroform (40 mL), washed with water (40 mL), dried
with MgSO4, and concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel at 4 °C (260 g; hex-
ane/EtOAc, 2:1) to yield 996 mg (31%) of (S,S) diastereomer 11a
(Rf = 0.54) as a yellow oil and 661 mg (21%) of (R,S) diastereomer
11b (Rf = 0.36) as yellow solid. Data for 11a: C22H25ClNO7P: Mr
1
lowing reaction. C11H12ClO5P: Mr = 290.64. H NMR (250 MHz,
3
CDCl3, 25 °C): δ = 3.80 (s, 3 H, 12-H3), 4.03 (d, JH,P = 14.1 Hz,
3
3
3 H, 11-H3), 6.39 (d, JH,H = 15.9 Hz, 1 H, 2-H), 7.29 [d, JH,H
=
3
8.4 Hz, 2 H, 6(8)-H], 7.54 [d, JH,H = 8.4 Hz, 2 H, 5(9)-H], 7.66 (d,
3JH,H = 15.9 Hz, 1 H, 3-H) ppm. 31P NMR (121.5 MHz, CDCl3,
25 °C): δ = 1.81 (br.) ppm.
Methyl (2E)-3-[4-(Aminomethoxyphosphoryloxy)phenyl]acrylate (1):
Crude product 9 from the reaction described above (586 mg) was
dissolved in dichloromethane (18 mL) and cooled to –15 °C. A
stream of gaseous ammonia was passed for 50 min through the
vigorously stirred solution, brine (20 mL) was added, and the layers
were separated. The aqueous phase was extracted with ethyl acetate
(3ϫ30 mL), and the combined organic layer was dried with
MgSO4 and concentrated in vacuo. The crude product was purified
by column chromatography on reverse-phase silica gel (Li-Chrop-
rep ®RP-18; MeOH/H2O, 6:4) to yield 252 mg (55% from 7) of the
title compound 1. All spectroscopic data were consistent with the
data of cinnamoylphosphoramide (1) isolated from Streptomyces
sp. JP90. C11H14NO5P (271.2): calcd. C 48.72, H 5.20, N 5.16;
found C 48.40, H 5.21, N 5.00.
3
= 481.87. 1H NMR (250 MHz, CDCl3, 25 °C): δ = 1.20 (t, JH,H
3
= 7.2 Hz, 3 H, CH2CH3), 3.03 (d, JH,P = 12.6 Hz, 3 H, 11-H3),
3
2
3.14 (dd, JH,H = 10.9 Hz, JH,H = 14.5 Hz, 1 H, 3Ј-HA), 3.37 (dt,
3JH,H = 3.6 Hz, JH,H = 14.5 Hz, 1 H, 3Ј-HB), 3.79 (s, 3 H, 12-H3),
2
3
4.09 (q, JH,H = 7.2 Hz, 2 H, CH2CH3), 4.91–4.99 (m, 1 H, 2Ј-H),
6.34 (d, 3JH,H = 15.9 Hz, 1 H, 2-H), 7.18–7.28 [m, 7 H, 6(8)-H, Ar-
H], 7.46 [d, 3JH,H = 7.2 Hz, 2 H, 5(9)-H], 7.63 (d, JH,H = 15.9 Hz,
3
1 H, 3-H) ppm. 13C NMR (63.9 MHz, CDCl3, 25 °C): δ = 14.0 (+,
CH2CH3), 40.4 (–, d, 3JC,P = 5.0 Hz, C-3Ј), 51.7 (+, C-12), 53.5 (+,
2
2
d, JC,P = 5.7 Hz, C-11), 61.7 (–, CH2CH3), 63.7 (+, d, JC,P
=
3
4.3 Hz, C-2Ј), 117.6 (+, C-2), 120.9 [+, d, JC,P = 5.0 Hz, C-6(8)],
126.9 (+, C-7Ј), 128.5 [+, C-6Ј(8Ј)*], 129.3 [+, C-5Ј(9Ј)*], 129.4 [+,
C-5(9)*], 131.3 (Cquat, C-4), 136.5 (Cquat, C-4Ј), 143.6 (+, C-3),
2
152.0 (d, JC,P = 7.4 Hz, C-7, Cquat), 167.3 (Cquat, C-1), 169.2
(Cquat, C-1Ј) ppm. MS (ESI): m/z (%) = 504 (18) [M + Na]+, 985
Methyl (S,S)- and (R,S)-(2E)-3-{4-[N-(1-Ethoxycarbonyl-2-phenyl-
(100) [2M + Na]+. Data for 11b: C22H25ClNO7P: Mr = 481.87. H
1
ethyl)aminomethoxyphosphoryloxy]phenyl}acrylate (10a,b): To
a
3
NMR (250 MHz, CDCl3, 25 °C): δ = 1.32 (t, JH,H = 7.2 Hz, 3 H,
solution of -phenylalanine ethyl ester hydrochloride (2.59 g,
11.3 mmol) dissolved in THF (4 mL) was added NEt3 (5.04 mL,
36.2 mmol) at 0 °C. After being stirred for 1 h the resulting suspen-
sion was added dropwise to a solution of 9 (3.28 g, 11.3 mmol) in
THF (48 mL), and the reaction mixture was vigorously stirred for
3 h at 0 °C. The mixture was filtered through a 2-cm pad of Celite,
the solids were washed with THF (2ϫ40 mL), and the filtrate was
concentrated in vacuo to a volume of approx. 20 mL. Then, the
solution was diluted with diethyl ether (60 mL), washed success-
ively with brine (40 mL), hydrochloric acid (1 , 40 mL), saturated
NaHCO3 solution (40 mL), and brine (40 mL), and dried with
MgSO4. The solvent was removed in vacuo, and the residue was
purified by column chromatography on silica gel (180 g; hexane/
EtOAc, 1:1) to yield 2.42 g (48%) of diastereomeric mixture 10a,b
(dr 13:7) as a colorless oil, which slowly started to crystallize.
CH2CH3), 3.06 (dd, 3JH,H = 10.9 Hz, 2JH,H = 14.5 Hz, 1 H, 3Ј-HA),
3.33 (dt, 3JH,H = 3.6 Hz, 2JH,H = 14.5 Hz, 1 H, 3Ј-HB), 3.83 (s, 3 H,
3
12-H3), 3.93 (d, JH.P = 12.6 Hz, 3 H, 11-H3), 4.21–4.33 (m, 2 H,
CH2CH3), 4.84–4.94 (m, 1 H, 2Ј-H), 6.34 (d, 3JH,H = 15.9 Hz, 1 H,
3
2-H), 6.86 [d, JH,H = 7.2 Hz, 2 H, 6(8)-H], 7.13 (mc, 5 H, Ar-H),
3
3
7.29 [d, JH,H = 7.2 Hz, 2 H, 5(9)-H], 7.61 (d, JH,H = 15.9 Hz,
1 H, 3-H) ppm. 13C NMR (63.9 MHz, CDCl3, 25 °C): δ = 14.1 (+,
CH2CH3), 40.4 (–, C-3Ј), 51.7 (+, C-12), 55.1 (+, d, 2JC,P = 6.0 Hz,
2
C-11), 61.8 (–, CH2CH3), 64.2 (+, d, JC,P = 4.0 Hz, C-2Ј), 117.5
(+, C-2), 120.4 [+, d, 3JC,P = 5.1 Hz, C-6(8)], 126.6 (+, C-7Ј), 128.4
[+, C-6Ј(8Ј)*], 129.0 [+, C-5Ј(9Ј)*], 129.3 [+, C-5(9)*], 131.1 (Cquat
,
2
C-4), 136.1 (Cquat, C-4Ј), 143.7 (+, C-3), 151.4 (d, JC,P = 6.9 Hz,
C-7, Cquat), 167.3 (Cquat, C-1), 169.6 (Cquat, C-1Ј) ppm. MS (DCI,
200 eV): m/z (%): 178 (9), 194 (100) [C10H9O3 + NH3], 211 (29).
C22H26NO7P: Mr = 447.43. Rf = 0.35 (hexane/EtOAc, 1:1). IR Methyl (S,S)- and (S,R)-(2E)-3-{4-[N-(1-Ethoxycarbonyl-1-meth-
(KBr): ν = 3446 (N–H), 2949 (C–H), 1734 (C=O), 1636 (N–H), oxy-2-phenylethyl)aminomethoxyphosphoryloxy]phenyl}acrylate
˜
1559 (C=C), 1508 (C=C), 1457, 1386 (P=O), 1170, 1044, 986, 845, (12): (S,S) Diastereomer 11a (288 mg, 0.598 mmol) was dissolved
759 cm–1. 1H NMR (250 MHz, CDCl3, 25 °C): δ = 1.14/1.23 (2ϫt,
in methanol (1.33 mL) and treated at –40 °C with sodium meth-
3JH,H = 7.0 Hz, 3 H, CH2CH3), 2.89–3.05 (mc, 2 H, 3Ј-H), 3.53/ oxide (1.0  in methanol, 0.60 mL, 0.60 mmol), and the solution
3.66 (2ϫd, 3JH,P = 11.5 Hz, 3 H, 11-H3), 3.76 (s, 3 H, 12-H3), 4.03– was stirred for 100 min at –40 °C. The reaction mixture was neu-
3
4.23 (m, 3 H, 2Ј-H, CH2CH3), 6.33 (d, JH,H = 16.0 Hz, 1 H, 2-H),
tralized with acetic acid and concentrated in vacuo. The residue
was extracted with ethyl acetate (3ϫ10 mL), and the combined
3
7.07–7.23 [m, 7 H, 6(8)-H, Ar-H], 7.42 [d, JH,H = 8.5 Hz, 2 H,
3
5(9)-H], 7.61 (d, JH,H = 16.0 Hz, 1 H, 3-H) ppm. 13C NMR organic phases were washed successively with aqueous solutions of
(63.9 MHz, CDCl3, 25 °C): δ = 14.1 (CH2CH3), 40.4 (C-3Ј), 51.7 citric acid (10%, 15 mL), NaHCO3 (1 , 15 mL), and brine
2
(C-12), 53.4/53.6 (d, JC,P = 5.7 Hz, C-11), 55.5/55.8 (C-2Ј), 61.4 (15 mL) and dried with MgSO4. Concentration in vacuo yielded
3
(CH2CH3), 117.4 (C-2), 120.5 [d, JC,P = 5.2 Hz, C-6(8)], 127.0 (C-
270 mg of 12 as a slightly impure mixture of both diastereomers
7Ј), 128.4 [C-6Ј(8Ј)*], 129.4 [C-5Ј(9Ј)*], 129.5 [C-5(9)*], 130.9 (C- (dr 1:1). C23H28NO8P: Mr = 477.44. Rf = 0.41 (hexane/EtOAc, 1:1).
2
3
4), 135.6/135.8 (C-4Ј), 143.7 (C-3), 152.2 (d, JC,P = 6.5 Hz, C-7),
1H NMR (250 MHz, CDCl3, 25 °C): δ = 1.23/1.32 (2ϫt, JH,H
=
167.3 (C-1), 172.1/172.2 (C-1Ј) ppm. MS (ESI): m/z (%) = 917 (100)
7.2 Hz, 3 H, CH2CH3), 3.26 (d, 2JH,H = 14.5 Hz, 1 H, 3Ј-HA), 3.27/
5122
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5117–5124

The New Metabolite (S)-Cinnamoylphosphoramide from Streptomyces sp.
3.28 (2ϫs, 3 H, OCH3), 3.36 (d, 2JH,H = 14.5 Hz, 1 H, 3Ј-HB), 3.78 (1.7 mL), and the reaction mixture was stirred for 7 h at room tem-
3
(2ϫs, 3 H, 12-H3), 3.74/3.86 (2ϫd, JH,P = 12.6 Hz, 3 H, 11-H3),
perature. Workup as described above and purification by column
chromatography on reverse-phase silica gel (Li-Chroprep ®RP-18;
MeOH/H2O, 6:4) yielded 22.8 mg (30% from 11b) of (R)-1 as a
colorless solid. All spectroscopic data were consistent with the data
3
2
4.12/4.23 (2ϫq, JH,H = 7.2 Hz, 2 H, CH2CH3), 4.69 (2ϫd, JH,P
= 8.0 Hz, 1 H, NH), 6.36/6.37 (2ϫd, JH,H = 15.6 Hz, 1 H, 2-H),
3
3
7.05–7.19 [m, 7 H, 6(8)-H, Ar-H], 7.47 [2ϫd, JH,H = 8.6 Hz, 2 H,
5(9)-H], 7.64/7.66 (2ϫd, JH,H = 15.6 Hz, 1 H, 3-H) ppm. 13C of cinnamoylphosphoramide (1) isolated from Streptomyces sp.
3
NMR (75.5 MHz, CDCl3, 25 °C): δ = 14.0/14.0 (+, CH2CH3), 43.1
JP90, and the optical rotation {[α]2D0 = +6.6 (c = 0.14, MeOH)}
indicated (R)-1 to be the nonnatural enantiomer.
(–, C-3Ј), 51.5/51.5 (+, OCH3), 51.7/51.7 (+, C-12), 54.2/54.3 (+, d,
2JC,P = 6.0 Hz, C-11), 62.7/62.8 (–, CH2CH3), 90.8/90.8 (d, JC,P
=
2
tert-Butyldimethylsilyl (2E)-3-(4-Hydroxyphenyl)acrylate (15): To a
solution of 4-hydroxycinnamic acid (1.65 g, 10.1 mmol) in THF
(70 mL) was added NEt3 (1.4 mL, 10 mmol) and tert-butyldi-
methylsilyl chloride (2.5  in toluene, 4.0 mL, 10 mmol). The solu-
tion was stirred for 30 min and then added to an aqueous phos-
phate buffer solution (pH 7, 75 mL) and extracted with diethyl
ether (3ϫ75 mL). The combined organic phase was dried with
3
5.3 Hz, C-2Ј, Cquat), 117.5/117.7 (+, C-2), 120.7/121.0 [+, d, JC,P
= 4.5 Hz, C-6(8)], 126.9/127.9 (+, C-7Ј), 128.0/128.1 [+, C-6Ј(8Ј)*],
129.5/129.5 [+, C-5Ј(9Ј)*], 130.2/130.3 [+, C-5(9)*], 131.0/131.3
(Cquat, C-4), 134.5/134.8 (Cquat, C-4Ј), 143.7/143.7 (+, C-3), 152.2/
152.6 (d, 2JC,P = 6.8 Hz, C-7, Cquat), 167.3 (Cquat, C-1), 172.0/172.1
(Cquat, C-1Ј) ppm.
Methyl (R,S)- and (R,R)-(2E)-3-{4-[N-(1-Ethoxycarbonyl-1-meth- Na2SO4 and concentrated in vacuo to yield 2.71 g (97%) of the
oxy-2-phenylethyl)aminomethoxyphosphoryloxy]phenyl}acrylate title compound as a colorless solid whose purity was Ͼ95%. An
(13): (R,S) Diastereomer 11b (136 mg, 0.282 mmol) was dissolved analytical sample was purified by column chromatography on silica
in methanol (10 mL) and treated at –35 °C with sodium methoxide
(1.0  in methanol, 0.28 mL, 0.28 mmol), and the solution was
stirred for 2.5 h at –35 °C. Due to incomplete conversion, ad-
ditional sodium methoxide (1.0  in methanol, 0.14 mL,
0.14 mmol) was added, and stirring was continued for 50 min. The
reaction mixture was then warmed to –20 °C, treated with sodium
methoxide (1.0  in methanol, 0.12 mL, 0.12 mmol), and stirred for
another 0.5 h. Workup as described above yielded 106 mg of 13 as
gel (hexane/EtOAc, 5:1 plus 2.5% MeOH), which, however, oc-
curred with partial desilylation. C15H22O3Si: Mr = 278.42. Rf =
0.28. M.p. 95 °C. IR (KBr): ν = 3244 (O–H), 2956 (C–H), 1659
˜
(C=O), 1472, 1462, 1411, 1296, 1275, 1253, 1173, 991, 873, 822,
1
793, 741 cm–1. H NMR (250 MHz, CDCl3, 25 °C): δ = 0.34 (s, 6
3
H, SiMe2tBu), 1.05 (s, 9 H, SiMe2tBu), 6.28 (d, JH,H = 15.9 Hz,
3
3
1 H, 2-H), 6.88 [d, JH,H = 8.5 Hz, 2 H, 6(8)-H], 7.41 [d, JH,H
=
8.5 Hz, 5(9)-H], 7.60 (d, JH,H = 15.9 Hz, 1 H, 3-H) ppm. 13C
3
a
slightly impure mixture of both diastereomers (dr 1:1).
NMR (63.9 MHz, CDCl3, 25 °C): δ = –4.7 (+, SiMe2tBu), 17.8
C23H28NO8P: Mr = 477.44. Rf = 0.39 (hexane/EtOAc, 1:1). 1H {Cquat, SiMe2[C(CH3)3]}, 25.6 {+, SiMe2[C(CH3)3]}, 115.9 [+, C-
3
NMR (250 MHz, CDCl3, 25 °C): δ = 1.23/1.32 (2ϫt, JH,H
=
6(8)], 116.9 (+, C-2), 126.8 (Cquat, C-4), 130.1 [+, C-5(9)], 145.5 (+,
C-3), 158.3 (Cquat, C-7), 168.1 (Cquat, C-1) ppm. MS (EI): m/z (%)
7.2 Hz, 3 H, CH2CH3), 3.26 (d, 2JH,H = 14.5 Hz, 1 H, 3Ј-HA), 3.27/
3.28 (2ϫs, 3 H, OCH3), 3.36 (d, 2JH,H = 14.5 Hz, 1 H, 3Ј-HB), 3.78 = 278 (Ͻ1) [M]+, 263 (2) [M – CH3]+, 221 (100) [M – tBu]+, 203
3
(2ϫs, 3 H, 12-H3), 3.74/3.86 (2ϫd, JH,P = 12.6 Hz, 3 H, 11-H3),
4.12/4.23 (2ϫq, JH,H = 7.2 Hz, 2 H, CH2CH3), 4.66 (2ϫd, JH,P
= 8.0 Hz, 1 H, NH), 6.36/6.37 (2ϫd, JH,H = 15.6 Hz, 1 H, 2-H),
(14), 177 (16), 147 (18), 119 (6), 75 (24). C15H22O3Si (278.4): calcd.
C 64.71, H 7.96; found C 64.88, H 7.62.
3
2
3
tert-Butyldimethylsilyl
(2E)-3-[4-(Chloromethoxyphosphoryloxy)-
3
7.05–7.19 [m, 7 H, 6(8)-H, Ar-H], 7.47 [2ϫd, JH,H = 8.6 Hz, 2 H,
phenyl]acrylate (16): Silyl ester 15 (555 mg, 1.99 mmol) was dis-
solved in dichloromethane (30 mL) and cooled to 0 °C, and sodium
hydride (60% in mineral oil, 111 mg, 2.78 mmol) was added. After
being stirred for 1 h at 0 °C, the suspension was cooled to –78 °C
and methoxy phosphoryl dichloride (8; 0.30 g, 2.0 mmol)[20] was
added dropwise under vigorous stirring. The reaction mixture was
warmed to room temperature, stirred for 16 h, and then trans-
formed in the next reaction without purification. An analytical
sample was concentrated in vacuo leading to the isolation of the
free carboxylic acid. 1H NMR (250 MHz, CDCl3, 25 °C): δ = 4.10
5(9)-H], 7.64/7.66 (2ϫd, JH,H = 15.6 Hz, 1 H, 3-H) ppm. 13C
3
NMR (75.5 MHz, CDCl3, 25 °C): δ = 14.0/14.0 (+, CH2CH3), 43.1
(–, C-3Ј), 51.5/51.5 (+, OCH3), 51.7/51.7 (+, C-12), 54.2/54.3 (+, d,
2JC,P = 6.0 Hz, C-11), 62.75/62.82 (–, CH2CH3), 90.8/90.8 (d, 2JC,P
= 5.3 Hz, C-2Ј, Cquat), 117.5/117.6 (+, C-2), 120.6/120.9 [+, d, 3JC,P
= 4.5 Hz, C-6(8)], 126.9/127.0 (+, C-7Ј), 128.0/128.1 [+, C-6Ј(8Ј)*],
129.5/129.5 [+, C-5Ј(9Ј)*], 130.2/130.3 [+, C-5(9)*], 131.1/131.3
(Cquat, C-4), 134.5/134.8 (Cquat, C-4Ј), 143.6/143.7 (+, C-3), 152.2/
152.6 (d, 2JC,P = 5.3 Hz, C-7, Cquat), 167.3 (Cquat, C-1), 172.0/172.1
(Cquat, C-1Ј) ppm.
3
3
(d, JH,P = 12.4 Hz, 3 H, 11-H3), 6.43 (d, JH,H = 15.8 Hz, 1 H, 2-
3
3
Methyl (S)-(2E)-3-[4-(Aminomethoxyphosphoryloxy)phenyl]acrylate
[(S)-1]: An aqueous solution of H2SO4 (5%, 2.1 mL) was added to
a solution of diastereomeric mixture 12 (1:1; 270 mg) in methanol
(4.1 mL), and the reaction mixture was stirred for 6 h at room tem-
perature. Water (15 mL) was added, and the solution was extracted
with chloroform (3ϫ15 mL). The combined organic layer was
washed with brine (20 mL), dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
reverse-phase silica gel (Li-Chroprep ®RP-18; MeOH/H2O, 6:4) to
yield 87.0 mg (54% from 11a) of the title compound (S)-1 as a
colorless solid. The optical rotation {[α]2D0 = –6.6 (c = 0.14,
MeOH)} and all spectroscopic data were consistent with the data
of cinnamoylphosphoramide (1) isolated from Streptomyces sp.
JP90.
H), 7.31 [d, JH,H = 7.9 Hz, 2 H, 6(8)-H], 7.58 [d, JH,H = 7.9 Hz,
2 H, 5(9)-H], 7.76 (d, JH,H = 15.8 Hz, 1 H, 3-H) ppm. 13C NMR
3
2
(63.9 MHz, CDCl3, 25 °C): δ = 56.3 (+, d, JC,P = 7.3 Hz, C-11),
118.0 (+, C-2), 120.9 [+, d, JC,P = 4.7 Hz, C-6(8)], 130.0 [+, C-
3
2
5(9)], 132.2 (Cquat, C-4), 145.2 (+, C-3), 151.5 (d, JC,P = 7.6 Hz,
C-7, Cquat), 171.6 (Cquat, C-1) ppm. 31P NMR (121.5 MHz, CDCl3,
25 °C): δ = 1.87 ppm.
(2E)-3-[4-(Aminomethoxyphosphoryloxy)phenyl]acrylic Acid (17):
Crude product 16 from the reaction described above was diluted
with dichloromethane (20 mL) and cooled to –17 °C. A stream of
gaseous ammonia was passed for 45 min through the vigorously
stirred solution, which was then added to an aqueous phosphate
buffer solution (pH 4, 25 mL). The aqueous phase was extracted
with ethyl acetate (3ϫ75 mL), and the combined organic layer was
dried with Na2SO4 and concentrated in vacuo. The crude product
was purified by column chromatography on silica gel (80 g; CHCl3/
MeOH, 6:1 gradually increased to 2:1) yielding 220 mg (43%) of
Methyl (R)-(2E)-3-[4-(Aminomethoxyphosphoryloxy)phenyl]acrylate
[(R)-1]: An aqueous solution of H2SO4 (5%, 0.82 mL) was added to
a solution of diastereomeric mixture 13 (1:1; 106 mg) in methanol
Eur. J. Org. Chem. 2008, 5117–5124
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5123

M. Quitschau, T. Schuhmann, J. Piel, P. von Zezschwitz, S. Grond
FULL PAPER
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phosphoramide 17 as a colorless solid. C10H12NO5P: Mr = 257.18.
Rf = 0.33 (CHCl3/MeOH, 4:1). Rf = 0.84 (RP; MeOH/H2O, 7:3).
M.p. 163–164 °C. IR (KBr): ν = 3363 (N–H), 2947 (C–H), 1713
˜
(C=O), 1639 (N–H), 1601 (C=C), 1507 (C=C), 1438, 1317, 1218
(P=O), 1173, 1060, 1002, 986, 931, 839, 793 cm–1. UV (MeOH):
λmax (log ε) = 272 (2.22) nm. 1H NMR (600 MHz, CD3OD, 25 °C):
δ = 3.80 (d, 3JH,P = 11.5 Hz, 3 H, 11-H3), 6.44 (d, 3JH,H = 16.0 Hz,
3
3
1 H, 2-H), 7.26 [d, JH,H = 8.5 Hz, 2 H, 6(8)-H], 7.61 [d, JH,H
=
8.5 Hz, 2 H, 5(9)-H], 7.62 (d, JH,H = 16.0 Hz, 1 H, 3-H) ppm. 13C
NMR (125.7 MHz, CD3OD, 25 °C): δ = 54.1 (+, d, 2JC,P = 5.7 Hz,
C-11), 120.3 (+, C-2), 122.0 [+, d, 3JC,P = 4.9 Hz, C-6(8)], 130.6 [+,
C-5(9)], 132.8 (Cquat, C-4), 144.4 (+, C-3), 153.9 (d, 2JC,P = 6.6 Hz,
C-7, Cquat), 171.1 (Cquat, C-1) ppm. 31P NMR (121.5 MHz, [D6]
DMSO, 25 °C): δ = 9.25 ppm. MS (ESI): m/z (%) = 537 (100) [2M
+ Na]+. HRMS (ESI): calcd. for C10H13NO5P [M + H]+ 258.05259;
found 258.05262.
3
Supporting Information (see footnote on the first page of this arti-
cle): Detailed experimental procedures; comparison of the spectro-
scopic data of the natural product and α,β-unsaturated carboxylic
esters related to 1 and related imido esters from the literature; chiral
HPLC traces; hypothesis for the biosynthetic pathway of cinna-
moylphosphoramide.
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Acknowledgments
The authors thank Prof. Ulrike Holzgrabe and Eva Kugelmann at
the University of Würzburg and Prof. Chris Meier and Nicolas
Gisch at the University of Hamburg for collaboration in per-
forming the cholinesterase inhibition assays and Prof. Bradley S.
Moore, Prof. Ron Quinn, Dr. Mark Butler, and Dr. Rohan Davis
for support with the collection of A. lenticulum.
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Received: July 2, 2008
Published Online: September 9, 2008
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