Structure and absolute configuration of auriculamide, a natural product from the predatory bacterium herpetosiphon aurantiacus

Article abstract of DOI:10.1002/ejoc.201500181

The genome of the filamentous, predatory bacterium Herpetosiphon aurantiacus harbors a plethora of genes that are predicted to be involved in natural product biosynthesis. Until now, however, no secondary metabolites have been described from this microorganism. Analysis of H. aurantiacus culture extracts by ¹H NMR spectroscopy now led to the discovery of a chlorinated amide, which we termed auriculamide. The configuration of the three chiral centers in auriculamide was solved by chromatographic comparison with stereospecifically prepared reference compounds following chemical degradation. Furthermore, a putative gene cluster for the biosynthesis of auriculamide was identified by genome mining. Chemical analysis of culture extracts from the predatory bacterium Herpetosiphon aurantiacus led to the discovery of the new natural product auriculamide, the structure of which was elucidated by MS and NMR analyses. The absolute configuration was determined, after hydrolysis, by chromatographic comparison with synthetic standards. A gene cluster for the biosynthesis of auriculamide is proposed.

Full text of DOI:10.1002/ejoc.201500181

FULL PAPER
DOI: 10.1002/ejoc.201500181
Structure and Absolute Configuration of Auriculamide, a Natural Product from
the Predatory Bacterium Herpetosiphon aurantiacus
Sebastian Schieferdecker,[a][‡] Nicole Domin,[a][‡] Christine Hoffmeier,[b] Donald A. Bryant,[c]
Martin Roth,[b] and Markus Nett*[a]
Keywords: Natural products / Structure elucidation / Configuration determination / Genome mining / Nonribosomal
peptides
The genome of the filamentous, predatory bacterium Herpe-
tosiphon aurantiacus harbors a plethora of genes that are
predicted to be involved in natural product biosynthesis. Un-
til now, however, no secondary metabolites have been de-
scribed from this microorganism. Analysis of H. aurantiacus
lamide. The configuration of the three chiral centers in auric-
ulamide was solved by chromatographic comparison with
stereospecifically prepared reference compounds following
chemical degradation. Furthermore, a putative gene cluster
for the biosynthesis of auriculamide was identified by ge-
nome mining.
1
culture extracts by H NMR spectroscopy now led to the dis-
covery of a chlorinated amide, which we termed auricu-
which many are unique to this micropredator.[11] Moreover,
H. aurantiacus appears to possess pathways to specific natu-
ral product building blocks, including the nonproteinogenic
amino acid 4-hydroxyphenylglycine, which is known as a
constituent of glycopeptide antibiotics.[12] Although the ge-
nus Herpetosiphon already demonstrated its potential for
natural product biosynthesis,[13] the secondary metabolites
of the sequenced type strain 114–95T have remained elusive
to date. Here, we report the discovery of the chlorinated
amide auriculamide (1, Figure 1), its structure elucidation
and stereochemical assignment. Furthermore, we propose a
gene cluster for the biosynthesis of auriculamide from the
genome of H. aurantiacus 114–95T.
Introduction
Predatory bacteria encompass a heterogeneous group of
prokaryotes that share the ability to consume living micro-
organisms.[1] The feeding strategy of predatory bacteria typ-
ically involves the secretion of lytic enzymes and small
bioactive molecules.[2,3] Antibiotics, which are produced
by predatory bacteria, such as roimatacene,[4] corallo-
pyronin,[5] or the recently described gulmirecins[6] and disci-
formycins,[7] are assumed to contribute to the killing of prey
organisms. A deficiency in the biosynthesis of these com-
pounds was shown to severely affect the predatory perform-
ance.[8] From a pharmaceutical perspective, predatory bac-
teria are hence a promising source for the discovery of anti-
microbial compounds. Except for the predatory myxobac-
teria, however, this resource has barely been exploited.[9,10]
Herpetosiphon aurantiacus represents an illustrative ex-
ample of a predatory bacterium with a little explored sec-
ondary metabolism. Analysis of its complete genome se-
quence revealed an abundance of biosynthesis genes, of
[a] Junior Research Group Secondary Metabolism of Predatory
Bacteria, Leibniz Institute for Natural Product Research and
Infection Biology, Hans-Knöll-Institute,
Figure 1. Structure of (A) auriculamide (1) and (B) selected COSY
(bold lines) and HMBC (arrows) correlations in 1 (B).
Adolf-Reichwein-Str. 23, 07745 Jena, Germany
E-mail: Markus.Nett@hki-jena.de
http://www.leibniz-hki.de/en/drug-discovery-from-predatory-
bacteria.html
[b] Bio Pilot Plant, Leibniz Institute for Natural Product Research
and Infection Biology, Hans-Knöll-Institute,
Results and Discussion
Adolf-Reichwein-Str. 23, 07745 Jena, Germany
[c] Department of Biochemistry and Molecular Biology,
The Pennsylvania State University,
For the production of auriculamide, H. aurantiacus 114–
95T was grown in a 30 L bioreactor in modified HP74
broth. After ten days of cultivation, the biomass was re-
moved by centrifugation. Metabolites that had been se-
creted during the fermentation were recovered from the su-
University Park, Pennsylvania 16802, USA
[‡] Both authors contributed equally to this study.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500181.
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M. Nett et al.
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pernatant by adsorption onto XAD-2 resin. Elution of the the benzene ring were deduced from an analysis of 13C
resin with methanol and subsequent concentration yielded chemical shifts.
a brownish residue, which was dissolved in 60% aqueous
The structure of 1 includes three chiral centers at C-3, C-
methanol (MeOH) and exhaustively extracted with di- 4, and C-6. For the stereochemical analysis, the amide bond
chloromethane (CH2Cl2). The combined organic layers of 1 was hydrolyzed with HCl and the two resulting frag-
were fractionated by ODS flash chromatography using a ments were analyzed by chromatographic comparison with
water/methanol gradient. Proton NMR analysis indicated synthetic standards. The configuration of the 2-hydroxy-3-
the 60% MeOH fraction to be of further interest due to the methylvaleric acid (HMVA) residue was determined by
presence of several signals in the aromatic region that were chiral thin-layer chromatography (TLC). To this end, the
absent in extracts originating from the pure growth me- four possible HMVA stereoisomers were prepared and indi-
dium. Subsequent purification by reverse-phase HPLC led vidually spotted on a chiral TLC plate together with the
to the isolation of 0.6 mg of auriculamide (1).
hydrolysate of 1.[14,15] This approach revealed that 1 con-
Compound 1 exhibits a pseudomolecular mass ion peak tains (2S,3S)-HMVA (Figure S1).
at m/z 340.1323 [M – H]–, which is consistent with a sum
To assign the stereochemistry of the 2-amino-1-(3-
formula of C17H24ClNO4 and accounts for six double bond chloro-4-hydroxyphenyl)pentan-3-one (CHPO) fragment,
equivalents. The 13C NMR spectrum of 1 indicated the the HCl hydrolysate was subjected to chiral HPLC. Refer-
presence of a ketone (C-14) and an amide (C-5), as well as ence compounds included the (S)-enantiomer of CHPO (6)
six additional sp2-hybridized carbon atoms (C-8 to C-13), as well as a racemic mixture of this building block (9). The
corresponding to three C–C double bonds (Table 1). It was stereospecific preparation of 6 started with Boc-protected
hence clear that 1 must feature a single ring structure to l-tyrosine (Scheme 1), which was initially converted into
comply with the required degrees of unsaturation. Analysis Boc-l-Tyr(Cbz)-OH (2). Subsequent conversion into Wein-
of the 1H NMR spectrum revealed 15 nonexchangeable sig- reb amide 3,[16] and Grignard reaction with ethylmagnesium
nals, accounting for a total of 21 protons. These protons bromide generated the desired pentan-3-one side-chain in
were attributed to their directly attached carbon atoms by 4. The cleavage of both protective groups with methane-
heteronuclear single quantum coherence (HSQC). Proton- sulfonic acid yielded enantiopure (S)-2-amino-1-(4-
proton correlation spectroscopy (COSY) together with
first-order multiplet analysis established four independent
spin systems, including a 1,2,4-trisubstituted benzene and
a 1-hydroxy-2-methylbutyl moiety. The secondary alcohol
function of the latter was concluded from the chemical shift
of C-4. All spin systems could be connected on the basis
of 1H,13C-long-range interactions (Figure 1). Heteronuclear
multiple bond correlation (HMBC) data also enabled the
placement of the amide and the ketone function. Finally,
the positions of the chlorine and hydroxy substituents on
Table 1. NMR spectroscopic data of auriculamide (1) in CDCl3.
Pos. δC [ppm] δH [ppm], M (J inHz) COSY
HMBC
1
2
11.8
26.2
0.91, t (7.5)
a: 1.41 m
b: 1.26 m
1.83 m
2a, 2b
1, 2b, 3
1, 2a, 3
2a, 2b, 4, 17 1, 2, 4, 5, 17
3
2, 3
1, 3, 4, 17
1, 3, 4, 17
3
4
5
6
7
38.3
74.5
173.1
58.1
36.7
4.09 d (2.8)
2, 3, 5, 17
4.84 dt (7.9, 6.8)
a: 3.01 dd (14.2, 6.8) 6, 7b
b: 2.88 dd (14.2, 6.8) 6, 7a
7a, 7b, NH
5, 7, 8, 14
6, 8, 9, 13, 14
6, 8, 9, 13, 14
8
9
129.2
129.1
116.4
150.5
119.9
129.5
209.0
34.4
6.94 dd (8.3, 2.0)
6.91 d (8.3)
10, 13
9
7, 11, 13
8, 11, 12
10
11
12
13
14
15
7.10 d (2.0)
9
7, 9, 11, 12
a: 2.44 dq (18.4, 7.2) 15b, 16
b: 2.41 dq (18.4, 7.2) 15a, 16
14, 16
14, 16
14, 15
2, 3, 4
7
16
7.3
1.02 t (7.2)
0.69 d (6.9)
6.98 d (7.9)
15a, 15b
3
6
17
NH
12.6
Scheme 1. Preparation of (S)-2-amino-1-(4-hydroxyphenyl)pentan-
3-one (6).
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Structure and Absolute Configuration of Auriculamide
hydroxyphenyl)pentan-3-one (5). Chlorination of the aro- could be involved in the final processing of the proposed
matic ring in the meta-position was achieved by addition of carboxylic acid intermediate (Figure 2).
SO2Cl2 in a mixture of Et2O and HOAc. Racemic 2-amino-
1-(4-hydroxyphenyl)pentan-3-one (8) was prepared from l-
tyrosine by a Dakin–West reaction with propionic an-
hydride and subsequent hydrolysis.[17] Chlorination of 8 was
achieved as described for the enantiopure compound 5.
Chromatographic comparison of the two reference com-
pounds 6 and 9 with the hydrolysis product of 1 indicated
C-6 to be S-configured (Figure S2).
Auriculamide is the first secondary metabolite to be re-
ported from the predatory bacterium H. aurantiacus 114–
95T. A retrobiosynthetic analysis was carried out to clarify
whether the structure of 1 could be associated with the pre-
viously annotated biosynthesis genes.[11] Auriculamide fea-
tures an α-hydroxy acid moiety in its structure. Such mono-
mers are known as constituents of various peptide natural
products and their incorporation typically involves nonri-
bosomal peptide synthetase (NRPS)-based chemistry.[18,19]
In brief, the adenylation (A) domain of an NRPS activates
an α-keto acid[20] in an ATP-driven process and tethers the
resulting acyl adenylate as a pantetheinyl thioester to a
peptidyl carrier protein (PCP) domain. The necessary re-
duction is subsequently carried out by a ketoreductase
(KR) domain that is integrated into the same NRPS.[21,22]
In case of 1, an amide bond links the HMVA moiety to a
tyrosine-like residue. Whereas the extension of HMVA with
an amino acid can be rationalized by an NRPS-based
mechanism,[18] the origin of the ethyl group next to the
ketone function in 1 was not clear. A possible scenario in-
volves the incorporation of a methylmalonyl-CoA-derived
C3 unit, mediated by a polyketide synthase (PKS). After
hydrolytic release from the NRPS/PKS complex, the re-
sulting carboxylic acid could be shortened by a decarboxyl-
ation reaction. Precedence for such a post-assembly-line
modification comes from tautomycetin biosynthesis.[23]
Figure 2. (A) Proposed auriculamide gene cluster and (B) as-
Taken together, a hypothetical auriculamide assembly
sembly-line biosynthesis of 1. The chlorination reaction might also
occur at a later biosynthetic stage.[34] Domain notation: A,
adenylation; KR, keto reductase; PCP, peptidyl carrier protein; C,
condensation; KS, β-ketoacyl synthase; AT, acyl transferase; ACP,
acyl carrier protein; TE, thioesterase. The gene haur_2417 was
annotated as a type-II thioesterase.
line should feature two discrete NRPS modules for the in-
corporation of HMVA and (3-chloro)tyrosine as well as an
additional PKS module for the final carbon extension.
Analysis of the annotated biosynthetic loci from the H. aur-
antiacus genome led to the identification of a putative auric-
ulamide gene cluster (Figure 2). The domain architecture of
the proteins encoded by haur_2414 – haur_2416 is consis-
tent with the proposed biosynthetic model. Furthermore, 2-
keto-3-methylvaleric acid and tyrosine were predicted as the
most likely substrates of Haur_2414 and Haur_2415,
thereby supporting an involvement of these proteins in the
assembly of 1 (Table S1).[24–26] It must be noted that Haur_
2414 is also the only NRPS-type enzyme of H. aurantiacus
114–95T featuring a KR domain. The chlorination is ex-
pected to occur after the tyrosine has been tethered to the
PCP domain of Haur_2415, thus paralleling the incorpora-
tion of chlorine atoms in the biosyntheses of balhimycin,
C-1027, and chondrochloren.[27–29] Homology searches
against the genome of H. aurantiacus 114–95T did not re-
According to a database search,[30] 1 shows only modest
structural similarity to other natural products. The closest
relatives are the depsipeptides ulongamide
E
and
guineamide C, which were isolated from cyanobacte-
ria.[31,32] Both compounds feature a HMVA moiety that is
amidated with tyrosine in their structures, but lack the dis-
tinctive chlorination and the terminal ethyl group of 1. This
finding substantiates the potential of unexplored bacterial
sources to produce new chemical scaffolds.[33]
Conclusion
The discovery of auriculamide provides the first evidence
veal a clear candidate gene for the halogenation. The prod- for the previously assumed biosynthetic potential of H. aur-
uct of haur_2404 was annotated as a decarboxylase and antiacus 114–95T.[11] Although a possible gene cluster for
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M. Nett et al.
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10% MeOH. Analytes were detected with vanadium(V) oxide spray
reagent.
the assembly of the structural scaffold of 1 could be iden-
tified in this study, the genetic basis for specific tailoring
reactions await further clarification. Since the lack of a ge-
netic system for H. aurantiacus impedes inactivation and
complementation studies, efforts are now underway to re-
constitute the biosynthesis of 1 in a heterologous host.
(S)-3-[4-(Benzyloxy)phenyl]-2-(tert-butoxycarbonylamino)propanoic
Acid (2): Boc-protected l-tyrosine (5 mmol) and sodium hydride
(5 mmol) were dissolved in anhydrous THF (5 mL) under an argon
atmosphere and stirred for 15 min at room temperature. Tetrabut-
ylammonium iodide (0.05 mmol) and benzyl bromide (5 mmol)
were then added and the solution was stirred for 3 h at room tem-
perature before brine (15 mL) was added and the solution was
exhaustively extracted with diethyl ether (Et2O). The organic layers
were combined, the solvent was evaporated, and the crude product
was purified by reversed-phase HPLC to give 2 (1690.3 mg, 91%).
Experimental Section
General Experimental Procedures: High-resolution mass analyses
were performed with an Exactive Mass Spectrometer (Thermo-
Scientific). NMR spectra were recorded at 300 K with Bruker spec-
trometers and [D4]MeOH (δH = 3.31 ppm; δC = 49.0 ppm) or
CDCl3 (δH = 7.24 ppm; δC = 77.0 ppm) as solvents and internal
standards. Preparative and analytical HPLC was conducted with a
Shimadzu HPLC system (LC-20AT, SPD-M20A).
(S)-3-[4-(Benzyloxy)phenyl]-2-(tert-butoxycarbonylamino)propanoic
1
Acid (2): H NMR (300 MHz, [D4]MeOH, 300 K): δ = 7.29–7.45
3
(m, 5 H, 12–14-CH), 7.14 (d, JH,H = 8.5 Hz, 2 H, 7-CH), 6.91 (d,
3
3JH,H = 8.5 Hz, 2 H, 8-CH), 5.05 (s, 2 H, 10-CH2), 4.29 (dd, JH,H
3
= 9.0, 5.0 Hz, 4-CH), 3.09 (dd, JH,H = 14.0, 5.0 Hz, 1 H, 5b-CH),
3
2.84 (dd, JH,H = 14.0, 9.0 Hz, 1 H, 5a-CH), 1.37 (s, 9 H, 1-
Production and Isolation of 1: H. aurantiacus strain 114–95T was
obtained from the German Collection of Microorganisms and Cell
Cultures (DSMZ). The bacterium was routinely cultured at 30 °C
in modified HP74 medium [sodium glutamate 1%, yeast extract
0.2%, MgSO4·7H2O 0.2%, d-(+)-glucose 1%, 50 mm phosphate
buffer, pH 6.6]. For the production of 1, fermentation was carried
out in a 30 L bioreactor (20 L working volume). Aeration was set
to 10 Lmin–1 and the culture broth was continuously stirred
(200 rpm). The glucose concentration was maintained at ca. 3 gL–1
by continuous feeding. The pH was adjusted to values between
6.8 and 7.2. After ten days of growth, the cells were removed by
centrifugation and the residual supernatant was mixed with 1.5 kg
Amberlite XAD-2 (Supelco). After two washing steps with distilled
water, the adsorbed metabolites were eluted from the resin with
10 L MeOH. The eluate was concentrated under vacuum to dryness
prior to resuspension in 200 mL of 60% aqueous MeOH and five
successive extractions with 200 mL of CH2Cl2. The organic layers
were combined and residual water was removed by the addition of
anhydrous Na2SO4. After filtration, the organic solvent was evapo-
rated under reduced pressure. Initial fractionation of the CH2Cl2
extract was accomplished by flash chromatography over 50 g of
Polygoprep 60–50 C18 (Macherey–Nagel) by using increasing con-
centrations of MeOH in H2O. Proton NMR spectroscopic analysis
indicated the 60% MeOH fraction to be of further interest. Final
purification of 1 was achieved by HPLC with a Zorbax-Eclipse
XDB C8 column (Agilent) with a linear gradient of MeOH in H2O
+ 0.1% trifluoroacetic acid and a flow rate of 2 mLmin–1.
CH3) ppm. 13C NMR (75 MHz, [D4]MeOH, 300 K): δ = 175.5 (C-
15), 159.1 (C-9), 157.8 (C-3), 138.9 (C-11), 131.3 (C-13), 130.9 (C-
14), 129.5 (C-7), 128.8 (C-6), 128.5 (C-12), 115.9 (C-8), 80.5 (C-2),
71.0 (C-10), 56.5 (C-4), 37.9 (C-5), 28.7 (C-1) ppm. HRMS (ESI):
m/z calcd. for C21H25NO5 [M – H]– 370.1660; found 370.1665.
tert-Butyl (S)-{3-[4-(Benzyloxy)phenyl]-1-[methoxy(methyl)amino]-
1-oxopropan-2-yl}carbamate (3): Tetrabromomethane, N,O-dimeth-
ylhydroxylamine, triethylamine, and 2 (3 mmol each) were dis-
solved in anhydrous CH2Cl2 (10 mL) under an argon atmosphere
at room temperature. Triphenylphosphine (3 mmol) was slowly
added and the solution was stirred for 10 min. Upon completion,
the CH2Cl2 solution was washed with brine (10 mL), dried under
reduced pressure, and purified by reversed-phase HPLC to give 3
(1056.2 mg, 85%).
tert-Butyl (S)-{3-[4-(Benzyloxy)phenyl]-1-[methoxy(methyl)amino]-
1-oxo-propan-2-yl}carbamate (3): 1H NMR (600 MHz, [D4]MeOH,
3
3
300 K): δ = 7.41 (d, JH,H = 7.3 Hz, 2 H, 12-CH), 7.35 (t, JH,H
=
7.3 Hz, 2 H, 13-CH), 7.28 (t, 3JH,H = 7.3 Hz, 1 H, 14-CH), 7.12 (d,
3JH,H = 8.2 Hz, 2 H, 7-CH), 6.90 (d, JH,H = 8.2 Hz, 2 H, 8-CH),
3
5.05 (s, 3 H, 10-CH3), 4.78 (m, 1 H, 4-CH), 3.70 (s, 3 H, 17-CH3),
3
3.14 (s, 3 H, 16-CH3), 2.92 (dd, JH,H = 13.5, 5.4 Hz, 1 H, 5b-
CH), 2.72 (m, 1 H, 5a-CH), 1.37 (s, 9 H, 1-CH3) ppm. 13C NMR
(150 MHz, [D4]MeOH, 300 K): δ = 174.5 (C-15), 159.1 (C-9), 157.6
(C-3), 138.8 (C-11), 131.4 (C-14), 130.8 (C-6), 129.5 (C-7), 128.8
(C-13), 128.5 (C-12), 115.9 (C-8), 80.5 (C-2), 70.9 (C-10), 62.0 (C-
17), 53.7 (C-4), 38.0 (C-5), 32.4 (C-16), 28.7 (C-1) ppm. HRMS
(ESI): m/z calcd. for C23H30N2O5 [M + H]+ 415.2227; found
415.2231.
Hydrolysis of 1: The amide bond in 1 was hydrolyzed by addition
of 500 μL 4 n HCl and subsequent heating to 120 °C for 24 h. The
acid was removed by evaporation and the residue was used directly
for the chromatographic analyses described below.
tert-Butyl (S)-{1-[4-(Benzyloxy)phenyl]-3-oxopentan-2-yl}carbamate
(4): Compound 3 (2 mmol) was dissolved in anhydrous Et2O under
an argon atmosphere. Ethylmagnesium bromide (1.5 equiv.) was
Configurational Assignment of the HMVA Moiety in 1: The four added and the solution was stirred at room temperature for 1 h.
possible stereoisomers of HMVA were prepared according to an
established protocol.[14] In brief, the four stereoisomers of iso-
leucine (0.2 mmol) were each dissolved in 1 m H2SO4 (10 mL) in
an ice bath. NaNO2 (2 mmol) was added and the solutions were
stirred at 0 °C for 10 h. Stirring was continued for two days at room
temperature, then the aqueous solutions were saturated with NaCl
and extracted with EtOAc (3ϫ 10 mL). The combined EtOAc ex-
tracts were dried under reduced pressure. Product formation was
confirmed by LC-HR-ESI-MS analyses. Subsequently, the HMVA
stereoisomers were subjected to TLC together with hydrolyzed 1
using ChiralPlate (Macherey–Nagel) as the stationary phase.[15]
The TLC plate was developed in a mixture of 90% CH2Cl2 and
The reaction was then quenched by the addition of H2O (500 μL),
the solvent was evaporated under reduced pressure, and the residue
was extracted with MeOH and purified by reversed-phase HPLC to
give 4 (620.8 mg, 81%). 1H NMR (500 MHz, [D4]MeOH, 300 K): δ
3
3
= 7.41 (d, JH,H = 7.3 Hz, 2 H, 12-CH), 7.35 (t, JH,H = 7.3 Hz, 2
3
3
H, 13-CH), 7.29 (t, JH,H = 7.3 Hz, 1 H, 14-CH), 7.11 (d, JH,H
=
3
8.5 Hz, 2 H, 7-CH), 6.90 (d, JH,H = 8.5 Hz, 2 H, 8-CH), 5.05 (s,
3
2 H, 10-CH2), 4.26 (dd, JH,H = 8.9, 5.8 Hz, 1 H, 4-CH), 2.98 (dd,
3JH,H = 14.0, 5.8 Hz, 1 H, 5b-CH), 2.72 (dd, JH,H = 14.0, 8.9 Hz,
3
3
1 H, 5a-CH), 2.52 (dq, JH,H = 14.1, 7.5 Hz, 1 H, 16b-CH), 2.40
(dq, 3JH,H = 14.1, 7.2 Hz, 1 H, 16a-CH), 1.37 (s, 9 H, 1-CH3), 0.97
(t, JH,H = 7.2 Hz, 3 H, 17-CH3) ppm. 13CNMR (125 MHz, [D4]-
3
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Structure and Absolute Configuration of Auriculamide
3
3
MeOH, 300 K): δ = 212.6 (C-15), 159.1 (C-9), 157.9 (C-3), 138.8
(C-11), 131.4 (C-14), 131.0 (C-6), 129.5 (C-7), 128.8 (C-13), 128.5
(C-12), 116.0 (C-8), 80.6 (C-2), 71.0 (C-10), 62.2 (C-4), 37.0 (C-5),
34.0 (C-16), 28.7 (C-1), 7.7 (C-17) ppm. HRMS (ESI): m/z calcd.
for C23H29NO4 [M + H]+ 384.2169; found 384.2173.
7-CH), 6.79 (d, JH,H = 8.5 Hz, 2 H, 8-CH), 4.13 (dd, JH,H = 7.9,
3
6.7 Hz, 1 H, 4-CH), 3.15 (dd, JH,H = 14.4, 6.7 Hz, 1 H, 5b-CH),
2.95 (dd, JH,H = 14.4, 7.9 Hz, 1 H, 5a-CH), 2.48 (q, JH,H
3
3
=
3
7.3 Hz, 2 H, 2-CH2), 1.02 (t, JH,H = 7.3 Hz, 3 H, 1-CH3) ppm.
13C NMR (75 MHz, [D4]MeOH, 300 K): δ = 207.7 (C-3), 158.4 (C-
9), 131.4 (C-7), 125.9 (C-6), 117.0 (C-8), 60.8 (C-4), 36.6 (C-5), 34.5
(C-2), 7.3 (C-1) ppm. HRMS (ESI): m/z calcd. for C11H15NO2 [M
+ H]+ 194.1176; found 194.1178.
(S)-2-Amino-1-(4-hydroxyphenyl)pentan-3-one (5): Compound
4
(1.5 mmol) was dissolved in CHCl3 (5 mL) and methanesulfonic
acid (2 mL) was added dropwise. After 30 min of stirring the reac-
tion was complete and H2O (10 mL) was added. The acid was neu-
tralized by the addition of solid NaHCO3 and the solvents were
removed under reduced pressure. The residue was extracted with
MeOH and the extracted salt that precipitated at 4 °C was removed
by filtration. The filtrate was purified by reversed-phase HPLC to
(R,S)-2-Amino-1-(3-chloro-4-hydroxyphenyl)pentan-3-one (9): Com-
pound 8 (1 mmol) was dissolved in a mixture of HOAc and Et2O
(1:1, 5 mL). The solution was cooled to 0 °C and SO2Cl2 (1 equiv.)
was added slowly. The reaction mixture was stirred for 2 h at 0 °C,
then the solvent was evaporated and the crude 2-amino-1-(3-
chloro-4-hydroxyphenyl)pentan-3-one (9, racemic CHPO) was
purified by reversed-phase HPLC to give 9 (141.2 mg, 62%). 1H
1
give 5 (139.1 mg, 48%). H NMR (500 MHz, [D4]MeOH, 300 K):
3
3
δ = 7.10 (dd, JH,H = 8.4 Hz, 2 H, 7-CH), 6.80 (d, JH,H = 8.4 Hz,
3
3
NMR (600 MHz, [D4]MeOH, 300 K): δ = 7.27 (d, JH,H = 2.1 Hz,
2 H, 8-CH), 4.31 (dd, JH,H = 8.1, 6.3 Hz, 1 H, 4-CH), 3.17 (dd,
3
3JH,H = 14.4, 6.3 Hz, 1 H, 5b-CH), 2.92 (dd, JH,H = 14.4, 8.1 Hz,
3
1 H, 7-CH), 7.04 (dd, JH,H = 8.3, 2.1 Hz, 1 H, 11-CH), 6.92 (d,
3JH,H = 8.3 Hz, 1 H, 10-CH), 4.34 (dd, JH,H = 8.6, 6.1 Hz, 1 H,
3
3
3
1 H, 5a-CH), 2.51 (q, JH,H = 7.1 Hz, 2 H, 2-CH2), 1.04 (t, JH,H
= 7.12 Hz, 3 H, 1-CH3) ppm. 13C NMR (125 MHz, [D4]MeOH,
300 K): δ = 207.6 (C-3), 158.5 (C-9), 131.4 (C-7), 125.8 (C-6), 117.0
(C-8), 60.9 (C-4), 36.6 (C-5), 34.4 (C-2), 7.4 (C-1) ppm. HRMS
(ESI): m/z calcd. for C11H15NO2 [M + H]+ 194.1176; found
194.1167.
4-CH), 3.21 (dd, 3JH,H = 14.6, 6.1 Hz, 1 H, 5b-CH), 2.89 (dd, 3JH,H
3
= 14.6, 8.6 Hz, 1 H, 5a-CH), 2.55 (qd, JH,H = 7.1, 2.5 Hz, 2 H, 2-
3
CH2), 1.06 (t, JH,H = 7.1 Hz, 3 H, 1-CH3) ppm. 13C NMR
(150 MHz, [D4]MeOH, 300 K): δ = 207.3 (C-3), 154.2 (C-9), 131.8
(C-7), 129.9 (C-11), 127.4 (C-6), 122.3 (C-8), 118.2 (C-10), 60.7 (C-
4), 36.0 (C-5), 34.3 (C-2), 7.4 (C-1) ppm. HRMS (ESI): m/z calcd.
for C11H14ClNO2 [M + H]+ 228.0786; found 228.0787.
(S)-2-Amino-1-(3-chloro-4-hydroxyphenyl)pentan-3-one (6): Com-
pound 5 (0.5 mmol) was dissolved in a mixture of Et2O and HOAc
(1:1, 2 mL). The solution was cooled to 0 °C and SO2Cl2
(0.5 mmol) was added slowly. The mixture was stirred for 2 h at
0 °C, then the solvent was evaporated and the crude product was
Configurational Assignment of the CHPO Moiety in 1: Chiral
HPLC analysis was carried out with a Nucleodur delta RP 5 col-
umn (Macherey–Nagel). The column was operated at a flow rate
of 0.5 mLmin–1 using a linear gradient of MeOH in H2O + 0.1%
trifluoroacetic acid. Analytes were detected with a diode array de-
tector.
purified by reversed-phase HPLC to give 6 (66.0 mg, 58%).
3
1H NMR (600 MHz, [D4]MeOH, 300 K): δ = 7.27 (d, JH,H
=
3
2.1 Hz, 1 H, 7-CH), 7.04 (dd, JH,H = 8.3, 2.1 Hz, 1 H, 11-CH),
6.92 (d, 3JH,H = 8.3 Hz, 1 H, 10-CH), 4.34 (dd, 3JH,H = 8.6, 6.1 Hz,
3
1 H, 4-CH), 3.21 (dd, JH,H = 14.4, 6.1 Hz, 1 H, 5b-CH), 2.89 (dd,
Acknowledgments
3JH,H = 14.4, 8.6 Hz, 1 H, 5a-CH), 2.54 (q, JH,H = 7.2 Hz, 2 H,
3
2-CH2), 1.06 (t, JH,H = 7.2 H, 3 H, 1-CH3) ppm. 13C NMR
3
Financial support was provided by the German Bundesministerium
für Bildung und Forschung (BMBF) (GenoMik-Transfer pro-
gramme; grant number 0315591A). The authors thank A. Perner
and H. Heinecke (Hans-Knöll-Institute, Department of Biomolec-
ular Chemistry) for recording HRMS (ESI) and NMR spectra,
respectively.
(150 MHz, [D4]MeOH, 300 K): δ = 207.3 (C-3), 154.2 (C-9), 131.8
(C-7), 129.9 (C-11), 127.4 (C-6), 122.3 (C-8), 118.2 (C-10), 60.7 (C-
4), 36.0 (C-5), 34.3 (C-2), 7.4 (C-1) ppm. HRME (ESI): m/z calcd.
for C11H14ClNO2 [M + H]+ 228.0786; found 228.0788.
4-(3-Oxo-2-propionamidopentyl)phenyl Propionate (7): l-Tyrosine
(5 mmol) was dissolved in anhydrous pyridine (10 mL), propionic
anhydride (5 equiv.) was added and the solution was heated to re-
flux at 120 °C for 8 h. The solvent was evaporated and the crude
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Published Online: March 17, 2015
3062
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Eur. J. Org. Chem. 2015, 3057–3062