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Photochemical Origin of the Immunosuppressive SNF4435CD and Formation of Orinocin through УPolyene SplicingФ.

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Spectinabilin (neoaureothin, 1)[5, 6] and aureothin (2)
represent unusual nitroaryl-substituted polyene metabolites
produced by Streptomyces thioluteus and Streptomyces spectabilis, respectively. Biosynthetic studies at the chemical,
biochemical, and genetic levels revealed that these cytotoxic
metabolites are assembled from nitrobenzoate,[7–11] malonate,
and methylmalonate[12] by modular polyketide synthases[13, 14]
followed by tailoring reactions (Scheme 1).[15, 16] Apparently
the diene moiety of aureothin is assembled from two
propionate units, while four propionates give rise to the
tetraene system of 1. The discovery of the intriguing constitutional isomers of 1, the immunosuppressant SNF4435C (3 a)
and SNF4435D (3 b),[17–19] from S. spectabilis (Scheme 2)
Natural Products
DOI: 10.1002/anie.200602840
Photochemical Origin of the
Immunosuppressive SNF4435C/D and
Formation of Orinocin through “Polyene
Markus Mller, Bjrn Kusebauch,
Guangxin Liang, Christopher M. Beaudry,
Dirk Trauner,* and Christian Hertweck*
Polyketides comprise a large group of structurally
diverse secondary metabolites, many of which are
endowed with medicinally relevant biological activities.[1] Irrespective of their diverse carbon frameworks
they all arise from repetitive Claisen condensations of
activated acetate or propionate building blocks in
analogy to the biosynthesis of fatty acids.[2] Depending
on the degree of b-keto procession, polyphenols,
polyenes, or highly reduced polyketides are produced.
Scheme 1. Structures of nitroaryl-substituted pyrone metabolites from Streptomyces sp.
It is known that polyketide-derived polyene systems,
CoA = coenzyme A, M = malonyl, MM = methylmalonyl, SAM = S-adenosylmethionine.
in particular those equipped with strong chromophores, easily undergo light-induced photoisomerization. This reaction can initiate fascinating rearrangement cascades, which may result in a plethora of reorganized
propelled substantial synthetic efforts.[20–26] Based on biosyn[3, 4]
carbon skeletons.
thetic considerations and biomimetic models it has been
suggested that the rare bicyclo[4.2.0]octadienes are formed
from an isomer of the tetraene 1 through an 8p–6p electro[*] M. M)ller, B. Kusebauch, Prof. Dr. C. Hertweck
cyclization cascade (Scheme 2).[20, 21, 25, 26] However, the origin
Department of Biomolecular Chemistry
of the putative biogenetic precursor, an E,Z,Z,Z-tetraene
Leibniz-Institute for Natural Product Research and
intermediate, has remained unclear to date.
Infection Biology, HKI
We thus reinvestigated the metabolic profile of S. orinoci,
Beutenbergstrasse 11a, 07745 Jena (Germany)
of spectinabilin, under various growth conditions.
Fax: (+ 49) 3641-656-705
Using synthetic 3 a as a reference, we noted that the
diastereoisomeric bicyclo[4.2.0]octadienes are formed only
G. Liang, C. M. Beaudry, Prof. Dr. D. Trauner
Department of Chemistry
when the culture is exposed to daylight (0.1–0.3 mg L 1; see
University of California, Berkeley
the Supporting Information). When fermentation and workup
Berkeley, CA 94720-1460 (USA)
were conducted in the dark, no formation of 3 a/b could be
Fax: (+ 1) 501-643-9480
observed. This result immediately implied that the spectinaE-mail:
bilin isomer is not formed enzymatically but is a result of a
[**] This project was generously funded by the DFG Priority Programme
photoinduced E/Z isomerization. We were able to confirm
SPP1152 “Evolution of Metabolic Diversity” (HE3468/2 for C.H.)
this observation in vitro by exposing pure spectinabilin
and supported by the National Institutes of Health grant R01
isolated from S. orinoci to daylight and artificial light at
GM067636 (to D.T.).
room temperature. In the HPLC profile of aliquots taken
Supporting information for this article is available on the WWW
from the mixture the occurrence of various isomers is clearly
under or from the author.
Angew. Chem. Int. Ed. 2006, 45, 7835 –7838
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 3. De novo synthesis of orinocin. a) 10 mol % [Pd(PPh3)4],
Me3Sn SnMe3, THF, 80 8C; b) 25 mol % [Pd2(dba)3], THF, 50 8C.
dba = trans,trans-dibenzylideneacetone.
Scheme 2. Photoinduced electrocyclic rearrangement cascade from 1
to 4 via 3.
detectable with subsequent formation of 3 a/b (see the
Supporting Information).
These results demonstrate unequivocally that 3 a and 3 b
are indeed derivatives of spectinabilin and result from a
nonenzymatic, photoinduced isomerization–electrocyclization sequence (see Scheme 2). Nonetheless, we were surprised
to find that this fascinating 8p–6p electrocyclic rearrangement is not the end of this intriguing reaction cascade.
In the course of our studies we noted by HPLC-MS
monitoring that minute amounts (0.3 mg mL 1) of another
nitroaryl-substituted metabolite (4) with m/z 357 are formed.
Surprisingly, this novel compound, named orinocin, occurred
mainly in fractions containing 3 a and 3 b. The structure of 4
was fully established by 1D- and 2D-NMR spectroscopy,
including NOESY experiments, and proved to be a truncated
homologue of 1 and 2.[27] In addition to spectroscopic
methods, the structure of 4 was unequivocally confirmed by
a synthetic reference. Our total synthesis of racemic orinocin
started from the vinyl iodide building block 5 (Scheme 3),
which was previously developed for our synthesis of aureothin
and related polyketides.[28] Iodine–tin exchange, followed by
Stille coupling with p-iodonitrobenzene afforded pure ( )-4
(see the Experimental Section).
The structure of orinocin suggests a biogenesis analogous
to that of 1 and 2. In contrast to its higher homologues, which
result from five and seven Claisen condensations, respectively, orinocin biosynthesis would correspond to only four
rounds of elongation. However, the simultaneous occurrence
of 4 and 3 prompted us to consider also an alternative route
that would involve the fragmentation of the bicyclo[4.2.0]octadienes. This hypothesis was proven by investigating
pure natural and synthetic 3 under fermentation conditions
(daylight, 28 8C).
As evident from the HPLC-MS profile (Figure 1), 4 is
directly derived from 3 a. The retro-[2+2]cycloaddition-type
fragmentation of 3 would lead to the concomitant production
of a side product composed of three propionate units,
mesitylene. The formation of this rather unusual “polyketide”
was in fact detected by GC-MS. In the GC profiles of both the
methanolic solution of 3 a and a mesitylene reference peaks
with identical retention time and m/z 121 and the characteristic fragmentation were observed. Consequently, mesitylene
is liberated directly from 3. It should be noted that this
reaction is also photoinduced and was not observed in the
dark. This finding is in full agreement with the Woodward–
Hoffmann rules, according to which thermal retro[2+2] cycloaddition would be symmetry forbidden.[29] The
reaction cascade with alternating photochemical and thermal
pericyclic steps is somewhat reminiscent of vitamin D biosynthesis.[3] However, the present reaction cascade, which
leads to a chain-contracted polyketide backbone as a result of
the excision of internal ketide units from a polyene precursor,
represents the first report of a process which we have termed
“polyene splicing”.
In summary, we have demonstrated that the formation of
3 a and 3 b from 1 by a 8p–6p electrocyclic rearrangement
cascade[30] is initiated by a photoinduced isomerization in vivo
and in vitro. This direct evidence also supports the reassignment of the configuration at C6 of 3 a and 3 b, which has been
made on the basis of biosynthetic considerations[31] and
synthetic studies.[23, 24] Furthermore we have isolated and
identified 4 as a new member of the aureothin/spectinabilin
family from S. orinoci and confirmed its structure by total
synthesis. The most surprising finding is that this novel
metabolite is not the immediate product of a polyketide
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 7835 –7838
Isolation of 1 and 4: Harvested mycelium and resin were
exhaustively extracted with ethanol. The crude product was purified
by open-column chromatography on silica using ethyl acetate/
hexanes (v/v = 1:1) and by preparative HPLC on a Kromasil C18
column (250 F 20 mm) using MeOH/H2O (v/v = 70:30) as the eluent
at a flow rate of 20 mL min 1. Yield: 450 mg of 1. Compound 4 was
obtained from repeated preparative reversed-phase HPLC using C18
Kromasil and acetonitrile/water 40:60 and 60 % MeOH as eluents.
Yield: 3.2 mg.
MS (ESI + ): 736.9 ([2 M+Na+], 380 [M+Na+], 358.2 (100,
M+H+); 1H NMR (500 MHz, CDCl3) see Table 1; 13C NMR
Table 1: 1H NMR data (d [ppm]) of isolated and synthetic 4.
C12, C14
C11, C15
Figure 1. Monitoring the formation of orinocin (4) and mesitylene
from 3. a) HPLC profile; b) top, middle: GC-MS detection of
mesitylene formation from SNF4435C (3 a), bottom: mesitylene reference; m/z 121 corresponds to [M + H]+, which arises at this high
fragmentation energy.
synthase but is derived from 1 by excision of three propionate
units. LC- and GC-MS analyses revealed that 4 originates
from 3 by a [2+2] cycloreversion with concomitant formation
of mesitylene. The entire sequence, which was termed
“polyene splicing”, is unprecedented for natural products
and represents an important example for the generation of
structural diversity through nonenzymatic downstream processes. Finally, we have devised a biosynthetic route to
mesitylene, which has not yet been described as a natural
product. Our results reveal that it is clearly composed of three
propionate units—and thus represents a polyketide.
Experimental Section
Fermentation of S. orinoci: Cultures of S. orinoci (21 F 0.5 L, each
supplemented with 2.5 g adsorber resin XAD4) were fermented for
5 d in analogy to the protocol reported.[15] One flask (reference) was
wrapped in aluminum foil and incubated with strict exclusion of light.
Angew. Chem. Int. Ed. 2006, 45, 7835 –7838
Isolated 4 (J in Hz)
Synthetic 4 (J in Hz)
8.21 (d, 8.8, 2 H)
7.25 (d, 8.8, 2 H)
6.59 (m, 2.2, 1 H)
5.21 (dd, 7.2, 6.5, 1 H)
4.86 (d, 14.3, 1 H)
4.76 (d, 14.3, 1 H)
3.81 (s, 3 H)
3.17 (br dd, 16.3, 7.6, 1 H)
3.05 (br dd, 16.5, 6.1, 1 H)
2.03 (s, 3 H)
1.83 (s, 3 H)
8.23 (d, 8.8, 2 H)
7.26 (d, 8.8, 2 H)
6.61 (m, 2.2, 1 H)
5.23 (dd, 7.3, 6.4, 1 H)
4.88 (d, 14.5, 1 H)
4.78 (d, 14.4, 1 H)
3.83 (s, 3 H)
3.20 (m, 1 H)
3.07 (m, 1 H)
2.04 (s, 3 H)
1.84 (s, 3 H)
(125 MHz, CDCl3): d = 6.9 (CH3, C2a), 9.5 (CH3, C4a), 38.4 (CH2,
C7), 55.2 (CH3, C16), 70.3 (CH2, C8a), 73.5 (CH, C6), 100.2 (C, C2),
120.2 (CH, C9), 120.3 (C, C4), 124.1 (CH, C12, C14), 128.3 (CH, C11,
C15), 143.0 (C, C8), 145.2 (CH, C10), 146.4 (C, C13), 154.4 (C, C5),
162.1 (C, C1), 180.5 ppm (C, C3); IR (ATR, solid): ñ = 2924, 1665,
1594, 1342 cm 1; UV (MeOH): l = 215, 315 nm; HRMS (EI) calcd.
for C19H20NO6 [M+H+]: 358.1291; found: 358.1318.
Monitoring formation of 4 and mesitylene from 3: Compound 4
was detected by HPLC–MS using an Agilent 1100 Series HPLC
System coupled to an LC/MSD Trap and additional UV detection
with a DAD (254 nm). A Zorbax Eclipse XDB C18 column (5 m,
150 F 4.6 mm) was used as the stationary phase. Eluent: MeOH/H2O
0–10 min: 20:80 to 100:0; 10–20 min: 100:0. Mesitylene was detected
by GC–MS using a TRACE GC ULTRA (Thermo) equipped with a
BPX 50 (SGE, 30 m F 0.25 mm ID) capillary column coupled in
parallel to a CTC Analytics FID detector and a POLARIS Q
(Thermo) quadrupole mass detector. GC parameters: injection in the
splitless mode, He carrier gas, constant flow rate 1.0 mL min 1, T =
40 8C (0.0–1.0 min), 40–280 8C (1.0–17.0 min, 15.0 8C min 1), 280 8C
(17.0–18.0 min). MS parameters: CI, methane reactant gas, flow rate
0.7 mL min 1, ion source 200 8C.
Synthesis of 4: Please see the Supporting Information.
Received: July 17, 2006
Published online: October 25, 2006
Keywords: electrocyclic reactions · natural products · polyenes ·
polyketides · streptomyces
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formation, splicing, immunosuppressive, snf4435cd, origin, уpolyene, orinocin, photochemical
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