Please do not adjust margins
ChemComm
Page 2 of 5
DOI: 10.1039/C7CC01929G
COMMUNICATION
Journal Name
Diels-Alder cycloaddition reaction is a powerful method for the
construction of cyclohexene ring, which has been wildly used
in organic synthesis and natural products total synthesis.10 In
another hand, the studies of natural enzymes as Diels-
Me
Me
Me
Me
N
O
Me
OMe
EtO
EtO
StBu
Me
O
O
P
O
Me
OH
6 steps
CHO
O
O
Me
(+)-citronellal (6)
CO2Me
10
Alderases has been
a long-term research goal in the
7
Ref. 10
biosynthesis field,11 which is a challenging project due to the
extremely hard identification, isolation, and characterization
O
P
MeONa
MeOH
100%
EtO
EtO
HO
NHMe
Me
Me
N
O
process. During the biosynthesis of pyrroindomycins (4 and 5;
8
9
OH
Me
Me
Figure 1), two cyclases PyrE3 and PyrI4 were found to catalyze
the tandem process for the formation of two cyclohexene
rings in the trans-decalin skeleton and the tetramate spiro-
conjugate of pyrroindomycins.12 As the first two
spirotetramates isolated from Streptomyces rugosporus,
Me
Me
N
O
O
11
O
OH
E
HO
H
H
O
Me
Me
Me
O
Me
N
Me
Me
B
A
OH
conditions
Me
O
pyrroindomycin A (
4
) and B (
5) contain the most complicated
H
O
12
equisetin (1)
structural feature in this family natural products.13 This
enzymatic [4+2] cyclization cascade proceed in a regio- and
stereoselective manner to establish the enantiomerically pure
pentacyclic core, which provides new solution for the
preparation of trans-decalin skeleton and spirotetramates
containing natural products and their derivatives.14 Recently,
Osada and co-workers reported a new gene fsa2 involved in
endo-selectivity
Me
O
N
OH
Me
Me
O
HO
H
N
OH
O
Me
O
3
Me
S
H
O
6
R
Me
Me
H
Me
13
(3S, 6R)-diastereomer of 1 (1')
exo-selectivity
the biosynthetic pathway of equisetin (1) and fusarisetin A (2)
in Fusarium sp. FN080326.,15 which showed no homology to
entry
acid
BF3•Et2O
AcOH
---
solvent
CH2Cl2
AcOH
temp.
-78 o
time
2 h[a]
yield
dr (1/1')
1:1
genes encoding proteins of known function. Notably, Osada
1
2
C
28%
55%
observed the formation of (3S, 6R)-diastereomer of
the ∆fsa2 mutant experiment. This might be caused by a
spontaneous exo-selective [4+2] cycloaddition of liner
1 (1') in
25 o
C
34 h[b]
1.3:1
3
PhMe
80 oC
2 h[c]
56%
2:1
a
diastereoselectivity. Directly using acetic acid as solvent, the
IMDA reaction of 11 gave the and 1’ in 55% yield, and the
polyene precursor, which also indicated fsa2 was an endo-
1
selective Diels-Alderase in the decalin formation of equisetin
diastereoselectivity was not significantly improved. Heating a
solution of 11 in toluene in 50 oC for 2 hours generated
(1). Based on this hypothesis, we were intrigued by the
possibility of the combination of chemical synthesis and
biosynthesis to confirm the function of the gene fsa2 in the
biosynthesis of equisetin. We also wanted to identify the
stereo-control capability by comparing the acid or
thermodynamic Diels-Alder reaction with enzyme-mediated
Diels-Alder reaction of the same precursor: polyenoyltretamic
equisetin (
1) and (3S, 6R)-diastereomer of
1
(
1') in 56%
combined yield
Scheme 1. Chemical Synthesis of Equisetin (
1). (a) Reactions
were performed with BF3
concentration of 0.054
·
Et2O (4.0 equiv.) at a substrate
M
in DCM. (b) Reactions were
performed at a substrate concentration of 0.054 M in AcOH.
(c) Reactions were performed at a substrate concentration of
0.01 M in PhMe and was slowly heated to 110 oC.
acid 11
.
The chemical synthesis started from the preparation of the
polyenoyltretamic acid 11, the precursor of the IMDA. We
have previously reported an efficient 6-step approach for the
scalable synthesizing polyenoylamino ester 10 from the easily
with a comparable diastereoselectivity (2:1). We reasoned that
the freely rotatable structure of polyene structure and the
tautomeric keto/enol isomers of the tetramic acid unit in
polyenoyltretamic acid 11 may result the poor
diastereoselectivity and efficiency of the IMDA reaction.
available building blocks, including (+)-citronellal
(6),
phosphonate 9 and (S)-serine derivative
7,
8
(Scheme 1).8b The
intramolecular Diels–Alder reaction of 10 was also studied, and
we found the Lewis-acid (2.0 equiv. BF3·Et2O) promoted IMDA
reaction delivered the desired trans-decalin in good
diastereoselectivity (d.r.=17:1) and moderate yield (63%).8b
Treatment of 10 with sodium methoxide in methanol led to
the quantitative desired polyenoyltretamic acid 11, which
could be used directly in the next step without further
purification. Then, the IMDA of 11 was explored by
investigating three classical pathways. Lewis-acid BF3·Et2O was
first used in the IMDA reaction of 11, and we observed that
Then we explored Fsa2-mediated [4+2] cycloaddition (Figure
2). The gene, fsa2, was optimized and synthesized based on
the sequence previously reported,15 and then cloned into a
pET-28a vector. The protein, Fsa2, with an N-terminal His6-tag
was heterologously expressed and purified from Escherichia
coli (Figure S1 in Supporting Information). The purified enzyme
Fsa2 was then used to treat with the polyenoyltretamic acid
11, and 11 was experienced an endo-selectivity and rapidly
equisetin (1) and (3S, 6R)-diastereomer of 1 (1') was produced
converted into equisetin (1) (Figure 2). In contrast, under the
as a 1:1 ratio of mixture in 28% yield. This IMDA proceeded
through the intermediates 12 (endo-selectivity) or 13 (exo-
selectivity) and furnished the trans-decalin unit in a poor
same condition no conversion was observed in the negative
control reaction which is in the absence of Fsa2. We also
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins
Li, Xiaojun
