Organic Letters
Letter
2a (R = CH3) that acts as a monomeric precursor for
dimerization to (−)-flavoskyrin. Hence the spontaneous
dimerization between two monomeric 2a in a stereocontrolled
exo-anti fashion following a [4 + 2] hetero-Diels−Alder
reaction forms (−)-flavoskyrin (1a).11 This simple chemo-
enzymatic approach not only gave access to the putative
biosynthetic intermediates (S)-2a and (R)-11a for the first
time but also implicated the role of (R)-10a as an intermediate
in the (bio)synthesis of (−)-flavoskyrin (1a) and (−)-rugulo-
sin (3a) (Scheme 1).11 Likewise, lunatin (9b, R = OCH3) and
citreorosein (9c, R = CH2OH) were used to obtain
(−)-lunaskyrin (1b)11 and (−)-flavoskyrin C (1c),12 respec-
tively (Scheme 1c). The spontaneous nature of stereo-
controlled dimerization in the above process motivated us to
investigate the catalyst-free oxidation of (R)-10a for the
synthesis of (−)-flavoskyrin (1a) under aqueous ambient
conditions. This might provide vital clues on how (−)-fla-
voskyrin (1a) is biosynthesized and why no flavoskyrin-type
molecule other than 1a has been isolated from natural sources.
Considering the involvement of the oxidation of hydro-
anthraquinone (R)-10a to an anthraquinone (R)-11a, we
asked if this could be achieved through the process of
autoxidation. The autoxidation of hydroquinone to quinone
plays a significant role in biological systems during redox
cycling13 as well as in biosynthesis.14 For example, during
melanin biosynthesis, the key metabolites such as flaviolin and
2-hydroxyjuglone have been proposed as being formed
through the oxidation of their precursors, 1,3,6,8-tetrahydrox-
ynaphthalene and 1,6,8-trihydroxynaphthalene, respectively,
without the involvement of an enzyme.14 In another example,
the process of the autoxidation of 2-alkyl-9,10-anthrahydro-
quinone to its anthraquinone in the presence of molecular
oxygen is used during the industrial production of hydrogen
peroxide.15 Likewise, we propose that similar autoxidation
might convert the hydroanthraquinone (R)-10a to the
anthraquinone (R)-11a using molecular oxygen, which on
tautomerization to (S)-2a under appropriate conditions will
spontaneously dimerize to form (−)-flavoskyrin (1a).
To prove our hypothesis, we used (R)-10a as a model
substrate, which was synthesized through the chemoenzymatic
reduction of emodin (9a) using an anthrol reductase from
Talaromyces islandicus (ARti) and NADPH in the presence of
Na2S2O4.16 At first, (R)-10a dissolved in acetonitrile (30% v/
v) was incubated in potassium phosphate buffer (50 mM KPi,
pH 7.0) under an oxygen atmosphere for 2 h. The reaction
mixture was analyzed using reverse-phase HPLC, which
revealed the formation of the expected deoxyanthraquinone,
chrysophanol (12a) and the oxidized product, emodin (9a), in
a 53:47 ratio with 77% conversion (Table 1, entry 1).
However, no (−)-flavoskyrin (1a) was formed, which could be
due to the nonmixing of molecular oxygen. Therefore, we
performed the above reaction by bubbling molecular oxygen
into the reaction system. This resulted in the formation of
(−)-flavoskyrin (1a) along with 12a and 9a in a 13:43:44 ratio
with 70% conversion (Table 1, entry 2). Also, considering the
role of the pH in tautomerism, which could affect the
dimerization to (−)-flavoskyrin (1a), we incubated (R)-10a in
buffer of pH 5.0, 5.5, 6.0, 6.5, and 8.0 (Table 1, entries 3−7)
and bubbled molecular oxygen into the reaction mixture for 2
h. We observed 45% conversion of (R)-10a to products at pH
5.0 (Table 1, entry 3). However, the reactions performed at
pH 5.5, 6.0, and 6.5 gave nearly 98% conversion with
(−)-flavoskyrin (1a) as a major product (Table 1, entries 4−
Table 1. Optimization of (−)-Flavoskyrin (1a) Synthesis
a
through Autoxidation of (R)-10a
entry
pH conc.(mg/mL) time (h) conv. (%) ratio (1a/12a/9a)
b
1
7
7
5
5.5
6
6.5
8
6
6
6
1
1
1
1
1
1
1
1
0.5
2
4
2
2
2
2
2
2
2
2
2
2
2
2
2
4
10
77
70
45
97
98
98
71
72
97
97
78
99
99
−:53:47
13:43:44
49:30:21
70:18:12
71:20:9
70:20:10
−:47:53
62:24:14
68:25:7
72:17:11
69:14:17
79:14:7
79:12:9
2
3
4
5
6
7
c
8
9
10
11
12
13
6
6
6
d
de
,
a
All conversions and ratios are based on reverse-phase HPLC using
b
c
questin as an internal standard. Under an oxygen atmosphere. tris-
HCl buffer (50 mM, pH 6). At 10 °C. 1 mmol scale reaction.
d
e
6). However, the further increase in pH to 8.0 did not yield
(−)-flavoskyrin (1a) in the reaction (Table 1, entry 7). This
indicates that an acidic pH facilitates the formation of tautomer
(S)-2a of (R)-3,4-dihydroemodin (11a) that is formed by the
oxidation of (R)-10a in the presence of molecular oxygen. This
is followed by the spontaneous cycloaddition between the two
monomeric (S)-2a to form dimeric (−)-flavoskyrin (1a).
Under a basic pH, (R)-3,4-dihydroemodin (11a) mainly
dehydrates to form chrysophanol (12a). We also used Tris-
HCl buffer (50 mM, pH 6.0) instead of KPi buffer, but it
resulted in a reduced conversion to products (Table 1, entry
8). In addition, we tested 2-propanol, 1,4-dioxane, acetone,
and DMSO as cosolvents in buffer (50 mM KPi, pH 6.0). The
results show reduced conversion to 1a in the case of 2-
propanol and 1,4-dioxane, whereas the use of acetone and
DMSO gave comparable conversion to that of acetonitrile (see
we look at the effect of concentration on the formation of 1a.
The reactions with concentration of 0.5 and 2 mg/mL of (R)-
10a gave 68 and 72% of 1a, respectively (overall conversion
97%) (Table 1, entries 9 and 10). However, the concentration
of 4 mg/mL gave 69% of 1a but reduced the overall conversion
to 78%, which could be due to the low solubility of (R)-10a
(Table 1, entry 11).
To further reduce the formation of side products, we
incubated (R)-10a under the same conditions as entry 10
(Table 1) but at a lower temperature (10 °C). This resulted in
the formation of (−)-flavoskyrin (1a), chrysophanol (12a),
and emodin (9a) in a 79:14:7 ratio with 99% overall
conversion (Table 1, entry 12). However, further lowering of
temperature reduces the overall conversion (see Table S1,
entry 6). Finally, a large-scale reaction was performed; taking 1
mmol of (R)-10a under the same conditions yielded 1a, 12a,
and 9a in a 79:12:9 ratio with 99% conversion after 10 h
(Table 1, entry 13). The purification using oxalic-acid-
impregnated silica gel resulted in the isolation of (−)-fla-
voskyrin (1a), chrysophanol (12a), and emodin (9a) in 72, 10,
and 6% yield, respectively. This is similar to the previously
B
Org. Lett. XXXX, XXX, XXX−XXX