X. Yu et al. / Tetrahedron Letters 53 (2012) 6861–6864
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Streptomyces nitrosporeus CQT14-24, were then incubated with nine
prenyltransferases from the DMATS superfamily in the presence of
dimethylallyl diphosphate (DMAPP). The tested enzymes included
five cyclic dipeptide prenyltransferases AnaPT, BrePT, CdpC3PT,
CdpNPT, and FtmPT1 with prenylation positions at C-2 or C-3 of the
indole ring,23–27 three dimethylallyltryptophan synthases FgaPT2,
5-DMATS, and 7-DMATS with prenylation positions at C-4, C-5, and
C-7,14,15,28 respectively. One tyrosine O-prenyltransferase SirD29
was also tested. HPLC analysis showed that 5-DMATS from Aspergillus
clavatus and FgaPT2 from Aspergillus fumigatus displayed more sub-
strate flexibilities toward the tested substances than other enzymes
(data not shown) and were studied in detail.
contains two doublets and two triplets with coupling constants in
the range of 7–9 Hz. In comparison, one set of signals in the 1H
NMR spectra of their enzyme products 2b, 2c, 3b, and 4b represent
merely three protons. Two of these protons couple with each other
with coupling constants of 7–9 Hz and the third one appears as a
singlet or doublet with a small coupling constant of less than
2 Hz. These changes indicated that the prenylation had taken place
at position C-2, C-3, C-9, or C-10. Similar phenomenon was also ob-
served in both sets of aromatic signals in the 1H NMR spectrum of
2d, suggesting that one prenylation took place at C-2 or C-3 and
the other at C-9 or C-10. Comparing the signals in the spectra of
2b, 3b, and 4b with those of the respective substrate (Supplemen-
tary Figs. S6–S8) revealed that the H-4 was changed from a doublet
with coupling constants of 7-9 Hz to another doublet with cou-
pling constants small than 2 Hz, which was found in the low field
in 1H NMR spectra at approximately dH 9 ppm due to a character-
istic deshielding effect from the lactam carbonyl,31 confirming the
prenylation at C-3 of 2b, 3b, and 4b. In the 1H NMR spectrum of 2c,
the signal of H-8 rather than that of H-4 was changed from a dou-
blet to a singlet (Figure S6),31 confirming that the prenylation had
taken place at C-9. The structure of 2d was assigned to a product
with two prenyl moieties at C-3 and C-9, since both H-4 and H-8
were altered from doublets to singlets (Supplementary Fig. S6).
This proved that both DMATS enzymes catalyzed the regiospecific
C-prenyltion on the indolocarbazole system, that is, the para-
position to the indole N-atom (C3, C9, or both) and function there-
fore as catalysts for Friedel–Crafts alkylations. A Friedel–Crafts
alkylation catalyzed by strong Lewis acids would involve an allyl
cation. This is also the case for the enzyme-catalyzed Friedel–Crafts
alkylation described in this study. The formation of a dimethylallyl
cation in an enzyme-catalyzed prenyl transfer reaction would be
facilitated by interactions of several basic amino acid residues of
the enzyme with pyrophosphate group of DMAPP.32,33
HPLC analysis of incubation mixtures with a 20
lg of 5-DMATS or
FgaPT2 in 100 l assay indicated that 1a was poor substrate for both
l
enzymes. 5-DMATS accepted 1a only with a total conversion yield
of 0.3%, while no product peak was detected in its incubation mixture
with FgaPT2. Other three indolocarbazoles (2a–4a) were clearly ac-
cepted by both 5-DMATS and FgaPT2 ( Fig. 2). Product formation
was only detected in the incubation mixtures with active, but not in
those with heat-inactivated proteins (by boiling for 20 min, data not
shown). This demonstrated the importance of the oxidation grade at
position C-7. Hydroxylation at this position seems better for accep-
tance by 5-DMATS than with a keto group. Detailed inspection of
the HPLC chromatograms with 2a–4a revealed that FgaPT2 showed
generally a lower activity than 5-DMATS, proving again that different
DMATS enzymes display different preference toward aromatic sub-
strates.17 Furthermore, more than one product peaks were detected
in the reaction mixtures and the main product of both enzymes for
a given substrate was proven to be identical (see below). For example,
2awasconvertedby5-DMATSinto2b, 2c, and2dwithyieldsof11, 11,
and 1.7 %, respectively. 2a was converted by FgaPT2 mainly into 2b
with a yield of 6.7 % (Fig. 2A). 3a/3a* were converted into 3b by 5-
DMATS and FgaPT2 with conversion yields of 42 and 25%, respec-
tively. The non-bridged intermediates of indolocarbazoles, that is,
arcyriarubin A and N-methylarcyriarubin A, were not accepted by 5-
DMATS and FgaPT2, indicating the importance of the presence of
the indolocarbazole skeleton. Glycosides of indolocarbazoles, that is,
staurosporine or K252d (Fig. 1), were also not prenylation substrates
for the enzymes of the DMATS superfamily (data not shown).
For structure elucidation, three enzyme products 2b, 2c, and 2d
were isolated from the incubation mixture of 5-DMATS with 2a
and one each, that is, 3b and 4b, from those with 3a and 4a, respec-
tively. 2b and 3b were also isolated from the reaction mixtures of
FgaPT2 with 2a and 3a. All isolated enzyme products were sub-
jected to MS and NMR analyses.
To elucidate the behavior of 5-DMATS and FgaPT2 toward indo-
locarbazoles, kinetic parameters were determined for the best ac-
cepted substrate 3a with both enzymes by Hanes–Woolf and
Eadia–Hofstee plots. Michaelis–Menten constants (KM) were calcu-
lated to be at 87 and 136 lM for 5-DMATS and FgaPT2, respec-
tively, while turnover numbers (kcat) were found at 6.8 and
7.3 minꢁ1. The catalytic efficiency (kcat/KM) of 5-DMATS toward
3a was 1302 sꢁ1 Mꢁ1, that is, 5.0 % of that of its best substrate
L-
tryptophan.14 Similarly, A kcat/KM value of 891 sꢁ1 Mꢁ1 was calcu-
lated for FgaPT2 toward 3a, which is 3.0 % of that of
-tryptophan.15
L
These data provided evidence that dimethylallyltryptophan syn-
thases could also be used for the production of C-prenylated
indolocarbazoles.
In conclusion, the present work demonstrated the acceptance of
indolocarbazoles by fungal dimethylallyltryptophan synthases of
the DMATS superfamily, which expands the potential usage of
these enzymes in the structural modifications. To the best of our
knowledge, this is the first report on the (chemoenzymatic) syn-
thesis of prenylated indolocarbazoles.
In the HPLC chromatograms of the incubation mixtures with 3a,
a minor product peak eluted after 3b was also observed (Fig. 2B),
which could be a prenylation product of 3a* or formed by tautom-
erism of 3b. Due to the low quantity, this minor product could not
be isolated and identified. For the same reason, no enzyme product
of 4a with FgaPT2 and 1a with 5-DMATS was isolated.
HR-EI-MS confirmed the monoprenylation in 2b, 2c, 3b, and 4b
and diprenylation in 2d, by detection of the molecular masses that
are 68 and 136 Da larger than those of the respective substrate
(Supplementary Table S1). The main enzyme products of FgaPT2
with 2a and 3a had identical 1H NMR spectra as those of 2b and
3b from the 5-DMATS assays, respectively, proving the same
structure of the enzyme products. The 1H NMR signals at dH
3.48–3.52 (d, 2H-10 or 2H-100), 5.40–5.44 (t sep, H-20 or H-200),
1.77–1.79 (d, 3H-40 or 3H-400), and 1.73–1.75 ppm (d, 3H-50 or
3H-500) in the spectra of 2b, 2c, 2d, 3b, and 4b (Supplementary Figs.
S1–S5, Table S2) revealed clearly the attachment of one regular
dimethylallyl moiety to a C-atom.17,30
Acknowledgments
This work was supported within the LOEWE program of the
State of Hessen (SynMikro to S.-M. Li). Xia Yu is a recipient of a fel-
lowship from China Scholarship Council. We thank Dr. Ortmann
and Laufenberg for taking NMR and mass spectra.
Supplementary data
In the 1H NMR spectra of the substrates 2a–4a (Supplementary
Figs. S6–S8), the aromatic signals appeared as two identical (4a) or
different sets of four vicinal coupling protons (2a and 3a). Each set
Supplementary data associated with this article can be found, in