Expansion of enzymatic Friedel-Crafts alkylation on indoles: Acceptance of unnatural β-unsaturated allyl diphospates by dimethylallyl-tryptophan synthases

Article abstract of DOI:10.1021/ol302207r

Prenyltransferases of the dimethylallyl-tryptophan synthase (DMATS) superfamily catalyze Friedel-Crafts alkylation with high flexibility for aromatic substrates, but the high specificity for dimethylallyl diphosphate (DMAPP) prohibits their application as biocatalysts. We demonstrate here that at least one methyl group in DMAPP can be deleted or shifted to the d-position. For acceptance by some DMATS enzymes, however, a double bond must be situated at the β-position. Furthermore, the alkylation position of an analogue can differ from that of DMAPP.

Full text of DOI:10.1021/ol302207r

ORGANIC
LETTERS
2012
Vol. 14, No. 18
4882–4885
Expansion of Enzymatic FriedelꢀCrafts
Alkylation on Indoles: Acceptance
of Unnatural β‑Unsaturated Allyl
Diphospates by Dimethylallyl-tryptophan
Synthases
Mike Liebhold,† Xiulan Xie,§ and Shu-Ming Li*,†
€
Institut fu€r Pharmazeutische Biologie und Biotechnologie, Philipps-Universitat
Marburg, Deutschhausstrasse 17a, 35037 Marburg, Germany, and Fachbereich Chemie,
€
Philipps-Universitat Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany
shuming.li@staff.uni-marburg.de
Received August 8, 2012
ABSTRACT
Prenyltransferases of the dimethylallyl-tryptophan synthase (DMATS) superfamily catalyze FriedelꢀCrafts alkylation with high flexibility for
aromatic substrates, but the high specificity for dimethylallyl diphosphate (DMAPP) prohibits their application as biocatalysts. We demonstrate
here that at least one methyl group in DMAPP can be deleted or shifted to the δ-position. For acceptance by some DMATS enzymes, however,
a double bond must be situated at the β-position. Furthermore, the alkylation position of an analogue can differ from that of DMAPP.
Prenyltransferases are a large family of enzymes found
in many living organisms such as bacteria, fungi, and
plants, which are responsible for transfer of a prenyl moiety
(nxC5) to another prenyl residue, a protein, or an aromatic
substrate.1 The reactions catalyzed by the last enzyme
group, known as “aromatic prenyltransferases”, are often
FriedelꢀCrafts alkylations on different aromatic systems
such as indoles, naphthalenes, flavonoids, or phenylpropa-
noids, resulting in the formation of biologically active
metabolites.1,2 One special group of the aromatic prenyl-
transferases shares a significant sequence similarity with
dimethylallyl-tryptophan synthase (DMATS) from Claviceps,3
and they are therefore classified asenzymes of the DMATS
superfamily.2a The members of this family are involved in
the biosynthesis of diverse important natural products
including ergot alkaloids and usually catalyze the transfer
of a dimethylallyl moiety from dimethylallyl diphosphate
(DMAPP) to different positions of the indole ring of
L-tryptophan and derivatives thereof.1c They showed
high flexibility toward their aromatic substrates and ac-
cepted even hydroxynaphthalenes, flavonoids, xanthones,
and tetracyclic napthacenediones as prenylation sub-
strates.2b,c,4 In contrast, these enzymes are usually highly
specific toward DMAPP and did not accept other prenyl
diphosphates as prenyl donors.2a One exception was found
† Institut f€ur Pharmazeutische Biologie und Biotechnologie.
§ Fachbereich Chemie.
(1) (a) Heide, L. Curr. Opin. Chem. Biol. 2009, 13, 171–179.
(b) Yazaki, K.; Tsurumaru, Y. Phytochemistry 2009, 70, 1739–1745.
(c) Li, S.-M. Nat. Prod. Rep. 2010, 27, 57–78.
(2) (a) Yu, X.; Liu, Y.; Xie, X.; Zheng, X.-D.; Li, S.-M. J. Biol. Chem.
2012, 287, 1371–1380. (b) Yu, X.; Xie, X.; Li, S.-M. Appl. Microbiol.
Biotechnol. 2011, 92, 737–748. (c) Yu, X.; Li, S.-M. ChemBioChem 2011,
12, 2280–2283. (d) Ozaki, T.; Mishima, S.; Nishiyama, M.; Kuzuyama,
T. J. Antibiot. 2009, 62, 385–92.
(3) Tsai, H. F.; Wang, H.; Gebler, J. C.; Poulter, C. D.; Schardl, C. L.
Biochem. Biophys. Res. Commun. 1995, 216, 119–125.
(4) (a) Sanchez, J. F.; Entwistle, R.; Hung, J. H.; Yaegashi, J.; Jain,
S.; Chiang, Y. M.; Wang, C. C.; Oakley, B. R. J. Am. Chem. Soc. 2011,
133, 4010–7. (b) Chooi, Y. H.; Wang, P.; Fang, J.; Li, Y.; Wu, K.; Wang,
P.; Tang, Y. J. Am. Chem. Soc. 2012, 134, 9428–37.
r
10.1021/ol302207r
Published on Web 09/07/2012
2012 American Chemical Society

for the newly characterized DMATS-type prenyltransfer-
ase VrtC, which only utilizes GPP.4b This feature prohibits
their usage as catalysts for FriedelꢀCrafts alkylation. We
expand in this study their potential application for alkyla-
tion with unnatural alkyl diphosphates.
Figure 1. DMAPP and its analogues.
For this purpose, four DMAPP analogues (Figure 1)
were successfully synthesized according to the method
described for GPP by Woodside et al.,5 which were con-
firmed by 1H and 31P NMR analyses. The four DMAPP
analogues were used as alkyl donors for enzyme assays
withFgaPT2and 7-DMATS fromAspergillus fumigatus as
well as with 5-DMATS from Aspergillus clavatus, which
catalyzed prenylation of L-tryptophan at C-4, C-7, and
C-5, respectively.2a,6 Enzyme assays with DMAPP were
used as positive controls (Figure 2). HPLC analysis of the
incubation mixtures revealed an almost complete conver-
sion of L-tryptophan to its prenylated products in the
three enzyme assays with DMAPP after incubation with
5 μg of protein at 37 °C for 16 h (Figure 2). Shifting one
methyl group of DMAPP from C-3 to C-4, as in the case of
2-pentenyl diphosphate (2-pen-PP), strongly reduced the
activity of 7-DMATS. No product formation was detected in
the incubation mixture under assay conditions (Figure 2C).
In contrast, FgaPT2 and 5-DMATS still accepted 2-pen-PP
well as a substrate (Figure 2A and 2B) and conversion yields
of 37% and 91% from the respective assays with DMAPP
were achieved under these conditions.
For structure elucidation, the enzyme products 3a and
3b were isolated from the incubation mixtures of FgaPT2
and 5-DMATS, respectively, and subjected to NMR and
MS analyses. HR-MS data clearly showed the alkylation
of both compounds by detection of [M]þ ions at m/z
272.1518 (3a) and 272.1509 (3b), which are 68 Da larger
than that of tryptophan.
In the 1H NMR spectrum of 3a, signals of a 2-pentenyl
moiety proved the alkylation at a C-atom in a regular
pattern.2a
Figure 2. HPLC analysis of L-tryptophan after incubation with
DMAPP or its analogues at 37 °C and 16 h.
The four aromatic protons of the indole moiety ap-
peared, according to their chemical shifts and coupling
pattern, in the same order as those of C5-prenylated indole
derivatives obtained from enzyme assays of 5-DMATS
with DMAPP2a and therefore can be assigned to H-4, H-7,
H-2, and H-6.
This indicated a C5-alkylation of L-tryptophan by FgaPT2
in the presence of 2-pen-PP and is somewhat unexpected
for a C4-prenyltransferase with its natural substrate DMAPP
(Scheme 1).6b Therefore, we obtained various 2D NMR
spectra for 3a to confirm the structure. In the HMBC
spectrum, connectivities were clearly observed for H-4 of
(5) Woodside, A. B.; Huang, Z.; Poulter, C. D. Org. Synth. 1988, 66,
211–215.
(6) (a) Kremer, A.; Westrich, L.; Li, S.-M. Microbiology 2007, 153,
€
3409–3416. (b) Unsold, I. A.; Li, S.-M. Microbiology 2005, 151, 1499–
1505.
Org. Lett., Vol. 14, No. 18, 2012
4883

Scheme 1. Proposed Reaction Mechanisms and Enzyme Products of FgaPT2 and 5-DMATS with DMAPP and Analogues
the indole ring with C-3 and C-8 as well as with C-10 of the
alkyl moiety, proving unequivocally the C5-akylation of
L-tryptophan in 3a.
The enzyme activities of FgaPT2 and 5-DMATS were
reduced, in comparison to those of DMAPP and 2-pen-
PP, but still clearly detectable. Two and one product peaks
were detected in the HPLC chromatograms of the incuba-
tion mixtures of L-tryptophan and MAPP with FgaPT2
and 5-DMATS, respectively. A conversion yield of 29% was
calculated for FgaPT2, and 44%, for 5-DMATS, respec-
tively. For structure elucidation, all three peaks were iso-
lated and subjected to NMR and MS analyses. HR-MS
data confirmed the presence of a butenyl moiety in their
structures by detection of [MþH]þ at m/z 259.1471 (4a) and
259.1434 (4b) or [M]þ ion at 258.1388 (4bþ4c), which are
54 Da larger than that of tryptophan.
In the 1H NMR spectrum of 3b, similar signals to those
of 3awere alsoobservedfor a 2-pentenyl moiety. However,
the 1H NMR spectrum of 3b differed clearly from that of
3a, especially in the aromatic region, although the same
number and similar coupling patterns were observed in
both spectra. The signals ofthe fouraromatic protons in3b
corresponded very well to those of C6-prenylated indole
derivatives obtained from enzyme assays of IptA with
DMAPP.7 This means that the alkylation of L-tryptophan
by 5-DMATS was shifted from C-5 in the presence of
DMAPP to C-6 in the presence of 2-pen-PP (Scheme 1).
The alkylation shifts from C-4 to C-5 for FgaPT2 and
from C-5 to C-6 for 5-DMATS could be explained by the
longer chain length and thus deeper extension of 2-pen-PP
into the reaction chamber in comparison to that of
DMAPP, so that C-10 of the alkyl residue was closer to
C-5 thanC-4 in FgaPT2and toC-6 thanC-5 in5-DMATS.
To prove the influence of chain length of the alkyl diphos-
phate on the enzyme activity and alkylation position,
the cis-methyl group of DMAPP was deleted to obtain
methylallyl diphosphate (MAPP) (Figure 1), which was
then used as an alkyl donor for enzyme assays of
L-tryptophan with FgaPT2, 5-DMATS, and 7-DMATS. Again,
7-DMATS showed no detectable conversion (Figure 2C).
1
The H NMR spectra of 4a and 4b are similar to each
otherinthe region of olefinic and aliphatic protons. Signals
for a 2-trans-butenyl moiety were detected for both 4a and
4b. The spectra however differed clearly from each other
by the chemical shifts and coupling pattern of the four
aromatic protons in the spectrum of 4a and 4b.
The features of the aromatic protons of 4a indicated an
alkylation at C-4 or C-7. The HMBC spectrum clearly
showed connectivities of H-10 of the butenyl residue8
to C-20 (δC 131.2) and C-30 (δC 125.0) as well as to C4
(δC 132.7) and C5 (δC 119.2) of the indole moiety. The
alkylation position at C-4 of L-tryptophan was confirmed
(8) The two protons at H-10 of the butenyl residue differ from each
other, probably due to the steric interaction with the side chain of
tryptophan. δH 3.75 (1H, dd, 15.4, 6.3, H-10) and δH 3.63 (1H, dd, 15.4,
6.3, H-10).
(7) Takahashi, S.; Takagi, H.; Toyoda, A.; Uramoto, M.; Nogawa,
T.; Ueki, M.; Sakaki, Y.; Osada, H. J. Bacteriol. 2010, 192, 2839–2851.
4884
Org. Lett., Vol. 14, No. 18, 2012

by detection of additional connectivities in the indole
moiety.9
apparently followed MichaelisꢀMenten kinetics. FgaPT2
accepted its natural substrate DMAPP with a KM value of
4 μM, much better than MAPP and 2-pen-PP with KM
values at 40 and 56 μM, respectively. In contrast, 5-DMATS
has no significant preference and even a higher affinity to
MAPP than to DMAPP. The turnover numbers of FgaPT2
and 5-DMATS toward unnatural alkyl diphosphates were
found to be less than 3% of those with DMAPP but are
still within a realized range for use in chemical reactions. For
both enzymes, 2-pen-PP was accepted with a higher reaction
velocity than MAPP.
The coupling pattern of the four aromatic protons in 4b
indicated a substitution at C-5 or C-6. The order of these
coupling protons was found to be identical to those in 3a
and other C5-prenylated derivatives.2a The alkylation at
C-5 of L-tryptophan in 4b (Scheme 1) was also confirmed
by the observation of connectivities in its HMBC
spectrum.
1
Inspection of the H NMR spectrum of the product
peak isolated from the incubation mixture of L-tryptophan
and MAPP with 5-DMATS revealed the presence of two
compounds with a ratio of 1:1. Unfortunately, it was
difficult to separate them from each other by HPLC under
different conditions. Fortunately, it was yet possible to
elucidate their structures based on the obtained spectra,
because one of them can be easily identified as 4b by
Table 1. Kinetic Parameters of FgaPT2 and 5-DMATS with
DMAPP and Analogues as Alkyl Donors
enzyme
FgaPT2
donor
KM (μM) kcat (sꢀ1
)
kcat/KM (sꢀ1 Mꢀ1
)
3
DMAPPa
MAPP
4
40
0.37
92 500
232
1
comparison of their H NMR spectra. The second one,
0.009
0.011
1.3
4c, showed similar signals and identical order in the
aromatic ranges to 3b and other C6-prenylated indole
derivatives7 and was therefore identified as a C6-alkylated
derivative. It seems that the smaller methylallyl group was
placed in the reaction site with a similar distance from
its C-10 to C-4 and C-5 in FgaPT2 or to C-5 and C6 in
5-DMATS, so that attacking from both positions was
possible.
2-pen-PP
56
198
5-DMATS DMAPPb
MAPP
76
17 105
136
40
0.005
0.023
2-pen-PP
131
174
a The data were adopted from ref 6b. b Thedatawereadoptedfromref2a.
In conclusion, the structure of the natural alkylation
reagent DMAPP can be modified and used as an alkyl
donor for some members of the DMATS superfamily, which
were successfully used for the prenylation of diverse aro-
matic substrates including indoles, hydroxynaphthalenes,
flavonoids, and tetracyclic napthacenediones.1c,2b,2c,4b One
methyl group in DMAPP can be deleted or shifted, but
the double bond at the β-position seems to be essential for
the stability of the formed carbocation. In comparison to
DMAPP, the alkylation of the smaller MAPP is in part
retained and in part shifted to one position, i.e. from C-4 to
C-5 by FgaPT2 and from C-5 to C-6 by 5-DMATS. With
2-pen-PP as an alkyl donor, the alkylation position was
completely shifted for one position. The acceptance of the
modified prenyl donors by prenyltransferases expanded
significantly the potential use of these enzymes as catalysts
in the chemoenzymatic synthesis. It would be interesting
to test the acceptance of the DMAPP analogues by other
prenyltransferases.
To test the importance of the double bond at the
β-position to diphosphate, we prepared 3-methylbutanyl
diphosphate 5 and 4-methyl-3-pentenyl diphosphate 6
(Figure 1) and used them as alkyl donors for FgaPT2,
5-DMATS, and 7-DMATS. As shown in Figure 2, no
product formation was detected for any of the enzyme
assays. This proved that the double bond at the β-position
is essential for the alkylation. Based on the protein struc-
tures of several prenyltransferases, it was proposed that a
carbocation will be first created with the help of several
basic amino acids.10 This carbocation must be then stabi-
lized by several amino acid residues, e.g. tyrosine, as well as
by the aromatic ring system of the substrate itself to protect
its reaction with undesired nucleophiles.10a Our data in this
study showed that stabilization of the carbocation by itself,
via a β-unsaturated CꢀC bond, is also essential for a
successful FriedelꢀCrafts alkylation. It would be of great
interest to create enzyme derivatives, e.g. by site directed
mutagenesis, which can stabilize such ions and accept also
saturated alkyl diphosphates as alkyl donors.
Acknowledgment. We thank Dr. Laufenberg and Lena
€
To compare the biochemical properties of FgaPT2 and
5-DMATS toward DMAPP and its analogues, kinetic
parameters were calculated from HanesꢀWoolf and
EadieꢀHofsteetransformations(Table 1). All thereactions
Ludwig (Philipps-Universitat Marburg) for acquiring
mass spectra and for the synthesis of DMAPP, respec-
tively. This work was supported in part by Deutsche
Forschungsgemeinschaft Grant Li844/4-1 (to S.-M.L.).
(9) Position C-5 (δC 119.2) is confirmed by connectivities of H-5
(δH 6.74) to C-7 (δC 109.5) and C-9 (δC 124.7) as well as connectivities of
H-10 (δH 3.29 and 2.90) and H-2 (δH 7.16) to C-9.
Supporting Information Available. Experimental pro-
cedures, HR-MS, NMR data and spectra. This material is
available free of charge via Internet at http://pubs.acs.org.
€
(10) (a) Metzger, U.; Schall, C.; Zocher, G.; Unsold, I.; Stec, E.; Li,
S.-M.; Heide, L.; Stehle, T. Proc. Natl. Acad. Sci. U.S.A 2009, 106,
14309–14314. (b) Lik, L. Y. P.; Tanner, M. E. J. Am. Chem. Soc. 2009,
131, 13932–13933.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 18, 2012
4885