Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides

Article abstract of DOI:10.1016/j.bmcl.2012.04.119

Fourteen tryptophan-containing cyclic dipeptides 1a-14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b-14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses. The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.

Full text of DOI:10.1016/j.bmcl.2012.04.119

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3866–3869
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Prenylation at the indole ring leads to a significant increase of cytotoxicity
of tryptophan-containing cyclic dipeptides
Beate Wollinsky a, Lena Ludwig a, Alexandra Hamacher b, Xia Yu a, Matthias U. Kassack b, Shu-Ming Li a,
⇑
a Institut für Pharmazeutische Biologie und Biotechnologie, Philipps Universität Marburg, Deutschhausstraße 17A, 35037 Marburg, Germany
b Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen tryptophan-containing cyclic dipeptides 1a–14a, including all four stereoisomers of cyclo-Trp-
Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b–14b in the pres-
ence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme
products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and
MS analyses.
Received 13 March 2012
Revised 26 April 2012
Accepted 29 April 2012
Available online 4 May 2012
The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia
K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly
shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipep-
tides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyc-
lic dipeptides showed less influence on the cytotoxicity of the tested compounds.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Chemoenzymatic synthesis
Cyclic dipeptides
Cytotoxicity
Prenyltransferase
Prenylated derivatives
The prenyltransferase FtmPT1 from Aspergillus fumigatus is in-
volved in the biosynthesis of fumitremorgin-type alkaloids and
catalyzes the prenylation of cyclo-L-Trp-L-Pro (brevianamide F,
1a) at C2 of the indole nucleus, resulting in the formation of try-
prostatin B (1b, Fig. 1).1,2 FtmPT1 showed high substrate specificity
towards its prenyl donor and accepted only dimethylallyl diphos-
phate (DMAPP), but not isopentenyl or geranyl diphosphate.2 By
contrast, five additional tryptophan-containing cyclic dipeptides
synthesis of the four stereoisomers 1b and 7b–9b.4,8,9 These ap-
proaches however consist of many steps and are not easy to apply
for diverse structures in this study. We chose therefore a combina-
tion of synthetic and chemoenzymatic approaches, that is chemical
synthesis of cyclic dipeptides, which are then prenylated regiospe-
cifically at C2 by using the purified recombinant FtmPT1 as catalyst
(Fig. 2).
For this purpose, all of the four stereoisomers of cyclo-Trp-Pro
(1a, 7a–9a) and cyclo-Trp-Ala (10a–13a) were synthesized accord-
including cyclo-
L-Trp-Gly (2a), cyclo-
L
-Trp-
L
-Leu (3a), cyclo-
L-Trp-
L
-Trp (4a), cyclo-
L
-Trp- -Tyr (5a) and cyclo-
L
L
-Trp- -Phe (6a) were
L
ing to the method published previously.10,11 Cyclo-
L
-Trp-
-Trp-OMeꢀHCl and
-Trp- -Pro from H-
-Pro-OH. Similarly, the two pairs
-Trp-OMeꢀHCl
-Ala-OH were used for the preparation of the four ster-
eoisomers of cyclo-Trp-Ala. Cyclo- -Trp- -His (14a) was synthe-
-Trp-OH and H-
-His-OMeꢀHCl according to
L-Pro and
accepted by FtmPT1 and converted to their C2-prenylated deriva-
tives 2b, 3b, 4b, 5b and 6b, respectively.2,3
cyclo-
N-Boc-
-Trp-OMeꢀHCl and N-Boc-
H-
and N-Boc-
L
-Trp-
D-Pro were synthesized from H-L
L
-Pro-OH, cyclo-
D
-Trp-
L
-Pro and cyclo-
D
D
The enzyme product of FtmPT1 1b was reported to exhibit
inhibitory effects on the cell cycle progression in the G2/M
D
D
L
-Trp-OMeꢀHCl and N-Boc-
L-Ala-OH as well as H-D
phase.4,5 MIC value of 4.4
lM and IC50 of 18.8
l
M were determined
D
with mouse tsFT210 cells.5,6 It has also been shown that the prenyl
moiety at C2 of 1b was essential for the cytotoxicity.7,8
L
L
sized from N-Boc-
L
L
Zhao et al.4 reported later that its diastereomer 7b (Fig. 1) was
much more active, while another diastereomer 8b and the enantio-
mer 9b were found to show similar effects on different cell lines. To
prove the influence of substitutions at the diketopiperazine ring
including the stereochemistry at C11 and C14 (Fig. 1) on the cyto-
toxicity, we decided to prepare diketopiperazines carrying a C2-
prenylated tryptophanyl moiety at C11 and aliphatic or aromatic
substituents at C14. Different approaches were described for the
the literature.11,12
The obtained cyclic dipeptides were purified and characterized
spectroscopically.13 The stereochemistry of the resulted stereoiso-
mers 1a, 7a–9a were confirmed by determination of optical
rotation.13 In total, 14 tryptophan-containing cyclic dipeptides14
were made available for the chemoenzymatic synthesis with
FtmPT1 in the presence of DMAPP, which was prepared according
to a protocol described for geranyl diphosphate.16
To get recombinant FtmPT1 for enzyme assays, Escherichia coli
M15 cells harbouring the expression plasmid pAG0122 were culti-
vated in 2 L Erlenmeyer flasks containing 1 L liquid Luria-Bertani
⇑
Corresponding author. Tel.: +49 6421 2822461; fax: +49 6421 2825365.
E-mail address: shuming.li@staff.uni-Marburg.de (S.-M. Li).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.119

B. Wollinsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3866–3869
3867
Figure 1. Production of several tryprostatin B analouges by FtmPT1.
Figure 2. Chemical synthesis and enzymatic conversion of tryptophan-containing cyclic dipeptides with proline (A), alanine (B) or L-histidine (C). Reagents and conditions:
(a) N-methylmorpholine (NMM), dimethylformamide (DMF) and dicyclohexyl carbodiimide (DCC), 0 °C, 3 h, 4 °C, 18 h; (b) 200 °C, 4 h; (c) 0.1 M HCl, 96% formic acid, rt,
30 min; (d) 0.1 M acetic acid, 2-butanol, NMM, 120 °C, 3 h.
medium supplemented with carbenicillin (50 l
g mlꢁ1) and grown
Table 1
Product yields FtmPT1 reactions
at 37 °C for about 4 h at 220 rpm until an absorption of 0.7 at
600 nm was reached. For induction, IPTG was added to the cultures
to a final concentration of 0.1 mM and the bacteria were cultivated
for further 16 h at 30 °C before harvest. Purification of the recom-
binant FtmPT1 was carried out according to the QIAexpressionist™
protocol from Qiagen.
Substrate
Product yield (%)
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
cyclo-
L
L
L
L
L
L
L
-Trp-
-Trp-Gly (2a)
L
-Pro (1a)
82.3
57.0
70.9
67.0
72.4
39.3
75.7
43.4
27.4
53.5
43.9
8.3
-Trp-
-Trp-
-Trp-
-Trp-
-Trp-
L
L
L
L
-Leu (3a)
-Trp (4a)
-Tyr (5a)
-Phe (6a)
HPLC analysis of the incubation mixtures in 100
taining 1 mM of 1a–14a, 2 mM of DMAPP, 10 mM of CaCl2, and
g of FtmPT1 in 50 mM Tris/HCl (pH 7.5) revealed clearly the for-
ll scales con-
D
-Pro (7a)
5
l
D
-Trp-
-Trp-
L-Pro (8a)
D
D
-Pro (9a)
-Ala (10a)
-Ala (11a)
-Ala (12a)
-Ala (13a)
mation of enzyme products (data not shown). The product yields
were found to be in the range of 8.3–82.3% with 1a as the best sub-
strate (Table 1) after incubation at 37 °C for 2 h.17 For structure elu-
cidation and determination of the cytotoxic activity, 1a–14a were
incubated in 50 ml scales under the same conditions with 1 mg
of FtmPT1. The reaction mixtures were subsequently extracted
L-Trp-
L
L-Trp-
D
D
-Trp-
-Trp-
D
D
L
46.4
68.1
L-Trp-L-His (14a)
The products were analyzed on a HPLC and detected at 296 nm.

3868
B. Wollinsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3866–3869
Table 2
IC50 values (
l
M) of non-prenylated and prenylated tryptophan-containing cyclic dipeptides against human leukemia cancer cell line K562, ovarian cancer cell lines A2780 sens
and A2780 CisR
K562
A2780 sens
A2780 CisR
Non-prenylated
Prenylated
Non-prenylated
Prenylated
Non-prenylated
Prenylated
1a
2a
3a
4a
5a
6a
7a
8a
>100
1b
2b
3b
4b
5b
6b
7b
8b
21.1
16.7
30.1
29.8
31.6
70.4
34.7
40.4
30.9
35.2
34.5
34.7
35.3
32.2
1a
2a
3a
4a
5a
6a
7a
8a
>100
>100
1b
2b
3b
4b
5b
6b
7b
8b
82.8
82.4
92.6
89.7
89.8
87.0
106
100
93.1
89.8
77.2
85.0
86.2
91.8
1a
2a
3a
4a
5a
6a
7a
8a
>100
>100
1b
2b
3b
4b
5b
6b
7b
8b
85.9
85.2
75.0
80.8
83.3
81.6
92.0
87.8
85.6
82.2
77.0
81.1
84.3
91.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9a
9b
9a
9b
9a
9b
10a
11a
12a
13a
14a
10b
11b
12b
13b
14b
10a
11a
12a
13a
14a
10b
11b
12b
13b
14b
10a
11a
12a
13a
14a
10b
11b
12b
13b
14b
with ethyl acetate and purified on HPLC18 resulting in 1.1–5.2 mg
pure enzyme products.
and 9b showed comparable cytotoxicity against PC-3 cell lines.4,8
In our assays, all four stereoisomers (1b, 7b–9b) displayed similar
IC50 values within a given cell line. Similar cytotoxicities were
detected for the stereoisomers of prenylated cyclo-Trp-Ala
(Table 2).
The obtained products were subsequently analyzed by 1H NMR
and MS analyses. The monoprenylation in 1b–14b was confirmed
by detection of [M+H]+ or [M+Na]+ ions, which were 68 daltons lar-
ger than the respective substrate.19 The structures of 1b–8b were
elucidated by comparison of the obtained spectroscopic data with
those published previously.2,3,8 The 1H NMR spectrum of cyclo-C2-
Acknowledgments
dimethylallyl-
D
-Trp-D-Pro (9b) was identical to that of its enantio-
This work was supported by a Grant from the Deutsche
Forschungsgemeinschaft (Li844/1-3 to S.-M.L.).
mer 1b.2
Inspection of the 1H NMR spectra of 10b–14b revealed the pres-
ence of signals for a regular dimethylallyl moiety at dH 3.38–3.49 (d
or m, 2H-10), 5.28–5.35 (t or tt, H-20), 1.75–1.77 (s, 3H-40) and 1.77–
1.80 (s, 3H-50) (Supplementary Table 1). In all spectra, the signal for
H-2 was disappeared, confirming the prenylation at C2 of the in-
dole ring.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
04.119.
The isolated enzyme products 1b–14b were tested for their
cytotoxicity towards human chronic myelogenous leukemia cell
line K562 and human ovarian carcinoma cell line A2780 sens and
CisR.20 The respective substrates 1a–14a were used as negative
controls.
The rate of cell-survival under the action of test substances was
evaluated by an improved MTT assay as previously described.21
The assay is based on the ability of viable cells to metabolize
yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide to violet formazane crystals that can be detected
spectrophotometrically.22,23
References and notes
1. Li, S.-M. J. Antibiot. 2011, 64, 45.
2. Grundmann, A.; Li, S.-M. Microbiology 2005, 151, 2199.
3. Wang, L.; Yin, W.-B.; Li, S.-M.; Liu, X.-Q. Chin. J. Biochem. Mol. Biol. 2009, 25, 580.
4. Zhao, S.; Smith, K. S.; Deveau, A. M.; Dieckhaus, C. M.; Johnson, M. A.;
Macdonald, T. L.; Cook, J. M. J. Med. Chem. 2002, 45, 1559.
5. Cui, C. B.; Kakeya, H.; Osada, H. J. Antibiot. 1996, 49, 534.
6. Cui, C. B.; Kakeya, H.; Okada, G.; Onose, R.; Osada, H. J. Antibiot. 1996, 49, 527.
7. Sanz-Cervera, J. F.; Stocking, E. M.; Usui, T.; Osada, H.; Williams, R. M. Bioorg.
Med. Chem. 2000, 8, 2407.
8. Jain, H. D.; Zhang, C.; Zhou, S.; Zhou, H.; Ma, J.; Liu, X.; Liao, X.; Deveau, A. M.;
Dieckhaus, C. M.; Johnson, M. A.; Smith, K. S.; Macdonald, T. L.; Kakeya, H.;
Osada, H.; Cook, J. M. Bioorg. Med. Chem. 2008, 16, 4626.
9. Yamakawa, T.; Ideue, E.; Shimokawa, J.; Fukuyama, T. Angew. Chem., Int. Ed.
Engl. 2010, 49, 9262.
Our results showed clearly that prenylation at the indole ring
led to a significant increase in cytotoxicity. Whereas the non-
prenylated compounds showed IC50 values > 100 lM, all of the
10. Caballero, E.; Avendaño, C.; Menéndez, J. C. Tetrahedron: Asymmetry 1998, 9,
967.
11. Cacciatore, I.; Cocco, A.; Costa, M.; Fontana, M.; Lucente, G.; Pecci, L.; Pinnen, F.
Amino. Acids 2005, 28, 77.
tested prenylated enzyme products were more toxic towards the
three cell lines with IC50 values in the lower to medium micromo-
lar range for K562 or the higher micromolar range for A2780 cells
(Table 2). With a few exceptions, for example 6b and 8b in the as-
says with K562, all prenylated substances showed similar toxicity
for a given cell line, indicating that the proline moiety in 1b is not
essential for this biological activity. Human leukemia cell line K562
had a higher sensitivity for prenylated substances than ovarian cell
lines. Interestingly, A2780 sens and A2780 CisR cell lines showed
similar sensitivity towards prenylated compound implying that
these prenylated compounds do not differentiate between the sen-
sitive and resistant phenotype of A2780 cells. It has been reported
that 7b exhibited the highest cytotoxic effect among the four ster-
eoisomers 1b, 7b–9b against human lung (H520), breast (MCF-7)
and prostate cancer (PC-3) cell lines.4,8 1b was more toxic against
H520 und MCF-7 than 8b and 9b and all three compounds 1b, 8b
12. Bivin, D. B.; Kubota, S.; Pearlstein, R.; Morales, M. F. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 6791.
13. The 1H NMR spectra were taken on
a JEOL ECA-500 spectrometer. The
spectroscopic data of 1a (250 mg) corresponded well to those reported
previously.2 The 1H NMR data of 9a (238 mg), were identical to those of its
enantiomer 1a. The 1H NMR data of 7a (560 mg) corresponded well to those
reported previously7 and the NMR data of the enantiomers 8a (267 mg) were
identical to those of 7a. The optical rotation were determined with 1% (m/v)
solutions in DMSO at 24 °C with a 2 ml cell, using a Jasco DIP-370 digital
polarimeter: 1a ½a D24
ꢂ
= ꢁ58; 9a ½a 2D4
ꢂ
= +57; 7a ½a 2D4
ꢂ
= +71 and 8a ½a 2D4
= ꢁ84.
ꢂ
The spectroscopic data of 10a (155 mg), 11a (165 mg), 12a (53 mg), 13a
(30 mg) and 14a (200 mg), corresponded very well to those reported
previously.10,15
14. Other cyclic dipeptides (2a–6a) were purchased from Bachem (Bubendorf,
Switzerland).
15. Sheinblatt, M.; Andorn, M.; Rudi, A. Int. J. Pept. Prot. Res. 1988, 31, 373.
16. Woodside, A. B.; Huang, Z.; Poulter, C. D. Org. Synth. 1988, 66, 211.

B. Wollinsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3866–3869
3869
17. The reactions were terminated by addition of an equal volume of methanol.
After removal of the protein by centrifugation (15,000 xg, 10 min, 4 °C), the
enzyme products were analyzed on an Agilent HPLC Series 1200 by using a
348.22; 13b [M+H]+ m/z 326.27 [M+Na]+ m/z 348.22 [2M+Na]+ m/z 673.49; 14b
[M+H]+ m/z 392 [M+Na]+ m/z 414 [2M+Na]+ m/z 805.
20. The human chronic myelogenous leukemia cell line K562 was obtained from
German Collection of Microorganisms and Cell Cultures (DSMZ, Germany) and
the human ovarian carcinoma cell line A2780 (A2780 sens) was obtained from
European Collection of Cell Cultures (ECACC, Salisbury, UK). A2780 cells were
MultospherÒ RP-18-5 column (250 ꢃ 4 mm,
5 lm, C+S Chromatographie
Water (solvent A) and methanol
-Trp- -His,
Service) at
a
flow rate of 1 ml minꢁ1
.
(solvent B) were used as solvents. For analysis of assays with cyclo-
L
L
0.5% (v/v) trifluoroacetic acid was added to the solvents. The assays were
analyzed with a gradient from 30 to 100% B over 30 min. The column was then
washed with 100% solvent B for 5 min equilibrated with 30% solvent B for
5 min. The substances were detected with a Photo Diode Array detector.
18. The ethyl acetate extracts of the enzyme assays were evaporated in vacuo. The
residues were dissolved in methanol and purified on the same Agilent
exposed to weekly cycles of 2 lmol/L cisplatin over a period of 24 weeks.
Cisplatin-resistant cells were denoted A2780 CisR. All three cell lines were
grown at 37 °C under humidified air supplemented with 5% CO2 in RPMI 1640
containing 10% fetal calf serum (PAN Biotech, Germany), 100 IU/mL penicillin
and 100 lg/mL streptomycin. Cells were grown to 80% confluency before
subjected to MTT cell viability assay.
equipment by using a MultospherÒ RP-18-5 column (250 ꢃ 10 mm, 5
lm,
21. Müller, H.; Kassack, M. U.; Wiese, M. J. Biomol. Screen. 2004, 9, 506.
22. The yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for
the MTT Assay was purchased from MTT, Applichem (Germany).
23. A2780 cells were seeded at a density of 8,000 cells/well and K562 at a density
of 30,000 cells/well in 96 well plates (Corning, Germany). After 24 h, cells were
exposed to increasing concentrations of the test compounds. Incubation was
stopped after 72 h and cell survival was determined by addition of MTT
solution (5 mg/mL in phosphate buffered saline). The formazan precipitate was
dissolved in DMSO. Absorbance was measured at 544 nm and 690 nm in a
FLUOstar microplate-reader (BMG LabTech, Offenburg, Germany). The
absorbance of untreated control cells was taken as 100% viability. All tests
were performed in triplicate.
C+S Chromatographie Service) at a flow rate of 2.5 ml minꢁ1. For isolation of
the most enzyme products, a linear gradient of 65 to 100% (v/v) solvent B in
15 min was used. The column was then washed with 100% solvent B for 5 min
and equilibrate with 65% (v/v) solvent B for 5 min. For isolation of 14b, 0.5%
trifluoroacetic (TFA) was added to solvent A and B. The fractions containing the
prenylated diketopiperazine derivatives were collected and evaporated in
vacuo.
19. The positive electrospray ionization mass spectrometry (ESI-MS) was carried
out on a Q-Trap Quantum (Applied Biosystem). ESI-MS data of the new enzyme
products: 10b [M+H]+ m/z 326.27 [M+Na]+ m/z 348.15 [2M+Na]+ m/z 673.14;
11b [M]+ m/z 325 [M+Na]+ m/z 348; 12b [M+H]+ m/z 326.25 [M+Na]+ m/z