Mitochondrial targeting of selective electron scavengers: Synthesis and biological analysis of hemigramicidin-TEMPO conjugates

Article abstract of DOI:10.1021/ja053679l

Synthetic hemigramicidin S-peptidyl TEMPO conjugates are effectively delivered into cells and mitochondria, where they act as electron scavengers and exert protection against apoptosis. Our delivery approach is based on the use of specific structural sign

Full text of DOI:10.1021/ja053679l

Published on Web 08/19/2005
Mitochondrial Targeting of Selective Electron Scavengers: Synthesis and
Biological Analysis of Hemigramicidin-TEMPO Conjugates
Peter Wipf,*,§ Jingbo Xiao,§ Jianfei Jiang,# Natalia A. Belikova,# Vladimir A. Tyurin,#
Mitchell P. Fink,$ and Valerian E. Kagan*,#
Departments of Chemistry, EnVironmental and Occupational Health, and Critical Care Medicine, UniVersity of
Pittsburgh, Pittsburgh, PennsylVania 15260
Received June 4, 2005; E-mail: pwipf@pitt.edu
The chemistry and biology of mitochondria, in particular, the
effects of intracellular reactive oxygen species (ROS, superoxide
radicals and H2O2) that are byproducts of the oxidative phospho-
rylation cascade, is under intense study.1 Cellular injury, aging, and
death, as well as suspended animation, neuro-, and cardioprotection
are influenced by events in the mitochondrial membrane that lead
to an imbalance in ATP production and O2 consumption.2 Recently,
dysregulated electron transport and generation of ROS were linked
to a mitochondria-specific phospholipid, cardiolipin (CL), and
involvement of CL oxidation products in apoptosis.3 Nitroxide
radicals prevent the formation of ROS, particularly superoxide, due
to their reduction by the mitochondrial electron transport to
hydroxylamine radical scavengers.4 Nitroxides also exert superoxide
dismutase and catalase activities,5 thus offering additional protective
benefits against oxidative stress. However, delivery of sufficient
amounts of nitroxides into mitochondria has proven difficult.6
A selective delivery of TEMPO7 to mitochondria could lead to
a therapeutically beneficial reduction of ROS; therefore, we have
investigated the use of conjugates8 of 4-amino-TEMPO (4-AT)9
and employed as targeting sequence fragments of the membrane-
active antibiotic GS as well as the corresponding alkene isosteres
(Figure 1).10 We selected the Leu-DPhe-Pro-Val-Orn fragment of
GS as the targeting sequence, because it encompasses the â-turn
motif that directs most of the polar functionality of the peptide
strand into the core, and acylated the amino functions of Leu and
Orn in order to reduce GS-related cytotoxicity.11
Figure 1. Gramicidin S (targeting sequence in blue) and 4-AT.
Scheme 1. Synthesis of Peptide Conjugatesa
The preparation of (E)-alkene dipeptide isostere 3 was based on
our Zr/Zn methodology (Scheme 1).12 Hydrozirconation13 of alkyne
114 with Cp2ZrHCl followed by transmetalation to Me2Zn and
addition of N-Boc-isovaleraldimine15 afforded diastereomeric allylic
amides, which were separated after desilylation and acetylation. A
two-step oxidation of 2 provided peptide isostere 3. The segment
assembly of 3 and tripeptide H-Pro-Val-Orn(Cbz)-OMe was ac-
complished using EDC as a coupling agent. Saponification of 4a
followed by coupling with 4-AT afforded the desired conjugate
5a, in which the Leu-DPhe peptide bond had been replaced with
an (E)-alkene. Conjugates 5b and 5c were prepared by coupling of
pentapeptide 4b16 and isostere 3 to 4-AT.
We used EPR spectroscopy to monitor the cellular delivery and
metabolic fate of 5a and 5b. Distinctive characteristic triplet signals
of nitroxide radicals (with hyperfine splitting constants of 16.6 G)
were detected in mouse embryonic cells (MECs) incubated with
10 µM 5a as well as in mitochondria isolated from these cells
(Figure 2). The cytosolic fraction did not elicit EPR signals of
nitroxide radicals. Similar results were observed with conjugate 5b
(data not shown). In contrast, 4-AT did not effectively partition
into either cells or mitochondria. Incubation of MECs in the
a Conditions: (a) (i) Cp2ZrHCl, Me2Zn, N-Boc-isovaleraldimine, then
TBAF, 74%; (ii) Ac2O, TEA, DMAP, 94%; (iii) K2CO3, MeOH, quant.;
(b) (i) Dess-Martin periodinane; (ii) NaClO2, NaH2PO4, 2-methyl-2-butene;
(c) H-Pro-Val-Orn(Cbz)-OMe, EDC, HOBt, DMAP, 94% from 2; (d) (i) 1
N NaOH; (ii) 4-AT, EDC, HOBt, DMAP; 5a, 99%; 5b, 99%; (e) 4-AT,
EDC, DMAP, 91%.
presence of 5a resulted not only in its integration but also in its
one-electron reduction, as evidenced by a significant increase in
the magnitude of the EPR signal intensity upon addition of a one-
electron oxidant, ferricyanide (Figure 2B). Thus, not only delivery
but also the reduction of 5a and 5b occurred in MEC mitochondria.
We tested the ability of 5a and 5b to prevent intracellular superoxide
generation (by flow cytometric monitoring of oxidation of dihy-
droethidium (DHE) to a fluorescent ethidium) and protect cells
against apoptosis triggered by actinomycin D (ActD). Both 5a and
5b (but not 4-AT) completely inhibited ActD-induced (∼2-fold)
increase of superoxide production in MECs (Figure 3A). Apoptotic
cell responses were documented using three biomarkers: (1)
Externalization of phosphatidylserine (PS) on the cell surface (by
flow cytometry using an FITC-labeled PS-binding protein, annexin
V); (2) Activation of caspase-3 (by cleavage of its specific substrate,
Z-DEVD-AMC); and (3) DNA fragmentation (by flow cytometry
of propidium iodide stained DNA). ActD effectively induced
apoptosis, as revealed by an increased number of annexin V-positive
§ Department of Chemistry.
# Department of Environmental and Occupational Health.
$ Department of Critical Care Medicine.
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J. AM. CHEM. SOC. 2005, 127, 12460-12461
10.1021/ja053679l CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
from specific interactions with cellular and mitochondrial mem-
branes, we tested nitroxide conjugate 5c, which is similarly
lipophilic (cLogP 5.5) but does not have a complete targeting
moiety. We found that 5c was ineffective in protecting MECs
against ActD-induced apoptosis (Figure 3B,C). Thus, the GS-
peptidyl targeting structure is required for anti-apoptotic activity
of nitroxide conjugates. Since the reduction of 5a and 5b could
also cause inhibition of mitochondrial oxidative phosphorylation,
we tested whether ATP levels were changed in cells treated with
these compounds. At concentrations at which anti-apoptotic effects
were maximal (5a, 10 µM, Figure 3E), nitroxide conjugates did
not cause significant changes in the cellular ATP level (Figure 3F).
Thus, synthetic GS-peptidyl conjugates migrate into cells and
mitochondria, where they are reduced (likely by electron-transport-
ing proteins) and exert protection against apoptosis. Previously, spin
trapping nitrones have demonstrated promise in aging research.17
Our radical scavenger delivery approach is based on the use of
specific GS-derived mitochondria targeting sequences11 and offers
similar potential for future anti-apoptotic interventions.6b,c,18
Acknowledgment. We thank DARPA (W81XWH-05-2-0026)
for financial support.
Figure 2. EPR-based analysis of integration and reduction of nitroxide
GS-peptidyl conjugates in MECs. Cells (10 million/mL) were incubated
with 10 µM of 4-AT or 5a for 15 min. Recovered nitroxide radicals in
whole cells, mitochondria, or cytosol fractions were resuspended in PBS
in the presence or absence of 2 mM K3Fe(CN)6 (JEOL-RE1X EPR
spectrometer under the following conditions: 3350 G center field; 25 G
scan range; 0.79 G field modulation, 20 mW microwave power; 0.1 s time
constant; 4 min scan time). (A) Representative EPR spectra of 5a in different
fractions of MECs in the presence of K3Fe(CN)6. (B) Assessment of
integrated nitroxides (n ) 3); *p < 0.01 vs K3Fe(CN)6; #p < 0.01 vs 5a
under the same conditions.
Supporting Information Available: Experimental procedures, 1H
and 13C spectra, and procedures for biological assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 3. Effect of nitroxide conjugates on ActD-induced apoptosis in
MECs. Cells were pretreated with 10 µM 4-AT, 5a, 5b, or 5c for 1 h, then
incubated with ActD (100 ng/mL). (A) Superoxide production: mean
fluorescence intensity from 10 000 cells. (B) PS externalization. (C) Cas-
pase-3 activation. (D) DNA fragmentation. (E) PS externalization at different
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cells (Figure 3B), caspase activation (Figure 3C), and DNA
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apoptotic effects resulted from unspecific lipophilicity rather than
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