Understanding enediyne-protein interactions: Diyl atom transfer results in generation of aminoacyl radicals

Article abstract of DOI:10.1021/ol0055566

Formula Presented The origin of the protein modulating capacity of enediynes has been probed. A series of synthetic enediyne-derived diyls participated in atom transfer chemistry with a labeled amino acid. Subsequent experiments suggest that diyl radicals may modulate protein architecture via formation of captodatively stabilized radicals.

Full text of DOI:10.1021/ol0055566

ORGANIC
LETTERS
2000
Vol. 2, No. 6
811-813
Understanding Enediyne−Protein
Interactions: Diyl Atom Transfer Results
in Generation of Aminoacyl Radicals
Graham B. Jones,* Gary W. Plourde II, and Justin M. Wright
Bioorganic and Medicinal Chemistry Laboratories, Department of Chemistry,
Northeastern UniVersity, Boston, Massachusetts 02115
grjones@lynx.neu.edu
Received January 14, 2000
ABSTRACT
The origin of the protein modulating capacity of enediynes has been probed. A series of synthetic enediyne-derived diyls participated in atom
transfer chemistry with a labeled amino acid. Subsequent experiments suggest that diyl radicals may modulate protein architecture via formation
of captodatively stabilized radicals.
Interest in the chemistry of the enediyne family of antitumor
antibiotics has grown steadily over the past decade, fueled
both by reports of their remarkable biological activity and
their intriguing and challenging molecular structures.1 While
the in vitro and in vivo effectiveness of enediynes against
certain cancers is unquestioned, the exact mechanism(s) of
biological activity remains to be clarified.2 Though DNA is
the recognized target of the naturally occurring enediyne
chromophores, studies involving calicheamicin suggest that
specific DNA strand scission is not directly related to
cytotoxicity in this potent antitumor agent but that subsequent
promotion of the DNA suicide repair enzyme poly(ADP-
ribose)polymerase results in cellular death by means of
depleting cellular NAD+ and ATP levels.3 Both the naturally
occurring kedarcidin and the related enediyne neocarzinosta-
tin (NCS) are chromoproteins that are capable of causing
damage to histones, in addition to DNA.4 In this class of
chromophore
(1) Reviews: Grissom, J. W.; Gunawardena, G. U.; Klingberg, D.;
Huang, D. Tetrahedron 1996, 52, 6453-6518. Maier, M. E. Synlett 1995,
13. Nicolaou, K. C.; Smith, A. L. Acc. Chem. Res. 1992, 25, 497.
(2) Xi, Z.; Goldberg, I. H. In ComprehensiVe Natural Products Chemistry;
Barton, D. H. R., Nakanishi, K.; Eds.; Elsevier Science: Oxford, 1999;
Vol. 7, pp 553-592. Lee, M. D.; Durr, F. E.; Hinman, L. H.; Haman, P.
R.; Ellestad, G. A. The Calicheamicins. In AdVances in Medicinal Chemistry;
Maryanoff, B. E., Maryanoff, C. A., Eds.; Jai Press: Greenwich, 1993;
Vol. 2.
enediyne antibiotic, proteolytic ability is derived from the
associated apoprotein component, tempting speculation that
this may serve as a dynamic delivery vehicle for the DNA
cleaving chromophore.4 In related studies, the Bristol-Myers
(4) Zein, N.; Schroeder, D. R. In AdVances in DNA Sequence Specific
Agents; Jones, G. B., Ed., JAI Press: Greenwich, 1998; Vol. 3, p 201. Zein,
N.; Reiss, P.; Bernatowicz, M.; Bolgar, M. Chem. Biol. 1995, 2, 451. Zein,
N.; Casazza, A. M.; Doyle, T. W.; Leet, J. E.; Schroeder, D. R.; Solomon,
W.; Nadler, S. G.; Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8009.
(3) Durkacz, B. W.; Omidiji, O.; Gray, D. A.; Shall, S. Nature 1980,
283, 593. Zhao, B.; Konno, S.; Wu, J. M.; Oronsky, A. L. Cancer Lett.
1990, 50, 141.
10.1021/ol0055566 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/01/2000

Squibb group identified protein targets of a synthetic
enediyne chromophore (1), which possesses the core frame-
work of the enediyne esperamicin.5 This agent, which has
demonstrated in vitro and in vivo antitumor activity, induced
damage to membrane extracts, tubulins, and histones,
presumably via its derived diyl radicals, whereas significantly
higher concentrations of 1 were required to achieve ap-
preciable DNA cleaving activity.6 In view of the potential
significance of a selective enediyne based protein modulator,7
we sought to examine the molecular basis for diyl-protein
interactions using atom transfer chemistry.8
(Hanovia 450 W, quartz vessel). Similar behavior was
observed for enediyne 8 (Scheme 2). Moving to more
Scheme 2. Deuterium Atom Transfer from Labeled Amino
Acid to Enediyne-Derived Diyl Radicals
Accordingly, we prepared a number of synthetic enediyne
core structures 2 designed to undergo cycloaromatization to
yield diyls 3 in the presence of the labeled amino acid 4,
readily prepared from glycine (Scheme 1).8 The enediynes
Scheme 1. Atom Transfer Chemistry Using Deuterated
Glycine Mimics
reactive precursors, enediyne 10 and eneyne allene 12, whose
half-lives for cycloaromatization are approximately 18 and
24 h at 37 °C, respectively,1,11 cyclized readily at physi-
ological temperature, giving low yields of labeled cycloaro-
matization adducts 11 and 13, which could be increased
slightly under photochemical conditions.
Finally, the hydroxymethyl enediyne 14 was employed.
The marked increase in chemical conversion to 15 suggests
that electrostatic effects between the donor (4) and pendant
functionality contribute to the efficiency of diyl capture.
Indeed, repeating the experiments using benzene-d6 as solvent
gave no increase in deuterium incorporation. Atom transfer
from 4 implies generation of a capdodatively stabilized
radical 16, which could be expected to either dimerize (17)
or react with molecular oxygen to form peroxides (e.g., 18)
which could ultimately result in fragmentation via a transient
iminium ion (Scheme 4).13 To probe these scenarios, an
authentic sample of 17 was prepared (tert-butyl peroxide,
hυ, PhH, 48 h, 55%). Reanalysis of the crude reaction
mixtures (Scheme 2) confirmed formation of 17 as the
principal byproduct following atom transfer. Not surprisingly,
since all reactions were conducted under deoxygenated
were selected on the basis of their differing modes of
activation, and propensity to undergo cycloaromatization, and
were all prepared using an efficient metallohalocarbenoid
route developed in our laboratories.9 Thermal cyclization of
Z enediyne 6 proceeded at 280 °C to give the corresponding
arenes 7 isolated as a mixture of labeled and unlabeled
species (Table 1). Alternatively, photochemical Bergman
cycloaromatization can be initiated with suitable substrates,10
and enediyne 6 likewise gave arenes 7 in moderate yield
following 3 h irradiation using a low-pressure mercury lamp
Table 1. Yields and Isotope Ratios of Atom Transfer Adducts
adduct
method
yield (%)a,b
D0:D1:D2c
7
thermal
hυ
thermal
hυ
thermal
hυ
thermal
hυ
10
18
8
20
11
20
10
15
40
50
31:2:1
17:2:1
19:2:1
14:2:1
3:2:1
2:2:1
2:2:1
7:2:1
4:2:1
(5) Zein, N.; Solomon, W.; Casazza, A. M.; Kadow, J. F.; Krishnan, B.
S.; Tun, M. M.; Vyas, D. M.; Doyle, T. W.; Bioorg. Med. Chem. Lett.
1993, 3, 1351. Lee, S.; Bain, A.; Sulikowski, G. A.; Solomon, W.; Zein,
N. Bioorg. Med. Chem. Lett. 1996, 6, 1261.
(6) Wittman, M. D.; Kadow, J. F.; Langley, D. R.; Vyas, D. M.; Rose,
W. C.; Solomon, W.; Zein, N. Bioorg. Med. Chem. Lett. 1995, 5, 1351.
(7) Jones, G. B.; Kilgore, M. W.; Pollenz, R. S.; Li, A.; Mathews, J. E.;
Wright, J. M.; Huber, R. S.; Tate, P. L.; Price, T. L.; Sticca, R. P. Bioorg.
Med. Chem. Lett. 1996, 6, 1971.
9
11
13
15
thermal
hυ
(8) Braslau, R.; Anderson, M. O. Tetrahedron Lett. 1998, 39, 4227.
(9) Jones, G. B.; Huber, R. S.; Mathews, J. E. J. Chem. Soc., Chem.
Commun. 1995, 1791. Jones, G. B.; Wright, J. M. Tetrahedron Lett. 1999,
40, 7605.
(10) Turro, N. J.; Evenzhav, A.; Nicolaou, K. C. Tetrahedron Lett. 1994,
35, 8089. Funk, R. L.; Young, E. R. R.; Williams, R. F.; Flanagan, M. F.;
Cecil, T. L. J. Am. Chem. Soc. 1996, 118, 3291.
(11) Myers, A G.; Dragovich, P. S. J. Am. Chem Soc. 1989, 111, 9130.
(12) Karel, K. J.; Brookhart, M.; Aumann, R. J. Am. Chem. Soc. 1995,
103, 2695.
3:2:1
a Yields based on GC analysis, calibrated using internal (n-decane) and
external standards. b Mass balance composed of recovered starting material
(10-30%) and unidentified oligomers. c Ratio determined by H/D NMR
and GC/MS analysis using authentic dideutero adduct (>95% D2 incorpora-
tion at 20% conversion) prepared by incubation of enediyne with cyclo-
12
hexadiene-d8 or THF-d8.
812
Org. Lett., Vol. 2, No. 6, 2000

more efficient using 4 rather than d5 (or H5) glycine as atom
transfer agent. This may reflect a preference for the
(hydrophobic) diyl to abstract from a neutral atom transfer
agent, and this has a positive impact on the rate of
cycloaromatization (Table 2, entry 6). Though a complex
Scheme 3. Atom Transfer Chemistry Using an Enediyne
Carboxylate
Table 2. Atom Transfer Chemistry of Enediyne Carboxylate
22
entry
solvent
CH3OH
CH3OD
H2O
H2O/(NH4)2HPO4 glycine
H2O/(NH4)2HPO4 glycine-d5
donor13
yieldof 24 (%) D0:D1:D2
15
1
2
3
4
5
6
17
<5
28
30
42
5:4:1
glycine
10:2:1
3:2:1
H2O/(NH4)2HPO4
4
mixture of other byproducts was formed in addition to dimer
17, both glyoxylate 19 and acetamide 20 were detected
(<10%), suggesting the intermediacy of peroxide 18 under
aerobic conditions. Repeating the incubations under deoxy-
genated conditions (triple freeze-pump-thaw cycles, N2),
the efficiency of formation of the peroxy-derived byproducts
was reduced (<1%), suggesting some control may be
possible in governing the fate of radical 16. On the basis of
these preliminary findings, a likely extension of this work
may be the design of enediynes capable of inducing inter-
and intramolecular peptide cross-links, via generation of
peptide radicals. Such radicals are not without precedent;14
indeed, families of “radical enzymes” exist, including the
well-studied examples from Escherichia coli ribonucleotide
reductase and pyruvate-formate lyase, both of which involve
intermediate glycyl radicals.15
conditions, products derived from hydroperoxide 18 were
not isolated. Though the experiments show clear evidence
of atom transfer chemistry from 4, the relevance of these
events in organic media remained in question. To address
this issue water-soluble enediyne 22 was prepared, by
reacting the shelf-stable cobalt complex 21 with isophthaloyl
chloride (Scheme 4),9 and then immediately unmasking the
Scheme 4. Potential Intermediates in the Activation of Amido
Acyl Radicals
In summary, a water-soluble enediyne has been prepared
and used to investigate amino acid-diyl interactions. Evi-
dence in support of diyl-mediated peptide damage at the
molecular level has been gleaned, which has ramifications
for the biology of enediyne antitumor agents. Application
of this methodology in the design of target specific protein
modulators will be reported in due course.16
OL0055566
(13) In all cases, 30 equiv of amino acid mimic was employed, since
control reactions using the conventional hydrogen donor 1,4-cyclohexadiene
gave optimal yields (>80%) under these conditions. Further increases in
stoichiometry resulted in only negligible increases in chemical yield of the
labeled (or control) adducts, but reduction (<10 equiv) resulted in a sharp
decrease. For discussion on the influence of quench concentration on rate
of abstraction, see: Semmelhack, M. F.; Neu, T.; Foubelo, F. J. Org. Chem.
1994, 59, 5038.
enediyne moiety. Though carboxylate 22 was freely soluble
in polar organic solvents, phosphate buffer was required to
achieve full homogeneity in aqueous conditions. To our
delight it was discovered that the cycloaromatization is
indeed viable: the corresponding atom-transfer adducts
formed at 37 °C under aerobic conditions. The process was
(14) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press: 1999.
(15) Marsh, E. N. G. BioEssays 1995, 17, 431.
(16) We thank the NIH [1RO1GM57123] for generous financial support.
Org. Lett., Vol. 2, No. 6, 2000
813