Synthesis and protein degradation capacity of photoactivated enediynes

Article abstract of DOI:10.1021/jo051403q

The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics.

Full text of DOI:10.1021/jo051403q

Synthesis and Protein Degradation Capacity of Photoactivated
Enediynes
Farid S. Fouad, Justin M. Wright, Gary Plourde II, Ajay D. Purohit, Justin K. Wyatt,
Ahmed El-Shafey, George Hynd, Curtis F. Crasto, Yiqing Lin, and Graham B. Jones*
Bioorganic and Medicinal Chemistry Laboratories, Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts, 02115
gr.jones@neu.edu
Received July 7, 2005
The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed
at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer
from diyl radicals produced from enediynes to form captodatively stabilized carbon centered
aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding,
a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were
prepared and used to target specific proteins, showing good correlation between affinity and
photoinduced protein degrading activity. The findings have potential applications in the design of
artificial chemical proteases and add to our understanding of the mechanism of action of the
clinically important enediyne antitumor antibiotics.
Introduction
cascade of reactions which results in the formation of a
postactivated diyl core, which abstracts hydrogen atoms
from the DNA backbone. On interception by molecular
oxygen, an intermediate peroxide is formed which ulti-
mately leads to strand scission by generation of a 5′
aldehyde.2
Depending on the local environment, however, it is also
possible for other events to occur and, in the case of the
enediyne C-1027, under anaerobic conditions, recombina-
tion of the ribosyl radical and enediyne-derived diyl has
been observed, leading to DNA adduction.1 Though the
generally accepted target of the naturally occurring
enediynes is believed to be DNA, there is often poor
correlation between enediyne-induced DNA damage and
observed antitumoral activity.3 This has prompted a
search for additional targets, the most likely being
proteins.4 Excepting water, proteins are the most abun-
dant constituents of cells and extracelluar fluids by
weight. Interest in this class of target was stimulated
The enediynes are a substantial class of antitumor
agents, with spectacular biological profiles and proven
clinical efficacy.1 Nearly 20 discrete enediynes have been
discovered, and clinical trials of a number of these are
ongoing. An antibody conjugate of one enediyne (cali-
cheamicin) was the first to gain approval by the FDA and
shows promise in the treatment of acute myeloid leuke-
mia (AML).2 Although the in vitro and in vivo effective-
ness of enediynes against certain cancers is unques-
tioned, the exact mechanism(s) of biological activity
remains to be fully resolved. Enediynes, per se, are
biologically inactive but undergo cycloaromatization reac-
tions which give rise to cytotoxic diyl radicals, which are
capable of inducing DNA strand scission at low concen-
tration.1 In the case of calicheamicin, this involves a
* To whom correspondence should be addressed. Phone: 617-373-
8619. Fax: 617-373-8795.
(1) Smith, A. L.; Nicolaou, K. C. J. Med. Chem. 1996, 39, 2103. Xi,
Z.; Goldberg, I. DNA Damaging enediyne compounds. In Comprehen-
sive Natural Products Chemistry; Barton, D. H. R., Nakanishi, K., Eds.;
Pergamon: Oxford, NY, 1999, 7, 553.
(2) For an overview of recent clinical findings, see: Schor, N. F. In
Cancer Therapeutics: Experimental and Clinical Agents; Teicher, B.,
Ed.; Humana Press: Totowa, NJ, 1996; pp 229-240. Schmitt, D. A.;
Kisanuki, K.; Kimura, S.; Oka, K.; Pollard, R. B.; Maeda, H.; Suzuki,
F. Anticancer Res. 1992, 12, 2219. Takahashi, T.; Yamaguchi, T.;
Kitamura, K.; Noguchi, A.; Mitsuyo, H.; Otsuji, E. Jpn. J. Cancer Res.
1993, 53, 976. Oda, M.; Sato, F.; Maeda, H. JNCI 1987, 79, 1205.
(3) Cell death induced by the suicide repair enzyme poly(ADP ribose
polymerase) has been suggested. See: 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. Nicolaou, K. C.;
Stabila, P.; Esmaeli-Azad, B.; Wrasidlo, W.; Hiatt, A. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 3142. Nicolaou, K. C.; Smith, A. L.; Yue, E. W.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5881. For an example of base-
selective DNA modification via synthetic enediynes, see: Tunti-
wechapikul, W.; David, W. M.; Kumar, D.; Salazar, M.; Kerwin, S. M.
Biochemistry 2002, 41, 5283.
10.1021/jo051403q CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/02/2005
J. Org. Chem. 2005, 70, 9789-9797
9789

Fouad et al.
SCHEME 1. Potential Pathways for Peptide
Degradation
would be a requirement if we were to be successful in
the development of enediyne-based proteases.6
Results and Discussion
In principle, there are several mechanisms by which
an enediyne-derived diyl radical could interact with a
protein. It is known that 1,4-diyls can undergo a variety
of atom transfer reactions to produce neutral arene
products, with hydrogen donors (including DNA), halo-
gens, or reactive carbon species.7 The analogous reaction
with peptides would result in generation of an intermedi-
ate peptide radical, whose fate would be determined by
environment (Scheme 1). Possibilities include scission,
via formation of an intermediate peroxy radical (as in
the case of DNA ribosyl radicals), peptide cross-linking,
or adduction with the radical source (X•). Inspection of
the chemical literature reveals that all of these pathways
are in fact precedented.8
At the most basic level, it is known that carbon cen-
tered glycyl radicals are generated on exposure of gly-
cine to peroxide sources and that they are relatively
stable, a consequence of captodative stabilization.8 In-
deed, it has been shown that glycyl radicals can be
selectively generated in preference to others, when pep-
tides are exposed to peroxy radicals.8 In one study, a
synthetic polypeptide composed of glycine/alanine showed
>30:1 selectivity for abstraction to form glycyl radicals,9
and a similar preference for glycine has also been
observed in proteins.10,11 Of additional relevance, glycyl
radicals themselves can persist under biological condi-
tions.12 In the anaerobic environment of Escherichia coli,
both pyruvate formate lyase and ribonuclease reductase
rely on generation of intermediate glycyl radicals for
proper function.13
by reports from the Bristol-Myers-Squibb laboratories,
which confirmed that a synthetic analogue of calicheami-
cin-induced5 (2) protein degradation, whereas its biologi-
cally inactive derivative, which instead targeted DNA (1),
did not.5
Though the prospect of developing protein-targeted
antitumor agents remains of interest, if the process could
be controlled on demand, an enediyne-based reagent cap-
able of inducing protein degradation could be of use as a
tool for molecular biology applications. Before engaging
in this, however, a number of background studies were
in order. Though enediyne-induced protein degradation
had been suggested,4,5 there were no studies confirming
the molecular mechanism of such a process, and this
Proof of Principle Studies. On the basis of the
reported susceptibility of glycine to radical-induced scis-
sion, we wished to demonstrate atom transfer to a diyl
from a labeled mimic of this amino acid. Though a variety
(6) Jones, G. B.; Plourde, G.; Wright, J. W. Org. Lett. 2000, 2, 811.
(7) Semmelhack, M. F.; Neu, T.; Foubelo, F. J. Org. Chem. 1994,
59, 5038. Grissom, J. W.; Gunawardena, G. U. Tetrahedron Lett. 1995,
36, 4951.
(8) Sperling, J. J. Am. Chem. Soc. 1969, 91, 5389. Sperling, J.; Elad,
D. J. Am. Chem. Soc. 1971, 93, 967. Elad, D.; Sperling, J. J. Chem.
Soc. C 1969, 1579.
(4) Zein, N.; Schroeder, D. R. In Advances in DNA Sequence Specific
Agents; Jones, G. B., Ed.; JAI Press Inc.: Greenwich, CT, 1998, 3, 201.
Zein, N.; Reiss, P.; Bernatowic, M.; Bolgar, M. Chem. Biol. 1995, 2,
451.
(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. 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, 1049.
(9) Elad, D.; Schwarzberg, M.; Sperling, J. J. Chem. Soc., Chem.
Commun. 1970, 617.
(10) Easton, C. J. Chem. Rev. 1997, 97, 53.
(11) Sperling, J.; Elad, D. J. Am. Chem. Soc. 1971, 93, 3839.
(12) Marsh, E. N. G. BioEssays 1995, 17, 431. Pedersen, J. Z.;
Finazzi-Agro, A. FEBS 1993, 325, 53.
(13) Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995, 2, 793. Free
Radicals in biology and medicine; Halliwell, B., Gutteridge, J. C., Eds.;
Oxford Science: New York, 1999.
9790 J. Org. Chem., Vol. 70, No. 24, 2005

Protein Degradation Capacity of Enediynes
TABLE 1. Yields and Isotopic Ratios for Atom
Transfera
SCHEME 2. Atom Transfer Using d2-Gly Mimic
entry
adduct
method
yield (%)
D0:D1:D2b
1
2
8
8
thermal
hν
thermal
hν
thermal
hν
thermal
hν
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
3
10
10
12
12
14
14
16
16
4
5
6
2:2:1
7
2:2:1
8
7:2:1
9
thermal
hν
4:2:1
SCHEME 3. d-Atom Transfer from 5 to Diyls
10
3:2:1
a Yields based on GC analysis, calibrated using internal (n-
decane) and external standards in reactions at 0.01 M (mesitylene)
using 10-100 equiv 5. b Ratio determined by H/D NMR and GC/
MS analysis using an authentic dideutero adduct prepared by
incubation of enediyne with cyclohexadiene-d8 or THF-d8.
SCHEME 4. Fate of Glycyl Radical
allene 13,16 whose half-lives for cycloaromatization are
approximately 18 and 24 h at 37 °C, respectively, cyclized
readily at physiological temperature, giving low yields
of labeled cycloaromatization adducts 12 and 14, which
could be increased slightly under photochemical condi-
tions. Finally, the hydroxymethyl enediyne 15 was
employed. The marked increase in chemical conversion
to 16 suggested that electrostatic effects between the
donor group and the pendant functionality might con-
tribute to the efficiency of diyl capture. The higher ratios
of D1 over D2 adducts observed presumably reflect the
increased stability and discriminatory capacity of the
diyl, in comparison to the more reactive monoradical
which may abstract from a more diverse range of (H)
atom donors.
Atom transfer from 5 implies generation of a captoda-
tively stabilized radical 17, which could be expected to
either dimerize (18) or react with molecular oxygen to
form peroxides which could ultimately result in fragmen-
tation via a transient iminium ion (Scheme 4). To probe
these scenarios, an authentic sample of 18 was prepared
using an organic peroxide. Reanalysis of the crude
reaction mixtures (Scheme 3) confirmed formation of 18
as the principal byproduct following atom transfer.
Unsurprisingly, because all reactions were conducted
under deoxygenated conditions, products derived from
hydroperoxides were not isolated. Though the experi-
ments showed clear evidence of atom-transfer chemistry
from 5, the relevance of these events in an aqueous
of options are available, we elected to use the dideu-
terioglycine 5 because an efficient synthesis is available14
and its solubility in organic media would allow flexibility
in the choice of enediynes. Accordingly, we prepared a
number of synthetic enediyne core structures 3 designed
to undergo cycloaromatization to yield diyls 4 in the
presence of 5 (Scheme 2). The enediynes were selected
on the basis of their differing modes of activation and
propensity to undergo cycloaromatization and are all
available using an efficient metallohalocarbenoid route
developed in our laboratories.15 Thermal cyclization of
Z-enediyne 7 proceeded at 280 °C to give the correspond-
ing arenes 8 isolated as a mixture of mono- and dideu-
terated species (Scheme 3, Table 1). Alternatively, pho-
tochemical Bergman cycloaromatization can be initiated
with suitable substrates, and enediyne 7 likewise gave
arenes 8 in moderate yield following 3 h of irradiation
using a low-pressure mercury lamp, presumably involv-
ing Z-E photoisomerization and subsequent capture.6
Similar behavior was observed for enediyne 9. By moving
to more reactive precursors, enediyne 11 and eneyne
(14) Braslau, R.; Anderson, M. O. Tetrahedron Lett. 1998, 39, 4227.
(15) Jones, G. B.; Wright, J. M.; Plourde II, G. W.; Hynd, G.; Huber,
R. S.; Mathews, J. E. J. Am. Chem. Soc. 2000, 122, 1937.
(16) Myers, A. G.; Parish, C. A. Bioconjugate Chem. 1996, 7, 322;
Myers, A. G. Tetrahedron Lett. 1987, 28, 4493.
J. Org. Chem, Vol. 70, No. 24, 2005 9791

Fouad et al.
SCHEME 6. Photo-Bergman Cyclization
SCHEME 5. Atom Transfer in Aqueous and
Organic Media
controlled process, the naturally occurring enediynes all
possess elaborate triggering functions,17 which induce
cycloaromatization on interaction with a cofactor. An
interesting variant is the photochemically activated
Bergman cyclization, as first demonstrated by Turro and
Nicolaou in the form of enediyne 23, which converts to
diyl 24 on irradiation (Scheme 6).18 Capitalizing on this
process, Funk and Williams designed the pyrene ene-
diyne 25, which by virtue of the aminoalkyl side chain
had affinity for DNA. Irradiation of this agent in the
presence of pBR322 DNA resulted in degradation, thus
constituting the first example of an enediyne-based
photocleaver.19 Spurred by these findings, a number of
other systems have been demonstrated,20 in what is
becoming a rapidly maturing field.21 Our challenge thus
became to design a similar system for photochemical
protein degradation.
de Novo Class of Photoenediynes. With the atom-
transfer chemistry of enediyne-derived diyls with peptide
mimic 5 proven, under both thermal and photochemical
activation conditions, we turned our attention to protein
targets. The preliminary results served to illustrate the
impact that hydrophilic functionality has on abstraction
efficiency, and it became clear that, for application to
protein targets, considerable flexibility would need to be
built into the system. We elected to design enediyne
systems where alkyne termini could be derivitized with
appropriate protein recognition functions and the pho-
toactivity of the Bergman cycloaromatization could be
tuned. Specifically, because most photoactivated ene-
diynes studied to date have the vinyl moiety embedded
in an arene, we wished to design a family of differentially
capped alicyclic enediynes 26 and investigate their
photochemical activation to diyls 27, as a function of ring-
strain and electronic effects (Scheme 7).22
TABLE 2. Isotopic Ratios for Atom Transfer with 20a
entry
solvent
donor
% 22
D0:D1:D2
1
2
3
4
5
6
CH3OH
15
17
<5
28
30
42
CH3OD
5:4:1
H2O
glycine
glycine
glycine-d5
5
H2O/(NH4)2HPO4
H2O/(NH4)2HPO4
H2O/(NH4)2HPO4
10:2:1
3:2:1
a Ratio determined by H/D NMR and GC/MS analysis using an
authentic dideutero adduct.
environment remained in question. To address this issue,
a water-soluble enediyne 20 was prepared, by reacting
the shelf-stable cobalt complex 19 with isophthaloyl
chloride (Scheme 5)15 and then immediately unmasking
the enediyne moiety. Cycloaromatization of 20 in neat
1,4-cyclohexadiene gave 22 in >70% yield; however, our
primary interest was with aqueous applications. Though
carboxylate 20 was freely soluble in polar organic sol-
vents, phosphate buffer was required to achieve full
homogeneity in aqueous conditions. To our delight, it was
discovered that the cycloaromatization is indeed viable
in aqueous media, and the corresponding atom-transfer
adducts formed at 37 °C under aerobic conditions. The
process was more efficient using 5 rather than d5 (or H5)
glycine as the atom-transfer agent, suggesting that the
abstracting ability in the diyls hydrophobic microenvi-
ronment is enhanced with this neutral donor and that
this has a positive impact on the rate of cycloaromatiza-
tion (Table 2).
Though a complex mixture of other byproducts was
formed in addition to dimer 18, both glyoxylate and
acetamide were confirmed, suggesting the intermediacy
of a peroxide under aerobic conditions (Scheme 4).
Through repetition of the incubations under deoxygen-
ated conditions (triple freeze-thaw cycles), the efficiency
of formation of the peroxy-derived byproducts was re-
duced, suggesting some control may be possible in
governing the fate of radical 17.
(17) Maier, M. E. Synlett 1995, 13.
(18) Turro, N. J.; Evenzahav, A.; Nicolaou, K. C. Tetrahedron Lett.
1994, 35, 8089. Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc. 1998,
120, 1835. Also see: Lewis, K. D.; Matzger, A. J. J. Am. Chem. Soc.
2005, 127, 9968. Kovalenko, S. V.; Alabugin, I. V. J. Chem. Soc., Chem.
Commun. 2005, 1444.
(19) Funk, R. L.; Young, E. R. R.; Williams, R. M.; Flanagan, M. F.;
Cecil, T. L. J. Am. Chem. Soc. 1996, 118, 3291.
(20) Kaneko, T.; Takahashi, M.; Hirama, M. Angew. Chem., Intl.
Ed. Engl. 1999, 38, 1267. Nahm, S.; Weinreb, S. M. Tetrahedron Lett.
1981, 22, 3815. Kim, C.-S.; Diez, C.; Russell, K. C. Chem.-Eur. J. 2000,
6, 1555. Ramakumar, D.; Kalpana, M.; Varghese, B.; Sankararaman,
S. J. Org. Chem. 1996, 61, 2247. Choy, N.; Blanco, B.; Wen, J.; Krishan,
A.; Russell, K. C. Org. Lett. 2000, 2, 3761. Clark, A.; Davidson, E. R.;
Zaleski, J. M. J. Am. Chem. Soc. 2001, 123, 2650. Alabugin, I. V.;
Kovalenko, S. V. J. Am. Chem. Soc. 2002, 124, 9052.
(21) Jones, G. B.; Russell, K. C. The Photo-Bergman Cycloaroma-
tization of Enediynes. In CRC Handbook of Photochemistry and
Photobiology; Horspool, W., Lenci, F., Eds; CRC Press: Boca Raton,
FL, 2004, 29-1.
Developing Photochemically Triggered Reagents.
Though the Bergman cycloaromatization is a thermally
(22) Jones, G. B.; Warner, P. M. J. Am. Chem. Soc. 2001, 123, 2134.
9792 J. Org. Chem., Vol. 70, No. 24, 2005

Protein Degradation Capacity of Enediynes
SCHEME 7. Photochemical Bergman
Cycloaromatization
SCHEME 9. Preparation and Photo-Bergman
Cyclization of Enediyne Hybrids
SCHEME 8. Synthesis of Alicyclic Photo-Bergman
Candidates
TABLE 3. Synthesis and Cycloaromatization of 31
entry
n
c-d (Å)a
time (h)b
convc
% 33d
1
2
3
4
5
6
2
3
4
4
5
6
4.941
4.284
4.018
4.018
3.947
3.893
3
3
100e
97
0(0)
13(13)
15(31)
21(22)
11(16)
9(14)
3
49
12
12
12
95
70
lecular “c-d” distances relative to the six-membered
analogue (Table 3).1 Though photo-Bergman cycloaro-
matization yields are modest, the synthesis of this class
of enediynes (four- through eight-membered) is notewor-
thy and may lead to many new applications in materials
and polymer chemistry.26
66
a Equilibrium geometry calculated using PM3 (PC Spartan Pro).
b Reactions conducted at 0.4 g/L enediyne n-2-propanol in a quartz
vessel and irradiated using a 450 W lamp. c % conversion as
determined by GC/MS. d Isolated yields (those based on recovered
starting material). e Decomposition and formation of polymeric
adducts ensues in <1 h.
Applications. (i) Bovine Serum Albumin. With a
route to photoactivated enediynes secure, we wished to
investigate interaction of the intermediate diyl radicals
27 with protein targets and, accordingly, sought to
prepare a hydrophilic variant, which was capable of
recognizing protein architecture. Our design was influ-
enced by the work of Kumar,27 who reported that an alkyl
pyrenyl derivative of phenylalanine (34) recognizes the
protein’s bovine serum albumin (BSA) and lysozyme,
inducing photocleavage following irradiation in the pres-
ence of an electron acceptor.27 Molecular modeling studies
indicated that diyls 35 would be close mimics of this
agent and could presumably be generated via irradiation
of an enediyne precursor. Accordingly, enediyne 40
became a logical candidate for proof-of-principle studies.
First, diketone 36 was prepared from adipoyl chloride
using a mixed coupling procedure. Subsequent functional
group interconversion and carbenoid coupling gave the
differentially functionalized C-6 enediyne, which was
unmasked to give 37 (Scheme 9).
Commencing from commercially available acyl chlo-
rides 28, we produced the corresponding diketones 29 via
the intermediate Weinreb amides (Scheme 8).23 Low
valent Ti-mediated coupling gave 31 directly, but the
yields were variable, in part, because of problems en-
countered recovering product from complex mixtures.
Alternatively, conversion to bromides 30 followed by
carbenoid coupling gave 31 (n ) 1-5) in good yield on a
preparative scale.24 Though stable at room temperature,
photochemical Bergman cycloaromatization gave adducts
33 in moderate yield, presumably via diyls 32 (Table 3).
As had been found previously, the nature of the hydrogen
donor plays an important role in the conversion to an
arene adduct.25 Thus, although consumption of enediyne
was often rapid using 1,4-cyclohexadiene, optimal yields
of cycloaromatization products were obtained using 2-pro-
panol, the balance of material typically composed of
uncharacterized polymeric byproducts. Evidently, ring-
strain effects play a role in the cycloaromatization, with
lower conversion efficiency observed with the C-7 and C-8
analogues despite the appreciable reduction in intramo-
(26) Modern Acetylene Chemistry; Stang, P. J., Diederich, F., Eds.;
VCH: Weinheim, Germany, 1995. John, J. A.; Tour, J. J. Am. Chem.
Soc. 1994, 116, 5011. Okamura, W. H.; Sondheimer, F. J. Am. Chem.
Soc. 1967, 89, 5991. Anthony, J. A.; Boudon, C.; Diederich, F.;
Gisselbrecht, J. P.; Gramlich, V.; Gross, M.; Hobi, M.; Seiler, P. Angew.
Chem., Int Ed. 1994, 33, 763. Gobb, L.; Seiler, P.; Diederich, F. Angew.
Chem., Int Ed. 1999, 38, 674.
(23) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
(24) Jones, G. B.; Wright, J.; Plourde, G.; Purohit, A.; Wyatt, J.;
Hynd, G.; Fouad, F. J. Am. Chem. Soc. 2000, 122, 9872.
(27) Kumar, C. V.; Buranaprapuk, A.; Opiteck, G. J.; Moyer, M. B.;
Jockusch, S.; Turro, N. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10361.
(25) Kim, C.-S.; Diez, C.; Russell, K. C. Chem.-Eur. J. 2000, 6, 1555.
J. Org. Chem, Vol. 70, No. 24, 2005 9793

Fouad et al.
FIGURE 1. 10% SDS polyacrylamide gel of the reaction
bovine serum albumin (BSA) (1 µM) with 42b. All assays were
conducted in 50 mM Tris-HCl, pH 7.0, at 37 °C. Lane 1:
molecular weight markers (kDa). Lane 2: BSA, no irradiation.
Lane 3: BSA, 3 h irradiation. Lane 4: BSA, 12 h irradiation.
Lane 5: BSA + 41 (10 µM), 12 h irradiation. Lane 6: BSA +
42b (10 µM), no irradiation. Lane 7: BSA + 42b (10 µM), 1 h
irradiation. Lane 8: BSA + 42b (10 µM), 12 h irradiation.
Photo-Bergman cycloaromatization of this enediyne (or
the triisopropylsilyl (TIPS) protected precursor) gave
<10% of the corresponding arene product, suggesting
that the aryl groups play a key role in the (reversible)
cyclization process.22 The alkyne was then coupled with
methyl-3-(3-iododphenyl)hexynoate 38, followed by hy-
drolysis to give carboxylate 39, which was then coupled
with L-phenylalanine methyl ester and subjected to
saponification to give conjugate 40 in good yield. In
contrast to the model series, photochemical activation
(450 W, 3 h) of this enediyne gave arene product 41 in
appreciable yield, together with unreacted starting mate-
rial, underscoring the influence of the conjugated arene
unit. Photochemically induced modification of BSA and
lysozyme was examined by photolyzing 40 in aqueous
buffer and using 41 and enediynes 39 and 31 as controls.
In the case of 66 kD protein BSA, subsequent analysis
(SDS-PAGE) showed some evidence of enediyne-induced
degradation with one principal fragment (∼40 kDa)
visible (<5% relative to parent) when a 5:1 ratio of
enediyne 40/BSA was employed.28 Encouraged by this
initial finding, we sought analogues with improved
affinity for BSA in the hope this would translate to
improved photocleavage capacity. Modification of the
synthesis could be affected, allowing preparation of alkyl-
linked conjugates. This required hydrogenation of inter-
mediate 38, followed by a procedure identical to that
employed for the preparation of 40, giving access to
enediynes 42a-c. Of these agents, 42b and 42c showed
enhanced affinity for BSA, and photolysis was con-
ducted.28 Significant cleavage into two principal frag-
ments (31 and 35 kDa) was observed, tempting the
correlation between affinity and cleavage efficiency (Fig-
ure 1).28 Consistent with the outcome of the photodeg-
radation experiment, Scatchard analysis suggested that
only one binding site is involved.28,27 In the case of the
14 kDa protein lysozyme, dimerization to form a species
with m/z 28.6 kDa was observed following irradiation
with 40 (Figure 2), suggesting the possibility of inter-
molecular diyl-mediated cross-coupling.28 These initial
findings encouraged us to search for photoproteases with
specific targets of biological relevance, and we were
encouraged to use carboxylate 39 as a building block.
FIGURE 2. 15% SDS polyacrylamide gel of the reaction of
lysozyme (Lyso) with 40 and 41. All reactions were conducted
in 50 mM Tris-HCl, pH 7.0, at 37 °C. Lane 1: molecular
weight markers. Lane 2: Lyso (0.15 µM). Lane 3: Lyso (0.15
µM), 3 h irradiation. Lane 4: Lyso (0.15 µM) and 40 (0.75 µM),
6 h irradiation. Lane 5: Lyso (0.15 µM) and 40 (0.75 µM), 3 h
irradiation. Lane 6: Lyso (0.15 µM) and 41 (15 µM), 12 h
irradiation. The running gel was overlaid with a 4% stacking
gel, and samples were run from top to bottom. Analogous
reactions with 31 (n ) 4) showed no change from the control
(data not shown).
SCHEME 10. Synthesis of Tri(Asp) Conjugate
(ii) Histone H1. In the case of the naturally occurring
chromoproteins kedarcidin, C-1027, and neocarzinostatin,
reports that the apoprotein components have proteolytic
ability have been made.4 In the case of kedarcidin, studies
suggested that the highly acidic apoprotein was capable
of inducing selective degradation of histone H1.4 As this
is the most basic histone (34% arginine/lysine content),
a natural conclusion was that affinity plays a role.
Following proteolysis of the histone, it was suggested that
the reactive enediyne chromophore could then more
efficiently deliver to exposed DNA, contributing to the
high efficiency of strand scission by kedarcidin.4 On the
basis of the present findings, we became interested in
preparing an acidic enediyne-peptide conjugate to de-
termine if enediyne-mediated histone degradation could
be achieved and, if so, with what specificity. To investi-
gate this, enediyne 39 was coupled with the triaspartic
acid derivative 43 (Scheme 10). Deprotection of the
(28) Plourde, G.; El-Shafey, A.; Fouad, F. S.; Purohit, A. S.; Jones,
G. B. Bioorg. Med. Chem. Lett. 2002, 12, 2985. Binding constants of
40 and 42 were determined following Scatchard analysis: BSA 40 )
1.6 × 105 dm3 mol-2; 42a ) 1.8 × 105 dm3 mol-2; 42b ) 6.7 × 105 dm3
mol-2; 42c ) 5.8 × 105 dm3 mol-2; and lysozyme 40 ) 0.3 × 105 dm3
mol-2; 42b ) 0.1 × 105 dm3 mol-2. Densitometric analysis of Coomassie
stained gels (processed with Adobe Photoshop/NIH-image software)
quantitated the BSA 29/37 kDa fragments (Figure 1) as 43% relative
to the parent and the lysozyme dimer (28 kDa, Figure 2) at 8% relative
to the parent. For procedures, see: Scatchard, G. Ann. N. Y. Acad.
Sci. 1949, 51, 660. MS analyses were performed using desalted
mixtures directly following incubation using a sinapinic acid (BSA) or
an R-cyano-4-hydroxycinnamic acid matrix (lysozyme). For a discussion
on binding sites in BSA, see ref 27.
9794 J. Org. Chem., Vol. 70, No. 24, 2005

Protein Degradation Capacity of Enediynes
SCHEME 11. High Affinity Carbocyclic
Enediyne-Estrogens
FIGURE 3. Scatchard plot of histone H1 with enediyne-
triaspartic acid conjugate 44. Solutions of enediyne 44 in a
minimum quantity of methanol were diluted with 25 mM
Tris-HCl (pH ) 7.0) buffer. Binding was assessed spectro-
photometrically over eight different concentrations of histone
(0-50 µmol range) with fixed 44 (20 µmol). A curve was
constructed via Scatchard analysis using: r/Cf ) Kb (n - r),
where r is the number of moles enediyne bound per mole of
protein, Cf is the free-enediyne concentration, n is the number
of binding sites, and Kb is the binding constant. This figure
illustrates r/Cf (y axis) vs r (x axis), revealing a Kb of 7.2 × 105
dm3 mol-2, and n ) 2.3.
FIGURE 4. 16.5% SDS polyacrylamide Tris-tricine gel of the
reaction of histone H1 (21.5 kDa, 1 µM) with 44. All reactions
were conducted in 50 mM Tris-HCl, pH 7.0. Lane 1: molec-
ular weight markers (kDa). Lane 2: histone H1, no irradiation.
Lane 3: histone H1, 1 h irradiation. Lane 4: histone H1, 12
h irradiation. Lane 5: histone H1 + 44 (10 µM), no irradiation,
12 h. Lane 6: histone H1 + 44 (10 µM), 1 h irradiation. Lane
7: histone H1 + 44 (10 µM), 12 h irradiation.
conjugate with LiOH gave the enediyne-triasp conjugate
44 in good yield. As expected, this conjugate had ap-
preciable solubility in aqueous buffer. Binding of this
agent to a variety of histones revealed that affinity, as
expected, was greater for H1 versus others, and Scat-
chard analysis suggested that (in the case of H1) two
binding sites are involved (Figure 3).29 More significantly,
irradiation of 44 in the presence of histone H1 (21.5 kDa)
led to degradation of the protein into principal compo-
nents in the 8-11 kDa range (Figure 4).29 The application
of a designed photoactivated enediyne with near micro-
molar affinity for a biological target is noteworthy and
suggested even greater affinity might be attainable by
coupling the photowarhead to the ligand with a compli-
mentary protein receptor.
(iii) Human Estrogen Receptor R. As a final ex-
ample of application, we elected to study agents with the
capability of degrading the human estrogen receptor
(hER), as lipophilic steroidal ligands with nanomolar
affinity are well recognized. Though a multitude of
substituted steroid templates are available via direct
synthesis, we were particularly interested in commencing
from a commercially available building block. Surveying
numerous options, we decided to utilize estrone, intro-
ducing the enediyne functionality at the 17R position.
Through quantitative structure-activity relationship
(QSAR) analysis, it has been shown that 17R-alkynyl
derivatives of estradiol show comparable hER affinity to
its endogenous ligand (estradiol),30 and we decided to
construct a hybrid with an alkynyl linker between the
enediyne and the steroidal template. Accordingly, ene-
diyne 37 was coupled to a protected (TBDMS) ether of
estrone via its organocerium derivative, then unmasked
to give 45 (Scheme 11). Unfortunately, this compound,
which had a relative binding affinity (RBA) of >100 nM
(Table 4), proved stable at physiological temperature and
did not undergo the expected photo-Bergman cycloaro-
matization to give 46 even on prolonged irradiation.
Following a strategy adopted previously, we elected to
insert a spacer unit in the system between the enediyne
and ligand and pursued m-disubstituted ligand 49.
(29) Binding constants determined following Scatchard analysis: 44
histone H1 ) 7.2 × 105 dm3 mol-2 and histone H2 ) 1.7 × 105 dm3
mol-2; 42b histone H1 ) 0.4 × 105 dm3 mol-2. Control reactions using
40 and 42 failed to produce any discernible photodegradation products
(histone H1) after 12 h irradiation (10 µM).
(30) Gao, H.; Katzenellenbogen, J. A.; Garg, R.; Hansch, C. Chem.
Rev. 1999, 99, 723.
J. Org. Chem, Vol. 70, No. 24, 2005 9795

Fouad et al.
TABLE 4. Relative Binding Affinity of
Steroid-Enediynes for ERa
entry
substrate
RBA (nM)
1
2
3
45
49
53
103
14
12
a Candidate + 3H â-estradiol incubated with cytosol at 4 °C.
Unbound agents were removed (DCC), and bound 3H â-estradiol
was measured by a scintillation counter. RBA corresponds to the
concentration required to reduce hER bound 3H â-estradiol by
50%.32
FIGURE 5. 10% SDS polyacrylamide gel of the reaction of
the estrogen receptor (ER) (1.5 µg/mL) with 53 (550 µM). All
reactions were conducted in 20 mM Tris-HCl buffer at pH
8.0. Lane 1: molecular weight markers (kDa). Lane 2: ER,
no irradiation. Lane 3: ER, 6 h irradiation. Lane 4: ER, 12 h
irradiation. Lane 5: ER + 53, no irradiation. Lane 6: ER +
52, 12 h irradiation. Lane 7: ER + 53, 1 h irradiation. Lane
8: ER + 53, 12 h irradiation.
SCHEME 12. High Affinity Photoactivated
Enediyne-Estrogens
Irradiation in the presence of recombinant hER (66 kDa)
resulted in marked degradation to produce two discrete
fragments within 12 h (31 and 35 kDa) and control
reactions confirming the effect of the enediyne warhead
(Figure 5). The ability to induce on demand degradation
of a nuclear receptor using a lipophilic warhead is of
significance and underscores the potential use of such
entities as molecular reagents.
In summary, the viability of proteins as targets of
enediynes has been confirmed at the molecular level. A
new series of photochemically activated enediynes has
been prepared using an intramolecular coupling strategy,
and derivatives are used for specific protein targeting.
On the basis of molecular architecture, three independent
classes of enediynes with defined protein targets have
been identified (albumin, histone, and estrogen receptor)
and show correlation between affinity and protein de-
grading activity. On the basis of these findings, coupled
with expeditious synthetic routes to the core warhead,
enediyne reagents capable of inducing specific protein
modulation can now be designed. Such affinity cleavage
systems may offer advantages over conventional methods
because they do not function via metal ion redox chem-
istry,33 presenting instead a lipophilic warhead. Further-
more, the biological significance of proteins as targets for
enediyne-derived diyls has been strengthened by recent
reports. Thorson has demonstrated that an organism
which produces one of the naturally occurring enediyne
chromophores is self-protected by an associated protein-
based suicide substrate, which intercepts diyls produced
by enediyne degradation resulting in formation of a glycyl
radical in the protein.34 Hirama has also demonstrated
that the C-1027 apoprotein is slowly degraded by the
Coupling of 37 with iodoalkyne 47 gave 48, which was
unmasked and subjected to 1,2 addition to estrone TBS
ether and, finally, followed by phenolic deprotection to
give 49. To our surprise, the affinity of 49 was greatly
superior to that of 45, supporting the notion that the
ligand binding domain (LBD) of hERR has considerable
tolerance for lipophilic functionality at the steroidal 17R
position.31 Though the binding affinity of 49 was spec-
tacular, to our disappointment, its photo-Bergman cy-
cloaromatization profile was poor, giving only trace
amounts of the expected cycloaromatization product even
after prolonged irradiation. We elected to improve the
efficiency of the critical process by extending the conjuga-
tion of the ligand and pursued 53 as a refined target.
Though this required complete resynthesis, an efficient
process was developed via preassembly of enediyne
template 52 (Scheme 12). This was coupled in turn to
iodoarene 51, produced by arylation of the unmasked
alkyne derived from 50, which could be produced from
either estrone TBS ether or the commercially available
17R-ethynyl estradiol. Though the binding affinity of 53
did not differ markedly from 49 (Table 4),32 this ligand
underwent smooth photocycloaromatization to give com-
plete conversion to cycloaromatized product within 12 h.
(31) Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.;
Bonn, T.; Engstrom, O.; Ohman, L.; Greene, L.; Gustafsson, J.-A.;
Carlquist, M. Nature 1997, 389, 753.
(32) Jones, G. B.; Wright, J. M.; Hynd, G.; Wyatt, J. K.; Yancisin,
M.; Brown, M. A. Org. Lett. 2000, 2, 1863. Jones, G. B.; Purohit, A.;
Wyatt, J.; Swamy, N.; Ray, R. Tetrahedron Lett. 2001, 42, 8579.
(33) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1991, 113, 1859.
Hoer, D.; Cho, H.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112, 3249.
Cuenoud, B.; Tarasow, T. M.; Schepartz, A. Tetrahedron Lett. 1992,
33, 895. Hegg, E. L.; Burstyn, J. N. J. Am. Chem. Soc. 1995, 117, 7015.
Wu, J.; Perrin, D. M.; Sigman, D.; Kaback, H. R. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 9186. Parac, T. N.; Kostic, N. M. J. Am. Chem.
Soc. 1996, 118, 5946. Kumar, C. V.; Buranaprapuk, A. J. Am. Chem.
Soc. 1999, 121, 4262. Miyake, R.; Owens, J. T.; Xu, D.; Jackson, W.
M.; Meares, F. J. Am. Chem. Soc. 1999, 121, 7453.
(34) Biggins, J. B.; Onwueme, K. C.; Thorson, J. S. Science 2003,
301, 1537.
9796 J. Org. Chem., Vol. 70, No. 24, 2005

Protein Degradation Capacity of Enediynes
diradical derived from its enediyne chromophore.35 Ac-
cordingly, the prospect of designing synthetic enediynes
to incapacitate specific protein targets resulting from the
human proteome project has become a realistic goal. The
findings may also prove important to our understanding
of the naturally occurring enediyne chromoproteins and,
specifically, to the interactions of chromophore and
apoprotein components of these clinically relevant anti-
biotics.1,36
CH2Cl2 (25 mL), the resulting solution was cooled to 0 °C, and
then bromine (0.753 g, 4.71 mmol, 0.243 mL) was added
dropwise over 5 min. The resulting suspension was allowed
to stir for 0.5 h, and then 2,6-lutidine (1.51 g, 14.13 mmol,
1.64 mL) was added. The resulting solution was stirred for an
additional 0.5 h, and then a solution of 1,10-diphenyl-1,9-
decadiyne-3,8-diol (0.500 g, 1.57 mmol), dissolved in CH2Cl2
(10 mL), was added dropwise via cannula. The reaction
mixture was allowed to slowly warm to 10 °C over 2 h, diluted
with n-pentane (30 mL), and stirred an additional 10 min at
0 °C. The suspension was filtered through a bed of silica gel
and rinsed with n-pentane (3 × 50 mL). The combined organic
eluents were concentrated in vacuo, and the resulting oil was
purified via SGC (100% hexanes) to yield the title compound
(0.48 g, 69%) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ
7.46 (m, 4H), 7.38 (m, 6H), 4.81 (2H, t, J ) 6.6 Hz), 2.15 (m,
4H), 1.71 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 132.0, 129.0,
128.5, 122.2, 88.0, 87.3, 39.6, 37.8, 26.7; IR (neat) 3010, 2960,
2240, 1430, 1330, 1209, 1145, 910, 621 cm-1. C22H20Br2
Requires: C, 59.49; H, 4.54. Found: C, 59.67; H, 4.82.
1,2-Di(2-phenyl-1-ethynyl)-1-cyclohexene (31, n ) 4).
In a 100 mL round-bottom flask was placed dibromide 30, n
) 4 (0.100 g, 0.226 mmol), THF (23 mL), and HMPA (1.01,
5.65 mmol, 0.983 mL). The resulting solution was cooled to
-78 °C, and LiHMDS (0.565 mmol, 0.565 mL of 1.0 M in THF)
in THF (5 mL) was added via cannula over the course of 2.0
h. On completion of the base addition, the reaction mixture
was allowed to stir for an additional 0.5 h and then poured
onto a saturated solution of NH4Cl (25 mL). The organic
material was extracted into Et2O (3 × 25 mL), and the
combined extracts were treated with 10% HCl (2 × 25 mL),
water (2 × 25 mL), saturated NaHCO3 (2 × 25 mL), and brine
(1 × 25 mL). The combined organic extracts were dried over
MgSO4 and concentrated in vacuo. The residue was purified
via radial chromatography (100% hexanes) to afford the title
compound (0.593 g, 94%) as a white solid, mp 72 °C, that was
spectroscopically identical to that reported:39 1H NMR (300
MHz, CDCl3) δ 7.50 (m, 4H), 7.31 (m, 6H), 2.39 (m, 4H), 1.71
(m, 4H); 13C NMR (75 MHz, CDCl3) δ 131.5, 128.3, 128.1, 126.5,
123.6, 93.6, 90.4, 30.0, 21.8.
Experimental Procedures15
1,10-Diphenyl-1,9-decadiyne-3,8-dione (29; n ) 4). A
suspension of N,O-dimethylhydroxylamine hydrochloride (3 g,
30.75 mmol) and Et3N (10 mL, 70 mmol) in CH2Cl2 (50 mL)
was cooled to 0 °C.15 Adipoyl chloride (14 mmol) was added,
and the mixture was left to stir at 0 °C for 1 h, warmed to 25
°C, stirred for an additional 1 h, and then quenched with brine
(50 mL). The product was extracted with CH2Cl2 (2 × 100 mL),
washed with brine (3 × 100 mL), and dried (MgSO4). Solvents
were removed in vacuo, and the residue was purified via a
short column (silica gel, 1% Et3N in ether) to give the
corresponding crude amide:37 1H NMR (300 MHz, CDCl3) δ
3.67 (s, 6H), 3.16 (s, 6H), 2.44 (bs, 4H), 1.67 (m, 4H); 13C NMR
(75 MHz, CDCl3) δ 174.0, 60.9, 31.9, 31.4, 24.1.
Phenyl acetylene (2.11 mL, 1.97 g, 19.27 mmol) was dis-
solved in THF (80 mL), and the solution was cooled to -10
°C. n-BuLi (19.27 mmol, 2.8 mL of 2.5 M in hexanes) was
added dropwise over 10 min, and the mixture was warmed to
room temperature (0.5 h), cooled to 0 °C, and cannulated onto
a cold solution (0 °C) of the crude Weinreb amide (8.76 mmol)
in THF (90 mL). The reaction was warmed to room temper-
ature (40 min) and quenched with HCl (5%, 50 mL), and then
brine (50 mL) was added. The mixture was extracted with
ethyl acetate (3 × 150 mL), and the organic layers washed
with brine (2 × 100 mL), dried (Na2SO4), and condensed in
vacuo. The residue was then purified with SGC (10% EtOAc/
hexanes) to give 29, n ) 4 (2.063 g, 75%), as a light brown
oil:38 1H NMR (300 MHz, CDCl3) δ 7.46 (m, 4H), 7.32 (m, 6H),
2.21 (m, 4H), 1.91 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 187.4,
133.2, 131.0, 128.9, 120.0, 90.59, 88.0, 43.2, 23.6. Anal. Calcd
for C22H18O2: C, 84.05; H, 5.77. Found: C, 84.10; H, 5.89.
1,10-Diphenyl-1,9-decadiyne-3,8-diol. To a 50 mL round-
bottom flask was added ketone 29, n ) 4 (1.0 g, 2.4 mmol),
and MeOH (10 mL). The resulting solution was cooled to -15
°C, and NaBH4 (0.181 g, 4.8 mmol) was added in one portion.
The mixture was allowed to warm to room temperature over
1.0 h, then carefully poured onto cold HCl (2%, 20 mL). The
layers were separated, and the aqueous layer was extracted
with ethyl acetate (3 × 20 mL). The combined organic layers
were treated with 5% HCl (2 × 20 mL) and brine (3 × 50 mL),
dried over MgSO4, and finally concentrated in vacuo. Purifica-
tion of the residual oil using SGC (30:70, EtOAc/hexanes)
afforded the title compound (0.600 g, 79%) as a colorless oil:
1H NMR (300 MHz, CDCl3) δ 7.42 (m, 4H), 7.25 (m, 6H), 4.60
6,7-Diphenyl-1,2,3,4-tetrahydronaphthalene (33, n )
4). A photochemical reaction vessel (250 mL) was charged with
bisphenylenediyne 31, n ) 4 (0.100 mg, 0.354 mmol), and
spectroscopic grade iPrOH (200 mL). The resulting solution
was deoxygenated with Ar for 30 min, then irradiated using a
450 W low-pressure mercury lamp, maintaining a slow purge
of Ar throughout the entire 12 h irradiation. The solution was
concentrated in vacuo, and the residue was purified by
preparative thin layer chromatography (TLC) (2 mm, 100%
hexanes) to yield the title compound (19 mg, 20%) as a white
solid, mp 109 °C, spectroscopically identical to that reported:
40
1H NMR (300 MHz, CDCl3) δ 7.06 (m, 6H), 6.99 (m, 4H),
2.86 (m, 4H), 1.85 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 141.8,
138.0, 136.7, 131.5, 130.0, 127.9, 126.3, 269.3, 23.4; MS (EI)
m/z 284 (M+), 254, 241, 228; HRMS calcd for C22H20 284.1565,
found 284.1520.
(t, J ) 5.7 Hz), 3.24 (br s, 2H), 1.82 (m, 4H), 1.55 (m, 4H); 13
C
Acknowledgment. We wish to acknowledge finan-
cial support from the NIH (5RO1GM57123), NSF
(MCB98661), PRF (33920AC1), American Cancer Soci-
ety, Elsa U. Pardee Foundation, and the Massachusetts
Department of Health.
NMR (75 MHz, CDCl3) δ 131.7, 128.3 (2C), 122.7, 90.4, 84.7,
62.69, 37.7, 24.9; IR (neat) 3350, 2936, 2250, 1450, 1010, 740
cm-1. C22H22O2 Requires: C, 82.99; H, 6.96. Found: C, 83.27;
H, 7.14.
3,8-Dibromo-1,10-diphenyl-1,9-decadiyne (30, n ) 4).
Triphenylphosphine (1.23 g, 4.71 mmol) was dissolved in
Supporting Information Available: Synthetic proce-
dures and spectroscopic data for the preparation of all reagents
(31 pages). This material is available free of charge via the
Internet at http://pubs.acs.org.
(35) Usuki, T.; Inoue, M.; Hirama, M.; Tanaka, T. J. Am. Chem.
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55, 8915. Satyamurthi, N.; Singh, J.; Aidhen, I., S. Synthesis 2000,
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J. Org. Chem, Vol. 70, No. 24, 2005 9797