Protein degradation with photoactivated enediyne-amino acid conjugates

Article abstract of DOI:10.1016/S0960-894X(02)00618-2

A series of photoactivated enediynes was prepared, and successfully employed for the selective degradation of target proteins.

Full text of DOI:10.1016/S0960-894X(02)00618-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2985–2988
Protein Degradation with Photoactivated
Enediyne-Amino Acid Conjugates
Gary Plourde, II, Ahmed El-Shafey, Farid S. Fouad, Ajay S. Purohit
and Graham B. Jones*
Department of Chemistry, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA
Received 21 May 2002; accepted 26 June 2002
Abstract—A series of photoactivated enediynes was prepared, and successfully employed for the selective degradation of target
proteins.
# 2002 Elsevier Science Ltd. All rights reserved.
The enediynes are an important class of antitumor
agents, with spectacular biological profiles.1 Nearly 20
discrete enediynes have been discovered, and clinical trials
of a number of these including calicheamicin 1 are
ongoing.2 Enediynes per se are biologically inactive, but
undergo cycloaromatization reactions which give rise to
cytotoxic diyl radicals, which are capable of inducing
DNA strand scission at low concentration.1 In the case
of calicheamicin this involves a cascade of reactions
which results in formation of a post-activated diyl core,
which abstracts hydrogen atoms from the DNA backbone.
On interception by molecular oxygen, an intermediate
peroxide is formed which ultimately leads to strand
scission by generation of a 50 aldehyde.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. This has prompted a
search for additional targets, including proteins. Interest
in protein targets was stimulated by reports from the
Bristol-Myers-Squibb laboratories, which confirmed
that a synthetic analogue of calicheamicin with anti-
tumoral activity, 2, induced protein degradation, pre-
sumably via its cycloaromatized diyl 3.3 To provide
proof of principle for the concept of proteinogenic sub-
strates for enediynes, we previously demonstrated that
the enediyne carboxylate derived from 4 undergoes
Bergman cycloaromatization in the presence of labeled
aminoacid 5, producing arene 6, and in the process
converting 5 to dimer.4 This implies generation of an
intermediate captodatively stabilized radical, and has
precedent based on the reported susceptibility of glycine
residues to radical induced scission.5 Though encourag-
ing, a limiting factor for application of enediynes as
reagents against protein targets would be the poor affi-
nity of such lipophilic agents. We therefore elected to
investigate enediyne-protein conjugates as a class, and
study both their affinity and protein degrading ability.
Our initial candidate was glycine derivative 7 (Scheme 1),
produced by unmasking the alkynes of 46 followed by
carbodiimide coupling with a glycine derivative, and
finally low temperature saponification (0 ꢀC) to avoid
*Corresponding author. Tel.: +1-617-373-8619; fax: +1-617-373-8795; e-mail: gr.jones@lynx.dac.neu.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00618-2

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Scheme 1. Atom transfer chemistry of thermally activated enediyne–amino acid conjugate.
Scheme 2. The photo-Bergman cycloaromatization.
premature cycloaromatization. At physiological tem-
perature 7 underwent Bergman cycloaromatization
(1,4-cyclohexadiene) to give the neutral arene (70%) with
an estimated t1/2 of 17 h. As previously the diyl could be
trapped with 5 (10 M, buffer) to give adduct 8 (3:2:1
D0:D1:D2).4 In contrast to the enediyne derived from 4
however, incubation of 7 with a panel of proteins indicated
that it is capable of inducing degradation at elevated con-
centration, yielding a combination of agglomerated
adducts and protein fragments.7 Though encouraging, a
drawback for use as a molecular biology reagent lies in
the prolonged half-life of the enediyne core, and we
elected to investigate photochemically activated variants.
Since pioneering work in the 1990s several examples of
the photo-Bergman cyclization have been reported,8
including the cyclohexylenediynes 9, which produce
arenes derived from 10 on irradiation (Scheme 2).9 We
thus sought to incorporate this motif into a substrate
with affinity for a specific protein target. In the case of
bovine serum albumin (BSA), binding of amphiphilic
substrates containing a single amino acid tethered to a
polyaromatic core has been reported, and on this basis
we elected to produce photoactivated enediyne precursors
of generic diyl 11.10 Accordingly, our initial targets
became enediyne 16, and its structural variants (Scheme
3, 19–22). Methyl-1-hex-5-ynoate 12 was coupled with
1,3-diiodobenzene, and subjected to selective hydrogena-
tion to give alkyl arene 13 (Scheme 3). Enediyne 14 was
then coupled,9 and the carboxylate function revealed to
give key building block 15. Aminoacids were introduced
using standard coupling methodology, followed by
saponification to give substrate(s) 16. Though stable
indefinitely at ambient temperature, photo-irradiation
(450 W medium pressure Hg lamp, 12 h) resulted in
smooth cycloaromatization, giving arenes 17 in the pre-
sence of an appropriate hydrogen atom donor (iPrOH,
MeOH). Likewise, modification of the synthesis allowed
preparation of alkynyl linked analogues 18. Of the
enediynes assembled, the lysine derivative 19, glutamic
acid derivative 20 and aspartic acid derivative 21 were
all assessed for binding affinity to BSA. Acid derivatives
20 and 21 had elevated affinity relative to 19,11 and
photo-induced protein cleavage was accordingly assessed.
The results were encouraging, with 21 capable of inducing
cleavage of BSA into two discrete fragments (Fig. 1).
Scatchard analysis10,11 suggested that a single binding
site exists, and presumably accounts for the single clea-
vage locus. Control studies confirmed that the photo-
activated enediyne is responsible for the cleavage, and,
when coupled with the binding data, suggests a tentative
correlation between affinity and cleavage. In an attempt
to extend the utility of the template, a number of more
elaborate derivatives were assembled. Of these, trias-
partic agent 22 was most promising, having been
designed with affinity for histones in mind. From the
work of Zein et al.12 it is known that histone H1, the
most basic of histones, is a preferential substrate for
members of the acidic family of naturally occuring ene-
diyne apoproteins, thus 22 was screened for affinity.
Near micromolar affinity for H1 was confirmed, and
photocleavage studies were then initiated. Remarkably,
following 12 h irradiation, H1 is degraded into one
principal component, which complements well the
results obtained with the enediyne chromoprotein
kedarcidin (Fig. 2).13 These findings are significant in
that they confirm that (i) controlled photodegradation
of proteins by designed enediynes can be accomplished
and (ii) that selectivity can be introduced by subtle
structural variation.14 Though preliminary, many
applications of this general method may be envisioned,
and the expeditious route to the templates 16 and 18 will
help accelerate the search.
In summary, routes to enediyne–amino acid conjugates
have been developed, and the conjugates show efficacy
in the photo-degradation of protein substrates. It is
expected that the ready availability of the templates will
now permit assembly and evaluation of both rationally
designed and combinatorial libraries of conjugates,
which may serve as valuable tools for proteomics.15

G. Plourde, II et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2985–2988
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Scheme 3. Preparation of variable linker photoactivated enediyne–amino acid conjugates.
Figure 2. 16.5% SDS polyacrylamide Tris–tricine gel of reaction of
histone H1 (1 mM) with 22. All reations were conducted in 50 mM
Tris–HCl, pH 7.0. Lane 1, molecular 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+22 (10 mM) no
irradiation 12 h; lane 6, histone H1+22 (10 mM) 1 h irradiation; lane 7,
histone H1 22 (10 mM) 12 h irradiation.
Figure 1. 10% SDS polyacrylamide gel of the reaction bovine serum
albumin (BSA) (1 mM) with 21. All assays conducted in 50 mM Tris–
HCl, pH 7.0. 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+21 (10 mM) no irradiation; lane 6, BSA+21
(10 mM) 1 h irradiation; lane 7, BSA+21 (10 mM) 12 h irradiation.

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118, 3291. Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc.
References and Notes
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51, 660). Ka (25 ꢀC) BSA 19: 1.58ꢂ105
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15. This work was supported by the National Institutes of
Health (R01GM57123).