Synthesis of orthogonally reactive FK506 derivatives via olefin cross metathesis

Article abstract of DOI:10.1016/j.bmc.2009.07.030

Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two s

Full text of DOI:10.1016/j.bmc.2009.07.030

Bioorganic & Medicinal Chemistry 17 (2009) 5763–5768
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of orthogonally reactive FK506 derivatives via olefin cross metathesis
Paul S. Marinec, Christopher G. Evans, Garrett S. Gibbons, Malloree A. Tarnowski, Daniel L. Overbeek,
*
Jason E. Gestwicki
Department of Pathology and The Life Sciences Institute, University of Michigan, 210 Washtenaw Ave, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications to con-
trol protein localization, modulate protein–protein interactions and improve drug lifetimes. These
bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity
for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses
of these bifunctional constructs, owing to the abundance and favorable distribution of their target,
FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syn-
theses and/or multiple protection–deprotection schemes prior to installation into CIDs. In this work, we
describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated,
cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified
with an array of functional groups, including primary amines and carboxylic acids, which make the
resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the syn-
thesis of a heterobifunctional HIV protease inhibitor.
Received 14 April 2009
Revised 14 July 2009
Accepted 15 July 2009
Available online 18 July 2009
Keywords:
Metathesis
Cross-coupling
Natural products
Chemical inducers of dimerization
Drug targeting
HIV protease
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ity and decreased affinity for FKBP (ꢀ10-fold compared to
FK506).24 In addition, SLF is challenging to use on a large scale be-
The natural product FK506 is a powerful immunosuppressant
that is used to prevent allograft rejection in transplant patients.1
This compound blocks immune cell function through an intriguing
mechanism in which it first interacts with the FK506-binding pro-
tein (FKBP) and then this complex binds tightly to the phosphatase
calcineurin.2,3 This unusual binding property has been mirrored in
synthetic tools termed chemical inducers of dimerization (CIDs).4–6
Like FK506, these compounds are composed of two, non-overlap-
ping chemical domains, which enables them to bridge two protein
targets. CIDs have been employed in a broad range of applications,
many focused on understanding signaling pathways, subcellular
localization and protein–protein interactions.7–14 In addition, this
platform has been used in drug discovery applications to modulate
specificity, lifetime and affinity.15–19
Despite this versatility, methods for the modular construction
of CIDs have not been extensively explored. One notable exception
is a study by the Verdine group that explored the systematic syn-
thesis of compounds coupled to the synthetic ligand for FKBP
(SLF).20 SLF is an attractive chemical component because it is a
non-immunosuppressive analog of FK506 that can be attached to
other molecules via pendant anilines18 or carboxylates.11,21–23
Although SLF has been used to synthesize many useful CIDs, there
are limitations to this approach, including modest aqueous solubil-
cause its synthesis requires ꢀ12 steps, with modest overall
yield.21,23,24 One alternative is to employ FK506 as a commercially
available FKBP-binding ligand. In fact, this strategy was used to
create the first CID, a homodimer of FK506 termed FK1012.7 How-
ever, because of its structural complexity, manipulations of FK506
are challenging; for example, reported methods for preparing reac-
tive intermediates require five steps with ꢀ50% yield.7
We reasoned that an improved entry into reactive FK506 deriv-
atives might involve selectively modifying the C39 alkene via cross
metathesis (CM) using Grubbs’ second generation ruthenium cata-
lyst (GII).25 The GII catalyst was chosen, in part, because it is toler-
ant of the many functional groups found in FK506, such as alcohols
and ketones. In support of this idea, both the Wender and Schreiber
groups have diversified complex natural products via CM at a ter-
minal alkene.26,27 Another attractive feature of this approach is
that the extracyclic C39 position of FK506 is crucial for binding cal-
cineurin. Thus, modifications at this site would be expected to
block immunosuppression without impacting the affinity for
FKBP.28 Consistent with this model, Clemons et al. used CM to
selectively append bulky groups to the C39 position on FK506 to
interrupt binding to calcineurin27 and Diver and Schreiber used
CM to produce FK1012-like homodimers, which are non-immuno-
suppressive but able to dimerize chimeric FKBP fusions.29 Based on
these studies, we sought to explore CM as a means to develop
modular building blocks for the rapid creation of FKBP-binding
CIDs.
* Corresponding author.
E-mail address: gestwick@umich.edu (J.E. Gestwicki).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.030

5764
P. S. Marinec et al. / Bioorg. Med. Chem. 17 (2009) 5763–5768
our selection criteria, as we hoped to produce a range of FK506
2. Results and discussion
intermediates containing orthogonally reactive electrophiles and
nucleophiles for the synthesis of CIDs. Accordingly, we prepared
a series of reactions using 21 different target olefins and 10% GII
catalyst irradiated in a sealed tube at 150 °C for 5 min in DCB
(Table 2). Based on the work of Chatterjee et al., we hypothesized
that target olefins bearing relatively bulky and electron-withdraw-
ing groups might provide the highest yielding CM reactions.31 In
agreement with this concept, some of the compounds with carbon-
yls or carboxylates close to the reacting olefinic center had the
highest conversion efficiency. For example, acrylate gave an excel-
lent yield (99%), as did olefins with acrylic or benzoic acids (79–
99%). Some of the halogenated olefins, such as allyl bromide, had
low yields, which MS experiments confirmed was due to the pro-
pensity of the unhindered allyl halide to undergo rapid, non-pro-
ductive homodimerization. Poor yields were also observed for
free amines, such as allylamine and 3-butenylamine (less than
5%), while protecting with Boc anhydride significantly improved
reaction yield for the allylamine reaction (50%). Finally, for a subset
of olefins that were only mildly soluble in DCB, we explored isopro-
panol as an alternative solvent. This strategy improved the solubil-
ity of all the chosen substrates and the yield of the CM reaction
involving acryloyl chloride improved from 6% to 88%; however, this
solvent did not significantly improve the reaction efficiency for the
other olefins.
Our initial efforts focused on optimizing the microwave-accel-
erated coupling between FK506 and
a representative olefin,
3,4-epoxy-1-butene (Fig. 1). Recently, Chapman et al. identified a
narrow window of conditions for efficient GII-mediated, micro-
wave-accelerated, ring-closing metathesis on peptide substrates.30
Guided by this effort, we systematically varied the reaction condi-
tions and found that microwave irradiation for 5 min yielded good
conversion rates (40–64%), while shorter (1–2 min) or longer (10–
20 min) irradiation times reduced the yields (Fig. 2A). Additionally,
by varying temperature, we identified a clear peak of efficiency at
150 °C. Higher temperatures resulted in lower yields and greater
side-product formation, while reactions at lower temperatures
consisted mostly of unreacted starting materials (Fig. 2B). Impor-
tantly, when a 20-fold molar excess of the olefin was used, these
reactions proceeded readily on unprotected FK506, while lower ra-
tios favored formation of FK1012-like homodimers.
As the success of microwave-accelerated reactions is dependent
on absorption of microwave energy by the reaction medium, we
examined a variety of solvents and monitored production of the
desired CM product (1) by liquid chromatography–mass spectrom-
etry (LC–MS). Using this screening approach, we found that 1,2-
dichlorobenzene (DCB) and 1,2-dichloroethane (DCE) produced
the highest yields (61–64%; Table 1). Other solvents with strong
dielectric constants, such as ethanol, were poor choices because
of reagent insolubility.
From these studies, we selected FK506 derivatives with the
most favorable combination of properties (e.g., yield, solubility
and reactive functionality) for re-synthesis and expanded studies.
No effort was made to separate E and Z isomers, because these
With these results in hand, our next goal was to evaluate a ser-
ies of target olefins. Structural and chemical diversity were key in
HO
HO
O
O
OH
OH
O
O
O
O
10 mol% GII
39
39
O
O
N
N
O
(20 equiv)
38
O
DCB, MW
38
O
O
O
HO
O
HO
1
O
O
O
O
FK506
O
O
Figure 1. Cross metathesis of 3,4-epoxy-1-butene to FK506 creates a reactive derivative (1). Grubb’s ruthenium catalyst (GII), FK506 and the epoxide-bearing olefin were
irradiated in 1,2-dichlorobenzene (DCB).
(B) Effects of temperature on yield of
CM product 1.
(A) Effects of irradiation time on yield
of CM product 1.
40
80
CM product (1)
30
60
side
products
CM product (1)
20
40
10
20
0
120 ºC
20
0
0
5
10
15
80
120
160
200
240
time (min)
temperature (ºC)
Figure 2. Varying the reaction time and temperature reveals optimal CM conditions. Results are the average of three independent reactions with the standard error shown.
Yields were estimated from quantitative LC–MS profiles, as described in Section 4. DCB was used as the solvent in all the reactions.

P. S. Marinec et al. / Bioorg. Med. Chem. 17 (2009) 5763–5768
5765
Table 1
that appending SLF to an HIV protease inhibitor partitions the
resulting molecule into blood cells and prolongs its lifetime 20-fold
in vivo.35 Evidence suggests that this improved persistence results
from the modified compound being sequestered into the cyto-
plasm of the blood cells, which is an environment that contains
high levels of FKBP but is nearly devoid of the P450 enzymes that
metabolize xenobiotics. Based on this model, we reasoned that cel-
lular partitioning and drug lifetime might be sensitive to the affin-
ity for FKBP.19 Therefore, as a proof-of-concept for the present
work, we sought to generate a fusion between FK506 and an HIV
protease inhibitor, which would allow us to directly compare
SLF- and FK506-conjugated molecules. Towards that goal, we cou-
pled the reactive FK506 derivative 2 in a single step to an ampre-
navir-like core to yield the bifunctional molecule 7 (Fig. 5A).
Coupling of the core inhibitor to the FK506 derivative occurred
through a pendant amine, which is known to be a well tolerated
attachment point (Fig. 5B).35,36
Summary of cross metathesis reactions between FK506 and 3,4-epoxy-1-butene in
different solvents (5 min, 150 °C)
Solvent
Yield (%)
1,2-DCB
1,2-DCE
DCM
DMF
ACN
Isopropanol
1-Butanol
EtOH
MeOH
DMSO
64
61
Trace
20
3
42
18
5
4
2
Table 2
Summary of cross metathesis reactions between FK506 and functionalized olefins
Olefin
Solvent
Yield (%)
To test the influence of FKBP binding on cellular partitioning,
we first examined the distribution of 4-methoxy amprenavir in
whole blood and, consistent with previous studies, we found that
it distributed evenly between the plasma and cellular compart-
ments (Fig. 6). Next, we confirmed that the corresponding SLF-
modified derivative 8 showed a strong (ꢀ9-fold) preference for
the cellular fraction (Fig. 6).35 Remarkably, the FK506-modified
derivative 7 was concentrated by more than ꢀ22-fold in the blood
cell compartment. Thus, replacing SLF with FK506 exaggerated cel-
lular partitioning by more than twofold, consistent with an impor-
tant role for FKBP-binding affinity. These results clearly illustrate
one advantage of using orthogonally reactive FK506 derivatives
to synthesize new CIDs.
None
Acrylamide
Acrylic acid
Acrylic acid, succinimidyl ester
Acryloyl chloride
DCB
DCB
DCB
DCB
DCB
Isopropanol
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
4 (FK1012)
99
99
25
6
88
Trace
11
12
44
27
22
50
39
5
Allylamine
Allylbromide
Allylchloroformate
2-Allylchlorohexanone
Allylisothiocyanate
Allylisocyanate
N-Boc allylamine
3-Butene-1-ol
3-Butenylamine
Isopropanol
DCB
DCB
DCB
DCB
2
3,4-Epoxy-1-butene
5-Hexenitrile
Methyl acrylate
56
36
97
8
3. Conclusions
The goal of this effort was to generate reactive building blocks
for CID applications. Accordingly, we systematically investigated
several reaction variables and identified conditions suitable for
microwave-accelerated CM and diversification at the C39 position
of FK506. These efforts afforded reactive analogs (1–6), which pres-
ent either electrophiles or nucleophiles. These derivatives are de-
signed for facile installation into parent compounds bearing the
corresponding functionality. One key feature of this strategy is that
the reactive intermediates are accessed in a single step using
unprotected FK506. During the course of these studies, we also ex-
plored a scavenger resin-based method for removing ruthenium
byproducts, which may be particularly advantageous for parallel
syntheses of CIDs. Finally, as demonstrated by the synthesis of
the bifunctional protease inhibitor 7, these methods are expected
to provide a convenient and modular platform for creating FKBP-
binding CIDs with interesting new properties.
5-Norbonene-2-carboxylic acid
Isopropanol
DCB
DCB
Isopropanol
DCB
DCB
7
4-Pentenoyl chloride
4-Pentynoic acid
25
Trace
Trace
5
79
9
4-Vinylaniline
4-Vinylbenzoic acid
Isopropanol
have been found to be equally active in biological assays.29 Follow-
ing HPLC purification to remove trace amounts of unmodified
FK506, a small collection of derivatives (1–6) suitable for the gen-
eration of CIDs was produced (Fig. 3).
Because these molecules are intended for use in the exploration
of biological systems, we were also interested in finding efficient
ways to remove ruthenium byproducts. Even trace amounts of
these organometallic complexes can lead to olefin isomerization,
product decomposition and cell toxicity. Several relevant purifica-
tion methods have already been explored, including the use of tris-
(hydroxymethyl)phosphine, triphenylphosphine oxide, and di-
methyl sulfoxide.32–34 However, these procedures require multiple
reagents, subsequent column chromatography, and relatively long
exposure times for maximal efficacy. Here, we explored a quick
and efficient strategy using silica-bonded equivalents of the metal
scavenger, 1-propanethiol. We found that treatment of the crude
reaction mixture with 5 equiv of resin for 30 min reduced the lev-
els of the ruthenium byproducts by greater than 50-fold (Fig. 4).
This simple method compared favorably to other approaches, espe-
cially in the minimal handling steps and short times required.
One of the potential advantages of using FK506 as a module in
the synthesis of bifunctional molecules is its superior affinity for
FKBP, especially when compared to SLF.24 We recently reported
4. Experimental methods
4.1. Synthetic methods
Solvents and olefins were purchased from Sigma or Thermo-
Fisher and used without further purification. FK506 was purchased
from LC Labs. All mass spectrometry results collected on a Shima-
dzu LC–MS using an APCI probe in negative mode. All NMR spectra
collected on a Varian 600 MHz system, with the samples dissolved
in DMSO-d.
4.2. General procedure for cross metathesis
To a solution of FK506 (2.0 mg; 0.00249 mmol) in dichloroben-
zene (2.0 mL) was added the target olefin (20 equiv; 0.0498 mmol)

5766
P. S. Marinec et al. / Bioorg. Med. Chem. 17 (2009) 5763–5768
HO
O
HO
O
OH
OH
O
O
O
O
10 mol% GII
R
O
N
O
N
R
DCB, MW
5 min.
150 ºC
(20 equiv)
O
O
O
O
HO
O
O
HO
O
O
O
O
yield
cmpd
R =
yield
cmpd
R =
1
55-60%
O
OH
4
5
75-80%
O
OH
2
3
>95%
>95%
O
O
CN
35-40%
45-50%
N
N
*
6
*following Boc deprotection
Figure 3. Synthesis of reactive FK506 derivatives. From the screen of 21 olefins, these FK506 derivatives were chosen for re-synthesis, scale up and characterization. Yields
are representative of at least two independent syntheses.
Version 2.05 using external standards. For the CM reactions that
were selected for further analysis, the scale was increased 10-fold
and the resulting crude reactions were treated with silica-immobi-
lized, 1-propanethiol (5 equiv; Biotage; Isolute Si-Thiol) for 30 min,
filtered and then the product was purified by preparative chroma-
2.5
2.0
1.5
1.0
0.5
0.0
N
N
resin
resin
Cl
Cl
Ru
CH
+
tography on
a
Waters Spherisorb S10 ODS2 column (10 Â
PCy3
250 mm). For compound 6, the crude product was additionally
treated with 5 equiv of 1:1 TFA:DCM, followed by dilution into
DCM, neutralization with 5% sodium bicarbonate and concentra-
tion by roto-evaporation prior to HPLC. See the Supplementary
data for characterization (1H NMR and mass spectrometry).
4.3. Synthesis of bifunctional HIV protease inhibitor (7)
As a test of the modular assembly of bifunctional molecules, the
reactive FK506 derivative 2 (1.0 equiv; 15.8 mg; 0.0186 mmol) was
added to the advanced HIV protease inhibitor (1.2 equiv; 9.1 mg;
0.0
2.5
5.0
7.5
10.0 min
retention time (min)
Figure 4. Removal of ruthenium byproducts by scavenger resin. LC–MS traces of a
CM reaction between FK506 and 3,4-epoxy-1-butene is shown before and after
treatment with 5 equiv of resin (see Section 4). The peak area of the ruthenium
byproducts was reduced by greater than 50-fold, without any appreciable reduc-
tions in the product peak. Results are representative of the traces from other
reactions.
0.0224 mmol) in 3.0 mL of DMF with DIC (10.0 equiv; 29.2 lL;
0.1863 mmol), HOBt (1.0 equiv; 2.5 mg; 0.1863 mmol) and DMAP
(1.0 equiv; 2.3 mg; 0.1863 mmol). This reaction was stirred at
room temperature overnight and the resulting product 7 purified
in 75% yield by HPLC as described above. The blood partitioning
experiments were performed as described.35 The synthesis of the
amprenavir core structure and compound 8 have been reported.35
See the Supplementary data for characterization (1H NMR and
mass spectrometry). FK506 coupled to HIV protease inhibitor (7);
molecular formula C66H97N3O17S; expected mass (m/z) = 1236.55;
actual mass (m/z) = 1235.95. d 7.74(d), d 7.63(s), d 7.57(d), d
7.51(s), d 7.44(d), d 7.36(s), d 7.28(t), d 7.19(s), d 7.11(t), d 6.9(d),
d 6.86(d), d 5.42(s), d 5.36(s), d 5.27(s), d 5.125(dd), d 5.04(d), d
4.8(m), d 4.7(d), d 4.6(s), d 4.48(s), d 4.24(d), d 3.9(s), d 3.87(d), d
3.81(s), d 3.78(s), d 3.71(s), d 3.62(d), d 3.54(d), d 3.41(dd), d
3.33(d), d 3.28(d), d 3.22(s), d 2.9–3.08(m), d 2.91(d), d 2.78(m), d
2.72(m), d 2.27(s), d 2.1(s), d 2.0(m), d 1.92(s), d 1.86(m), d 1.8(d),
d 1.72(d), d 1.65(d), d 1.62(s), d 1.55(s), d 1.48(m), d 1.39(s),
d1.26(t), d 1.21(t), d 1.14(t), d 1.03(m), d 0.86(d), d 0.81(d), d
0.79(d), d 0.73(d).
followed by catalyst (0.1 equiv; 0.211 mg; 0.249 lmol) in a micro-
wave vial. The vial was then sparged with nitrogen, sealed, and
placed in a Biotage Initiator EXP microwave reactor. The mixture
was stirred for 30 s, followed by irradiation at the desired temper-
ature and time. To achieve the desired temperature, the microwave
power was typically ꢀ20 W. To quantify reaction progress, crude
mixtures were placed in 1.5 ml vials and analyzed with a Shimazdu
(Columbia, MD) series 2010EV Liquid Chromatograph Mass Spec-
trometer equipped with an APCI probe to minimize ion suppres-
sion. Samples (5.0 lL) were injected onto 1.8 lm ZORBAX Eclipse
XDB-C18 column (Agilent), and elution was performed with a step-
wise 10–100% gradient of acetonitrile over 11 min. Products eluted
between 5.5 and 6 min, while the peak for unreacted FK506 was
found at 6.7 min. Quantification was performed using LCMSolution

P. S. Marinec et al. / Bioorg. Med. Chem. 17 (2009) 5763–5768
5767
(B) Modified compound 7 retains
anti-HIV-1 protease activity
(A) Synthesis of an FK506-modified HIV protease inhibitor
HO
O
HO
O
1.0
0.8
0.6
0.4
OH
OH
O
O
O
O
O
O
H
N
S
OH
N
N
O
N
O
O
O
O
OH
O
O
DMF, DIC,
2
O
HO
O
O
+
µ
M
IC50 = 6.3 1.7
O
HOBt, DMAP HO
rt, 18 hr, 75%
O
FK506-modified
amprenavir (7)
O
0.2
0
S
H2N
O
O
N
O
O
O
OH
-9
-8
-7
-6
-5
-4
compound 7 (M)
Figure 5. Modular synthesis of a modified HIV protease inhibitor by installation of FK506. (A) Reactive intermediate 2 was coupled to the known core of an HIV protease
inhibitor to produce the bifunctional molecule 7 in good purified yield. (B) The FK506-modified inhibitor 7 retains anti-protease activity in vitro. Results are the average of
triplicates.
O
N
O
O
O
O
O
O
O
O
O
O
H
N
S
S
MeO
MeO
N
H
N
O
O
O
N
H
N
OH
O
OH
4-methoxy-amprenavir
SLF-modified amprenavir (8)
25
20
plasma
blood cell
unmodified
inhibitor
15
10
5
FKBP-binding
inhibitor
FKBP
0
4-methoxy-
amprenavir
8
7
Figure 6. An FK506-coupled HIV protease inhibitor is partitioned into blood cells. Fresh whole blood from male C57BL/6 mice was treated with either 4-methoxyamprenavir,
SLF-conjugated amprenavir (8) or FK506-modified amprenavir (7) at 100 M. After 6 h at 37 °C, whole blood was separated into plasma and cellular components by
l
centrifugation and the compound in each fraction quantified by LC–MS. The fold partitioning was determined by dividing the cellular concentration by the concentration in
the plasma. Error of triplicate experiments was approximately 10%. The schematic shows the model for how high affinity binding to cytoplasmic FKBP might deplete modified
compounds from plasma.
4.4. HIV protease assay
ing Grant (GM008353-18). J.E.G. acknowledges ThermoFisher Sci.
and the McKnight Foundation for financial support.
To determine the inhibitory potency of compound 7, we used a
commercially available, FRET-based assay (Bachem, Torrance, CA),
as previously reported.35 Briefly, HIV-1 protease was preincubated
with inhibitor for 60 min at 37 °C, followed by addition of 30 nM
Supplementary data
Synthesis and 1H NMR spectra of modified FK506 derivatives
and a complete record of all the reactions. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.07.030.
HIV-1 protease substrate
1 (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-
Pro-Ile-Val-Gln-Lys(dabcyl)-Arg; Molecular Probes, Eugene OR).
Substrate cleavage was measured for 60 min. at 37 °C by monitor-
ing fluorescence at 490 nm on a SpectraMax M5 plate reader
(Molecular Devices, Mountain View, CA).
References and notes
1. Dumont, F. J. Curr. Med. Chem. 2000, 7, 731.
2. Liu, J.; Albers, M. W.; Wandless, T. J.; Luan, S.; Alberg, D. G.; Belshaw, P. J.;
Cohen, P.; MacKintosh, C.; Klee, C. B.; Schreiber, S. L. Biochemistry 1992, 31,
3896.
3. Griffith, J. P.; Kim, J. L.; Kim, E. E.; Sintchak, M. D.; Thomson, J. A.; Fitzgibbon, M.
J.; Fleming, M. A.; Caron, P. R.; Hsiao, K.; Navia, M. A. Cell 1995, 82, 507.
4. Lin, H.; Cornish, V. W. Angew. Chem., Int. Ed. 2001, 40, 871.
Acknowledgments
The authors thank R. Owen and E. Vedejs for helpful comments,
J. Bauer and T. Almeida for technical assistance. C.G.E. is supported
by a predoctoral fellowship from the Cellular Biotechnology Train-

5768
P. S. Marinec et al. / Bioorg. Med. Chem. 17 (2009) 5763–5768
5. Clackson, T. Chem. Biol. Drug Des. 2006, 67, 440.
6. Gestwicki, J. E.; Marinec, P. S. Comb. Chem. High Throughput Screening 2007, 10,
667.
22. Keenan, T.; Yaeger, D. R.; Courage, N. L.; Rollins, C. T.; Pavone, M. E.; Rivera, V.
M.; Yang, W.; Guo, T.; Amara, J. F.; Clackson, T.; Gilman, M.; Holt, D. A. Bioorg.
Med. Chem. 1998, 6, 1309.
7. Spencer, D. M.; Wandless, T. J.; Schreiber, S. L.; Crabtree, G. R. Science 1993, 262,
1019.
8. Belshaw, P. J.; Ho, S. N.; Crabtree, G. R.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 4604.
9. Pownall, M. E.; Welm, B. E.; Freeman, K. W.; Spencer, D. M.; Rosen, J. M.; Isaacs,
H. V. Dev. Biol. 2003, 256, 89.
10. de Graffenried, C. L.; Laughlin, S. T.; Kohler, J. J.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 16715.
11. Gestwicki, J. E.; Crabtree, G. R.; Graef, I. A. Science 2004, 306, 865.
12. Karpova, A. Y.; Tervo, D. G.; Gray, N. W.; Svoboda, K. Neuron 2005, 48, 727.
13. Haruki, H.; Nishikawa, J.; Laemmli, U. K. Mol. Cell 2008, 31, 925.
14. Geda, P.; Patury, S.; Ma, J.; Bharucha, N.; Dobry, C. J.; Lawson, S. K.; Gestwicki, J.
E.; Kumar, A. Yeast 2008, 25, 577.
23. Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M.; Amara, J. F.; Rollins, C. T.;
Stevenson, L. F.; Magari, S. R.; Wood, S. A.; Courage, N. L.; Lu, X.; Cerasoli, F., Jr.;
Gilman, M.; Holt, D. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10437.
24. Holt, D. A.; Luengo, J. I.; Yamashita, D. S.; Oh, H.-J.; Konialian, A. L.; Yen, H.-K.;
Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy, M. A.; Eggelston, D. S.; Liang, J.;
Schultz, L. W.; Stout, T. J.; Clardy, J. J. Am. Chem. Soc. 1993, 115, 9925.
25. Hoveyda, A. H.; Zhugralin, A. R. Nature 2007, 450, 243.
26. Wender, P. A.; Hilinski, M. K.; Skaanderup, P. R.; Soldermann, N. G.; Mooberry,
S. L. Org. Lett. 2006, 8, 4105.
27. Clemons, P. A.; Gladstone, B. G.; Seth, A.; Chao, E. D.; Foley, M. A.; Schreiber, S.
L. Chem. Biol. 2002, 9, 49.
28. Dumont, F. J.; Staruch, M. J.; Koprak, S. L.; Siekierka, J. J.; Lin, C. S.; Harrison, R.;
Sewell, T.; Kindt, V. M.; Beattie, T. R.; Wyvratt, M., et al J. Exp. Med. 1992, 176, 751.
29. Diver, S. T.; Schreiber, S. L. J. Am. Chem. Soc. 1997, 119, 5106.
30. Chapman, R. N.; Arora, P. S. Org. Lett. 2006, 8, 5825.
15. Briesewitz, R.; Ray, G. T.; Wandless, T. J.; Crabtree, G. R. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 1953.
16. Pollock, R.; Giel, M.; Linher, K.; Clackson, T. Nat. Biotechnol. 2002, 20, 729.
17. Baker, K.; Bleczinski, C.; Lin, H.; Salazar-Jimenez, G.; Sengupta, D.; Krane, S.;
Cornish, V. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16537.
18. Braun, P. D.; Barglow, K. T.; Lin, Y. M.; Akompong, T.; Briesewitz, R.; Ray, G. T.;
Haldar, K.; Wandless, T. J. J. Am. Chem. Soc. 2003, 125, 7575.
19. Marinec, P. S.; Lancia, J. K.; Gestwicki, J. E. Mol. Biosyst. 2008, 4, 571.
20. Koide, K.; Finkelstein, J. M.; Ball, Z.; Verdine, G. L. J. Am. Chem. Soc. 2001, 123,
398.
31. Chatterjee, A. K.; Choi, T. L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem. Soc. 2003,
125, 11360.
32. Ahn, Y. M.; Yang, K.; Georg, G. I. Org. Lett. 2001, 3, 1411.
33. Hong, S. H.; Grubbs, R. H. Org. Lett. 2007, 9, 1955.
34. Haack, K.; Ahn, Y. M.; Georg, G. I. Mol. Divers. 2005, 9, 301.
35. Marinec, P. S.; Chen, L.; Barr, K. J.; Mutz, M. W.; Crabtree, G. R.; Gestwicki, J. E.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1336.
36. Surleraux, D. L.; Tahri, A.; Verschueren, W. G.; Pille, G. M.; de Kock, H. A.;
Jonckers, T. H.; Peeters, A.; De Meyer, S.; Azijn, H.; Pauwels, R.; de Bethune, M.
P.; King, N. M.; Prabu-Jeyabalan, M.; Schiffer, C. A.; Wigerinck, P. B. J. Med.
Chem. 2005, 48, 1813.
21. Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.; Keenan, T.; Natesan, S.; Pollock,
R.; Yang, W.; Courage, N. L.; Holt, D. A.; Gilman, M. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 10618.