Effects on polo-like kinase 1 polo-box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position

Article abstract of DOI:10.1016/j.bmc.2012.05.036

Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pS

Full text of DOI:10.1016/j.bmc.2012.05.036

Bioorganic & Medicinal Chemistry 21 (2013) 3996–4003
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects on polo-like kinase 1 polo-box domain binding affinities
of peptides incurred by structural variation at the phosphoamino
acid position
a,
Wenjian Qian a, , Jung-Eun Park b, , Fa Liu a, Kyung S. Lee b, , Terrence R. Burke Jr.
⇑
⇑
a Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
b Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 26 May 2012
Protein–protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1)
serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides
and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been
little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities
of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the
role of methyl functionality at the pThr b-position and by replacing the phosphoryl group by phosphonic
acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for
PBD recognition of phosphoamino acid mimetics.
Keywords:
Plk1
Polo-like kinase
Polo-box domain
Phosphoamino acid mimetic
Published by Elsevier Ltd.
1. Introduction
an alternative PPI-directed approach to down-regulating Plk1 func-
tion.25,37–43
The central roles that protein-protein interactions (PPIs) play in
biochemical processes1,2 and their frequent reliance on binding ‘hot
spots’3–6 have made them attractive therapeutic targets.7–14 Many
components of cellular signal transduction employ sub-classes of PPIs
that involve the recognition of either phosphotyrosine (pTyr) or phos-
phothreonine (pThr)/phosphoserine (pSer)-containing sequences.15–
19 Because phosphoryl groups serve as key elements of high affinity
interactions, these phospho-dependent PPIs are potentially amenable
to disruption by small-to-moderate size molecules.
The polo-like kinases (Plks) are a subfamily of serine-threonine
kinases that play critical roles in cellular proliferation.20–24 Of the
known Plks (Plk1–Plk5), Plk1 has been identified as an anticancer
target due to its ability to promote tumorigensesis.25–27 In addition
to its catalytic domain, Plk1 contains a C-terminal polo-box do-
main (PBD), which recognizes pThr/pSer-containing sequences
and directs the enzyme to specific sub-cellular locations.28 While
significant effort has been devoted to developing inhibitors target-
ing the catalytic activity of Plk1,29–36 targeting the PBD may afford
The 5-mer peptide ‘Pro-Leu-His-Ser-pThr’ (1, Fig. 1) represents a
high affinity PBD-binding sequence derived from the pT78 region
of the polo-box interacting protein 1 (PBIP1), which offers a start-
ing point for the design of PBD-directed inhibitors.38 Although the
phosphoryl moiety provides an essential PBD recognition motif, it
presents particular challenges to bioavailability due to its dianionic
charge and the lability of the phosphoryl ester bond to phospha-
tases. Similar problems are also encountered in the development
of inhibitors directed against pTyr-binding domains. However, un-
like the latter family of compounds, for which a large number of
pTyr mimetics have been examined,44,45 significantly fewer pThr/
pSer mimetics have been reported.46–50 Our current paper reports
the synthesis and PBD-binding affinities of peptides containing a
variety of pThr/pSer mimetics, many of which have not yet been
examined within the context of PBD-binding peptides.
2. Results and discussion
2.1. Design of phosphomimetic-containing peptides
⇑
Corresponding authors. Addresses: National Cancer Institute, National Insti-
Recently, we discovered that introduction of long-chain aryl–al-
kyl groups onto the d1 nitrogen (N3) of the histidine imidazole ring
of parent peptide 1 could significantly increase Plk1 PBD-binding
affinity.42 From the X-ray co-crystal structure of a high affinity
peptide containing a C6H5(CH2)8–His adduct (2a, Fig. 1), we found
that the potency enhancement occurred through new binding
tutes of Health, 9000 Rockville Pike, Building 37, Room 3118, Bethesda, MD 20892,
USA. Tel.: +1 301 496 9635; fax: +1 301 496 8419 (K.S.L.); National Cancer Institute,
National Institutes of Health, Building 376, Boyles St., NCI-Frederick, Frederick, MD
21702, USA. Tel.: +1 301 846 5906; fax: +1 301 846 6033 (T.R.B.).
E-mail addresses: kyunglee@mail.nih.gov (K.S. Lee), tburke@helix.nih.gov (T.R.
Burke).
These authors contributed equally to this work.
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.05.036

W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
3997
2.2. Preparation of protected amino acid analogs and their use
in solid-phase peptide synthesis
OH
Ac
N
O
O
N
O
H
N
H
N
N
H
N
H
NH2
Peptides 2a–2k were prepared by solid-phase techniques using
Fmoc-based protocols. The C-terminal residues of peptides 2a–2g
were incorporated using the protected amino acid analogs 4a–4g,
respectively (Fig. 2). Commercially-available monobenzyl
phosphoryl esters (4a, 4e and 4g) were used to introduce pThr,
pSer and p(allo-Thr) respectively. Reagent 4b, which was used to
introduce the (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid
(Pmab) residue, was prepared according to literature procedures.50
The remaining amino acid analogs (4c, 4d and 4f) were synthesized
as outlined below.
O
O
O
N
O
P
OH
H
OH
1
OH
Ac
N
O
O
O
H
N
H
N
N
H
N
H
NH2
O
O
X
N
N
(CH2)7
2.2.1. Preparation of N-Fmoc-(2S,3R)-3-methyl-4-sulfonylbutanoic
acid (4c)
X =
2
Preparation of peptide 2c, containing the sulfonic acid-based
pThr mimetic, (2S,3R)-2-amino-3-methyl-4-sulfonylbutanoic acid
(Smab), was achieved using reagent 4c, which has a sulfonic acid
moiety. Mitsunobu treatment of known 550 with thioacetic acid,
triphenylphosphane, and diisopropyl diazodicarboxylate (DIAD)
in dry THF gave 6 in good yield (Scheme 1). The N-benzyl carba-
mate group of 6 was converted to acetyl amide (7) using sodium
iodide and acetyl chloride. Oxidative chlorination of 7 to the corre-
sponding sulfonyl chloride was achieved in good yield using N-
chlorosuccinimide (NCS) in aqueous HCl–MeCN52 and this was fol-
lowed by treatment with aqueous sodium hydroxide to provide the
free sulfonic acid (8). Finally, removal of N-acetyl and O-methyl
groups by refluxing in 6 N HCl yielded the free amino acid, which
was converted to the N-Fmoc-protected 4c using Fmoc-OSu
(Scheme 1).
O
O
O
C
O
P
OH
OH
O
S
O
OH
O
O
P
OH
OH
O
P
OH
OH
O
P
OH
OH
O
OH
a
b
c
d
e
f
(pThr)
(Pmab)
(Smab)
(pSer)
O
HO
HO
C
C
P
OH
O
OH
O
NH2
O
OH
g
h
i
j
k
[p(allo-Thr)]
(Glu)
(Gln)
(Asp)
(Thr)
Figure 1. Structures of peptides discussed in the text.
interactions in a previously occluded hydrophobic channel on the
PBD surface. Although our original His-adduct-containing peptides
were obtained in small quantities as synthetic by-products, we de-
vised a route to synthesize protected d1-alkyl-containing amino
acid analogs of type 3 (Fig. 2) that allowed direct Fmoc-based so-
lid-phase synthesis His-modified peptides such as 2a.51 In our cur-
rent study, we replaced the pThr phosphate group in 2a with
phosphonic acid (2b), sulfonic acid (2c) and carboxylic acid (2d)
functionality. We also replaced the pThr residue with pSer (2e),
the b,b-bis-methyl variant of pSer (2f) and p(allo-Thr) (2g) as well
as with Glu (2h), Gln (2i) and Asp (2j) residues (Fig. 1) and deter-
mined their Plk1 PBD binding affinities.
2.2.2. Preparation of N-Fmoc-(2S,3S)-2-amino-5-(benzyloxy)-3-
methyl-5-oxopentanoic acid (4d)
Introduction of the C-terminal pThr mimicking residue in pep-
tide 2d employed the protected amino acid 4d. The synthesis of
4d is shown in Scheme 2. Conversion of the protected D-serine ana-
log 9–14 was according to literature procedures (Scheme 2).53
Compound 14 was first converted from the ethyl ester to the ben-
zyl ester (15) and then subjected to Jones’ oxidation to afford the
protected amino acid 16. The desired reagent 4d was obtained by
removing the N-Boc group (4 M HCl in dioxane) and installing
the N-Fmoc group (Fmoc-OSu) (Scheme 2).
O
O
2.2.3. Preparation of N-Fmoc (2S)-2-amino-3-(benzyloxyphos-
phoryloxy)-3-methylbutanoic acid (4f)
FmocHN
FmocHN
OH
OH
The preparation of phosphopeptide 2f employed the protected
amino acid 4f, which was prepared as shown in Scheme 3. Grig-
nard reaction of 10 with MeMgI in diethyl ether at 0 °C proceeded
to give 17, which was converted to 18 by oxidation with Jones’ re-
agent in acetone at 0 °C.54 Compound 18 was converted to its allyl
ester and then the N-Boc group was exchanged for N-Fmoc
X
N
N
(CH2)7
4
3
X =
O
P
OBn
O
O
O
OBut
O
O
S
O
OH
OH
O
O
OH
C
O
P
O
OBn
CbzHN
RHN
S
AcHN
OBut
O
O
O
c
a
d
4c
a
c
b
d
OH
O
S
OH
Ac
O
5
8
6 R = Cbz
7 R = Ac
b
O
O
O
P
O
P
OH
P
OH
Scheme 1. Reagents and conditions: (a) AcSH (2.0 equiv), PPh3 (2.0 equiv), DIAD
(2.0 equiv), THF, 0 °C–rt, 3 h, 98%; (b) NaI (10.0 equiv), AcCl (10.0 equiv), MeCN,
reflux, 7 h, 85%; (c) (1) NCS (4.0 equiv), 2 N HCl/MeCN (v/v, 1/4), 10 °C, 1 h; (2) 0.1 N
NaOH/dioxane (1:1, v/v), rt, 0.5 h; (d) (1) 6 N HCl, reflux, 12 h; (2) Fmoc-OSu, 0.1 N
NaOH/dioxane, overnight, 14% for four steps.
OBn
OBn
OBn
e
g
f
Figure 2. Protected amino acids used to prepare peptides 2a–2g.

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W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
O
O
O
lysates.38,42 Results are shown in Figures 3 and 4. As previously re-
Boc
N
Boc
N
ported, addition of the C6H5(CH2)8-group to the histidine d1-nitro-
gen (peptide 2a) significantly enhanced binding affinity relative to
the parent parent peptide 1 (Fig. 3). We first examined dianionic
functionality. Consistent with previous results,38,42,43,50 high affin-
ity was maintained when pThr was replaced by Pmab- (compare
peptide 2a with 2b, Fig. 3). This indicates that the phosphoryl ester
a
b
BocHN
HO
O
O
O
N
O
O
11
9
10
O
P
OEt
O
O
O
EtO
Boc
N
Boc
N
OEt
oxygen in the pThr residue is not
recognition.
a critical component of
c
OEt
d
O
O
Next, we directed our attention toward modifying the 3-posi-
tion of pThr. We observed that deletion of the (3R)-methyl group
to give pSer (peptide 2e) resulted in an approximate order-of-mag-
nitude loss of affinity (Fig. 3). This was in agreement with previous
work that had shown an approximate sevenfold preference for
pThr over pSer.56 However, it should be noted that high affinity
pSer-containing peptides are known that include a Pro residue C-
terminal to the pSer.57 Placement of a second methyl group at
the 3-position (2f) led to a further loss of potency, but the 3,3-di-
methyl analog did retain moderately good affinity. In contrast,
reversal of the pThr stereochemistry at the 3-position (p-alloThr,
2g), resulted in a nearly three orders-of-magnitude loss of affinity.
The relatively good affinities of the desmethyl (pSer, 2e) and
dimethyl (2f) analogs indicate that both the absence of the (3R)-
methyl and the presence of a pro-(3S)-methyl are tolerated within
the binding pocket. Therefore, the dramatic loss of affinity ob-
served with 2g may be related to unfavorable conformational
geometries of the p-alloThr residue.
12
13
4d
O
O
BocHN
g
e
h
BocHN
OH
C
O
C
O
OBn
OR
16
14 R = Et
f
15 R = Bn
Scheme 2. Reagents and conditions: (a) 2,2-Dimethoxypropane (10 equiv), BF3
Et2O (0.1 equiv), acetone, rt, 2 h, 91%; (b) (1) LiOH H2O (2.0 equiv), MeOH/H2O, rt,
4 h; (2) BOP (1.2 equiv), NH(OMe)Me HCl (1.25 equiv), Et3N (2.0 equiv), rt, 3 h, 70%
for two steps; (c) MeLi (1.7 equiv), THF, ꢀ78 °C, 1 h, 59%; (d) triethyl phosphono-
acetate (1.5 equiv), NaH (1.5 equiv), 0 °C, 24 h, 74%; (e) 10% PdꢁC, H2, MeOH/AcOEt,
rt, 12 h, 97%; (f) (1) LiOH H2O (3.0 equiv), THF/H2O, rt, 12 h.; (2) BnBr (1.5 equiv),
NaHCO3 (2.0 equiv), DMF, rt, 24 h, 67% for two steps; (g) 2.7 M Jones’ reagent
(1.5 equiv), acetone, rt, 30 min, 89%; (h) (1) 4 M HCl in dioxane (10 equiv), 3 h; (2)
Fmoc-OSu (1.5 equiv) , NaHCO3 (2.0 equiv), THF/H2O (v/v 1:1), 12 h, 81% for two
steps.
2.3.2. Monoanionic and uncharged species
Unlike SH2 domains, in which recognition of tyrosyl phosphates
involves salt bridges between both anionic phosphoryl oxygens
and two conserved Arg residues,58 interaction of the PBD with
the pThr phosphoryl group of 2a involves only a single salt bridge
with Lys540 (PBD accession code 3RQ742). Additional PBD-phos-
phoryl interactions are hydrogen bonding in nature (with His538
and several water molecules). Therefore, while SH2 domain-direc-
ted pTyr mimetics often require maintenance of dianionic charac-
ter for high affinity binding,45 it may be reasonable to expect that a
single anionic charge could be sufficient for ligands to bind with
high affinity to PBD domains. The Smab-containing peptide (2c)
can be viewed as a monoanionic isostere of Pmab-containing 2b,
in which a sulfonic acid moiety is employed rather than a phos-
phonic acid group. Sulfonic acids are known as monoanionic phos-
phoryl mimetics.59,60 However, peptide 2c exhibited almost four
orders-of-magnitude loss of affinity relative to the reference pep-
tide 2a (Fig. 4). This unexpectedly large shift to the right in Fig. 4
O
O
FmocHN
HO
BocHN
HO
BocHN
HO
CO2H
OAllyl
a
c
b
10
17
18
19
O
FmocHN
O
OAllyl
e
d
4f
O
P
OBn
OBn
20
Scheme 3. Reagents and conditions: (a) Mg (6.0 equiv), MeI (7.5 equiv), Et2O, 0 °C,
30 min, 92%; (b) Jones’ reagent (2.7 M, 1.5 equiv), acetone, 0 °C to rt, 1 h, 78%; (c) (1)
Allyl bromide (1.5 equiv), NaHCO3 (2.0 equiv), DMF, rt, 24 h, 60%; (2) 1 M HCl in
Et2O (10.0 equiv), rt, 3 h; (3) Fmoc-OSu (1.5 equiv), NaHCO3 (2.0 equiv), THF/H2O
(1:1 v/v), rt, 12 h, 85% for two steps; (d) dibenzyl diisopropyl phosphoramidite
(2.0 equiv), N-methylaniline trifluoroacetate (4.0 equiv), t-butyl hydroperoxide in
n-octane (1.9 equiv), rt, 1 h, 90%; (e) Pd(PPh3)4 (0.1 equiv), N-methylaniline
(3.0 equiv), THF, rt, 1 h, 91%; (2) 10% PdꢁC, 2,20-bipyridyl (0.5 equiv), H2, rt, 0.5 h,
60%.
protection (19) by first deprotecting using 1 M HCl and then react-
ing with Fmoc-Osu. Following a similar strategy as previously re-
ported for the synthesis of 4a,55 19 was converted first to the
dibenzyl ester (20) and then to the desired reagent 4f (Scheme 3).
2.3. Plk1 PBD-binding affinities of peptides 2a–2k
2.3.1. Dianionic species
We determined the Plk1 PBD binding affinities using an ELISA-
based 96-well assay that measured the ability of the synthetic
peptides to compete with support-bound pT78-derived phospho-
peptide for binding to Plk1 expressed in mitotic 293A cell
Figure 3. ELISA-based Plk1 PBD-binding results. Representative graphs are shown
from three independent experiments.

W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
3999
able with 1.42 Therefore, the unexpected SAR data for certain ser-
ies-2 pThr mimetics potentially indicate that something
unexpected may be going on in the pThr binding pocket: Just what
this ‘something’ is, is not clear. In spite of these uncertainties, the
current work represents the first report of PBD binding affinities
of peptides containing a series of pThr/pSer mimetics. The SAR
parameters derived from our work may be of value in the further
design of Plk1 PBD-binding peptides, including those that do not
contain the unusual His-adduct moiety.
3. Experimental
3.1. General experimental
Figure 4. ELISA-based Plk1 PBD-binding results. Representative graphs are shown
from three independent experiments.
All experiments involving moisture-sensitive compounds were
conducted under dry conditions (positive argon pressure) using
standard syringe, cannula, and septa apparatus. Solvents: All sol-
vents were purchased anhydrous (Aldrich) and used directly.
HPLC-grade hexanes, EtOAc, CH2Cl2, and MeOH were used in chro-
matography. TLC: analytical TLC was performed on Analtech pre-
resulted in 2c binding less efficaciously than peptide 1, in which
the His-adduct group is lacking.
Carboxylic acids can serve as monoanionic phosphoryl mimet-
ics in a number of physiological contexts. Aryl-containing carbox-
ylic acids have shown widespread value in the design of constructs
targeting pTyr-dependent interactions.44 Glutamic acid has a side
chain length that places its carboxylic acid group at a distance from
coated plates (Uniplate, silica gel GHLF, 250 lm) containing a fluo-
rescence indicator; NMR spectra were recorded using a Varian Ino-
va 400 MHz spectrometer. Coupling constants are reported in
Hertz, and peak shifts are reported in d (ppm) relative to TMS.
Low-resolution mass spectra (ESI) were measured with an Agilent
1200 LC/MSD-SL system. High resolution, accurate mass measure-
ments for confirmation of elemental compositions were obtained
by positive ion, ESI analysis on a Thermo Scientific LTQ-XL Orbitrap
mass spectrometer with HPLC sample introduction using a short
narrow-bore C18 reversed-phase column and standard CH3CN/
H2O gradient. Reported m/z values are the average of 8 or more
scans over the chromatographic peak of interest.
the
pSer (e.g., see61–67) and pThr (e.g., see61,68–71). Although the side
chain length of Asp situates its carboxylic acid closer to the -car-
a-carbon that approximates the phosphoryl groups of both
a
bon than Glu, it has also frequently been used as a pSer mimetic
(e.g., see62–67). In our current work, we observed that the Glu-con-
taining peptide (2h) bound with affinity equivalent to the sulfonic
acid-containing 2c. This is somewhat unexpected, since the Smab
residue in 2c exhibits greater structural similarity to pThr (or
Pmab) than does 2h. The Asp-containing peptide (2j) bound with
slightly less affinity than 2h and the uncharged Gln-containing
peptide (2i) bound nearly as well as 2j. Based on the results pre-
sented in Figure 3 showing that a (3R)-methyl group enhances
the binding of phosphate-containing species, we anticipated that
a (3R)-methyl-containing Glu residue should serve as a better pThr
mimetic than unmodified Glu. Therefore, it was surprising to find
that introduction of a (3R)-methyl group onto the Glu residue in
2h resulted in a significant loss of affinity (peptide 2d, Fig. 4).
Peptide 2d bound with even less affinity than the non-phosphory-
lated peptide (2k), which contained no phosphoryl-mimicking
functionality.
The differences in binding affinities of mono-anionic and un-
charged species discussed above are minor in comparison to their
shared greater than three orders-of-magnitude loss of affinity
when compared to the parent pThr-containing peptide 2a. None
of the sulfur or carbon-based phosphoryl mimetics was as effective
as even the least potent phosphorus-based analog (2g). It was
noteworthy that 3-methyl functionality can play an important role
on binding recognition of phosphoryl-containing species.
3.2. Synthesis of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-sulfo-
butanoic acid 4c from 5
3.2.1. (2S,3R)-Methyl N-Cbz-2-amino-3-methyl-4-acetylthio-
butanoate (6)
Starting from readily prepared amino acid 550 DIAD (0.95 mL,
4.83 mmol) was added at 0 °C under dry nitrogen to a solution of
triphenylphosphine (1.27 g, 4.83 mmol) in dry THF (15 mL) in a
flame-dried round-bottomed flask, and the mixture was stirred un-
til a white solid appeared. Stirring was continued for 10 min at
0 °C, after which compound 5 (0.68 g, 2.42 mmol) in dry THF
(5 mL) was added. After the mixture had been stirred for 45 min,
thioacetic acid (0.35 mL, 4.83 mmol) was added and stirring was
continued for 3 h. Diethyl ether was added, and the mixture was
washed with H2O, followed by drying of the organic layer over
MgSO4. The mixture was filtered and concentrated. The final resi-
due was purified by silica gel column chromatography (EtOAc/hex-
anes; from 1:10 to 1:2) to afford product 6 (0.8 g) as s colorless
gum (98%). ½a 1D8
ꢂ
24.6 (c 0.77, CHCl3); 1H NMR (400 MHz, CDCl3) d
7.39–7.27 (m, 5H), 5.62 (d, J = 8.0 Hz, 1H), 5.10 (s, 2H), 4.40 (dd,
J1 = 8.0 Hz, J2 = 4.0 Hz, 1H), 3.75 (s, 3H), 3.04 (dd, J1 = 12.0 Hz,
J2 = 8.0 Hz, 1H), 2.68 (dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 2.32 (s,
3H), 2.76–2.12 (m, 1H), 1.00 (d, J = 8.0 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 195.6, 171.9, 156.2, 136.3, 128.6, 128.2, 67.2,
57.7, 52.4, 37.2, 31.7, 30.6, 16.3; HR-ESI MS calcd for C16H22NO5S
(M+H)+: 340.1219, found: 340.1207.
Our reasoning in selecting platform 2 as a display vehicle relied
on the high affinity of the parent peptide 2a, which is approxi-
mately three orders-of-magnitude greater than 1. This added affin-
ity reflects extended ligand–protein interactions outside the pThr
binding pocket that are afforded by the C6H5(CH2)8-His adduct
moiety. These interactions could potentially lessen the relative
importance of binding within the pThr pocket. For example, while
the non-phosphorylated version of 1 (PLHST) exhibits near negligi-
ble affinity, the corresponding non-phosphorylated form of 2 (pep-
tide 2k) displays measurable affinity arising from a shift to the left
in its binding curve that is consistent with the shift seen in going
from 1 to 2a (Fig. 4). It should also be noted that X-ray co-crystal
data show that the binding orientation of 2a is nearly superimpos-
3.2.2. (2S,3R)-Methyl N-acetyl-2-amino-3-methyl-4-acetylthio-
butanoate (7)
To a mixture of the 6 (0.322 g, 0.95 mmol) and sodium iodide
(1.42 g, 9.49 mmol) in dry acetonitrile (19 mL) was slowly added
at 0 °C acetyl chloride (0.67 mL, 9.49 mmol). The resulting mixture

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W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
was heated at 60 °C for 7 h with stirring under a nitrogen atmo-
sphere. After addition of saturated aqueous sodium hydrogen sul-
fite and saturated aqueous sodium hydrogen carbonate with ice
cooling, the mixture was thoroughly extracted with chloroform.
The extract was washed with brine, dried over potassium carbon-
ate, and evaporated under reduced pressure. Purification of the
product using silica gel column chromatography (EtOAc/hexanes;
from 1:2 to 2:1) gave the product 7 (0.2 g) as a white foam
with EtOAc (3ꢃ). The combined organic phases were washed with
brine, dried (MgSO4) and filtered and concentrated and the crude
product was used directly in next step.
The crude material from above was dissolved in DMF (10 mL) at
room temperature under argon and the NaHCO3 (326 mg,
3.88 mmol) and BnBr (0.346 mL, 2.91 mmol) were added sequen-
tially. The mixture was stirred until the starting material was con-
sumed (TLC). The mixture was diluted with EtOAc and then
washed with H2O, brine, dried (MgSO4) and filtered and concen-
trated. The residue was purified by silica gel column chromatogra-
phy (EtOAc/hexanes; from 1:15 to 1:4) to afford product 15 (0.5 g)
as colorless oil (67% for two steps). [Note: Because it was found
that starting 14 was obtained as ratio a 3:1 of diastereomers, which
were difficult to separate, the diastereomeric mixture was used in
the synthesis of 15, resulting in a 3:1 diastereomeric product ration
for 15]: 1H NMR (400 MHz, CDCl3) d 7.38–7.29 (m, 5H), 5.12 (s, 2H),
3.90 (d, J = 8.0 Hz, 2H), 3.79 (t, J = 8.0 Hz, 1H), 2.62–2.47 (m, 2H),
2.27–2.04 (m, 1H), 1.67–1.41 (m, 15H), 0.93 (d, J = 8.0 Hz, 3H);
(85%). ½a 1D8
ꢂ
30.14 (c 0.75, CHCl3); 1H NMR (400 MHz, CDCl3) d
6.34 (d, J = 8.0 Hz, 1H), 4.60 (dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 3.76
(s, 3H), 3.08 (dd, J1 = 16.0 Hz, J2 = 8.0 Hz, 1H), 2.62 (dd,
J1 = 16.0 Hz, J2 = 8.0 Hz, 1H), 2.34 (s, 3H), 2.24–2.10 (m, 1H), 2.07
(s, 3H), 1.00 (d, J = 8.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
196.3, 172.2, 170.3, 55.9, 52.5, 37.4, 32.0, 30.7, 23.4, 16.7; HR-ESI
MS calcd for C10H18NO4S (M+H)+: 248.0957, found: 248.0950.
3.2.3. N-Fmoc-(2S,3R)-2-amino-3-methyl-4-sulfobutanoic acid
(4c)
At 0 °C, to a mixture of 7 (0.093 g, 0.38 mmol) in MeCN and 2 N
HCl (1 mL:0.2 mL, 5/1 v/v) was added N-chlorosuccinimide
(0.201 g, 1.50 mmol) in portions. The mixture was stirred at
<20 °C for 10–30 min. The solution was diluted with Et2O and
washed with 12% aqeuous NaCl (3ꢃ). The organic layer was dried
(Na2SO4) and concentrated to give product as an off-white solid.
The crude product was added 0.1 N NaOH and dioxane (v/v 1/1).
After 30 min, the mixture was concentrated and H2O was added.
The aqueous solution was washed with dichloromethane. After
all, acidified it by concentrated HCl and lyophilized to get com-
pound 8, then used it directly in next step.
HR-ESI MS calcd for
378.2279.
C
21H32NO5 (M+H)+: 378.2280, found:
3.3.2. N-Fmoc (2S,3S)-2-amino-5-(benzyloxy)-3-methyl-5-oxo-
pentanoic acid (4d)
To a stirred solution of 15 (0.242 g, 0.64 mmol) in acetone
(5.0 mL) at 0 °C was added freshly prepared Jones’ reagent
(2.7 M, 0.356 mL, 0.96 mmol). The mixture was allowed to warm
to room temperature over 30 minutes, and then stirred at room
temperature (1 h). Celite and isopropyl alcohol (1.48 mL) were
added to the mixture and the resulting precipitate was removed
by filtration and the filtrate was concentrated and extracted with
EtOAc (3ꢃ). The combined extracts were washed with brine (2ꢃ),
dried (Na2SO4) and concentrated in vacuo to give 16 (0.2 g) as col-
orless gum (89%).
Compound 8 was refluxed in 6 N HCl (3.8 mL) for 12 h. The H2O
was added (4 mL), and then the aqueous solution was washed with
dichloromethane (3ꢃ). The final solution was lyophilized to dry
and used in next step.
The residue obtained above and NaOH (35 mg, 0.88 mmol) were
dissolved in H2O (3.5 mL) cooled in an ice bath. A solution of Fmoc-
OSu (130 mg, 0.38 mmol) in dioxane (3.5 mL) was added in one
portion. The mixture was stirred at room temperature (4 h), then
the mixture was evaporated under reduced pressure and H2O
(7.0 mL) was added. The aqueous solution was washed with Et2O
and acidified with concentrated aqueous HCl. The aqueous solution
was lyophilized to yield crude product, which was purified by
preparation HPLC (flow rate10 mL/min, acetonitrile/H2O with
0.1% TFA, acetonitrile from 30% to 100% in 30 min; retention
time = 17.2 min) to provide pure 4c as white foam 20 mg (14%
Crude product 16 (0.16 g, 0.46 mmol) was dissolved in HCl in
dioxone (4 M, 1.1 mL, 4.55 mmol) and the reaction mixture was
stirred at room temperature (3 h), then the solvent was removed
by evaporation and the residue was dissolved in THF and H2O
(4.5 mL:4.5 mL, 1:1 v/v) and cooled in an ice bath. Sodium bicar-
bonate (69 mg, 0.82 mmol) and FmocOSu (0.23 g, 0.68 mmol) were
added and the mixture was stirred at room temperature for (10 h).
At this time, 1 N HCl was added to adjust pH to 2–3, and then the
THF was removed by evaporation. The resulting aqueous solution
was extracted with EtOAc, and the combined organic extracts
was washed with brine, dried (Na2SO4), filtered and the filtrate
concentrated under reduced pressure. The resulting crude product
was purified by silica gel column chromatography (dichlorometh-
ane/MeOH, from 20:1 to 10:1) to yield 4d (0.17 g) as a semisolid
(81% for two steps). [As noted above, starting 15 was present as
a diastereomeric mixture, 4d was also obtained as a diastereomeric
mixture]: 1H NMR (400 MHz, CDCl3) d 7.76 (d, J = 4.0 Hz, 2H), 7.59
(t, J = 4.0 Hz, 2H), 7.45–7.28 (m, 9H), 5.44 (d, J = 8.0 Hz, 1H), 5.15 (s,
2H), 4.62 (dd, J1 = 8.0 Hz, J2 = 4.0 Hz, 1H), 4.42 (d, J = 8.0 Hz, 2H),
4.22 (t, J = 8.0 Hz, 1H), 2.77–2.22 (m, 3H), 0.96 (d, J = 8.0 Hz, 3H);
for four steps). ½a D18
ꢂ
3.0 (c 0.95, MeOH); 1H NMR (400 MHz, CD3OD)
d 7.76 (d, J = 8.0 Hz, 2H), 7.66 (t, J = 8.0 Hz, 2H), 7.40–7.20 (m, 4H),
4.30 (d, J = 4.0 Hz, 2H), 4.20 (t, J = 8.0 Hz, 2H), 3.02 (dd, J1 = 16.0 Hz,
J2 = 4.0 Hz, 1H), 2.80–2.70 (m, 1H), 2.62–2.50 (m, 1H), 1.19 (d,
J = 8.0 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 173.6, 158.9, 145.4,
145.3, 142.7, 128.9, 128.3, 126.4, 121.0, 68.3, 60.5, 55.1, 34.3,
17.5; HR-ESI MS calcd for C20H22NO7S (M+H)+: 420.1117, found:
420.1115.
3.3. Synthesis of N-Fmoc-(2S,3S)-2-amino-5-(benzyloxy)-3-me-
thyl-5-oxopentanoic acid (4d) from 9
HR-ESI MS calcd for
474.1904.
C
28H28NO6 (M+H)+: 474.1917, found:
Conversion of the protected
to literature procedures.53
D-serine analog 9–14 was according
3.4. Synthesis of N-Fmoc (2S)-2-amino-3-(benzyloxyphosphory-
loxy)-3-methylbutanoic acid (4f) from 10
3.3.1. (3S,4S)-Benzyl N,O-isopropylidenyl-4-(N-tert-butyloxycar-
bonylamino)-3-methyl-5-oxo-pentanoate (15)
Conversion of the protected D-serine analog 10 to the Boc-pro-
tected amino acid 18 was according to literature procedures.54
To a solution of 14 (0.612 g, 1.94 mmol) in THF–H2O (8/2 mL) at
0 °C was added LiOHꢁH2O (244 mg, 5.82 mmol) and the mixture
was stirred at room temperature until the starting material was
consumed (TLC). To the mixture was added 1 N HCl until pH = 2–
3, and then the THF was evaporated and the residue was extracted
3.4.1. Allyl N-Fmoc (2S)-2-amino-3-hydroxyl-3-methylbutano-
ate (19)
To a solution of 18 (0.503 g, 2.16 mmol) in DMF (10 mL) at room
temperature under argon were sequentially added NaHCO3

W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
4001
(0.362 g, 4.31 mmol) and allyl bromide (0.28 mL, 3.23 mmol) and
the mixture was stirred until the starting material had been con-
sumed (TLC). The mixture was diluted with EtOAc and the solution
was washed with H2O, brine and dried by (MgSO4) and then fil-
tered and the filtrate concentrated. The resulting residue was puri-
fied by silica gel column chromatography (EtOAc/hexanes; from
1:10 to 1:2) to afford a white foam (0.35 g, 60%). This was dissolved
in a 1 M solution of HCl in Et2O (12.44 mL, 12.44 mmol) and the
reaction mixture stirred at room temperature (3 h). The solvent
was then removed by evaporation and the residue was dissolved
in a solution of THF/H2O (12 mL:12 mL, 1:1 v/v) and cooled in an
ice bath. Sodium bicarbonate (0.21 g, 2.49 mmol) and Fmoc-OSu
(0.63 g, 1.87 mmol) were added and the mixture was stirred at
room temperature (10 h). The THF was then removed by evapora-
tion and the aqueous phase was extracted with EtOAc and the
combined organic layer was washed with brine and dried (Na2SO4),
then filtered and concentrated under reduced pressure. The result-
ing residue was purified by silica gel column chromatography
(EtOAc/hexanes; from 1:10 to 1:2) to yield product 19 (0.42 g) as
dichloromethane/MeOH (50:1) to remove aniline impurities. Sub-
sequent elution with dichloromethane:MeOH/AcOH (25:1:0.1)
provided the free acid as foam (0.12 g, 91%).
A portion of this material (0.06 g, 0.097 mmol) and 2,20-dipyri-
dyl (7.6 mg, 0.049 mmol) were dissolved in MeOH (1 mL) and
10%Pd/C (10 mg) was added and the flask was flushed with H2
and subsequently stirred under a balloon of H2. After the reaction
had achieved completion (TLC), the mixture was filtered and evap-
orated to dryness. The resulting oily solid was purified by prepara-
tive HPLC (flow rate 10 mL/min, acetonitrile/H2O with 0.1% TFA,
acetonitrile from 30% to 100% in 20 min; retention time = 21.9 -
min) and lyophilized to provide 4f as white foam 30 mg (60%).
½
a 1D8
ꢂ
ꢀ1.06 (c 0.45, CHCl3); 1H NMR (400 MHz, CDCl3) d 9.85 (br
s, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 8.0 Hz, 2H), 7.41–7.27
(m, 9H), 6.00 (br s, 1H), 5.05 (d, J = 8.0 Hz, 2H), 4.50–4.30 (m,
3H), 4.20 (t, J = 8.0 Hz, 1H), 1.71 (s, 3H), 1.49 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 173.6, 156.6, 144.0, 143.8, 141.5, 135.8,
128.8, 128.6, 128.0, 127.4, 125.4, 124.9, 120.2, 85.2, 69.6, 67.6,
62.8, 62.1, 47.3, 26.1, 25.5; HR-ESI MS calcd for C27H29NO8P
(M+H)+: 526.1631, found: 526.1620.
a foam (85% for two steps). ½a D18
ꢂ
ꢀ17.0 (c 1.3, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.78 (d, J = 4.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H),
7.41 (t, J = 8.0 Hz, 2H), 7.32 (t, J = 8.0 Hz, 2H), 6.0–5.86 (m, 1H),
5.72 (d, J = 8.0 Hz, 1H), 5.38 (d, J = 16.0 Hz, 1H), 5.29 (d, J = 8.0 Hz,
1H), 4.76–4.62 (m, 2H), 4.50–4.37 (m, 2H), 4.32 (d, J = 8.0 Hz,
1H), 4.24 (t, J = 8.0 Hz, 1H), 2.55 (s, 1H), 1.31 (s, 3H), 1.29 (s, 3H);
13C NMR (100 MHz, CDCl3) d 171.6, 156.5, 144.0, 143.9, 141.5,
131.4, 127.9, 127.3, 125.3, 125.2, 120.21, 120.19, 119.7, 72.1,
67.4, 66.4, 61.8, 47.4, 27.2, 26.6; HR-ESI MS calcd for C23H26NO5
(M+H)+: 396.1811, found: 396.1810.
3.5. Solid-phase peptide synthesis
Fmoc protected amino acids 4a, 4e, 4g, and other amino acids
whose syntheses were not described above, were purchased from
Novabiochem. Reagent 4b was synthesized according to litera-
ture.50 Reagent 3 was readily prepared as recently reported.51
Peptides were synthesized on NovaSynÒTGR resin (Novabiochem,
cat. no. 01-64-0060) using standard Fmoc solid-phase protocols
in N-methyl-2-pyrrolidone (NMP). 1-O-Benzotriazole-N,N,N0,N0-
tetramethyl-uronium-hexafluoro-phosphate (HBTU) (5.0 equiv),
hydroxybenzotriazole (HOBT) (5.0 equiv) and N,N-diisopropyleth-
ylaminutese (DIPEA) (10.0 equiv) were used as coupling reagents.
Amino terminal acetylation was achieved using 1-acetylimida-
zole. Finished resins were washed with DMF, MeOH, dichloro-
methane and Et2O and then dried under vacuum (overnight).
Peptides were cleaved from the resin by treatment with TFA/
H2O/triisopropylsilane (95:2.5:2.5) (4 h). The resins were then fil-
tered and the filtrate was concentrated under vacuum, then pre-
cipitated with cold ether and the precipitate washed with cold
ether. The resulting solid was dissolved in 50% aqueous acetoni-
trile 5 mL) and purified by reverse phase preparative HPLC using
a Phenomenex C18 column (21 mm dia ꢃ 250 mm, cat. no: 00G-
4436-P0) with a linear gradient from 0% aqueous acetonitrile
(0.1% trifluoroacetic acid) to 100% acetonitrile (0.1% trifluoroace-
tic acid) over 30 min at a flow rate of 10.0 mL/min. Lyophilization
gave product peptides as white powders. Peptides 2a–2c and 2e–
2k were synthesized directly by standard Fmoc-based solid-
phase protocols. Because peptide 2d is obtained with the benzyl
ester intact arising from amino acid 4d, following HPLC purifica-
3.4.2. N-Fmoc (2S)-2-amino-3-(dibenzyloxyphosphoryloxy)-3-
methylbutanoic acid (20)
A solution of 19 (0.2 g, 0.51 mmol), dibenzyl-diisopropyl-phos-
phoramidite (0.35 g, 1.01 mmol) and N-methylaniline-trifluoroace-
tate (0.45 g, 2.02 mmol) in DMF (5 mL) were stirred at room
temperature under argon (1 h). Oxidation was performed by addi-
tion of t-butyl-hydroperoxide in n-octane (5.0 M, 0.192 mL,
0.96 mmol). The solution was diluted with H2O and extracted with
EtOAc and the combined organic layer was washed with H2O and
brine, dried (MgSO4), then filtered and the filtrate concentrated.
The resulting residue was purified by silica gel column chromatog-
raphy (EtOAc/hexanes; from 1:4 to 2:1) to provide 20 (0.3 g) as
semisolid (90%). ½a D18
ꢂ
ꢀ8.4 (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.76 (d, J = 4.0 Hz, 2H), 7.65–7.60 (m, 2H), 7.43–7.28 (m, 14H),
6.40 (d, J = 8.0 Hz, 1H), 5.92–5.80 (m, 1H), 5.31 (d, J = 16.0 Hz,
1H), 5.20 (d, J = 12.0 Hz, 1H), 5.10–4.96 (m, 4H), 4.62 (d,
J = 4.0 Hz, 2H), 4.43 (d, J = 2.0 Hz, 1H), 4.40 (d, J = 8.0 Hz, 2H),
4.25 (t, J = 8.0 Hz, 1H), 1.65 (s, 3H), 1.60 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 169.2, 156.5, 144.1, 143.9, 141.5, 136.0,
131.6, 128.8, 128.7, 128.0, 127.9, 127.3, 125.4, 120.2, 119.3, 84.4,
69.5, 67.5, 66.4, 62.5, 47.4, 29.9, 26.5, 25.4; HR-ESI MS calcd for
C
37H38NO8P (M+H)+: 656.2413, found: 656.2408.
Table 1
ESI-mass spectral data and HPLC purity of synthetic peptides
No
Expected (M + H)+
Observed (M + H)+
HPLC purity (%)
3.4.3. N-Fmoc (2S)-2-amino-3-(benzyloxyphosphoryloxy)-3-meth-
ylbutanoic acid (4f)
2a
2b
2c
2d
2e
2f
2g
2h
2i
863.4
861.4
861.4
825.5
849.4
877.5
863.5
811.5
810.5
797.4
767.5
863.3
861.3
861.4
825.4
849.3
877.4
863.4
811.4
810.4
797.4
767.4
100
100
100
90
100
98
96
100
98
To a solution of 20 (0.141 g, 0.22 mmol) in THF (1.1 mL) was
added N-methylaniline (70
lL, 0.64 mmol) followed by the addi-
tion of Pd(PPh3)4 (0.012 g, 10.75
lmol). The mixture was protected
from light and stirred under at room temperature under nitrogen
(45 min). The mixture was then diluted with EtOAc and washed
with saturated aqueous NH4Cl. The NH4Cl layer was back-
extracted with EtOAc and the combined EtOAc extracts were dried
(Na2SO4) filtered and concentrated. The resulting residue was
purified by silica gel chromatography eluting first with
2j
2k
99
100

4002
W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
15. Yaffe, M. B. Nat. Rev. Mol. Cell Biol. 2002, 3, 177.
16. Pawson, T. Cell 2004, 116, 191.
tion the product was dissolved in MeOH and subjected to
hydrogenolysis (10% PdꢁC, H2) to afford the completely deprotec-
ted 2d. ESI-mass spectral data and HPLC purity of the synthetic
peptides are provided in Table 1.
17. Yaffe, M. B.; Elia, A. E. H. Curr. Opin. Cell Biol. 2001, 13, 131.
18. Yaffe, M. B.; Smerdon, S. J. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 225.
19. Mayer, B. J. Methods Mol. Biol. 2006, 332, 79.
20. Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429.
21. Dai, W. Oncogene 2005, 24, 214.
3.6. ELISA-based PBD-binding inhibition assays
22. Lowery, D. M.; Lim, D.; Yaffe, M. B. Oncogene 2005, 24, 248.
23. van de Weerdt, B. C. M.; Medema, R. H. Cell Cycle 2006, 5, 853.
24. Archambault, V.; Glover, D. M. Nat. Rev. Mol. Cell Biol. 2009, 10, 265.
25. Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer 2006, 6, 321.
26. Eckerdt, F.; Yuan, J.; Strebhardt, K. Oncogene 2005, 24, 267–276.
27. Takai, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene 2005, 24, 287.
28. Elia, A. E.; Rellos, P.; Haire, L. F.; Chao, J. W.; Ivins, F. J.; Hoepker, K.;
Mohammad, D.; Cantley, L. C.; Smerdon, S. J.; Yaffe, M. B. Cell 2003, 115, 83.
29. Goh, K. C.; Wang, H.; Yu, N.; Zhou, Y.; Zheng, Y.; Lim, Z.; Sangthongpitag, K.;
Fang, L.; Du, M.; Wang, X.; Jefferson, A. B.; Rose, J.; Shamoon, B.; Reinhard, C.;
Carte, B.; Entzeroth, M.; Ni, B.; Taylor, M. L.; Stuenkel, W. Drug Dev. Res. 2004,
62, 349.
30. McInnes, C.; Mezna, M.; Fischer, P. M. Curr. Top. Med. Chem. 2005, 5, 181.
31. Gumireddy, K.; Reddy, M. V. R.; Cosenza, S. C.; Nathan, R. B.; Baker, S. J.;
Papathi, N.; Jiang, J.; Holland, J.; Reddy, E. P. Cancer Cell 2005, 7, 275.
32. Lansing, T. J.; McConnell, R. T.; Duckett, D. R.; Spehar, G. M.; Knick, V. B.;
Hassler, D. F.; Noro, N.; Furuta, M.; Emmitte, K. A.; Gilmer, T. M.; Mook, R. A.;
Cheung, M. Mol. Cancer Ther. 2007, 6, 450.
Peptide pull-down assays were carried out essentially as de-
scribed previously.38,42 A biotinylated p-T78 peptide was first di-
luted with 1ꢃ coating solution (KPL Inc., Gaithersburg, MD) to a
final concentration of 0.3 lM, and then 100 lL of the resulting
solution was immobilized onto a 96-well streptavidin-coated
plate (Nalgene Nunc, Rochester, NY). The wells were washed
once with PBS plus 0.05% Tween20 (PBST), and incubated with
200 lL of PBS plus 1% BSA (blocking buffer) for 1 h to prevent
non-specific binding. Mitotic 293A lysates expressing HA-EGFP-
Plk1 were prepared in TBSN {20 mM Tris–HCl (pH 8.0), 150 mM
NaCl, 0.5% NP-40, 5 mM EGTA, 1.5 mM EDTA, 20 mM p-nitrophe-
nylphosphate and protease inhibitor cocktail (Roche)} buffer
33. Lenart, P.; Petronczki, M.; Steegmaier, M.; Di Fiore, B.; Lipp, J. J.; Hoffmann, M.;
Rettig, W. J.; Kraut, N.; Peters, J.-M. Curr. Biol. 2007, 17, 304.
34. Lu, L.-Y.; Yu, X. Cell Div. 2009, 4, 12.
35. Reindl, W.; Yuan, J.; Kraemer, A.; Strebhardt, K.; Berg, T. ChemBioChem 2009, 10,
1145.
36. Strebhardt, K. Nat. Rev. Drug Disc. 2010, 9, 643–659.
37. Reindl, W.; Yuan, J.; Kraemer, A.; Strebhardt, K.; Berg, T. Chem. Biol. 2008, 15,
459–466.
38. Yun, S.-M.; Moulaei, T.; Lim, D.; Bang, J. K.; Park, J.-E.; Shenoy, S. R.; Liu, F.;
Kang, Y. H.; Liao, C.; Soung, N.-K.; Lee, S.; Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka,
A.; Appella, E.; McMahon, J. B.; Nicklaus, M. C.; Burke, T. R., Jr.; Yaffe, M. B.;
Wlodawer, A.; Lee, K. S. Nat. Struct. Mol. Biol. 2009, 16, 876–882.
39. Wipf, P.; Arnold, D.; Carter, K.; Dong, S.; Johnston, P. A.; Sharlow, E.; Lazo, J. S.;
Huryn, D. H. Curr. Top. Med. Chem. 2009, 9, 1194–1205.
40. Li, L.; Wang, X.; Chen, J.; Ding, H.; Zhang, Y.; Hu, T.-C.; Hu, L.-H.; Jiang, H.-L.;
Shen, X. Acta Pharmacol. Sinica 2009, 30, 1443.
41. Watanabe, N.; Sekine, T.; Takagi, M.; Iwasaki, J.-I.; Imamoto, N.; Kawasaki, H.;
Osada, H. J. Biol. Chem. 2009, 284, 2344.
42. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Graber, M.; Berg, T.; Yaffe, M. B.; Lee, K.
S., ; Burke, T. R., Jr. Nat. Chem. Biol. 2011, 7, 595.
43. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K.
S.; Burke, T. R. ACS Chem. Biol. 2012, 7, 805.
44. Burke, T. R., Jr.; Lee, K. Acc. Chem. Res. 2003, 36, 426–433.
45. Costantino, L.; Barlocco, D. Cur. Med. Chem. 2004, 11, 2725.
46. Berkowitz, D. B.; Bose, M. J. Fluorine Chem. 2001, 112, 13–33.
47. Otaka, A.; Mitsuyama, E.; Watanabe, J.; Watanabe, H.; Fujii, N. Biopolymers
2004, 76, 140–149.
(ꢄ60
lg total lysates in 100 lL buffer), mixed with the indicated
amount of peptide ligands and applied immediately onto the bio-
tinylated p-T78 peptide-coated ELISA wells, and then incubated
with constant rocking for 1 h at 25 °C. Following incubation, the
ELISA plates were washed 4 times with PBST. To detect bound
HA-EGFP-Plk1, the plates were probed for 2 h with 100
of anti-HA antibody at a concentration of 0.5 g/mL in blocking
buffer and then washed five times. The plates were further
probed for 1 h with 100 L/well of HRP-conjugated secondary
lL/well
l
l
antibody (GE Healthcare, Piscataway, NJ) at a 1:1000 dilution in
blocking buffer. The plates were washed five times with PBST
and incubated with 100 l
L/well of 3,30,5,50-tetramethylbenzidine
(TMB) substrate solution (Sigma, St. Louis, MO) until a desired
absorbance was achieved. The reactions were stopped by the
addition of 100 lL/well of stop solution (Cell Signaling Technol-
ogy, Danvers, MA) and the optical densities (OD) were measured
at 450 nm using an ELISA plate reader (Molecular Devices, Sun-
nyvale, CA). Results are shown in Figures 3 and 4.
Acknowledgments
48. Fernandez, M. C.; Diaz, A.; Guillin, J. J.; Blanco, O.; Ruiz, M.; Ojea, V. J. Org. Chem.
2006, 71, 6958.
49. Panigrahi, K.; Eggen, M.; Maeng, J.-H.; Shen, Q.; Berkowitz, D. B. Chem. Biol.
2009, 16, 928.
This work was supported in part by the Intramural Research
Program of the NIH, Center for Cancer Research, NCI-Frederick
and the National Cancer Institute, National Institutes of Health.
We thank Drs. Christpher C. Lai and James A. Kelley of the CBL
for obtaining the high-resolution mass spectra and for assistance
in the interpretation of the data. The content of this publication
does not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the
U.S. Government.
50. Liu, F.; Park, J.-E.; Lee, K. S.; Burke, T. R. Tetrahedron 2009, 65, 9673.
51. Qian, W.; Liu, F.; Burke, T. R. J. Org. Chem. 2011, 76, 8885.
52. Nishiguchi, A.; Maeda, K.; Miki, S. Synthesis 2006, 4131.
53. Konno, H.; Takebayashi, Y.; Nosaka, K.; Akaji, K. Heterocycles 2010, 81, 79.
54. Yonezawa, Y.; Shimizu, K.; Yoon, K.-S.; Shin, C.-G. Synthesis 2000, 634.
55. Vorherr, T.; Bannwarth, W. Bioorg. Med. Chem. Lett. 1995, 5, 2661.
56. Elia, A. E. H.; Cantley, L. C.; Yaffe, M. B. Science 2003, 299, 1228.
57. Johmura, Y.; Soung, N.-K.; Park, J.-E.; Yu, L.-R.; Zhou, M.; Bang, J. K.; Kim, B.-Y.;
Veenstra, T. D.; Erikson, R. L.; Lee, K. S. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
11446.
58. Bradshaw, J. M.; Waksman, G. Adv. Protein Chem. 2003, 61, 161.
59. Kotoris, C. C.; Chen, M.-J.; Taylor, S. D. Bioorg. Med. Chem. Lett. 1998, 8,
3275.
References and notes
60. Beaulieu, P. L.; Cameron, D. R.; Ferland, J.-M.; Gauthier, J.; Ghiro, E.; Gillard, J.;
Gorys, V.; Poirier, M.; Rancourt, J.; Wernic, D.; Llinas-Brunet, M.; Betageri, R.;
Cardozo, M.; Hickey, E. R.; Ingraham, R.; Jakes, S.; Kabcenell, A.; Kirrane, T.;
Lukas, S.; Patel, U.; Proudfoot, J.; Sharma, R.; Tong, L.; Moss, N. J. Med. Chem.
1999, 42, 1757.
61. Koitabashi, N.; Aiba, T.; Hesketh, G. G.; Rowell, J.; Zhang, M.; Takimoto, E.;
Tomaselli, G. F.; Kass, D. A. J. Mol. Cell Cardiol. 2010, 48, 713.
62. Lee, S.; Shuman, J. D.; Guszczynski, T.; Sakchaisri, K.; Sebastian, T.; Copeland, T.
D.; Miller, M.; Cohen, M. S.; Taunton, J.; Smart, R. C.; Xiao, Z.; Yu, L.-R.;
Veenstra, T. D.; Johnson, P. F. Mol. Cell. Biol. 2010, 30, 2621.
63. Wang, G.-L.; Shi, X.; Haefliger, S.; Jin, J.; Major, A.; Iakova, P.; Finegold, M.;
Timchenko, N. A. J. Clin. Invest. 2010, 120, 2549.
1. del Sol, A.; Carbonell, P. PLoS Comput. Biol. 2007, 3, 2446.
2. Keskin, O.; Gursoy, A.; Ma, B.; Nussinov, R. Chem. Rev. 2008, 108, 1225.
3. Clackson, T.; Wells, J. Science 1995, 267, 383.
4. Ofran, Y.; Rost, B. PLoS Comput. Biol. 2007, 3, 1169.
5. Moreira, I. S.; Fernandes, P. A.; Ramos, M. J. Proteins: Struct. Funct. Bioinform.
2007, 68, 803–812.
6. Geppert, T.; Hoy, B.; Wessler, S.; Schneider, G. Chem. Biol. 2011, 18, 344.
7. Yin, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 4130.
8. Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001.
9. Zinzalla, G.; Thurston, D. E. Future Med. Chem. 2009, 1, 65.
10. Meireles, L. M. C.; Mustata, G. Curr. Top. Med. Chem. 2011, 11, 248.
11. Garner, A. L.; Janda, K. D. Curr. Top. Med. Chem. 2011, 11, 258.
12. Morelli, X.; Bourgeas, R.; Roche, P. Curr. Opin. Chem. Biol. 2011, 15, 475.
13. Rechfeld, F.; Gruber, P.; Hofmann, J.; Kirchmair, J. Curr. Top. Med. Chem. 2011,
11, 1305.
64. Farghaian, H.; Chen, Y.; Fu, A. W. Y.; Fu, A. K. Y.; Ip, J. P. K.; Ip, N. Y.; Turnley, A.
M.; Cole, A. R. J. Biol. Chem. 2011, 286, 19724.
65. O’Leary, H.; Liu, W. H.; Rorabaugh, J. M.; Coultrap, S. J.; Bayer, K. U. J. Biol. Chem.
2011, 286, 31272.
14. Mullard, A. Nat. Rev. Drug Disc. 2012, 11, 173.

W. Qian et al. / Bioorg. Med. Chem. 21 (2013) 3996–4003
4003
66. Maheswaranathan, M.; Gole, H. K. A.; Fernandez, I.; Lassegue, B.; Griendling, K.
K.; San, M. A. J. Biol. Chem. 2011, 286, 35430.
67. Mielgo, A.; Seguin, L.; Huang, M.; Camargo, M. F.; Anand, S.; Franovic, A.; Weis,
S. M.; Advani, S. J.; Murphy, E. A.; Cheresh, D. A. Nat. Med. 2011, 17, 1641.
68. Moss, N. M.; Wu, Y. I.; Liu, Y.; Munshi, H. G.; Stack, M. S. J. Biol. Chem. 2009, 284,
19791.
69. Romanova, L. Y.; Holmes, G.; Bahte, S. K.; Kovalchuk, A. L.; Nelson, P. J.; Ward,
Y.; Gueler, F.; Mushinski, J. F. J. Cell Sci. 2010, 123, 1567.
70. Ng, Y.-W.; Raghunathan, D.; Chan, P. M.; Baskaran, Y.; Smith, D. J.; Lee, C.-H.;
Verma, C.; Manser, E. Structure 2010, 18, 879.
71. Weidenfeld-Baranboim, K.; Koren, L.; Aronheim, A. Biochem. J. 2011, 436,
661.