Enhancing polo-like kinase 1 selectivity of polo-box domain-binding peptides

Article abstract of DOI:10.1016/j.bmc.2017.02.063

An important goal in the development of polo-like kinase 1 (Plk1) polo-box domain (PBD) binding inhibitors is selectivity for Plk1 relative to Plk2 and Plk3. In our current work we show that Plk1 PBD selectivity can be significantly enhanced by modulating

Full text of DOI:10.1016/j.bmc.2017.02.063

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhancing polo-like kinase 1 selectivity of polo-box domain-binding
peptides
⇑
Xue Zhi Zhao, David Hymel, Terrence R. Burke Jr.
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
An important goal in the development of polo-like kinase 1 (Plk1) polo-box domain (PBD) binding inhi-
bitors is selectivity for Plk1 relative to Plk2 and Plk3. In our current work we show that Plk1 PBD selec-
tivity can be significantly enhanced by modulating interactions within a previously discovered ‘‘cryptic
pocket” and a more recently identified proximal ‘‘auxiliary pocket.”
Received 26 January 2017
Revised 24 February 2017
Accepted 27 February 2017
Available online xxxx
Published by Elsevier Ltd.
Keywords:
Plk1
Selectivity
Polo-box domain
Cryptic binding pocket
1. Introduction
possibility of qualitatively different therapeutic profiles or syner-
gism with KD inhibitors.10 Accordingly, significant effort has been
Members of the polo family of serine/threonine kinases (polo-
like kinases; Plks 1–5) play important roles in mammalian cellular
physiology.1 Overexpression of the cell cycle regulatory Plk1
occurs in a number of cancers, where it is associated with a poor
prognosis and Plk1 is recognized as a potentially promising anti-
cancer target.2 In addition to their amino-terminal catalytic kinase
domains (KD) (not present in Plk53), the Plks are characterized by
C-terminal polo-box domains (PBDs), which mediate protein-pro-
tein interactions (PPIs) by recognizing and binding to phosphoser-
ine (pSer) and phosphothreonine (pThr)-containing sequences.4
Development of Plk1 inhibitors is made complicated by a number
of factors. Among these is the fact that Plks 2 and 3 may function as
tumor suppressors, thereby making Plk1-selectivity an important
objective.5 Issues of collateral cytotoxicity have arisen for KD-
directed inhibitors, potentially due to similarities among the KDs
of Plk1, 2 and 3 as well as high homology among protein kinases
in general. Because both the KD and PBD are essential for proper
Plk function,6 PBD-binding antagonists offer a potentially attrac-
tive alternative to KD-directed inhibitors for down-regulating Plk
function.7 Such agents may offer advantages in terms of selectivity,
since PBDs are unique to the Plks and the domains exhibit differ-
ences in their binding preferences for phosphoepitope
sequences.8,9 Additionally, the results of blocking PBD-binding
may not be the same as inhibiting kinase activity, opening the
devoted to develop Plk1 PBD-binding antagonists.2,7
Competitive PBD-binding inhibitors have been approached
using peptides or peptide mimetics, which are based on cognate
recognition sequences. Starting from the polo-box interacting pro-
tein 1 (PBIP) pT78-derived peptide PLHSpT (1),11 we have previ-
ously reported that appending alkylphenyl groups from different
positions on the peptide, including the His N(p)-position [the
sequence having a (CH2)8Ph group appended is designated as
PLH⁄SpT (2a)],12 the 4-position on the Pro residue (3)13 and an
amino-terminal N-alkyl Gly residue (4),14 can result in up to one
thousand-fold enhancement in Plk1 PBD-binding affinity (Fig. 1).
We found in each case that the alkylphenyl groups bind within a
‘‘cryptic binding pocket” on the PBD surface, which is defined by
PBD residues Y417, Y421, Y481, L478, F482 and Y485 and revealed
by rotation of the Y481 side chain (Fig. 2). Abell has also shown
that the same cryptic pocket can be accessed by the phenyl ring
of the terminal Phe residue in the longer PBIP1 pT78-derived
sequence FDPPLHSpTA15 and by the phenyl ring of 3-phenylpropy-
lamide on the Pro residue of 1.16 This group has also shown that
higher affinity can be realized by replacing the terminal Phe resi-
due in the FDPPLHSpTA sequence with a residue that contains a
more lipophilic 2-benzothiophene ring (5), which is consistent
with the highly lipophilic nature of the cryptic pocket (Fig. 1).17
Recently, we examined access to the cryptic pocket from the His
N(p)-position in peptide 1 using an oxime-based post-solid phase
peptide diversification strategy18 and found that replacing the phe-
nyl ring with motifs having two aryl rings arranged in an angular
⇑
Corresponding author.
E-mail address: burkete@helix.nih.gov (T.R. Burke Jr.).
http://dx.doi.org/10.1016/j.bmc.2017.02.063
0968-0896/Published by Elsevier Ltd.
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

2
X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Structures of peptides discussed in the text.
orientation, can result in several fold greater Plk1 PBD-binding
affinity relative to the parent phenyl ring.19 We hypothesized that
enhanced affinity may result from interactions within an auxiliary
region that is contiguous with the canonical cryptic pocket. Yet,
beyond the above, very little has been reported to exploit binding
within the cryptic pocket to greater advantage. Therefore, as we
report herein, we used the simple tethered phenyl ring of peptide
2a and our recently disclosed 2-fluoro-6-phenyloxylphenyl pep-
tide 2b as starting points for the design of peptides 6a and 6b,
which include hydroxyls at different locations. Our thought was
that this added functionality could potentially afford interactions
with hydroxyl or carbonyl groups of residues Y421 and Y481 or
other residues proximal to the Plk1 PBD cryptic pocket. The benzyl
protected precursors 7a and 7b also provided the opportunity to
extend aromatic interactions in this region. Our ultimate goal
was to more fully explore what could be achieved by altering
known binding motifs, particularly effects related to Plk1, 2 and
3 selectivity.
alcohol 2-(4-bomophenyl)ethan-1-ol (8). Coupling of the aryl bro-
mides 9(a, b) and oct-7-yn-1-ol,21 followed by hydrogenation cat-
alyzed by PdÁC (for 10a) or Wilkinson’s catalyst (for 10b) afforded
alcohols 11(a, b) having t-butyl and benzyl protecting groups,
respectively. Separately, SNAr reaction of 2,6-difluorbenzaldehyde
(12) with 3-(tert-butoxy)phenol or 3-(benzyloxy)phenol gave the
2-fluoro-6-phenoxybenzaldehydes 13(a, b), respectively.22 Reduc-
tion of the aldehyde functionality in 13(a, b) and replacement of
the hydroxyls in the resulting 14(a, b) with bromide gave the cor-
responding benzyl bromides 15(a, b). Coupling of 15(a, b) with
hept-6-yn-1-ol or ((hept-6-yn-1-yloxy)methyl)benzene afforded
the alkynes 16(a, b). Hydrogenation catalyzed by PdÁC or Wilkin-
son’s catalyst afforded the alcohols 17(a, b). Finally, reaction of
alcohols 11(a, b) and 17(a, b) with either the 2,4-dimethoxylbenzyl
a
ester or the benzyl ester of N -Fmoc-His using our previously
reported methodology afforded the key orthogonally protected
His N(p
)-alkylated analogs 18(a–d).19,20 Employing these reagents,
we prepared peptides 19(a–d) on NovaSyn TGR resin. Cleavage of
the peptides from the resin using a cocktail solution of TFA/H2O/
TIS (95/2.5/2.5) and purification by HPLC gave the desired peptide
products 6(a, b) and 7(a, b) (Scheme 1).
2. Results and discussion
2.1. Chemistry
2.2. Biological evaluation
Peptides 6(a, b) and 7(a, b) were obtained by Fmoc-based
solid-phase peptide synthesis using reagents 18(a–d), which were
prepared as shown in Scheme 1.20 The synthesis began with the
t-butyl and benzyl-protected alcohols 9(a, b), which were synthe-
sized by protecting the free hydroxyl of the commercially available
2.2.1. PBD-binding activity in full-length Plk1
In order to evaluate PBD-binding affinities, we employed an
ELISA assay, which measures the ability of the synthetic peptides
to compete with an immobilized phosphopeptide ‘‘PMQSpTPLN”
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
2.2.2. Binding affinities measured using isolated PBDs of Plk1, 2 and 3
An important consideration in the development of PBD-binding
inhibitors is selectivity for the PBD of Plk1 relative the PBDs of Plk2
and Plk3. While, there are no PBD-binding assays currently avail-
able that employ full-length Plk2 and Plk3, fluorescence polariza-
tion (FP) assays have been reported, which measure the abilities
of inhibitors to compete with fluorescently labeled pThr-contain-
ing peptides for binding to the isolated PBDs of Plk1, 2 and 3.8,9
These assays employ fluorescently labeled peptides that have been
optimized for binding to each individual PBD isoform.8,9 It is worth
noting that the IC50 values measured against the isolated Plk1 PBD
are more potent than those measured against full length Plk1.23,24
This difference can be attributed to inhibitory interactions between
the PBD and KD, which affect equilibrium binding to phosphopep-
tides. We measured the abilities of 6(a, b) and 7(a, b) as well as 2(a,
b) to compete with fluorescently-labeled phosphopeptide probes
for binding to isolated PBDs of Plks 1–3 using FP assays (Table 1).
While the parent peptide 2a showed approximately 20-fold
selectivity for the Plk1 PBD relative to the Plk2 and approximately
50-fold selectivity relative to the Plk3 PBD, introduction of func-
tionality onto the cryptic pocket-binding phenyl ring of 2a was
capable of further increasing selectivity for both domains (Table 1).
Fold-selectivities for Plk1 relative to Plk2 and Plk3, respectively
upon introduction of the indicated functionality were: 4-(2-
hydroxylethyl) (6a) 50 and 175; 2-fluoro, 6-phenlyoxy (2b) 40
and 200; 2-fluoro, 6-(3-hydroxy)phenyloxy (6b) 60 and 350. Inter-
estingly, introducing a benzyl group onto 6b to give 2-fluoro, 6-(3-
benzyloxy)phenoxy-containing 7b, increased Plk1 PBD selectivity
versus the Plk2 PBD to 300-fold and 2500-fold relative to the
Plk3 PBD. This data showed that modification of the phenyl group
in 2a could result in significant improvements in selectivity for the
Plk1 PBD relative to the Plk2 PBD and that near complete selectiv-
ity over the Plk3 PBD could be realized.
2.3. Comparison of the cryptic pocket of the Plk1 PBD with potential
cryptic pockets in the PBDs of Plk2 and Plk3
Fig. 2. Plk1 PBD electrostatic surfaces (blue = positive; red = negative). (A) PLHSpT
with bound 1(PDB accession code: 3HIK); (B) PLH⁄SpT with bound 2a (PDB
accession code: 3RQ7) having the portion of the peptide accessing the cryptic
pocket shown in yellow and the cryptic pocket overlain yellow mesh and a recently
identified ‘‘auxiliary pocket” shown with overlain red mesh.
A major focus of our current study was to examine the effects
incurred by modifying interactions within the cryptic binding
pocket. Although we prepared only a very limited number of ana-
logs, we found that changes to the cryptic pocket-binding phenyl
ring could have a profound impact on the selectivity for the Plk1
PBD relative to the Plk2 and Plk3 PBDs. We speculated whether
there might be differences among the PBDs of Plk1, 2 and 3, which
could potentially contribute to modulation of selectivity. The exis-
tence of ‘‘cryptic binding pockets” in the Plk2 and Plk3 PBDs have
not been reported. Crystal structures of the Plk2 PBD are
known,25,26 yet these structures do not contain bound ligands
and to date, no crystal structures of the Plk3 PBD have been
reported. Therefore, we set out to define putative interacting resi-
dues within potential cryptic pockets of Plk2 and Plk3 and to com-
pare them with corresponding residues in the Plk1 PBD.
Lacking a crystal structure for the Plk3 PBD, we aligned the
sequences of Plk1 with Plk3 and identified residues of Plk3 that
corresponded to the Plk1 PBD. We then generated a homology
model of the Plk3 PBD based on the Plk1 PBD (PDB accession code:
3FVH) through the online Robetta server.27 Using the crystal struc-
ture of the Plk1 PBD having ligand 2a bound (PDB accession code:
3RQ7) we superimposed the crystal structure of the apo Plk2 PBD
(PDB accession code: 4RS6) and the Plk3 PBD homology model.
Ligand 2a was oriented into the Plk2 and Plk3 PBDs as in the
Plk1 PBD and residues proximal to the ligands were minimized
(Fig. 3). We found that all three PBDs were highly similar in the
region of the cryptic pocket. Several of the residues from Plk1
(creme; Y417, Y421, Y481, F482 and Y485) and Plk2 (orange;
Y510, Y514, Y574, F575 and Y578) are closely oriented. Although
for binding to full-length Plk1.19 As we have recently reported, the
parent peptide 1, which lacks an N(
p)-alkyl group, shows almost
no inhibitory potency in this assay (IC50 > 300
l
M), while introduc-
tion of a –[CH2]8Ph at this position results in greater than an
approximate 1000-fold enhancement in binding affinity (2a,
IC50 = 180 nM, Table 1).19 Replacement of the phenyl ring in 2a
with a 2-fluoro-6-phenoxybenzy group resulted in enhanced bind-
ing affinity (2b, IC50 = 127 nM).19 The focus of our current study
was to examine the effects of introducing additional oxygen func-
tionality into cryptic pocket-binding motifs. For this purpose, we
prepared 6a as a 4-(2-hydroxyethyl)-containing analog of 2a and
6b as an analog of 2b, which included a 3-hydroxyl group at the
3-position of the phenyl ether moiety. Both 6a and 6b exhibited
IC50 values (220 nM and 190 nM, respectively), which were
approximately equal to 2a, indicating that the presence of the
hydroxyl groups was compatible with maintenance of high affinity
binding (Table 1). Given the large number of aromatic side chains
bordering the cryptic pocket, we were also interested in observing
the effects incurred by introducing additional aryl functionality.
Peptides 7a and 7b, which are intermediates in the synthesis of
6a and 6b, respectively served this purpose. While the added ben-
zyl group was well tolerated in 7a (IC50 = 150 nM), its presence in
7b caused an approximate fourfold loss of potency (IC50 = 860 nM)
(Table 1).
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

4
X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 1. Preparation of peptides 6(a, b) and 7(a, b). Reagents and Conditions: i) Boc2O, Mg(ClO4)2; ii) Pd(PPh3)2Cl2, CuI, CH„C(CH2)6OH, 70 °C; iii) H2, (Ph3P)3RhCl or Pd/C;
iv) ArOH, K2CO3, DMA, 165 °C; v) NaBH4, MeOH; vi) CBr4, PPh3, CH3CN; vii) CuI, TBAI, Cs2CO3, CH„C(CH2)5OH or nBuLi, CH„C(CH2)5OBn; viii) CH„CCH2OtBu, CuI, DIEA, Pd
a
s
a
s
(PPh3)2Cl2; ix) N -Fmoc-His(N -Trt)-OBn or N -Fmoc-His(N -Trt)-ODMP, Tf2O; x) TFA/TIS or H2, Pd/C; xi) TFA/TIS/H2O.
Table 1
Inhibitory binding potencies of peptides determined using full-length Plk1 and isolated PBDs of Plk1, Plk2 and Plk3.i
No.
Ar
IC50 (nM)ii
Plk1 ELISA
181 13
Plk1 PBD FP
5.0 0.2
Plk2 PBD FP
Plk3 PBD FP
2a
130 10 (26Â)iv
280 100 (56Â)
6a
218 11
5.5 0.2
310 25 (56Â)
960 280 (175Â)
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 1 (continued)
No.
Ar
IC50 (nM)ii
Plk1 ELISA
152
Plk1 PBD FP
9.7 0.7
Plk2 PBD FP
Plk3 PBD FP
7a
4
250 20 (25Â)
920 100 (95Â)
2biii
127 21
6.6 0.3
290 45 (44Â)
1450 530 (220Â)
6b
188 22
5.5 0.4
3400 60 (62Â)
1900 540 (345Â)
7b
862 113
11.2 0.3
3500 800 (312Â)
29,500 11,300 (2634Â)
i
Assays were conducted as described in Section 4.
ii
iii
iv
Values represent the average SEM from three independent experiments and fit using nonlinear regression in GraphPad Prism 6.
See Ref. 19.
Selectivity compared with Plk1.
these residues were also found in the Plk3 PBD, certain side chains
were rotated or slightly displaced in the latter domain (blue; Y470,
F474, Y534, F535 and Y538) (Fig. 3). Notable differences among the
tree domains appeared to be a single residue at the terminus of the
pocket, which consisted of L478, V571 and I531 for Plk1, Plk2 and
Plk3, respectively. Additionally, while similar orientations were
observed for K513 and K473 in Plk2 and Plk3, respectively, the cor-
responding K420 in Plk1 was considerably displaced. The most sig-
nificant variations were in residues bordering a recently identified
‘‘auxiliary binding region”, which is contiguous with the canonical
cryptic pocket.19 Here the differences for Plk1, Plk2 and Plk3 were
found to be N484, H577 and S537; E488, E581 and Q541 and H489,
N582 and H542, respectively. In spite of the general overall high
similarity of residues proximal to the cryptic pocket among the
three PBDs, it is possible that the minor differences noted as well
as the significant variations in the auxiliary pocket could poten-
tially contribute to significant modulation of ligand recognition.
Although beyond the scope of the current work, replacing select
residues identified above in the PBDs of Plk2 and Plk3 could help
to resolve speculation regarding the roles that these may play in
ligand selectivity.
Achieving selectivity is an important consideration in the develop-
ment of Plk1 PBD-binding inhibitors. In our current work we
demonstrate that structural variation of functionality intended to
interact within the cryptic pocket can have a significant impact
on the relative affinities of peptides for the PBDs of Plk2 and Plk3
relative to Plk1. In the case of Plk3, specificity can exceed three
orders-of-magnitude. We have found subtle differences in residues
proximal to the putative cryptic pockets and particularly in resi-
dues forming a recently identified proximal auxiliary binding
pocket. We suggest that these differences may contribute to ligand
selectivity. Introducing extended functionality that permits access
to the auxiliary pocket may be a valuable approach to increasing
differences in binding affinity among the three domains.
A further significant challenge in developing clinically relevant
PBD-binding inhibitors arises from the fact that PBD-binding pep-
tides and peptide mimetics, even those showing extremely high
affinities in extracellular assays, have frequently shown disap-
pointing biological efficacies in cell-based systems.12 The discrep-
ancies in potencies may result from poor cellular uptake.
Accordingly, increasing cellular bioavailability of PBD-binding
inhibitors represents an active area of investigation.28 Our current
peptides are not clinically relevant, due in part to poor bioavailabil-
ity. However, given the extreme enhancement in overall ligand
affinity that can be achieved by accessing the cryptic pocket, we
believe that optimizing interactions within this region may be
highly relevant to developing more bioavailable agents.
3. Conclusions
Targeting the PBD offers a conceptually attractive alternative to
KD-directed inhibitors for down-regulating Plk1 function.2,7
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

6
X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. Superposition of residues judged to potentially impact interactions in the cryptic pocket as well as in the ‘‘auxiliary pocket” (encircled in red). Residue carbons and
residue numbering are color keyed as shown in the legend.
Accordingly, our current findings may facilitate the further devel-
opment of clinical agents.
(10 mL) was stirred under H2 (16 h). The crude mixture was puri-
fied by silica gel chromatography to provide alcohols (11a, 17a).
4.1.3. General procedure C for the synthesis of alcohols (11b, 17b)
using PdÁC
4. Experimental section
To a solution of alkynes (10b, 16b) (10 mmol) in MeOH (10 mL)
was added PdÁC (30 mg, 10%) and the mixture was hydrogenated
under H2. When all starting material had been consumed (TLC),
the reaction mixture was collected by filtration and concentrated.
The crude residue was purified by silica gel chromatography to
provide alcohols (11b, 17b).
4.1. General chemical methods
Proton (1H) and carbon (13C) NMR spectra were recorded on a
Varian 400 MHz spectrometer or a Varian 500 MHz spectrometer
and are reported in ppm relative to TMS and referenced to the sol-
vent in which the spectra were collected. Solvent was removed by
rotary evaporation under reduced pressure and anhydrous solvents
were obtained commercially and used without further drying.
Purification by silica gel chromatography was performed using
Combiflash with EtOAc–hexanes or CH2Cl2-MeOH solvent systems.
Electrospray ionization-mass spectrometra (ESI-MS) were acquired
with an Agilent LC/MSD system equipped with a multimode ion
source. High resolution mass spectrometric (HRMS) were acquired
with a LTQ-Orbitrap-XL at 30 K resolution by LC/MS-ESI.
4.1.4. General procedure D for the synthesis of aldehydes (13a, b)22
Potassium carbonate (100 mmol) was added to a solution of
2,6-difluorobenzaldehydes (12a) (102 mmol) and 3-(benzyloxy)
phenol or 3-(tert-butoxy)phenol (100 mmol) in dimethylacetamide
(DMA) (50 mL). The reaction mixture was stirred and refluxed
(2 h). The crude mixture was partitioned between CH2Cl2 and
H2O, dried (Na2SO4), concentrated and purified by silica gel chro-
matography to provide aldehydes (13a, b).
4.1.1. General procedure A for the synthesis of alkynes (10a, b) using
Sonogashira reaction21
Appropriate terminal alkynes (0.45 mmol) were added to a mix-
ture of bromides (9a, b) (0.3 mmol) and bis(triphenylphosphine)-
4.1.5. General procedure E for the synthesis of benzylalcohols (14a, b)
NaBH4 (20 mmol) was added portion-wise to the solution of
aldehydes (13a, b) (20 mmol) in MeOH (100 mL) (0 °C). The reac-
tion mixture was stirred (0 °C, 0.5 h) and concentrated. The crude
mixture was partitioned between EtOAc and H2O, dried (Na2SO4),
concentrated and purified by silica gel chromatography to provide
benzylalcohols (14a, b).
palladium (II) dichloride (9 lmol), DIEA (0.3 mmol) and copper
(I) iodide (0.03 mmol) in DMF (1.5 mL). The reaction mixture was
sealed and heated under argon (70 °C, 18 h). The crude mixture
was purified by silica gel chromatography to provide alkynes
(10a, b).
4.1.6. General procedure F for the synthesis of benzylbromide (15a, b)
PPh3 (30 mmol) was added to a solution of benzylalcohols (14a,
b) (20 mmol) in acetonitrile (100 mL). CBr4 (30 mmol) was added
(0 °C). The formed suspension was stirred (30 min). The mixture
was filtered and the filtrate was concentrated. The crude residue
4.1.2. General procedure B for the synthesis of alcohols (11a, 17a)
using Wilkinson’s catalyst29
A mixture of alkynes (10a, 16a) (1.0 mmol), tris(triphenylphos-
t
phine)rhodium(I) chloride (0.1 mmol) in THF (10 mL) and BuOH
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
was purified by silica gel chromatography to provide benzylbro-
mides (15a, b).
7.17–7.11 (m, 4H), 3.65 (t, J = 6.6 Hz, 2H), 3.55 (t, J = 7.7 Hz, 2H),
2.82 (t, J = 7.7 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 11.63–1.56 (m, 4H),
1.40–1.30 (m, 8H), 1.21 (s, 9H). 13C NMR (101 MHz, CDCl3) d
140.60, 136.39, 128.85 (2C), 128.27 (2C), 72.90, 63.25, 63.03,
37.08, 35.56, 32.78, 31.53, 29.47, 29.36, 29.25, 27.55 (3C), 25.74.
APCI-MS m/z: 307.2 (MNa+).
p
4.1.7. General procedure G for the synthesis of N -alkylated His
residues (18a–d)19,20
According to our previous reports,19,20 a solution of alcohols
(11a, b and 17a, b) (2 mmol) and DIEA (2 mmol) in CH2Cl2
(1.5 mL) was added dropwise to a solution of trifluoromethanesul-
fonic anhydride (2 mmol, 1.0 M in CH2Cl2) in CH2Cl2 (2.5 mL) under
argon (À78 °C) and stirred (20 min). A solution of 2,4-dimethoxy-
4.1.13. 8-(4-(2-(Benzyloxy)ethyl)phenyl)octan-1-ol (11b)
Treatment of 10b as outlined in general procedure B provided
11b as a colorless oil (29% yield). 1H NMR (400 MHz, CDCl3) d
7.39–7.29 (m, 5H), 7.23–7.22 (m, 1H), 7.18–7.12 (m, 3H), 4.56
(s, 2H), 3.72 (q, J = 7.1 Hz, 2H), 3.65 (td, J = 6.6, 4.5 Hz, 2H), 2.94
(t, J = 7.2 Hz, 2H), 2.62–2.58 (m, 1H), 1.65–1.50 (m, 6H), 1.40–
a
s
benzyl N -Fmoc-N -Trt-
L
-histidinate (2 mmol) or benzyl
a
s
N -Fmoc-N -Trt-
L
-histidinate (2 mmol) in CH2Cl2 (5 mL) was
added (À78 °C). The mixture was stirred (16 h) and concentrated.
The residue was deprotected using TFA:TIS (10:1, 10 mL) or hydro-
genated using PdÁC (10%) in MeOH. The resultant crude residue
1.30 (m, 7H). 13C NMR (101 MHz, CDCl3)
d 140.72, 138.45,
136.04, 128.80 (2C), 128.38 (2C), 128.36 (2C), 127.64 (2C), 127.53
(2C), 72.95, 71.41, 63.05, 35.96, 35.57, 32.79, 31.54, 29.48, 29.37,
29.26, 25.75. DUIS-MS m/z: 341 (MH+).
p
was purified by silica gel chromatography to provide N -alkylated
His residues (18a–d).
4.1.8. 1-Bromo-4-(2-(tert-butoxy)ethyl)benzene (9a)30
4.1.14. 2-(3-(tert-Butoxy)phenoxy)-6-fluorobenzaldehyde (13a)
Treatment of commercial available 2,6-difluorobenzaldehyde
12 and 3-(tert-butoxy)phenol as outlined in general procedure D
provided 13a as a yellow oil (78% yield). 1H NMR (400 MHz, CDCl3)
d 10.51 (s, 1H), 7.46–7.40 (m, 1H), 7.31–7.27 (m, 1H), 6.89–6.80 (m,
3H),6.73 (t, J = 2.2 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 1.37 (s, 9H). 13C
Magnesium perchlorate (0.96 g, 4.3 mmol) and commercial
available 2-(4-bromophenyl)ethanol 8 (8.6 g, 43 mmol) were dis-
solved in DCM (50 mL). (Boc)2O (21 g, 98 mmol) was added to
the suspension. The mixture was stirred (35 °C, 16 h) and concen-
trated. The crude residue was purified by silica gel column chro-
matography to provide 9a as a colorless oil (7.6 g, 69% yield). 1H
NMR (101 MHz, CDCl3)
d
186.78 (d, J = 2.4 Hz), 162.90
NMR (400 MHz, CDCl3)
d
7.42 (d, J = 8.3 Hz, 2H), 7.13 (d,
(d, J = 263.4 Hz), 160.43 (d, J = 5.2 Hz), 157.14, 155.93, 135.69
(d, J = 11.6 Hz), 129.90, 120.42, 115.95 (d, J = 9.4 Hz), 115.65,
114.64, 113.41 (d, J = 3.8 Hz), 110.77 (d, J = 21.2 Hz), 79.24, 28.84
(3C). ESI-MS m/z: 271.1 (MH+-OH), 311.1 (MNa+). [3-(tert-butoxy)
phenol was prepared from commercial available 3-(benzyloxy)
phenol through two steps below.30 Magnesium perchlorate
(0.56 g, 2.5 mmol) and 3-(benzyloxy)phenol (5 g, 25 mmol) were
dissolved in DCM (50 mL). (Boc)2O (12.5 g, 57 mmol) was added.
The formed brown solution was stirred (35 °C, 16 h) and concen-
trated. The crude residue was purified by silica gel chromatogra-
J = 8.3 Hz, 2H), 3.54 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.1 Hz, 2H), 1.18
(s, 9H). 13C NMR (100 MHz, CDCl3) d 138.57, 131.22 (2C), 130.79
(2C), 119.85, 72.90, 62.56, 36.78, 27.50 (3C).
4.1.9. 1-(2-(Benzyloxy)ethyl)-4-bromobenzene (9b)
2-(4-Bromophenyl)ethanol
8 (3.2 g, 16 mmol) and (bro-
momethyl)benzene (1.9 mL, 16 mmol) were dissolved in THF
(50 mL). NaH (0.45 g, 18 mmol) was added (0 °C). The reaction
mixture was stirred (from 0 °C to room temperature, 16 h),
quenched by pouring into water, extracted by EtOAc, dried (Na2
SO4) and concentrated. The crude residue was purified by silica
gel chromatography to provide 9b as a colorless oil (1.9 g, 41%
yield). 1H NMR (400 MHz, CDCl3) d 7.45–7.43 (m, 2H), 7.39–7.31
(m, 5H), 7.15–7.13 (m, 2H), 4.55 (s, 2H), 3.70 (t, J = 6.9 Hz, 2H),
2.91 (t, J = 6.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) d 138.26,
138.13, 131.38 (2C), 130.72 (2C), 128.40 (2C), 127.61 (3C),
120.03, 73.03, 70.77, 35.80.
phy to provide 1-(benzyloxy)-3-(tert-butoxy)benzene as
a
colorless oil (4.6 g, 72% yield). 1H NMR (400 MHz, CDCl3) d 7.49–
7.34 (m, 5H), 7.21 (t, J = 8.2 Hz, 1H), 6.77 (dd, J = 7.9, 1.9 Hz, 1H),
6.69–6.66 (m, 2H), 5.08 (s, 2H), 1.39 (s, 9H). 13C NMR (100 MHz,
CDCl3) d 159.36, 156.62, 137.06, 129.15, 128.59 (2C), 127.96,
127.54 (2C), 116.73, 111.10, 109.87, 78.64, 70.08, 28.93 (2C).
Hydrogenation of 1-(benzyloxy)-3-(tert-butoxy)benzene (4.0 g,
15.9 mmol) using PdÁC (10%, 60 mg) in MeOH (50 mL) (4 h) pro-
vided 3-(tert-butoxy)phenol a yellow solid (2.4 g, 93% yield). 1H
NMR (400 MHz, CDCl3) d 7.10 (t, J = 8.1 Hz, 1H), 7.05 (brs, 1H),
6.61 (ddd, J = 8.1, 2.4, 1.5 Hz, 2H), 6.55 (t, J = 2.3 Hz, 1H), 1.35
(s, 9H). 13C NMR (101 MHz, CDCl3) d 156.37, 155.67, 129.51,
116.52, 111.83, 111.31, 79.73, 28.79 (3C). ESI-MS m/z: 167.1 (MH+).]
4.1.10. 8-(4-(2-(tert-Butoxy)ethyl)phenyl)oct-7-yn-1-ol (10a)
Treatment of 9a and commercial available oct-7-yn-1-ol as out-
lined in general procedure A provided 10a as a yellow oil (41%
yield). 1H NMR (400 MHz, CDCl3) d 7.32 (d, J = 8.1 Hz, 2H), 7.15
(d, J = 8.1 Hz, 2H), 3.66 (td, J = 6.6, 5.3 Hz, 2H), 3.53 (t, J = 7.3 Hz,
2H), 2.81 (t, J = 7.3 Hz, 2H), 2.42 (t, J = 7.0 Hz, 2H), 1.66–1.58 (m,
5H), 1.54–1.48 (m, 2H), 1.47–1.40 (m, 2H), 1.17 (s, 9H). 13C NMR
(101 MHz, CDCl3) d 139.02, 131.36 (2C), 128.90 (2C), 121.67,
89.66, 80.67, 72.92, 62.86, 62.72, 37.24, 32.66, 28.73, 28.68, 27.49
(3C), 25.32, 19.36. ESI-MS m/z: 325.2 (MNa+).
4.1.15. 2-(3-(Benzyloxy)phenoxy)-6-fluorobenzaldehyde (13b)
Treatment of commercial available 2,6-difluorobenzaldehyde
(12) with 3-(benzyloxy)phenol as outlined in general procedure
D provided 13b as a brown oil (18% yield). 1H NMR (400 MHz,
CDCl3) d 10.40 (s, 1H), 7.37–7.18 (m, 7H), 6.82–6.77 (m, 1H), 6.75
(dd, J = 8.3, 2.3 Hz, 1H), 6.61–6.58 (m, 3H), 4.97 (s, 2H). 13C NMR
4.1.11. 8-(4-(2-(Benzyloxy)ethyl)phenyl)oct-7-yn-1-ol (10b)
Treatment of 9b with commercial available oct-7-yn-1-ol as
outlined in general procedure A provided 10b as a yellow oil
(62% yield). 1H NMR (400 MHz, CDCl3) d 7.37–7.29 (m, 7H), 7.16
(d, J = 8.1 Hz, 2H), 4.53 (s, 2H), 3.68 (dt, J = 9.2, 6.8 Hz, 4H), 2.94–
2.91 (m, 2H), 2.43 (t, J = 7.0 Hz, 2H), 1.74 (bs, 1H), 1.68–1.60 (m,
4H), 1.55–1.48 (m, 2H), 1.47–1.41 (m, 2H).
(101 MHz, CDCl3) d 186.76, 162.90 (d, J = 263.5 Hz), 160.28,
160.08 (d, J = 5.2 Hz), 156.88, 136.42, 135.69 (d, J = 11.6 Hz),
130.61, 128.65 (2C), 128.14, 127.51 (2C), 116.21 (d, J = 9.5 Hz),
113.97 (d, J = 3.7 Hz), 111.97, 111.25, 111.05 (d, J = 21.2 Hz),
106.67, 70.26.
4.1.16. (2-(3-(tert-Butoxy)phenoxy)-6-fluorophenyl)methanol (14a)
Treatment of 13a as outlined in general procedure E provided
14a as a colorless oil (93% yield). 1H NMR (400 MHz, CDCl3) d
7.24 (t, J = 8.1 Hz, 1H), 7.21–7.17 (m, 1H), 6.87–6.80 (m, 2H),
6.77–6.74 (m, 1H), 6.69 (t, J = 2.3 Hz, 1H), 6.65 (d, J = 8.5 Hz, 1H),
4.1.12. 8-(4-(2-(tert-Butoxy)ethyl)phenyl)octan-1-ol (11a)
Treatment of 10a as outlined in general procedure C provided
11a as a yellow solid (54% yield). 1H NMR (400 MHz, CDCl3) d
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

8
X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.83 (d, J = 1.0 Hz, 2H), 2.32 (brs, 1H), 1.36 (s, 9H). 13C NMR
(101 MHz, CDCl3) d 161.68 (d, J = 247.3 Hz), 156.97 (2C), 156.91
(d, J = 7.4 Hz), 129.67, 129.53 (d, J = 10.4 Hz), 119.59, 119.34
(d, J = 18.1 Hz), 114.93, 113.89, 113.71 (d, J = 3.3 Hz), 110.44
(d, J = 22.6 Hz), 79.06, 53.97 (d, J = 4.9 Hz), 28.84 (3C). ESI-MS
m/z: 291.1 (MH+), 313.1 (MNa+).
(m, 4H), 1.36 (s, 9H). 13C NMR (101 MHz, CDCl3) d 161.50 (d,
J = 246.8 Hz), 157.63, 156.78, 155.95 (d, J = 7.3 Hz), 138.69,
129.36, 128.34 (2C), 127.95 (d, J = 10.2 Hz), 127.61 (2C), 127.46,
119.00, 117.51 (d, J = 18.2 Hz), 114.56, 114.46 (d, J = 3.3 Hz),
113.58, 110.52 (d, J = 22.3 Hz), 79.75, 78.84, 76.71, 72.86, 70.34,
29.28, 28.87 (3C), 28.70, 25.43, 18.74, 12.99 (d, J = 4.8 Hz).
4.1.17. (2-(3-(Benzyloxy)phenoxy)-6-fluorophenyl)methanol (14b)
Treatment of 13b as outlined in general procedure E provided
14b as a colorless oil (79% yield). 1H NMR (400 MHz, CDCl3) d
7.46–7.34 (m, 5H), 7.30–7.26 (m, 1H), 7.25–7.20 (m, 1H), 6.92–
6.87 (m, 1H), 6.80 (ddd, J = 8.4, 2.4, 0.9 Hz, 1H), 6.72–6.63 (m,
3H), 5.06 (s, 2H), 4.83 (d, J = 1.4 Hz, 2H), 2.18 (S, 1H). 13C NMR
4.1.21. 8-(2-(3-(tert-Butoxy)phenoxy)-6-fluorophenyl)octan-1-ol
(17a)
Treatment of 16a as outlined in general procedure C provided
17a as a yellow solid (95% yield). 1H NMR (400 MHz, CDCl3) d
7.21 (t, J = 8.1 Hz, 1H), 7.09 (td, J = 8.2, 6.5 Hz, 1H), 6.83
(t, J = 8.8 Hz, 1H), 6.77–6.74 (m, 1H), 6.70–6.66 (m, 2H), 6.61
(t, J = 2.3 Hz, 1H), 3.62 (td, J = 6.6, 1.3 Hz, 2H), 2.67 (td, J = 7.6,
1.6 Hz, 2H), 1.61–1.51 (m, 4H), 1.36 (s, 9H), 1.33–1.26 (m, 8H).
13C NMR (101 MHz, CDCl3) d 162.04 (d, J = 244.6 Hz), 158.02,
156.74, 155.84 (d, J = 8.3 Hz), 129.40, 126.85 (d, J = 10.4 Hz),
122.17 (d, J = 18.7 Hz), 118.68, 114.70 (d, J = 3.1 Hz), 114.02,
113.00, 110.50 (d, J = 23.1 Hz), 78.91, 63.00, 32.77, 29.43, 29.32,
29.25, 29.22, 28.84 (3C), 25.69, 22.98 (d, J = 2.7 Hz). ESI-MS m/z:
389.2 (MH+), 333.1 (MH+-Bu).
(101 MHz, CDCl3)
d 161.71 (d, J = 247.6 Hz), 160.20, 157.94,
156.52 (d, J = 7.3 Hz), 136.57, 130.44, 129.63 (d, J = 10.4 Hz),
128.64 (2C), 128.11, 127.54 (2C), 119.67 (d, J = 17.9 Hz), 114.34
(d, J = 3.3 Hz), 111.18, 110.75 (d, J = 22.5 Hz), 110.41, 105.81,
70.20, 54.04 (d, J = 5.1 Hz).
4.1.18. 2-(Bromomethyl)-1-(3-(tert-butoxy)phenoxy)-3-
fluorobenzene (15a)
Treatment of 14a as outlined in general procedure F provided
15a as a colorless oil (90% yield). 1H NMR (400 MHz, CDCl3) d
7.29–7.25 (m, 1H), 7.21 (td, J = 8.4, 6.5 Hz, 1H), 6.86–6.80 (m,
3H), 6.73 (t, J = 2.3 Hz, 1H), 6.63 (d, J = 8.5 Hz, 1H), 4.67 (s, 2H),
1.38 (s, 9H). 13C NMR (101 MHz, CDCl3) d 161.51 (d, J = 250.4 Hz),
156.95, 156.82 (d, J = 6.5 Hz), 156.73, 130.11 (d, J = 10.6 Hz),
129.65, 119.87, 117.08 (d, J = 17.2 Hz), 115.38, 114.44, 113.50
(d, J = 3.3 Hz), 110.11 (d, J = 21.6 Hz), 79.07, 28.87 (3C), 20.03
(d, J = 5.4 Hz).
4.1.22. 8-(2-(3-(Benzyloxy)phenoxy)-6-fluorophenyl)octan-1-ol (17b)
Treatment of 15b with hept-6-yn-1-ol using CuI, Cs2CO3 and
TBAI in acetonitrile (70 °C, 20 h) as the reported method31 followed
by using the synthesis as outlined in general procedure B provided
17b as a colorless oil (9% yield). 1H NMR (400 MHz, CDCl3) d 7.45–
7.35 (m, 5H), 7.28–7.22 (m, 1H), 7.14–7.08 (m, 1H), 6.88–6.83
(m, 1H), 6.76–6.70 (m, 2H), 6.61–6.56 (m, 2H), 5.05 (s, 2H), 3.64
(t, J = 6.6 Hz, 2H), 2.67 (td, J = 7.6, 1.6 Hz, 2H), 1.62–1.52 (m, 4H),
1.33 (qt, J = 8.3, 3.2 Hz, 8H). 13C NMR (101 MHz, CDCl3) d 162.06
(d, J = 244.7 Hz), 160.10, 158.87, 155.56 (d, J = 8.3 Hz), 136.68,
130.18, 128.61 (2C), 128.06, 127.56 (2C), 126.93 (d, J = 10.3 Hz),
122.42 (d, J = 18.8 Hz), 115.12 (d, J = 3.1 Hz), 110.71
(d, J = 23.0 Hz), 110.47, 109.29, 105.03, 70.15, 63.06, 32.78, 29.44,
29.35, 29.29 (2C), 29.26, 25.71, 23.06, 23.04. ESI-MS m/z: 423.2
(MH+), 435.2 (MNa+).
4.1.19. 1-(3-(Benzyloxy)phenoxy)-2-(bromomethyl)-3-fluorobenzene
(15b)
Treatment of 14b as outlined in general procedure F provided
15b as a colorless oil (87% yield). 1H NMR (400 MHz, CDCl3) d
7.33–7.23 (m, 5H), 7.19–7.14 (m, 1H), 7.12–7.06 (m, 1H), 6.76–
6.68 (m, 2H), 6.60–6.52 (m, 3H), 4.94 (s, 2H), 4.53 (d, J = 1.4 Hz,
2H). 13C NMR (101 MHz, CDCl3) d 161.54 (d, J = 250.6 Hz), 160.18,
157.57, 156.51 (d, J = 6.5 Hz), 136.59, 130.40, 130.18
(d, J = 10.4 Hz), 128.65 (2C), 128.11, 127.57 (2C), 117.34
(d, J = 17.1 Hz), 113.93 (d, J = 3.3 Hz), 111.80, 110.74, 110.35
(d, J = 21.6 Hz), 106.37, 70.21, 20.04 (d, J = 5.4 Hz). APCI-MS m/z:
387.0, 389.0 (MH+).
a
p
4.1.23. N -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N -(8-(4-(2-(tert-
butoxy)ethyl)phenyl)octyl)- -histidine (18a)
L
Treatment of 11a as outlined in general procedure G provided
18a as a yellow oil (30% yield). DUIS-MS m/z: 667 (MH+).
a
p
4.1.24. N -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N -(8-(2-(3-(tert-
butoxy)phenoxy)-6-fluorophenyl)octyl)- -histidine (18b)
4.1.20. 2-(8-(Benzyloxy)oct-2-yn-1-yl)-1-(3-(tert-butoxy)phenoxy)-
3-fluorobenzene (16a)31
L
Treatment of 17a as outlined in general procedure G provided
A solution of butyllithium in hexanes (10 mmol, 2.5 M) was
added dropwise to a solution of ((hept-6-yn-1-yloxy)methyl)ben-
zene (10 mmol) [which was prepared from commercial available
hept-6-yn-1-ol using BnBr and NaH in THF (0 °C) (78% yield). 1H
NMR (400 MHz, CDCl3) d 7.37–7.35 (m, 4H), 7.33–7.29 (m, 1H),
4.53 (s, 2H), 3.50 (t, J = 6.5 Hz, 2H), 2.22 (td, J = 6.8, 2.6 Hz, 2H),
1.96 (t, J = 2.6 Hz, 1H), 1.71–1.61 (m, 2H), 1.60–1.47 (m, 4H). 13C
NMR (101 MHz, CDCl3) d 138.65, 128.36 (2C), 127.62, 127.50
(2C), 84.53, 72.92, 70.23, 68.29, 29.29, 28.34, 25.42, 18.39. ESI-
MS m/z: 203.1 (MH+)] in THF (20 mL) (À78 °C). The mixture was
stirred (À78 °C, 1 h). The solution of benzylbromides (15a)
(3 mmol) in THF (10 mL) was added. The reaction mixture was stir-
red (À78 °C to room temperature, 16 h), quenched by pouring into
ice, extracted by EtOAc and concentrated. The crude residue was
purified by silica gel chromatography to provide benzylalkynes
(16a) as a colorless oil (77% yield). 1H NMR (400 MHz, CDCl3) d
7.38–7.3 (m, 4H), 7.33–7.27 (m, 1H), 7.21 (t, J = 8.2 Hz, 1H), 7.15
(td, J = 8.3, 6.5 Hz, 1H), 6.85 (t, J = 8.7 Hz, 1H), 6.79–6.72 (m, 2H),
6.67 (dd, J = 5.3, 3.0 Hz, 2H), 4.51 (s, 2H), 3.59 (brs, 2H), 3.46 (t,
J = 6.6 Hz, 2H), 2.11–2.07 (m, 2H), 1.64–1.57 (m, 2H), 1.47–1.39
18b as a yellow solid (30% yield). ESI-MS m/z: 748.3 (MH+).
a
p
4.1.25. N -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N -(8-(4-(2-
(benzyloxy)ethyl)phenyl)octyl)- -histidine (18c)
L
Treatment of 11b as outlined in general procedure G provided
18c as a colorless oil (64% yield). ESI-MS m/z: 700.3 (MH+).
a
p
4.1.26. N -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N -(8-(2-(3-
(benzyloxy)phenoxy)-6-fluorophenyl)octyl)- -histidine (18d)
L
Treatment of 11b as outlined in general procedure G provided
18d as a yellow oil (65% yield). ESI-MS m/z: 782.3 (MH+).
4.1.27. General solid-phase peptide synthesis (SPPS)
Protected amino acids used were Fmoc-Thr(PO(OBzl)OH)-OH,
Fmoc-Ser(OtBu)-OH, N(
p)-alkylated Fmoc-His-OH (18a–d), Fmoc-
Leu-OH, and Fmoc-Pro-OH (purchased from Novabiochem). Pep-
tides were synthesized on a NovaSynÒ TGR resin (Novabiochem
Cat#. 855009) using standard Fmoc solid-phase protocols in N-
methyl-2-pyrrolidone (NMP). 1-[Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063

X.Z. Zhao et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
(HATU) (5.0 equivalents), and N,N-diisopropylethylamine (DIPEA)
3.8–5/MacOSX-IL64bit/g4.2/Release/HardwareGL/Gui264199/
WebKit/Video/FlexLM built Oct 17, 2016 14:22. 4.8.7.
(10 equivalents) were used as coupling reagents. Unnatural amino
acid residues were coupled using 2.5 equivalents. Deprotection
was performed using 20% piperidine in DMF (15 min, twice).
Amino-terminal acetylation was performed using 1-acetylimida-
zole. Finished resins were washed with NMP, MeOH, CH2Cl2, and
Et2O, dried under vacuum, and then cleaved by treatment with a
solution of TFA:H2O:TIS (95:2.5:2.5) (5 h). The resin was removed
by filtration, and the filtrate was concentrated under vacuum and
the resulting residue dissolved in 50% aqueous acetonitrile (5 mL)
and purified by reverse-phase preparative high pressure liquid
chromatography (HPLC) using a Waters 2535 system having pho-
todiode array detection and Phenomenex C18 columns (Cat. No.
4.3.2. Sequence alignment and identification of cryptic pocket residues
ICM Modeling was preformed using Plk1 (PDB accession code:
3RQ7), Plk2 (PDB accession code: 4RS6) and Plk3 [(Robetta homol-
ogy model based on PDB accession code: 3FVH (see Supplementary
data)]. Sequences were aligned using 3RQ7 as the static reference.
The 2a ligand from the 3RQ7 structure was placed in each structure
based on the protein alignments, then all neighboring residues
were minimized using the ICM standard protocol. Results are
shown in Fig. 3.
00G-4436-P0-AX, 250 mm  21.2 mm 10
l
m particle size, 110 Å
Acknowledgements
pore) at a flow rate of 10 mL/min. Binary solvent systems consist-
ing of A = 0.1% aqueous TFA and B = 0.1% TFA in acetonitrile were
employed with gradients as indicated. Products (6a, b and 7a, b)
were obtained as amorphous solids following lyophilization.
This work was supported in part by the Intramural Research
Program, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, United States.
4.1.28. Peptides
A. Supplementary data
4.1.28.1. Peptide 6a. Title peptide 6a was prepared using 18a as
outlined in SPPS and purified by preparative HPLC (linear gradient
of 0% B to 80% B over 30 min, retention time = 17.7 min). ESI-MS m/
z: 907.4 (MH+). HRMS calcd for C42H68N8O12P(MH+) 907.4689,
found 907.4676.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.063.
References
1. Zitouni S, Nabais C, Jana SC, Guerrero A, Bettencourt-Dias M. Nat Rev Mol Cell
Biol. 2014;15:433–452.
2. Lee KS, Burke TR, Park J-E, Bang JK, Lee E. Trends Pharmacol Sci.
2015;36:858–877.
3. de Carcer G, Escobar B, Higuero AM, et al. Mol Cell Biol. 2011;31:1225–1239.
4. Elia AEH, Cantley LC, Yaffe MB. Science. 2003;299:1228–1231.
5. Strebhardt K. Nat Rev Drug Discovery. 2010;9:643–659.
4.1.28.2. Peptide 6b. Title peptide 6b was prepared using 18b as
outlined in SPPS and purified by preparative HPLC (linear gradient
of 0% B to 80% B over 30 min, retention time = 19.7 min). ESI-MS m/
z: 989.2 (MH+). HRMS calcd for C46H67FN8O13P(MH+) 989.4544,
found 989.4516.
6. Park J-E, Erikson RL, Lee KS. Proc Natl Acad Sci USA. 2011;108:8200–8205.
7. Berg A, Berg T. ChemBioChem. 2016;17:650–656.
4.1.28.3. Peptide 7a. Title peptide 7a was prepared using 18c as
outlined in SPPS and purified by preparative HPLC (linear gradient
of 0% B to 80% B over 30 min, retention time = 21.6 min). ESI-MS m/
z: 997.3 (MH+).
8. Reindl W, Strebhardt K, Berg T. Anal Biochem. 2008;383:205–209.
9. Reindl W, Graeber M, Strebhardt K, Berg T. Anal Biochem. 2009;395:189–194.
10. Archambault V, Lepine G, Kachaner D. Oncogene. 2015;34:4799–4807.
11. Yun S-M, Moulaei T, Lim D, et al. Nat Struct Mol Biol. 2009;16:876–882.
12. Liu F, Park J-E, Qian W-J, et al. Nat Chem Biol. 2011;7:595–601.
13. Liu F, Park J-E, Qian W-J, et al. ACS Chem Biol. 2012;7:805–810.
14. Liu F, Park J-E, Qian W-J, et al. ChemBioChem. 2012;13:1291–1296.
15. Sledz P, Stubbs CJ, Lang S, et al. Angew Chem Int Ed. 2011;50:4003–4006.
16. Tan YS, Sledz P, Lang S, et al. Angew Chem Int Ed. 2012;51:10078–10081.
17. Sledz P, Lang S, Stubbs CJ, Abell C. Angew Chem Int Ed Engl. 2012;51:7680–7683.
18. Liu F, Stephen AG, Waheed AA, et al. ChemBioChem. 2008;9:2000–2004.
19. Zhao XZ, Hymel D, Burke Jr TR. Bioorg Med Chem Lett. 2016;26:5009–5012.
20. Qian W, Liu F, Burke Jr TR. J Org Chem. 2011;76:8885–8890.
21. Sonogashira K, Tohda Y, Hagihara N. Tetrahedron Lett. 1975;16:4467–4470.
22. Fish PV, Ryckmans T, Stobie A, Wakenhut F. Bioorg Med Chem Lett.
2008;18:1795–1798.
4.1.28.4. Peptide 7b. Title peptide 7b was prepared using 18d as
outlined in SPPS and purified by preparative HPLC (linear gradient
of 0% B to 80% B over 30 min, retention time = 22.3 min). ESI-MS m/
z: 1079.2 (MH+). HRMS calcd for C53H73FN8O13P (MH+) 1079.5013,
found 1079.4993.
4.2. Biological experimental procedures
23. Elia AE, Rellos P, Haire LF, et al. Cell. 2003;115:83–95.
Experimental procedures used to obtain the IC50 values shown
in Table 1 have been previously reported24 and are described in
detail in the Supplementary data.
24. Hymel D, Burke Jr TR. ChemMedChem. 2017;12:202–206.
25. Shan H-M, Wang T, Quan J-M. Biochem Biophys Res Commun.
2015;456:780–784.
26. Kim JH, Ku B, Lee KS, Kim SJ. Proteins Struct Funct Bioinf. 2015;83:1201–1208.
27. Kim DE, Chivian D, Baker D. Nucleic Acids Res. 2004;32:W526–W531.
28. Qian W-J, Park J-E, Lim D, et al. Biopolym Pept Sci. 2014;102:444–455.
29. Jourdant A, González-Zamora E, Zhu J. J Org Chem. 2002;67:3163–3164.
30. Bartoli G, Bosco M, Locatelli M, Marcantoni E, Melchiorre P, Sambri L. Org Lett.
2005;7:427–430.
4.3. Comparison of the PBDs of Plk1, Plk2 and Plk3 in the region
corresponding to the cryptic binding pocket of Plk1
4.3.1. ICM Modeling protocols
Standard Molsoft parameters and procedures were used
as defined in the unmodified ICM Chemist Pro version
31. Kwon Y, Cho H, Kim S. Org Lett. 2013;15:920–923.
Please cite this article in press as: Zhao X.Z., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.063