Preparation of orthogonally protected (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) as a phosphatase-stable phosphothreonine mimetic and its use in the synthesis of polo-box domain-binding peptides

Article abstract of DOI:10.1016/j.tet.2009.09.093

Reported herein is the first stereoselective synthesis of (2S,3R)-4-[bis-(tert-butyloxy)phosphinyl]-2-[(9H-fluoren-9-ylmethoxy)car bonyl]amino-3-methylbutanoic acid [(N-Fmoc, O,O-(bis-(tert-butyl))-Pmab), 4] as a hydrolytically-stable phosphothreonine mim

Full text of DOI:10.1016/j.tet.2009.09.093

Tetrahedron 65 (2009) 9673–9679
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Preparation of orthogonally protected (2S,3R)-2-amino-3-methyl-4-
phosphonobutyric acid (Pmab) as a phosphatase-stable phosphothreonine
mimetic and its use in the synthesis of polo-box domain-binding peptides
,
b
b
a,
*
Fa Liu a , Jung-Eun Park , Kyung S. Lee , Terrence R. Burke, Jr.
*
a Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, NCI-Frederick, PO Box B, Bldg.
376 Boyles St., Frederick, MD 2170, 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:
Reported herein is the first stereoselective synthesis of (2S,3R)-4-[bis-(tert-butyloxy)phosphinyl]-2-[(9H-
fluoren-9-ylmethoxy)carbonyl]amino-3-methylbutanoic acid [(N-Fmoc, O,O-(bis-(tert-butyl))-Pmab), 4] as
a hydrolytically-stable phosphothreonine mimetic bearing orthogonal protection compatible with standard
solid-phase protocols. The synthetic approach used employs Evans’ oxazolidinone for chiral induction. Also
presented is the application of 4 in the solid-phase synthesis of polo-like kinase 1 (Plk1) polo box domain
(PBD)-binding peptides. These Pmab-containing peptides retain PBD binding efficacy similar to a parent
pThr containing peptide. Reagent 4 should be a highly useful reagent for the preparation of signal trans-
duction-directed peptides.
Received 20 July 2009
Received in revised form
22 September 2009
Accepted 23 September 2009
Available online 26 September 2009
Published by Elsevier Ltd.
1. Introduction
Although a significant body of literature exists concerning the de-
velopment and application of phosphotyrosyl (pTyr) mimetics,18 fewer
Phosphorylation of proteins facilitates critical protein–protein
binding interactions that may result in signal propagation or mod-
ulation of enzyme activity.1–4 Changes in normal post-translational
modification of proteins through phosphorylation of tyrosine, serine
and threonine residues is a central paradigm in oncogenic trans-
formation.5–7 In light of this, development of kinase-directed signal
transduction inhibitors is a promising approach toward new anti-
cancer therapeutics.8–10 Synthetic phosphopeptides based on short-
ened sequences derived from phosphoproteins, can retain significant
binding affinities and they can serve as competitive antagonists of
cognate protein–protein interactions. In this fashion they can provide
initial starting points for the design of peptidomimetic-based ther-
apeutics. Typically, a key component of the recognition provided by
phosphoamino acids is derived from the phosphoryl group itself.11
However the hydrolytic lability of phosphoryl esters to phosphatases
limits the use of phosphopeptides in cellular contexts. Development
of hydrolytically-stable mimetics, in which the labile phosphoryl
ester oxygen has been replaced non-hydrolyzable methylene or
difluoromethylene groups, offers one approach to circumvent this
limitation. Peptides containing metabolically stable analogues have
proven to be useful biological tools that may serve as potential leads
for further therapeutic design.12–17
examples can be found dealing with mimetics of phosphothreonine
(pThr,1, Fig.1). Stereoselective synthesis of the pThr mimetic (2S,3R)-2-
amino-3-methyl-4-phosphonobutanoic acid (Pmab, 2) has been
reported using Schollkopf’s bislactim ether. This has provided deriv-
atized Pmab bearing O,O-(bis-allyl) protection of the phosphonic acid
group along with N-Fmoc protection.19 Synthesis of the corresponding
4,4-difluoro analogue (F2Pmab, 3) bearing O,O-(bis-ethyl) phosphonic
acid and N-Boc protection groups, has been approached using both
(R)-isopropylideneglycerol as a chiral synthon20 and Oppolzer’s sultam
chiral auxiliary.21 To date, there have been no stereoselective syntheses
reported of Pmab bearing orthogonal O,O-[bis-(tert-butyl)] phos-
phonic acid and N-Fmoc groups. This protection scheme would allow
facile use in standard solid-phase protocols on acid-labile resins.
Therefore, we report herein the first synthesis of (2S,3R)-4-[bis-(tert-
butyloxy)phosphinyl]-2-[(9H-fluoren-9-ylmethoxy)carbonyl]amino-
3-methylbutanoic acid [(N-Fmoc, O,O-(bis-(tert-butyl))-Pmab), 4]
O
P
O
O
P
O
HO
X
OH
O
OH
Fmoc
OH
O
NH2
N
H
pThr: X = O
4
1
2 Pmab: X = CH2
3
F2Pmab: X = CF2
* Corresponding authors. Tel.: þ301 846 5906; fax: þ301 846 6033.
E-mail addresses: liuf@ncifcrf.gov (F. Liu), tburke@helix.nih.gov (T.R. Burke, Jr.).
Figure 1. Structures of pThr and pThr mimetics discussed in the text.
0040-4020/$ – see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2009.09.093

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F. Liu et al. / Tetrahedron 65 (2009) 9673–9679
by a route using Evans’ oxazolidinone for chiral induction. We also
present the applicationof this reagentin thesolid-phase synthesisof
a biologically active peptide.
reduction of the g-carboxyl of 13 using DIBAL provided the alcohol
14 (Scheme 2). Key to this reaction was the use of substrate con-
centrations less than 0.03 M. Similar to above, N-deprotection of 14
by hydrogenation in a mixture of AcOH and MeOH and subsequent
Cbz protection gave the lactone 10 and the alcohol 11 in a 1:4 ratio.
1.1. Chemistry
O
O
Stereoselective synthesis of orthogonally-protected Pmab (4) be-
gan with the Swern oxidation of tert-butyldimethylsilyl (TBDMS)
mono-protected (2E)-2-butene-1,4-diol 522 followed by sodium chlo-
rite oxidation. This provided acid 6 with Z-double bond geometry
(previouslyreportedastheE-isomer23)(Scheme 1). Acid 6was coupled
with Evan’s chiral auxiliary, (4R)-4-phenyl-2-oxazolidinone24 and the
Z-doublebond geometry was isomerizedbytreatment withtri-n-butyl
O
O
CO2H
NH2
12
HO2C
N
phosphine in THF to give the desired E-isomer (7). Both a and b stereo-
genic centers of 9were constructed bya tandem sequence consistingof
13
O
HO
an asymmetric Cu(I)-catalyzed 1,4-Michael addition of methyl-
O
magnesium chloride followed by electrophilic
a
-bromination.25
a
b
The crude (2R)-bromidewas then converted tothe corresponding (2S)-
azide by nucleophilic SN2 replacement using sodium azide. A single
(2S,3R)-diastereomer (9) was obtained by column chromatographic
purification and crystallization. Assignment of absolute stereochemis-
tries was based on well-established literature precedence.25–27
Removal of the TBDMS group by treatment with catalytic p-toluene-
sulfonic acid was followed by cyclization to release Evan’s auxiliary
group and provide the five-membered lactone. The azide was reduced
by hydrogenation ina mixture ofAcOH and MeOHandprotectedin situ
to provide the lactone 10 as well as the ring-open alcohol 11 in a 1:4
ratio. Lactone 10 was further converted to 11 (Scheme 1).
+
11
10
N
14
Scheme 2. Reagents and conditions: (a) DIBAL, THF, ꢀ40 ꢁC to 0 ꢁC, 4 h, 61%. (b) 1.
1 atm H2, 10% Pd$C (10%), MeOH/AcOH, rt, overnight; 2. Benzyl chloroformate, NaHCO3,
THF/H2O, 0 ꢁC, 4 h.
Swern oxidation of alcohol 11 gave the corresponding aldehyde
(15). This aldehyde was subjected to a phospho-Mukaiyama aldol
reaction with freshly-prepared di-tert-butyltrimethylsilyl phos-
phite (17) to yield the aldehyde 17 (Scheme 3).29,30 Subsequent
treatment with citric acid gave the free alcohol (18), which was
derivatized as the phenylthiocarbonate 19 and subjected to Barton–
O
O
Si
Si
OH
a
CO2H
5
6
O
P
16
O
O
Si
O
O
O
c
b
d
N
N
O
a
O
O
O
11
O
O
H
O
N
b
H
O
Cbz
O
7
O
Si
Si
15
8
O
P
O
P
Si
O
OH
O
O
O
c
d
O
O
O
O
O
O
N
N3
O
N
H
Cbz
O
N
H
Cbz
e
N
10
H
Cbz
O
17
18
O
+
9
Si
HO
O
N
S
O
O
H
Cbz
O
P
O
P
O
11
O
O
e
O
O
f
Scheme 1. Reagents and conditions: (a) 1. Oxalyl chloride, DMSO, DCM, ꢀ78 ꢁC, 2 h; 2.
NaClO2, KH2PO4, 2-methyl-2-butene, tert-butanol/H2O, rt, overnight, 97% for 2 steps.
(b) 1. Trimethylacetyl chloride, triethylamine, THF, ꢀ78 ꢁC–0 ꢁC, 20 min; 2. (R)-
(þ)-Phenyl-2-oxazolidione lithium salt, THF, ꢀ78 ꢁC–0 ꢁC, overnight, 100%. (c) Tribu-
tylphosphine, THF, rt, 1 h, 84%. (d) 1. Methylmagnesium chloride, copper(I) bromide
dimethyl sulfide, dimethyl sulfide/THF, ꢀ78 ꢁC–ꢀ40 ꢁC, 2 h; 2. NBS, ꢀ78 ꢁC, 1.5 h; 3.
NaN3, DMF, 0 ꢁC, 2 h, 79% for 2 steps. (e) 1. p-Toluenesulfonic acid monohydrate, MeOH,
rt, 6 h; 2. 1 atm H2, 10% Pd$C (10%), MeOH/AcOH, rt, overnight; 3. Benzyl chloroformate,
NaHCO3, THF/H2O, 0 ꢁC, 4 h, 49% for 11 over 3 steps.
4
O
O
N
O
H
Cbz
N
H
19
Cbz
20
Scheme 3. Reagents and conditions: (a) Oxalyl chloride, DMSO, DCM, ꢀ78 ꢁC, 2 h.
(b) 16, DCM, rt, 3 h. (c) citric acid, MeOH/H2O, rt, overnight, 88% over
3 steps.
(d) O-phenylchlorothionoformate, DMAP (cat.) and N,N-diisopropylethylamine, DCM,
rt, overnight. (e) Tributyltin hydride, AIBN, toluene, 100 ꢁC, 20 min, 58% over 2 steps.
(f) 1. LiOH, THF/H2O, rt, overnight; 2. 1 atm H2, 10% Pd$C (10%), MeOH, rt, overnight; 3.
FmocOSu, NaHCO3, dioxane/H2O, rt, overnight, 100% over 3 steps.
It is of note that alcohol 11 can also be prepared from
L-aspartic
acid through the known bis-methyl ester 13.28 Selectively

F. Liu et al. / Tetrahedron 65 (2009) 9673–9679
9675
McCombie deoxygenation to yield 20.31 Hydrolysis of the methyl
ester, then hydrogenation and re-protection using Fmoc-OSu
provided the orthogonally protected Pmab derivative 4.
O
R
O
O
O
H
N
H
N
N
N
H
N
H
X
1.2. Application of reagent 4 to the synthesis of polo
box domain-binding peptides
O
O
Y
N
HN
The polo-like kinase 1 (Plk1) functions as an important mitotic
regulator that phosphorylates serine and threonine residues.32 Its
over-expression in a number of cancers33 and its association with poor
prognosis have made it a potential anticancer therapeutic target.34,35
A main focus of Plk1 inhibitor development has been directed at the
kinase catalytic domain.36–43 However, Plk1 contains modular C-ter-
minal ‘Polo-box domains’ (PBDs) that bind specific phosphoserine and
phosphothreonine-containing sequences to provide critical localiza-
tion of Plk1.32,44,45 Competitive PBD binding antagonists could serve as
inhibitors of Plk1 function that are distinct from kinase-directed
agents.44 A starting point for the development of PBD-binding an-
tagonists is given by short pThr-containing peptides modeled on
consensus binding sequences derived from the p-Thr78 region (p-T78)
of the PBD-binding protein, PBIP1.46–48 By examining various PBD-
binding phosphopeptides, it has recently been shown that a 5-mer
phosphopeptide ‘PLHSpT’ (21, Fig. 2) specifically interacts with the
21 R = OH; Y = NH2; X = OH
22 R = OH; Y = NH2; X = O-PO(OH)2
23 R = OH; Y = NH2; X = CH2-PO(OH)2
24 R = H; Y = NH2; X = CH2-PO(OH)2
NH2
NH2
25 R = OH; X = CF2-PO(OH)2; Y =
N
H
O
O
26 R = H; X = CF2-PO(OH)2; Y =
N
H
Figure 2. Structures of synthetic peptides used in the study.
To examine the ability of Pmab- and F2Pmab-containing pep-
tides to inhibit PBD-dependent interactions, Plk1 PBD-binding in-
hibition assays were conducted in the presence of various
concentrations of synthetic peptides. It was found that ‘PLHS–
Pmab’ (23) inhibits the interaction of the Plk1 PBD with an
immobilized p-T78 peptide as effectively as the wild-type peptide,
‘PLHSpT’ (22, Fig. 3A). In contrast, the peptide, ‘PLHS-F2Pmab-A’
(25, Fig. 2), inhibits the interaction at a somewhat reduced level
(data not shown). Replacement of the critical (pThr-1) Ser residue
with an alanine (equivalent to S77A mutation) is known to signif-
icantly attenuate PBD binding affinity.49 The non-phosphorylated
control peptide ‘PLHST’ (21, Fig. 2) and the S77A mutants of the
Pmab- and the F2Pmab-containing peptides (24 and 26, re-
spectively, Fig. 2), did not inhibit PBD binding even at 1000-fold
higher molar concentrations (Fig. 3A and data not shown).
Plk1 PBD with high affinity (Kd¼0.45
m
M).49 In order to provide
phosphatase-stable peptides for in vivo studies, F2Pmab (3) was also
incorporated into a 6-mer T78 peptide, ‘PLHSTA’, to give the corre-
sponding peptide 25.49 (Note: The 6-mer sequence ‘PLHS-F2Pmab-A’
(25) was synthesized due to inefficient synthesis of the 5-mer
sequence, ‘PLHS-F2Pmab’). It was found that 25 showed much weaker
PBD-binding affinity than the respective p-T78 peptide, ‘PLHSpTA’,
and it exhibited significant toxicity in cell-based experiments. The
toxicity can potentially be attributed to the highly acidic CF2PO3H
moiety. Therefore, using solid-phase techniques and standard Fmoc-
based protocols, we employed reagent 4 to synthesize the Pmab-
containing peptides 23 and 24 (Fig. 2).
Figure 3. Measurement of the ability of synthetic peptides 21–24 to inhibit PBD-dependent interactions. (A) PBD-binding inhibition assays were carried out in the presence of
different concentrations of the indicated inhibitory peptides.49 The level of the remaining interaction between a p-T78 peptide and full-length Plk1 was quantified by optical density
(O.D.) at 450 nm (error bars represent standard deviation). (B) Representative images of green fluorescence in EGFP plasmid-containing HeLa cells following microinjection with
PLHS–Pmab (23) or the PBD-binding defective peptides, PLHST (21) and PLHA–Pmab (24), are shown (procedure described in Experimental Section). Note induction of mitotically-
arrested, rounded-up, morphologies associated with the PBD-binding competent PLHS–Pmab.

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F. Liu et al. / Tetrahedron 65 (2009) 9673–9679
Evidence suggests that the PBD plays critical roles in the proper
3.3. Cell microinjection and confocal microscopy
sub-cellular localization and mitotic functions of Plk1. Disruption
of PBD-dependent Plk1 functions by expressing a dominant-
negative form of PBD results in a mitotic arrest that ultimately
leads to apoptotic cell death.50 To investigate the effects of
inhibiting Plk1 PBD interactions peptides 21, 23 and 24 were in-
troduced into HeLa cells. In order to overcome poor membrane
permeability of the negatively charged Pmab-containing peptides,
microinjection was employed. The Pmab-containing peptide (23),
but not the non-phosphorylated peptide 21 or the respective S77A
mutant (24), induced mitotically arrested, rounded-up, morphol-
ogy in approxi-mately 50% of the microinjected, green fluorescent
protein (GFP)-positive population (Fig. 3B). These results dem-
onstrate that inhibition of PBD function by the Pmab-containing
p-T78 mimetic peptide is sufficient to interfere with the mitotic
functions of Plk1.
Similar to procedures described in reference 49, HeLa cells were
arrested at the G1/S boundary by double thymidine treatment and
released into fresh medium. Six hours after release, the cells were
microinjected with a mixture of 3 mM of peptides 21, 23 or 24 and
30 ng/mL of pEGFP-C1 vector and the cells were then photographed
15 h after G1/S release. Co-injected EGFP plasmid provided a con-
venient marker to identify the microinjected cells. Results are
shown in Figure 3B.
3.3.1. Synthetic (E)-4-[(tert-butyldimethylsilyl)oxy]-2-buten-1-ol (5).
To a solution of (2E)-2-butene-1,4-diol (8.22 mL, 0.10 mol) and
imidazole (8.50 g, 0.125 mol) in DMF (50 mL) at 0 ꢁC, was added
tert-butyldimethylsilyl chloride (7.50 g, 0.050 mol) in several
portions over 10 min. The resulting mixture was warmed to room
temperature and stirred (2 h), then poured into H2O (200 mL) and
extracted with EtOAc (2ꢂ150 mL). The organic layer was washed
(brine), dried (Na2SO4) and purified by silica gel column chroma-
tography (hexanes:EtOAc) to yield 5 as a colorless oil (9.0 g, 89%
2. Conclusions
yield). 1H NMR (400 MHz, CDCl3)
4.09 (m, 2H), 2.76 (br, 1H), 0.83 (s, 9H), 0.01 (s, 6H). 13C NMR
(100 MHz, CDCl3) 136.3, 135.4, 64.8, 63.8, 31.1, 23.6.
d 5.60–5.50 (m, 2H), 4.18 (m, 2H),
Although a significant body of literature exists concerning the
development and application of pTyr mimetics, fewer examples can
be found dealing with mimetics of pThr. Presented herein is the first
stereoselective synthesis of the hydrolytically-stable phospho-
threonine mimetic Pmab (4), bearing (O,O)-bis-tert-butyl protection
of the phosphonic acid group along with N-Fmoc derivatization. This
orthogonal protection scheme allows facile use in standard solid-
phase protocols on acid-labile resins, where side chain protecting
groups can be removed during TFA-mediated resin cleavage. Our
synthetic approach to Pmab utilizes Evans’ oxazolidinone for chiral
induction. We also present the application of 4 in the solid-phase
synthesis of biologically active peptides directed against the Plk1
PBD. Here we show that Pmab-containing peptides retain PBD
binding efficacy similar to a parent pThr-containing peptide, while
retaining the ability to inhibit PBD-dependent interactions in whole
cells. In summary, reagent 4 should be a highly useful reagent for the
preparation of signal transduction-directed peptides.
d
3.3.2. Z-4-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-2-butenoic
acid
(6). To a solution of oxalyl chloride (3.55 mL, 40.8 mmol) in CH2Cl2
(100 mL) at ꢀ78 ꢁC, was added a solution of DMSO (5.80 mL,
81.7 mmol) in CH2Cl2 (40 mL) and the mixture was stirred (15 min).
Alcohol 5 (5.50 g, 27.2 mmol) in dry CH2Cl2 (40 mL) was added over
5 min, then the mixture was stirred at ꢀ75 ꢁC (2 h) then triethyl-
amine (31 mL, 0.22 mol) was added. The mixture was warmed
room temperature and saturated NH4Cl (50 mL) was added and the
mixture was extracted with Et2O (2ꢂ100 mL). The combined
organic layers were washed (brine), dried (Na2SO4) and evaporated
to yield the intermediate aldehyde as a pale yellow liquid. Without
purification, to a mixture of the aldehyde, potassium phosphate
monobasic (5.55 g, 40.8 mmol) and 2-methyl-2-butene (14.4 mL,
136 mmol) in tert-butanol (150 mL) and H2O (30 mL) at 0 ꢁC, was
added sodium chlorite (9.23 g, 81.6 mmol, 80% technical grade) in
several portions over 10 min. The mixture was warmed to room
temperature slowly and stirred (night). After cooling to 0 ꢁC, a so-
lution of sodium bisulfate (31.8 g, 0.30 mol) in H2O (100 mL) was
added slowly and the mixture was stirred (30 min) and extracted
with EtOAc (2ꢂ150 mL). The combined organic layer was washed
(brine), dried (Na2SO4) and purified by silica gel column chromato-
graphy (hexanes:EtOAc) to yield acid 6 as a colorless oil (5.70 g, 97%
3. Experimental procedures
3.1. General
All experiments involving moisture-sensitive compounds were
conducted under dry conditions (positive argon pressure) using
standard syringe, cannula, and septa apparatus. Solvents: All solvents
were purchased anhydrous (Aldrich) and used directly. HPLC-grade
hexanes, EtOAc, CH2Cl2, and MeOH were used in chromatography.
TLC: analytical TLC was performed on Analtech precoated plates
(Uniplate, silica gel GHLF, 250 mm) containing a fluorescence in-
dicator; NMR spectra were recorded using a Varian Inova 400 MHz
spectrometer. The coupling constants are reported in Hertz, and the
yield). 1H NMR (400 MHz, CDCl3)
(dt, J¼12.0, 2.6 Hz,1H), 4.65 (dd, J¼4.6, 2.4 Hz, 2H), 0.83 (s, 9H), 0.00
d
6.40 (dt, J¼11.6, 4.6 Hz, 1H), 5.68
(s, 6H). 13C NMR (100 MHz, CDCl3)
d
176.0, 159.7, 123.0, 67.1, 31.0,
23.0, 0.00. APCI (ꢀVE) m/z: 215.2 (MꢀH)ꢀ. HR-ESI MS ca.lcd for
C10H19O3Si (MꢀH)ꢀ: 215.1109, found: 215.1103.
peak shifts are reported in the
d (ppm) scale. Low resolution mass
3.3.3. (4R)-3-[(2Z)-[4-[(1,1-Dimethylethyl)dimethylsilyl]oxy]-1-oxo-
2-buten-1-yl]-4-phenyl-2-oxazolidinone (7). To a solution of acid 6
(6.0 g, 28.2 mmol) in THF (40 mL) at ꢀ78 ꢁC, was added triethyl-
amine (4.00 mL, 28.2 mmol) followed by trimethylacetyl chloride
(3.46 mL, 28.2 mmol) drop-wise. The mixture was warmed to 0 ꢁC
over 20 min, then the anhydride mixture was cooled to ꢀ78 ꢁC.
Separately, to a solution of (R)-(þ)-phenyl-2-oxazolidione (Aldrich)
(4.60 g, 28.2 mmol) in THF (40 mL) at ꢀ78 ꢁC was carefully added
n-BuLi (2.50 M in THF, 11.3 mL, 28.2 mmol) and the mixture was
stirred (30 min) then transferred to the anhydride solution at
ꢀ78 ꢁC. The final reaction mixture was warmed to room tempera-
ture and stirred (overnight). The mixture was diluted with EtOAc
(200 mL), washed (H2O and brine), dried (Na2SO4), and purified by
silica gel column chromatography (hexanes:EtOAc) to yield 7 as
spectra (ESI) was measured with Agilent 1200 LC/MSD-SL system,
and high resolution mass spectra (ESI or APCI) was measured by UCR
Mass Spectrometry Facility, Department of Chemistry, University of
California, 3401 Watkins Dr., Riverside CA 92521. Optical rotations
were measured on a Jasco P-1010 polarimeter at 589 nm. IR spectra
were obtained neat with a Jasco FTIR/615 spectrometer.
3.2. ELISA-based PBD-binding inhibition assays
An ELISA-based PBD-binding inhibition assay was carried out
using an immobilized p-T78 peptide and cellular lysates expressing
HA-EGFP-Plk1 as described in Ref.
Figure 3A.
49
Results are shown in

F. Liu et al. / Tetrahedron 65 (2009) 9673–9679
9677
a colorless oil (10.2 g, 100% yield). 1H NMR (400 MHz, CDCl3)
7.20 (m, 5H), 7.10 (dt, J¼11.6, 2.6 Hz, 1H), 6.50 (dt, J¼12.0, 4.6 Hz,
1H), 5.44 (dd, J¼8.8, 4.0 Hz, 1H), 4.68–4.59 (m, 3H), 4.22 (dd, J¼8.8,
4.0 Hz, 1H), 0.85 (s, 9H), 0.00 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
7.36–
column chromatography (hexanes:EtOAc) to yield the intermediate
azide-containing lactone as a colorless liquid (270 mg, containing
a small amount EtOAc). 1H NMR (400 MHz, CDCl3)
d
4.36 (dd, J¼8.8,
6.4 Hz, 1H), 4.26 (d, J¼7.2 Hz, 1H), 4.02 (dd, J¼9.2, 4.0 Hz, 1H), 2.75 (m,
1H), 1.13 (d, J¼7.2 Hz, 3H). A suspension of this lactone and Pd$C (10%,
60 mg) in MeOH (9.0 mL) and acetic acid (1.0 mL) was stirred under H2
(1 atmosphere) at room temperature (overnight). The catalyst was
removed by filtration though a Celite pad under argon and the filtrate
was concentrated. The residue was re-dissolved in THF (10.0 mL)
containing H2O (10 mL) and then cooled to 0 ꢁC. To this was added
benzyl chloroformate (0.32 mL, 2.25 mmol) and NaHCO3 (840 mg,
10.0 mmol) and the mixture was stirred (4 h). The mixture was diluted
with EtOAc (150 mL), washed (H2O and brine), dried (Na2SO4), and
purified by silica gel column chromatography (hexanes:EtOAc) to
yield 10 as a white crystalline solid (90 mg, 25% yield over 3 steps) and
11 as a viscous colorless oil (250 mg, 49% yield over 3 steps). For (10):
d
169.3, 160.6, 158.8, 144.3, 134.5, 134.0, 131.0, 122.0, 75.2, 67.9, 62.8,
31.1, 23.4, 0.00. ESI (þVE) m/z: 384.1 (MþNa)þ. HR-ESI calcd for
C19H28NO4Si (MþNa)þ: 362.1782, found: 362.1789.
3.3.4. (4R)-3-[(2E)-[4-[(1,1-Dimethylethyl)dimethylsilyl]oxy]-1-oxo-
2-buten-1-yl]-4-phenyl-2-oxazolidinone (8). To
a solution of 7
(5.00 g, 13.9 mmol) in anhydrous THF (70 mL) at room temperature
was added tributylphosphine (0.34 mL, 1.39 mmol). The resulting
solution was stirred at room temperature (60 min), then diluted
with EtOAc (200 mL), washed (H2O and brine), dried (Na2SO4), and
purified by silica gel column chromatography (hexanes:EtOAc) to
20
yield 8 as a white solid (4.20 g, 84% yield). [
a]
ꢀ54.5 (c 1.40,
D
CHCl3). Mp 79–81 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.48 (dt, J¼15.2,
mp 125–127 ꢁC. 1H NMR (400 MHz, CDCl3)
d 7.40–7.30 (m, 5H), 5.33
2.4 Hz, 1H), 7.30–7.21 (m, 5H), 7.02 (dt, J¼15.2, 3.4 Hz, 1H), 5.39 (dd,
J¼8.6, 3.8 Hz, 1H), 4.60 (t, J¼8.8 Hz, 1H), 4.28 (dd, J¼3.4, 2.2 Hz, 2H),
4.17 (dd, J¼8.8, 4.0 Hz, 1H), 0.85 (s, 9H), 0.00 (s, 6H). 13C NMR
(m, 1H), 5.10 (s, 2H), 4.53 (t, J¼6.8 Hz, 1H), 4.35 (dd, J¼9.2, 5.2 Hz, 1H),
4.05 (d, J¼9.2 Hz, 1H), 2.92 (m, 1H), 0.95 (d, J¼7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3)
d 174.5, 156.1, 135.9, 128.5, 128.3, 128.1, 72.4, 67.3,
(100 MHz, CDCl3)
d
170.0,159.0,155.4,144.5,134.6,134.1,131.4,124.1,
54.5, 34.1, 12.7. ESI (þVE) m/z: 272.1 (MþNa)þ. HR-ESI calcd for
75.3, 68.1, 63.2, 31.3, 23.8, 0.00. IR (KBr) nmax: 2927, 2855, 1759, 1693,
1324, 1201, 1104, 951, 834, 715 cmꢀ1. ESI (þVE) m/z: 384.1 (MþNa)þ.
HR-ESI calcd for C19H28NO4Si (MþNa)þ: 362.1782, found: 362.1790.
C13H16NO4 (MþH)þ: 250.1074, found: 250.1081.
For (11): 1H NMR (400 MHz, CDCl3)
d 7.29–7.28 (m, 5H), 5.98
(d, J¼8.4 Hz, 1H), 5.04 (s, 2H), 4.34 (m, 1H), 3.65 (s, 3H), 3.54 (dd,
J¼11.2, 4.4 Hz, 1H), 3.44 (dd, J¼11.2, 6.0 Hz, 1H), 2.92 (s, 1H), 2.14
(m, 1H), 0.92 (d, J¼7.2 Hz, 3H). ESI (þVE) m/z: 304.2 (MþNa)þ. HR-
ESI MS calcd for C14H20NO5 (MþH)þ: 282.1336, found: 282.1343.
3.3.5. (4R)-3-[(2S,3R)-[2-Azido-4[(1,1-dimethylethyl)dimethylsilyl]
oxy]-3-methyl-1-oxo-butyl]-4-phenyl-2-oxazolidinone (9). To a solu-
tion of copper (I) bromide dimethyl sulfide complex (2.56 g,
12.45 mmol) in dimethyl sulfide (20 mL) and THF (30 mL) at ꢀ78 ꢁC
was added a solution of methylmagnesium chloride (3.0 M in THF,
5.50 mL, 16.4 mmol). The suspension was stirred at ꢀ78 ꢁC (20 min),
then warmed to 0 ꢁC (20 min) and cooled to ꢀ78 ꢁC. The mixture was
then transferred to a pre-cooled (ꢀ78 ꢁC) solution of 8 (1.80 g,
4.98 mmol) in THF (16.0 mL) and CH2Cl2 (8.0 mL) using a cannula. The
resulting mixture was kept at ꢀ78 ꢁC (60 min) then warmed to ꢀ40 ꢁC
(60 min) and cooled again to ꢀ78 ꢁC. To the mixture was added a pre-
cooled (ꢀ78 ꢁC) solution of N-bromosuccinimide (4.45 g, 25.0 mmol)
in THF (50 mL) and the mixture was stirred at ꢀ78 ꢁC (90 min). The
reaction was quenched by addition of saturated NaHSO3 (50 mL),
extracted with EtOAc (100 mLꢂ2). The combined organic phase was
washed (H2O and brine), dried (Na2SO4), and purified by silica gel
column chromatography (hexanes:EtOAc) to yield the requisite
3.3.7. (2S,3R)-4-Hydroxy-N-(9-phenylfluoren-9-yl)-N-benzyl-L-va-
line methyl ester (14). To a solution of 13 (4.00 g, 7.91 mmol) in
anhydrous THF (260 mL) at ꢀ40 ꢁC, was added DIBAL (1.0 M in
hexanes, 19.8 mL, 19.8 mmol). The mixture was stirred for 4 h
(ꢀ40 ꢁC to 0 ꢁC) before cooled down to ꢀ78 ꢁC, quenched by ace-
tone (10 mL), warmed to rt, stirred with 1 N KH2PO4 (500 mL) and
sodium potassium tartrate (30.0 g) overnight, filtered through the
Celite. The filtrate was extracted with EtOAc, washed (H2O and
brine), dried (Na2SO4), and purified by silica gel column chroma-
tography (hexanes:EtOAc) to yield alcohol 14 as a white wax
(2.30 g, 61% yield, quantitative yield based on recovered starting
material)and recycled 13 as a white wax (1.60 g). 1H NMR
(400 MHz, CDCl3) d 7.76–7.60 (m, 8H), 7.35–7.20 (m, 10H), 4.70 (AB,
JAB¼13.6 Hz, 1H), 4.38 (AB, JAB¼13.6 Hz, 1H), 3.84 (dd, J¼10.8,
3.6 Hz, 1H), 3.33 (dd, J¼10.8, 6.4 Hz, 1H), 3.04 (d, J¼8.4 Hz, 1H), 2.93
(s, 3H), 1.40 (m, 1H), 0.34 (d, J¼6.8 Hz, 3H). 13C NMR (100 MHz,
a
-bromo-containing intermediate as a white solid (1.93 g). To a solu-
tion of the
a
-bromo compound (1.93 g) in DMF (25 mL) at 0 ꢁC, was
added sodium azide (1.00 g, 15.4 mmol) and the mixture was stirred
(2 h). The mixture was diluted with EtOAc (150 mL), washed (H2O and
brine), dried (Na2SO4), and purified by silica gel column chromato-
CDCl3) d 171.7, 148.3, 144.8, 144.0, 142.0, 141.3, 139.7, 129.7, 128.6,
128.4, 128.0, 127.7, 127.3, 127.2, 127.1, 127.0, 125.3, 120.2, 80.3, 65.5,
63.3, 50.6, 36.3, 14.2. ESI (þVE) m/z: 478.2 (MþH)þ. HR-ESI MS calcd
for C32H32NO3 (MþH)þ: 478.2377, found: 478.2385.
graphy (hexanes:EtOAc) then crystallized (EtOAc:petroleum ether) to
20
yield azide 9 as a white solid (1.65 g, 79% yield). [
a]
D ꢀ73.0 (c 1.10,
CHCl3). Mp 80–82 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.40–7.30 (m, 5H),
3.3.8. (2S,3R)-4-[Di-(tert-butyl)-oxyphosphinyl]-(4-hydroxy-N-phenyl-
5.49 (dd, J¼8.8, 4.0 Hz,1H), 5.17 (d, J¼8.8 Hz,1H), 4.75 (t, J¼9.0 Hz,1H),
4.34 (dd, J¼8.8, 4.0 Hz, 1H), 3.65 (dd, J¼10.2, 5.4 Hz, 1H), 3.48 (dd,
J¼10.2, 3.4 Hz, 1H), 2.14 (m, 1H), 0.89 (s, 9H), 0.83 (d, J¼6.8 Hz, 3H),
methoxycarbonyl)-L-valine methyl ester (18). To a solution of oxalyl
chloride (0.96 mL, 10.1 mmol) in CH2Cl2 (40 mL) at ꢀ78 ꢁC, was
added a solution of DMSO (1.60 mL, 20.2 mmol) in CH2Cl2 (5 mL) and
the mixture was stirred (15 min). To this was added alcohol 11
(0.63 g, 2.24 mmol) in dry CH2Cl2 (5 mL) over 5 min and the mixture
was stirred at ꢀ75 ꢁC (2 h). triethylamine (8.40 mL, 53.8 mmol) was
added and the mixture was warmed to room temperature. To this
was added saturated NH4Cl (50 mL) and the mixture was extracted
with Et2O (100 mLꢂ2) and the combined organic phase was washed
(brine), dried (Na2SO4), and purified by silica gel column chroma-
tography (hexanes:EtOAc). Aldehyde 15 was obtained as a viscous
colorless oil (450 mg, 96% yield based on recovered starting material)
along with starting alcohol 11 (160 mg). To a solution of di-tert-butyl
phosphite (0.30 mL, 1.50 mmol) and triethylamine (0.21 mL,
1.50 mmol) in CH2Cl2 (5 mL) at 0 ꢁC, was added chlorotrimethylsilane
(0.19 mL, 1.50 mmol) and the mixture was stirred (5 min) and then
0.03 (s, 3H), 0.00 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 175.7, 158.7,
143.9, 134.8, 134.6, 131.9, 75.7, 69.3, 66.7, 63.4, 43.5, 31.4, 23.8, 19.4,
0.00. IR (KBr) nmax: 2930, 2359, 2106, 1786, 1710, 1206, 1097, 833,
778 cmꢀ1. ESI (þVE) m/z: 441.1(MþNa)þ. HR-ESI MS calcd for
C20H31N4O4Si (MþH)þ: 419.2109, found: 419.2114.
3.3.6. [(3S,4R)-Tetrahydro-4-methyl-2-oxo-3-furanyl]-carbamic acid
phenylmethyl ester (10) and (2S,3R)-4-hydroxy-N-(phenylmethoxy-
carbonyl)- -valine methyl ester (11). To a solution of 9 (600 mg,
L
1.44 mmol) in MeOH (20 mL) at room temperature was added p-tol-
uenesulfonic acid monohydrate (14 mg, 0.07 mmol). The solution was
stirred at room temperature (6 h), then diluted with EtOAc (150 mL),
washed (H2O and brine), dried (Na2SO4), and purified by silica gel

9678
F. Liu et al. / Tetrahedron 65 (2009) 9673–9679
transfer to a solution of aldehyde 15 (300 mg, 1.08 mmol) in CH2Cl2
(5 mL) at room temperature and the mixture was stirred (3 h). The
mixture was diluted with EtOAc (150 mL), washed (brine), dried
(Na2SO4) and concentrated. The resulting crude silyl-protected 17
was re-dissolved in MeOH (10 mL), to this was added H2O (1.0 mL)
and citric acid (200 mg) and the mixture was stirred at room tem-
perature (overnight). The mixture was diluted with EtOAc (200 mL),
washed (saturated NaHCO3 and brine), dried (Na2SO4) and purified
by silica gel column chromatography (hexanes:EtOAc) to yield 18 as
J¼7.2 Hz, 2H), 7.41 (t, J¼7.4 Hz, 2H), 7.31 (t, J¼7.6 Hz, 2H), 4.30–4.19
(m, 4H), 3.84 (m, 1H), 2.31 (m, 1H), 1.80–1.55 (m, 2H), 1.42 (s, 18H),
0.96 (d, J¼6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 155.8, 143.9,
140.7, 127.6, 127.0, 125.1, 120.0, 80.7, 65.4, 60.5, 46.7, 31.5, 30.0, 16.9.
ESI (þVE) m/z: 554.2 (MþNa)þ. HR-ESI MS calcd for C28H38NO7NaP
(MþNa)þ: 554.2278, found: 554.2277.
3.4. Peptide synthesis 21–24
a white wax epimeric at the
(400 MHz, CDCl3)
g
-carbon (450 mg, 88% yield). 1H NMR
Fmoc-Thr(PO(OBzl)OH)-OH and other Fmoc protected amino acids
were purchased from Novabiochem. Peptides were synthesize on
NovaSynÒTGR resin (Novabiochem, cat. no. 01-64-0060) using stan-
dard Fmoc solid-phase protocols in N-Methyl-2-pyrrolidone (NMP).
1-O-Benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phos-
phate (HBTU) (5.0 equiv), hydroxybenzotriazole (HOBT) (5.0 equiv)
and N,N-Diisopropylethylamine (DIPEA) (10.0 equiv) were used as
coupling reagents. The N-terminal was acetylated by 1-Acetylimid-
azole. The final resinwas washed with N,N-dimethylformamide (DMF),
methanol, dichloromethane and ether then dried under vacuum
(overnight). Peptides were cleaved from resin (200 mg) by treatment
with 5 mL of trifluoroacetic acid:triisbutylsilane:H2O (90:5:5) (4 h).
The resin was filtered off and the filtrate was concentrated under
vacuum, then precipitated with ether and the precipitate washed with
ether. The resulting solid was dissolved in 50% aqueous acetonitrile
5 mL and purified by reverse phase preparative HPLC using a Phe-
nomenex C18 column (21 mm diaꢂ250 mm, cat. no: 00G-4436-P0)
with a linear gradient from 0% aqueous acetonitrile (0.1% trifluoroacetic
acid) to 50% acetonitrile (0.1% trifluoroacetic acid) over 35 min at a flow
rate of 10.0 mL/min. Peptide 21: ESI (þVE) m/z: 595.3 (MþH)þ. Peptide
22: ESI (þVE) m/z: 675.3 (MþH)þ. Peptide 23: ESI (þVE) m/z:
673.3 (MþH)þ. Peptide 24: ESI (þVE) m/z: 657.3 (MþH)þ. Analytical
HPLC [By using Phenomenex C18 column (4.60 mm diaꢂ250 mm, cat.
no: 00G-4435-E0) with a linear gradient from 5% aqueous acetonitrile
(0.1% trifluoroacetic acid) to 100% acetonitrile (0.1% trifluoroacetic acid)
over 25 min at a flow rate of 1.0 mL/min indicated the purity of peptide
21: 100%, peptide 22: 100%, peptide 23: 87%, peptide 24: 83%.
d
7.30–7.29 (m, 5H), 6.30 (d, J¼8.0 Hz, 0.7H), 5.30
(m, 0.3H), 5.10–5.05 (m, 2H), 4.30 (m, 0.7H), 4.09 (m, 0.3H), 3.75–3.55
(m, 4H), 2.51 (m, 0.7H),1.51–1.40 (m,18H),1.15–1.00 (m, 3H).13C NMR
(100 MHz, CDCl3)
d 172.3, 156.7, 136.4, 128.4, 128.0, 70.5, 68.8, 67.3,
66.8, 59.8, 53.9, 53.1, 52.2, 36.6, 35.4, 30.3, 24.1, 14.7, 11.5, 9.4. ESI
(þVE) m/z: 496.2 (MþNa)þ. HR-ESI MS calcd for C22H36NO8NaP
(MþNa)þ: 496.2071, found: 496.2065.
3.3.9. (2S,3R)-4-[Di-(tert-butyl)-oxyphosphinyl]-N-(phenyl-
methoxycarbonyl)-
18 (250 mg, 0.53 mmol), O-phenylchlorothionoformate (215
1.60 mmol), 4-(dimethylamino) pyridine (DMAP) (15 mg,
0.20 equiv) and N,N-diisopropylethylamine (363 L, 2.10 mmol) in
L
-valine methyl ester (20). A solution of alcohol
mL,
m
anhydrous CH2Cl2 (8.0 mL) was stirred at room temperature
(overnight). The mixture was diluted with EtOAc (100 mL), washed
(satd NaHCO3 and brine), dried (Na2SO4) and purified by silica gel
column chromatography (hexanes:EtOAc) to give the intermediate
thiocarbonate 19 as a pale brown wax (225 mg). Crude 19 was
dissolved in toluene (10 mL) and to this was added tributyltin hy-
dride (0.42 mL, 1.59 mmol) and azobisisobutyronitrile (AIBN) (one
spatula tip). The mixture was maintained at 100 ꢁC (20 min), then
cooled to room temperature and concentrated under vacuum.
The residue was purified by silica gel column chromatography
(hexanes:EtOAc) to give 20 as viscous colorless oil (140 mg, 58%
20
yield for 2 steps). [
CDCl3)
a]
Dþ2.4 (c 0.85, CHCl3). 1H NMR (400 MHz,
d
7.27–7.20 (m, 5H), 5.80 (d, J¼8.4 Hz, 1H), 5.07 (AB,
JAB¼12.4 Hz, 1H), 5.02 (AB, JAB¼12.4 Hz, 1H), 4.23 (m, 1H), 3.67 (s,
3H), 2.33 (m,1H),1.69–1.10 (m, 20H),1.05 (d, J¼6.8 Hz, 3H). 13C NMR
Acknowledgements
(100 MHz, CDCl3)
d 172.1, 156.2, 136.3, 128.4, 128.1, 82.1, 66.9, 59.4,
52.2, 32.2, 30.3, 29.6, 27.8, 26.8, 17.5, 13.5. IR (KBr) nmax: 2976, 1720,
1535, 1322, 1252, 975 cmꢀ1. ESI (þVE) m/z: 480.3 (MþNa)þ. HR-ESI
MS calcd for C22H36NO7NaP (MþNa)þ: 480.2122, found: 480.2126.
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. 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.
3.3.10. (2S,3R)-4-[Di-(tert-butyl)-oxyphosphinyl]-N-[(9H-fluoren-9-
ylmethoxy)carbonyl]- -valine (4). To a solution of 20 (140 mg,
L
0.31 mmol) in THF (3.0 mL) and H2O (3.0 mL) at 0 ꢁC, was added
LiOH$H2O (26 mg, 0.62 mmol) and the mixture was stirred room
temperature (overnight). The THF was removed by rotary evapo-
ration and the residual aqueous phase was neutralized by addition
of saturated aqueous NH4Cl (20 mL) and extracted with EtOAc
(3ꢂ50 mL). The combined organic extract was washed with H2O
(50 mL), brine (50 mL), dried (Na2SO4) and concentrated. The
resulting residue was dissolved in MeOH (20 mL) and hydroge-
nated (1 atmosphere H2) over 10% Pd$C (40 mg) at room temper-
ature (overnight). The catalyst was removed by filtration and the
filtrate was concentrated. The resulting residue was dissolved in
dioxane (5.0 mL) and H2O (5.0 mL) and 9-fluorenylmethyl–succi-
nimidyl carbonate Fmoc–OSu (173 mg, 0.465 mmol) and NaHCO3
(62 mg, 0.62 mmol) were added and the mixture was stirred at
room temperature (overnight). The reaction mixture was neutral-
ized by addition of saturated NH4Cl (20 mL) and extracted with
EtOAc (3ꢂ50 mL). The combined EtOAc layer was washed with H2O
(50 mL), brine (50 mL), dried (Na2SO4) and purified by silica gel
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