Design and synthesis of a reagent for solid-phase incorporation of the phosphothreonine mimetic (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) into peptides in a bio-reversible phosphonyl-bis-pivaloyloxymethyl (POM) prodrug form

Article abstract of DOI:10.1007/s00726-013-1567-0

Reported herein are the synthesis and solid-phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl phosphoryl ester [Fmoc-Pmab(POM)₂-OH, 2] as a phosphatase-stable phosphothreonine (pThr) mimetic bearing orthogonal protection suitable for the synthesis of Pmab-containing peptides having bio-reversible protection of the phosphonic acid moiety. This represents the first report of a bio-reversibly protected pThr mimetic in a form suitable for facile solid-phase peptide synthesis.

Full text of DOI:10.1007/s00726-013-1567-0

Amino Acids
DOI 10.1007/s00726-013-1567-0
ORIGINAL ARTICLE
Design and synthesis of a reagent for solid-phase incorporation
of the phosphothreonine mimetic (2S,3R)-2-amino-3-methyl-4-
phosphonobutyric acid (Pmab) into peptides in a bio-reversible
phosphonyl-bis-pivaloyloxymethyl (POM) prodrug form
•
Wen-Jian Qian Terrence R. Burke Jr.
Received: 7 June 2013 / Accepted: 18 July 2013
Ó Springer-Verlag Wien (outside the USA) 2013
Abstract Reported herein are the synthesis and solid-
phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-
3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl
phosphoryl ester [Fmoc-Pmab(POM)2-OH, 2] as a phos-
phatase-stable phosphothreonine (pThr) mimetic bearing
orthogonal protection suitable for the synthesis of Pmab-
containing peptides having bio-reversible protection of the
phosphonic acid moiety. This represents the first report of a
bio-reversibly protected pThr mimetic in a form suitable
for facile solid-phase peptide synthesis.
ESI
Electrospray ionization
EtOAc
F2Pmp
Fmoc
Ethyl acetate
Difluorophosphonomethylphenylalanine
9-Fluorenylmethoxycarbonyl
Fmoc-OSu 9-Fluorenylmethyl succinimidyl carbonate
HBTU
1-O-Benzotriazole-N,N,N0,N0-tetramethyl-
uromium- hexafluoro-phosphate
Hydrochloric acid
HCl
His
Histidine
HOBt
HPLC
HRMS
Leu
1-Hydroxybenzotriazole
High-performance liquid chromatography
High resolution mass spectra
Leucine
Keywords Phosphothreonine mimetic ꢀ Prodrug ꢀ
Pmab ꢀ Solid-phase peptide synthesis ꢀ Signal
transduction
LiOH
MeOH
MgSO4
Mtt
Lithium hydroxide
Methanol
Abbreviations
Ac
Magnesium sulfate
Acetyl
Methyltrityl
[a]D
BnBr
But
Specific rotation
Benzyl bromide
tert-Butyl
m/z
Mass-to-charge ratio
Sodium bicarbonate
N-Methyl-2-pyrrolidone
Nuclear magnetic resonance
Polo box domain
NaHCO3
NMP
NMR
PBD
Cbz
Benzyloxycarbonyl
Dimethyl chloride
Acetonitrile
CH2Cl2
CH3CN
DIPEA
DMF
PdꢀC
PLE
Palladium on carbon
Porcine liver esterase
Polo-like kinase 1
Diisopropylethylamine
Dimethylformamide
Plk1
Pmab
(2S,3R)-2-Amino-3-methyl-4-
phosphonobutyric acid
Pivaloyloxymethyl
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-013-1567-0) contains supplementary
material, which is available to authorized users.
POM
POMI
PPIs
pSer
pThr
pTyr
Ser
Pivaloyloxymethyl iodide
Protein-protein interactions
Phosphoserine
W.-J. Qian ꢀ T. R. Burke Jr. (&)
Chemical Biology Laboratory, Molecular Discovery Program,
Center for Cancer Research, National Cancer Institute-Frederick,
National Cancer Institute, National Institutes of Health,
Frederick, MD 21702, USA
Phosphothreonine
Phosphotyrosine
Serine
SPPS
Solid-phase peptide synthesis
e-mail: burkete@helix.nih.gov
123

W.-J. Qian, T. R. Burke
the broader sense, there is a paucity of reagents available for
the solid-phase synthesis of polypeptides containing pro-
drug-protected pThr mimetics.
t
Half life
‘
TFA
THF
TLC
TMS
Trt
Trifluoroacetic acid
Tetrahydrofuran
Thin layer chromatography
Trimethylsilane
Trityl
The pivaloyloxymethyl (POM) group is an esterase-
labile moiety that has been widely applied to phosphoryl
prodrug protection of nucleotides (Hecker and Erion 2008)
and phosphate and phosphonic acid functionality in small
molecules and peptide mimetics (Stankovic et al. 1997;
Mandal et al. 2009, 2011; Zhao and Etzkorn 2007). How-
ever, in spite of its usefulness in these latter contexts, there
are no prior reports of polypeptides containing pThr, pSer
or their phosphonic acid-based mimetics, bearing POM
protection. Given the importance of pThr in a large number
of biological processes (Elia and Yaffe 2005), a reagent
that would permit the solid-phase synthesis of polypeptides
containing POM-protected pThr mimetics would be highly
desirable.
Introduction
Protein phosphorylation is a fundamental form of post-
translational modification that affects approximately one-
third of all proteins. The presence of phosphothreonine
(pThr), phosphoserine (pSer) and phosphotyrosine (pTyr)-
can introduce unique recognition features, which often
facilitate specific protein–protein interactions (PPIs) (Yaffe
2002; Ladbury 2005; Elia and Yaffe 2005). Synthetic
phosphopeptides modeled on recognition sequences can
serve as useful pharmacological tools for studying these PPIs
(Eisele et al. 1999; Lu et al. 2012). However, in cellular
systems the bioavailability of phosphopeptides may be
limited by the enzymatic lability of the phosphoryl ester
bond toward phosphatases and poor membrane transport of
the phosphoryl di-anionic species. While replacement of the
phosphoryl ester oxygen by methylene or fluoromethylenes
has addressed issues related to phosphatase hydrolysis for
pTyr (Burke and Lee 2003), pSer (Shapiro et al. 1993; Perich
1994; Nair et al. 1995; Panigrahi et al. 2009) and pThr
(Berkowitz et al. 1996; Otaka et al. 2000; Liu et al. 2009), cell
membrane transit can still be limited by the di-anionic charge
of the resulting phosphonic acids.
We had previously reported the preparation of N-Fmoc-
(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-
À
Á
tert-butyl phosphoryl ester [Fmoc-Pmab But2 -OH, 1,
Fig. 1] as a hydrolytically-stable pThr mimetic bearing
orthogonal protection suitable for the standard Fmoc-based
synthesis of peptides containing Pmab. However this reagent
yields peptides in which the Pmab residue has its phosphonic
acid moiety in the free di-anionic form (Liu et al. 2009). To
date, there have been no reports of Pmab suitably derivatized
for the solid-phase synthesis of polypeptides that maintained
the phosphonic acid in a prodrug-protected form. In the
current paper we describe the first preparation of Fmoc-
Pmab having the phosphonic acid group masked as its POM
bis-esters [Fmoc-Pmab(POM)2-OH, 2, Fig. 1] and demon-
strate its use in the solid-phase synthesis of a peptide bearing
full POM protection of the Pmab residue.
A general strategy for increasing the cell membrane
permeability of phosphates and phosphonic acids is to mask
their acidic functionality with neutral ‘‘prodrug’’ groups
that can be removed enzymatically once the agent is within
the cell. Although a variety of general prodrug strategies
have been for phosphonic acids (Schultz 2003; Hecker and
Erion 2008), there are few reports detailing incorporation of
these functionalities into protected amino acid derivatives
that are amenable to solid-phase peptide synthesis. Recent
examples of the latter include the application of 50-nitro-
furyl-20-methyl N-(400-chlorobutyl)phosphonamido ester
prodrug strategy to the pTyr mimetic difluoropho-
sphonomethylphenylalanine (F2Pmp) (Boutselis et al. 2007)
and to the pSer mimetic, difluorophosphonoaminobutyric
acid (Arrendale et al. 2012). In these two cases, the prodrug-
protected reagents were used to prepare dipeptides for
examination in whole cell studies. The use of these reagents
for the solid-phase synthesis of longer peptides is poten-
tially limited by chemical instability of the phosphonamido
group to repetitive cycles of piperidine treatment needed for
Fmoc-based solid-phase protocols (Boutselis et al. 2007). In
Materials and methods
General methods
All experiments involving moisture-sensitive compounds
were conducted under anhydrous conditions. Fmoc-
Ser(Trt)-OH, and Fmoc-His(Mtt)-OH were purchased from
Fig. 1 Structures of key reagents
123

Design and synthesis of a reagent for solid-phase
NovaChioChem. All solvents were purchased in anhydrous
form (Aldrich) and used directly. Analytical TLCs were
performed using Analtech precoated plates (Uniplate, silica
gel GHLF, 250 nm) containing a fluorescence indicator.
NMR spectra were recorded using a Varian Inova
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 260 1200 LC/MSD-SL system. High res-
olution mass spectra (HRMS) 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 with
CH3CN–H2O gradients. Reported m/z values are the
average of eight or more scans over the chromatographic
peak of interest.
The mixture was diluted with EtOAc and washed with H2O
and brine, dried (MgSO4) and filtered and concentrated.
Purification by silica gel column chromatography (CH2Cl2
: MeOH from 100:1 to 30:1) afforded 4 as a colorless
oil (0.25 g, 80 % for two steps, Scheme 1). [a]2D1.6 -0.59
1
(c 1.0, CHCl3); H NMR (400 MHz, CDCl3) d 7.40–7.28
(m, 10H), 5.91 (d, J = 8.0 Hz, 1H), 5.20–5.06 (m, 4H),
4.38 -4.31 (m, 1H), 2.42 (brs, 1H), 1.84–1.69 (m, 1H),
1.57 – 1.48 (m, 1H), 1.47–1.45 (m, 18H), 1.10 (d, J =
8.0 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) d 171.7,
156.4, 136.5, 135.4, 128.8, 128.7, 128.6, 128.3, 82.4
(d, 2JCP = 10 Hz), 82.3 (d, 2JCP = 10 Hz), 67.4, 67.2, 59.8
3
(d, JCP = 10 Hz), 34.0, 32.6, 30.59, 30.56, 17.6 ppm;
ESI-HRMS m/z calcd for C28H41NO7P (M?H)?:
534.2621, found: 534.2594.
((((2R,3S)-4-(Benzyloxy)-3-(((benzyloxy)carbonyl)amino)-
2-methyl-4-oxobutyl)phosphoryl)bis(oxy))bis(methylene)
Bis(2,2-dimethylpropanoate) (5) A solution of 4 (0.27 g,
0.5 mmol) in 10 % TFA (CH2Cl2) was stirred at room
temperature (1 h), then volatiles were removed under
vacuum and the residue was taken up in DMF with
pivaloyloxymethyl iodide (POMI) (Bandgar et al. 2011)
(0.32 mL, 2.0 mmol) and DIPEA (0.35 mL, 2.0 mmol)
and stirred at room temperature under argon (overnight).
The mixture was diluted with H2O, extracted with EtOAc
and the combined organic extracts were washed with
H2O, and brine, dried (MgSO4), filtered and concentrated.
Purification by silica gel chromatography (EtOAc : hex-
anes from 1:4 to 1:1) afforded 5 as a white semi-solid
(0.24 g, 74 % yield for two steps, Scheme 1). [a]2D1.9 2.88
Synthesis of Fmoc-Pmab(POM)2-OH (2)
(2S,3R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-4-(di-tert-
butoxyphosphoryl)-3-methylbutanoate (4) To a solution of
3 (Liu et al. 2009) (0.26 g, 0.57 mmol) in THF–H2O (4:1;
5 mL) at 0 °C was added a solution of LiOHꢀH2O (48 mg,
1.14 mmol) and the mixture was stirred at room tempera-
ture until all starting material was consumed as indicated
by TLC. The pH was adjusted to 2–3 by the addition of 1 N
aqueous HCl and THF was removed by evaporation. The
residue was extracted (EtOAc) and the combined organic
phase was washed with brine, dried (MgSO4), filtered and
concentrated. To a solution of the crude residue in DMF
(5.0 mL) were added sequentially, NaHCO3 (95 mg,
1.14 mmol) and BnBr (0.10 mL, 0.85 mmol) at room
temperature under argon and the mixture was stirred until
the starting material was consumed as indicated by TLC.
1
(c 0.7, CHCl3); H NMR (400 MHz, CDCl3) d 7.42–7.18
(m, 10H), 5.70–5.60 (m, 4H), 5.56 (d, J = 12.0 Hz, 1H),
5.19 (s, 2H), 5.11 (s, 2H), 4.42–4.34 (m, 1H), 2.45 (brs,
1H), 2.04–1.91 (m, 1H), 1.78–1.67 (m, 1H), 1.22 (s, 9H),
Scheme 1 Synthesis of title
reagent 2
O
O
CbzHN
CbzHN
1. LiOH•H2O, THF, H2O,
0 °C – rt (2 h)
OMe
OBn
2. BnBr, NaHCO3, DMF,
rt (12 h)
O P O
O P O
80% (2 steps)
O
O
3
4
1.10% Pd•C, H2 MeOH,
rt (1 h)
2. Fmoc-OSu, NaHCO3,
dioxane : H2O [1:1 (v/v)]
(12 h)
O
1. TFA : CH2Cl2 [1:10 (v/v)],
rt (1 h)
2. POMI, DIPEA, DMF,
rt (12 h)
CbzHN
OBn
O
2
O
O
O P O
O
55% (4 steps)
O
5
123

W.-J. Qian, T. R. Burke
1.21 (s, 9H), 1.08 (d, J = 4.0 Hz, 3H) ppm; 13C NMR
(100 MHz, CDCl3) d 177.1, 171.1, 156.3, 136.3, 135.2,
128.9, 128.8, 128.7, 128.44, 128.35, 81.7 (d,
Use of reagent 2 for the solid-phase synthesis of peptide 6
Standard Fmoc-protected amino acids were purchased from
Novabiochem. Peptides were synthesized on NovaSynÒ TG
Sieber resin (Novabiochem, cat. no. 01-64-0092) using
Fmoc-basedsolid-phaseprotocolsinN-methyl-2-pyrrolidone
(NMP). 1-O-Benzotriazole-N,N,N0,N0-tetramethyl-uronium-
hexafluoro-phosphate (HBTU) (5.0 eq.), hydroxybenzotria-
zole (HOBT) (5.0 eq.) and N,N-diisopropylethylamine
(DIPEA) (10.0 eq.) were used as coupling reagents. Reagent
2 was employed for introduction of the first residue. Fol-
lowing completion of peptide elongation and Fmoc-depro-
tection, amino terminal acetylation was achieved using
1-acetylimidazole. The finished resin (7, Scheme 2) was
washed with DMF, MeOH, CH2Cl2 and diethyl etherand then
dried under vacuum (overnight). Peptide 6 was cleaved from
the resin using 1 % TFA in CH2Cl2. The resin was removed
by filtration and the filtrate was concentrated under vacuum,
then precipitated with cold ether and the precipitate washed
with cold ether. The resulting solid was dissolved in 50 %
aqueous acetonitrile (5 mL) and purified by reverse phase
preparative HPLC using a Phenomenex C18 column
(21 mm dia 9 250 mm, cat. no: 00G-4436-P0) with a linear
gradient from 0 % aqueous acetonitrile (0.1 % TFA) to
100 % acetonitrile (0.1 % trifluoroacetic acid) over 30 min at
aflowrateof10.0 mL/min. Lyophilizationgavepeptide6asa
white powder ([99 % pure by HPLC, Scheme 2). ESI-MS
m/z calcd for C39H66N8O14P (M?H)?: 901.4, found: 901.4.
3
2JCP = 20 Hz), 67.6, 67.4, 59.2 (d, JCP = 10 Hz), 38.9,
1
31.9, 30.3, 29.9, 29.6 (d, JCP = 140 Hz), 27.0, 17.5 ppm;
ESI-HRMS m/z calcd for 650.2725, found: 650.2704.
(2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-
4-(bis((pivaloyloxy)methoxy)phosphoryl)-3-methylbutanoic
Acid [N-Fmoc-Pmab(POM)2-OH] (2) A solution of 5
(0.18 g, 0.28 mmol) in MeOH was hydrogenated over
10 % PdꢀC (30 mg) until the reaction was complete as
indicated by TLC. The mixture was filtered, evaporated to
dryness and reacted with Fmoc-OSu (0.19 g, 0.55 mmol)
and NaHCO3 (70 mg, 0.83 mmol) in dioxane : H2O (1:1,
5.5 mL) at room temperature (overnight). The mixture was
acidified with 1 N HCl, extracted with EtOAc and the
combined organic phases were washed with brine, dried
(MgSO4), filtered and the filtrate concentrated. Purification
by silica gel column chromatography (CH2Cl2:MeOH from
20:1 to 4:1) provided 2 as a colorless oil (0.14 g, 75 %
yield for two steps, Scheme 1). [a]2D1.0 12.8 (c 0.66,
CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.77 (d,
J = 8.0 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.40 (t,
J = 8.0 Hz, 2H), 7.32 (td, J1 = 8.0 Hz, J2 = 4.0 Hz, 2H),
5.78–5.64 (m, 5H), 4.51–4.44 (m, 1H), 4.40 (d,
J = 8.0 Hz, 2H), 4.22 (t, J = 8.0 Hz, 1H), 2.54 (brs 1H),
2.18–2.03 (m, 1H), 1.97–1.83 (m, 1H), 1.24 (s, 9H), 1.23
(s, 9H), 1.13 (d, J = 8.0 Hz, 3H) ppm; 13C NMR
(100 MHz, CDCl3) d 177.2, 172.8, 156.4, 144.0, 143.8,
141.5, 128.0, 127.3, 125.3, 120.2, 82.0, 67.5, 58.3 (d,
Pig liver esterase hydrolysis studies on peptide 6
1
3JCP = 10 Hz), 47.3, 39.0, 31.4, 29.4 (d, JCP = 140 Hz),
3
27.0, 17.6 (d, JCP = 7 Hz) ppm; ESI-HRMS m/z calcd for
Following literature procedures (Srivastva and Farquhar
1984), 0.05 M potassium phosphate buffer (pH 7.4) was
C32H42NO11PNa (M?Na)?: 670.2393, found: 670.2376.
Scheme 2 Synthesis of bis-
POM-protected peptide 6 using
reagent 2
123

Design and synthesis of a reagent for solid-phase
et al. 2008). The syntheses of Ac-Pro-Leu-His-Ser-
Pmab(POM)2-amide (6) was accomplished on NovaSyn
TG Sieber resin using standard Fmoc protocols. Histidine
and serine were employed in their 4-methoxytrityl (Mtt)
and trityl (Trt) side chain-protected forms, respectively, to
allow their cleavage under mildly acidic conditions
(Scheme 2). Following synthesis completion, the resin-
bound Ac-Pro-Leu-His(Mtt)-Ser(Trt)-Pmab(POM)2-amide
(7) was subjected to treatment with 1 % TFA, which
resulted in removal of histidine and serine protecting
groups and cleavage of the peptide from the resin with
retention of Pmab-bis-POM functionality. Purification by
HPLC provided Ac-Pro-Leu-His-Ser-Pmab(POM)2-amide
(6) as a white solid (Scheme 2).
Fig. 2 Results of pig liver esterase treatment of peptide 6
In order to examine the bio-reversibility of the Pmab
POM protection of 6, we performed in vitro assays using
porcine liver esterase (PLE) in phosphate buffer at pH 7.4
(Srivastva and Farquhar 1984). We observed that the parent
Pmab bis-POM-containing 6 was rapidly converted to the
mono-POM-containing product (t1/2 & 5 min) with further
deprotection to the free Pmab-containing peptide occurring
at a slower rate (20 % conversion over 8 h) (Fig. 2).
Reduction in the rate of enzymatic cleavage of a second
POM group is known (Srivastva and Farquhar 1984).
placed in a centrifuge tube and a solution of peptide 6 in
MeOH was added to achieve a concentration of 200 lM of
6 with MeOH being less than 1 %. To a 1 mL aliquot of
the above solution was added pig liver esterase (57.6 units)
and the reaction mixture was incubated at 37 °C with
gentle agitation. At various time points, aliquots of the
reaction mixture (50 lL) were transferred to an Eppendorf
tube containing MeCN (50 lL). Following filtration, the
hydrolysis product was monitored by LC-MS. Data are
shown in Fig. 2.
Conclusions
Results and discussion
Our current paper presents the synthesis of a reagent
[Fmoc-Pmab(POM)2-OH (2)] that allows the facile syn-
thesis of peptides containing the phosphatase-stable pThr
mimetic, Pmab, bearing bio-reversible POM protection.
This represents a rare example of a reagent that allows the
solid-phase synthesis of polypeptides having a pThr
mimetic in bio-reversible prodrug form. Reagent 2 should
find utility in a variety of pharmacological applications.
À
Á
Preparation of reagent 2 began with Cbz-Pmab But2 -OMe
(3), which is an intermediate in our previously reported
synthesis of 1 (Liu et al. 2009). Benzyl trans-esterification
of 3–4 was accomplished by initial LiOH-mediated
hydrolysis of the methyl ester followed by reaction of the
free acid with benzyl bromide (NaHCO3 in DMF, 80 %
yield for two steps, Scheme 1). Subsequent treatment of 4
with pivaloyloxymethyl iodide (POMI) (Bandgar et al.
2011) and diisopropylethyl amine (DIPEA) in DMF pro-
vided the corresponding bis-POM-protected intermediate
(5). Hydrogenolytic removal of amino and carboxyl pro-
tecting groups and introduction of N-Fmoc-protection by
treatment with 9-fluorenylmethyl succinimidyl carbonate
(Fmoc-OSu) in aqueous THF with NaHCO3, gave the
desired reagent 2 (55 % yield from 4) (Scheme 1).
Acknowledgments This research was supported by the Intramural
Research Program of the NIH, Center for Cancer Research, NCI-
Frederick and the National Cancer Institute, National Institutes of
Health.
Conflict of interest The authors declare that they have no conflict
of interest.
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