Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form

Article abstract of DOI:10.1002/cbdv.201300202

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Full text of DOI:10.1002/cbdv.201300202

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Design and Synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the Preparation of
Peptide Prodrugs Containing Phosphothreonine in Fully Protected Form
by Wen-Jian Qian, Christopher C. Lai, James A. Kelley, and Terrence R. Burke Jr.*
Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, P.O. Box B, Frederick, MD 21702, USA
(phone: þ1-301-8465906; fax: þ1-301-8466033; e-mail: burkete@helix.nih.gov)
The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyl-
oxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported.
This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols.
It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl
protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having
bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.
Introduction. – Post-translational modification by phosphorylation plays central
roles in cellular signaling. Phosphoryl esterification of proteins on the side-chain OH
groups of threonine, serine, and tyrosine residues to yield the corresponding
phosphothreonine (pThr), phosphoserine (pSer), and phosphotyrosine (pTyr) species,
respectively, can introduce unique molecular features that facilitate signaling cascades,
often by promoting specific proteinꢀprotein interactions (PPIs) [1–3]. The contribu-
tions that aberrations in protein phosphorylation can make to the etiology of a number
of diseases, including cancers [4][5], have rendered synthetic phosphopeptides as
valuable pharmacological tools for studying both normal and pathological processes
[6][7]. However, in cellular systems, the bioavailability of such phosphopeptides may
be limited by poor membrane transport due to the dianionic character of the
phosphoryl species [8–10]. Many approaches have been investigated to enhance
cellular bioavailability of organic phosphates. The most common methodologies
involve masking the phosphoryl moiety with ꢀprodrugꢁ protecting groups that can be
removed biologically within the cell [11]. The (pivaloyloxy)methyl (POM) moiety
is an esterase-cleavable group that has found wide utility in phosphoryl prodrug
protection, particularly in nucleotides [12], and in phosphate and phosphonic acid
functionality in peptide mimetics [13–16]. In spite of its usefulness, there have been no
reports of pThr or pSer reagents that utilize phosphoryl-POM protection in forms
suitable for direct solid-phase synthesis. This may be one reason why there have been
no reports of pThr or pSer-containing polypeptides bearing POM phosphoryl
protection. Furthermore, to the best of our knowledge, there are no reports of
polypeptides containing pThr bearing bioreversible protecting groups of any kind.
Given the physiological importance of pThr-containing sequences [3], there is a
considerable need for a reagent that would permit the solid-phase synthesis of peptides
containing POM-protected pThr residues. Accordingly, we report herein the prepara-
ꢂ 2014 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
785
tion of Fmoc-Thr[PO(OH)(OPOM)]-OH (1) as a new reagent for the synthesis of
peptides containing pThr with its phosphoryl group in fully protected bis-POM prodrug
form, i.e., Thr[PO3(POM)2].
Results and Discussion. – Reagent Design. When considering protocols for the
incorporation of Thr[PO3(POM)2] into peptides, we noted that the solid-phase
synthesis of pTyr-containing peptides can be achieved in the absence of phosphoryl
protection using Fmoc-Tyr(PO3H2)-OH [17]. Accordingly, we envisioned that one
potential route to the synthesis of Thr[PO3(POM)2]-containing peptides could involve
using unprotected Fmoc-Thr(PO3H2)-OH, which would result in resin-bound peptides
bearing full side-chain protection except for a free pThr phosphoryl group. In this
approach, in situ introduction of phosphoryl POM protection (ꢀPOMylationꢁ) would be
carried out immediately prior to cleavage of the peptide from the resin. However, when
we investigated this approach, we found that, although initial coupling of unprotected
Fmoc-Thr(PO3H2)-OH occurred cleanly, further peptide synthesis through the
introduction of additional amino acid residues, followed by resin cleavage, led to very
low yields of isolated pThr-containing peptides. This indicated that peptide-chain
elongation in the presence of unprotected Thr(PO3H2) was problematic.
In further studies, we observed that treatment of resin-bound Fmoc-Thr(PO3H2)
with excess iodomethyl pivalate (POMI) [18] and EtNiPr2, followed by resin cleavage
(1% TFA in CH2Cl2) cleanly provided Fmoc-Thr[PO3(POM)2]-amide. This demon-
strated both the ability to perform on-resin POM protection of the phosphoryl group
and the stability of the resulting Thr[PO3(POM)2] residue to the conditions of resin
cleavage. However, we found that piperidine-mediated Fmoc deprotection of resin-
bound Fmoc-Thr[PO3(POM)2], followed by 1% TFA resin cleavage, gave the
Thr[PO(OH)(OPOM)]-containing peptide, indicating that mono-POM deprotection
had occurred in the presence of piperidine. This is consistent with the reported ability of
piperidine to selectively cleave a single POM group from the triester PO(OPOM)3 to
yield PO(OH)(POM)2 [19]. The instability of pThr phosphoryl bis-esters to piperidine
treatment is one reason why incorporation of pThr and pSer residues on acid-labile
resins is normally achieved using commercially-available Fmoc derivatives bearing
mono-Bn protection of the phosphoryl group, i.e., [Fmoc-Thr[PO(OH)(OBn)]-OH
and Fmoc-Ser[PO(OH)(OBn)]-OH, respectively (reviewed in [20–22]). Based on this
information, we concluded that synthesis of Thr[PO3(POM)2]-containing peptides
could best be accomplished using Fmoc-Thr[PO(OH)(OPOM)]-OH (1) as a reagent,

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
with conversion of the Thr[PO(OH)(OPOM)] residue to the desired fully-protected
Thr[PO3(POM)2] form by POMI/EtNiPr2 treatment after completion of peptide
synthesis and Fmoc deprotection, but immediately prior to final resin cleavage. It
should be noted that the current protocol provides terminal-N-acetylated peptides. To
obtain peptides with a free terminal amines, the procedure can be altered through the
use of terminal N-Boc protection, in which ꢀPOMylationꢁ is undertaken with
maintenance of Boc protection. Desired peptides bearing a free terminal amine can
then be obtained by cleavage under conditions that are compatible with Boc removal
and retention of phosphoryl POM protection (95% TFA; unreported results).
Synthesis of Fmoc-Thr[PO(OH)(OPOM)]-OH (1) from Commercially-Available
Fmoc-Thr[PO(OH)(OBn)]-OH. Benzyl esterification of the carboxylic acid of
commercially available Fmoc-Thr[PO(OH)(OBn)]-OH was achieved by treatment
with BnOH and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI) in the presence of 4-(dimethylamino)pyridine (DMAP; Scheme 1). Reaction
of the resulting Fmoc-Thr[PO(OH)(OBn)]-OBn (2) with excess POMI (formed from
the corresponding commercially available chloride by halide exchange using NaI in
MeCN [18]) and EtNiPr2 resulted in esterification of the free phosphoryl OH group to
yield Fmoc-Thr[PO(OBn)(OPOM)]-OBn (3) as a 1:1 mixture of phosphoryl diaste-
reoisomers. Subsequent hydrogenolytic cleavage of all Bn groups cleanly yielded the
desired reagent 1 (Scheme 1).
Utilization of Fmoc-Thr[PO(OH)(OPOM)]-OH (1) for the Synthesis of Ac-Pro-
Leu-His-Ser-Thr[PO3(POM)2]-amide (4). To demonstrate the applicability of 1 for the
synthesis of Thr[PO3(POM)2]-containing peptides, we chose as a target the pentapep-
tide ꢀPro-Leu-His-Ser-pThrꢁ, which has been reported as a high affinity ligand of the
polo-like kinase 1 (Plk 1) polo box domain (PBD) [23–25]. PBD-Binding ligands may
potentially serve as anticancer agents by blocking the spatial organization required for
Plk 1 to function in oncogenic processes [26]. The synthesis of Ac-Pro-Leu-His-Ser-
Thr[PO3(POM)2]-amide (4) was accomplished on NovaSyn TG Sieber resin using
reagent 1 according to standard Fmoc protocols. Histidine and serine were employed in
their methyltrityl (Mtt) and trityl side chain-protected forms, respectively, to allow
selective on-resin cleavage using 1% TFA (Scheme 2). Following peptide formation
and terminal amino acetylation of the Pro residue, the resin 5 was treated with POMI
(10 equiv.) and EtNiPr2 (10 equiv.) in DMF (4 h). Reaction products were then cleaved
Scheme 1
i) BnOH (1.5 equiv.), 4-(Dimethylamino)pyridine (DMAP; 1.0 equiv.), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide·HCl (EDCI; 2.5 equiv.), CH2Cl2, 08 to r.t., 2 h; 70%. ii) Iodomethyl pivalate
(POMI; 2.0 equiv.), EtNiPr2 (2.0 equiv.), DMF, r.t., 12 h; 60%. iii) 10% Pd/C, MeOH, r.t., 1 h; 68%.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
787
Scheme 2
NovaSynꢄTG Sieber resin; i) POMI, EtNiPr2, DMF, 4 h; ii) 1% CF3COOH (TFA), CH2Cl2.
from the resin (1% TFA) and purified by reversed-phase (RP) HPLC to provide two
peptides with retention times (tR) of 18.8 (peptide 6) and 20.9 min (peptide 4),
respectively. Both peptides exhibited molecular weights and elemental composi-
tions (HR-MS) corresponding to the desired product, i.e., Ac-Pro-Leu-His-Ser-
Thr(PO3POM2)-amide (4).
MS/MS Analysis of Peptides 4 and 6. High-resolution (HR) MS/MS analysis of the
[MþH]þ ions (m/z 903.4) of peptides 4 and 6 indicated that, while both peptides
contained two POM groups, the locations of one of the POM moieties differed for each.
The MS/MS fragmentation pattern of the slower-eluting peptide (Fig.,a) was consistent
with product 4, in which both POM groups are on the phosphoryl moiety. With this
peptide, the primary MS/MS product-ion peak at m/z 559.2997 resulted from concerted
loss of the elements of (HO)PO3(POM)2 and H2O from the [MþH]þ ion, which can
only occur if the phosphate is ꢀdi-POMylatedꢁ. In addition, b3 and b4 product-ions peaks
were also observed, providing strong evidence for an unaltered Ac-Pro-Leu-His-Ser-
sequence (Fig.). The MS/MS of the faster-eluting peptide (Fig.,b) strongly suggested
that both the phosphoryl group and histidine residue each contained a single POM
adduct (i.e., 6; Scheme 2). In this spectrum, the most abundant MS/MS product-ion
peak at m/z 771.3430 resulted from loss of t-C4H9CO2H and CH2O from the [MþH]þ

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Figure. MS/MS of peptides a) of 4 and b) of 6, obtained by collision-induced dissociation (CID) of the
[MþH]þ ion (m/z 903.4, 3.0-Da selection window) of each peptide
ion, which can only occur for a ꢀPOMylatedꢁ phosphate and not for a ꢀPOMylatedꢁ
histidine. The subsequent loss of HPO3 (m/z 691.3774), and of HPO3 and H2O (m/z
673.3670) further corroborated the presence of only one POM moiety on the phosphate
(Fig.). The absence of b3 and b4 product-ion peaks, expected for an unaltered Ac-Pro-

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
789
Leu-His-Ser sequence, was additional evidence that the histidine residue had been
modified.
The ꢀPOMylationꢁ of histidine to yield peptide 6 was not unexpected, since we had
previously observed alkylation of the imidazole N(p) atom in histidine residues bearing
standard trityl protection of the N(t) atom [24][27]. While alkylation of histidine does
represent an unwanted source of side-product formation, a search in the literature
reveals that, in general, alkyl halide-mediated modification of side chain-protected
peptides on solid support is not a significant concern. Of greater consideration
regarding the chemistry reported herein is that it presents the first and so far the only
method of preparing polypeptides with pThr residues in fully-protected biocleavable
form. This uniqueness overcomes limitation associated with the methodology.
Conclusions. – We report the design, synthesis, and application of Fmoc-
Thr[PO(OH)(OPOM)]-OH (1) as a reagent for the facile solid-phase preparation of
peptides bearing pThr residues in the fully-protected Thr[PO3(POM)2] form. To the
best of our knowledge, this represents the first methodology for preparing biolabile-
protected pThr residues within a polypeptide platform. Our approach should have a
significant utility in a variety of biological applications.
This work 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.
Experimental Part
General. All experiments involving moisture-sensitive compounds were conducted under anh.
conditions (positive Ar pressure) using standard syringe, cannula, and septa apparatus. Fmoc-Ser(Trt)-
OH, Fmoc-His(Mtt)-OH, and Fmoc-Thr[PO(OH)((OBn)]-OH were purchased from Novabiochem. All
solvents were purchased in anhy. form (Aldrich) and used directly. HPLC-Grade hexanes, AcOEt,
CH2Cl2, MeCN, and MeOH were used for both column chromatography (CC) and prep. high-
performance liquid chromatograhy (HPLC). LC/MS-Grade solvents (Burdick and Jackson Brand,
Honeywell), ultra-high-purity acids (HCO2H, AcOH), and buffers (NH4HCO3, NH4OAc) were
employed for all LC/MS analyses. Anal. TLC: Analtech precoated plates (Uniplate, silica gel GHLF,
250 nm) containing a fluorescence indicator. NMR Spectra: Varian Inova 400 MHz spectrometer;
coupling constants, J, reported in Hz, and chemical shifts d in ppm rel. to TMS. Low-resolution,
electrospray ionization mass spectra (LR-ESI-MS): Agilent 1200/1100 LC/MSD single quadrupole
system, equipped with an in-line UV diode-array detector (DAD), to assess compound identity and
homogeneity. HR-MS: Thermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer system operated at a
resolution of 30,0000 (FWHM) in either positive- or negative-ion mode, depending on which mode was
the most suitable based on previous LR analyses. For LC/MS analyses on the Orbitrap, a narrow-bore
(50ꢁ2.1 mm), Zorbax Rapid-Resolution RP C18 column coupled with a C18 guard column (12.5ꢁ
2.1 mm) was eluted at 250 ml/min with a 5–90% gradient of MeCN/H2O containing either 0.1% HCOOH
or 1 mm NH4HCO3. The resulting accurate mass measurement of a molecular species ([MþH]þ or [Mꢀ
H]ꢀ ) was then used to determine a unique elemental composition for each particular compound. Where
appropriate, 1H- and 13C-NMR data were used to set elemental constraints for this calculation. For
peptides 4 and 6, MS/MS of [MþH]þ ions were obtained at a resolution of 15,000 (FWHM) by both CID
and HCD fragmentation. The resulting HR product-ion spectra were used to confirm peptide sequences
and determine structural assignments.
Benzyl O-[(Benzyloxy)(hydroxy)phosphoryl]-N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-l-threoni-
nate (2). Commercially available Fmoc-Thr[PO(OH)((OBn)]-OH (1.0 g, 1.96 mmol), BnOH (0.30 ml,
2.93 mmol), and DMAP (0.24 g, 1.96 mmol) were dissolved in CH2Cl2 (20 ml), and the mixture was

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
cooled to 08. To the cold soln. was added EDCI (0.94 g, 4.89 mmol), and the mixture was warmed to r.t.
and stirred (2 h). The reaction was quenched by the addition of 1n aq. HCl soln. (10 ml), and the mixture
was extracted with CH2Cl2. The combined org. layer was washed with 1n HCl and brine, dried (MgSO4),
and concentrated, and the residue was purified by CC (SiO2; CH2Cl2/MeOH 20 :1!4 :1) to afford 2
(0.82 g, 70%). White semi-solid. [a]D ¼24.8 (c¼1.65, CHCl3). UV (DAD, MeCN/H2O): 264, 289, 299
(Fmoc). 1H-NMR (400 MHz, CDCl3): 8.81 (br. s, 1 H); 7.78 (d, J¼8.0, 2 H); 7.62 (dd, J¼12.0, 8.0, 2 H);
7.41 (t, J¼8.0, 2 H); 7.38–7.27 (m, 12 H); 5.91 (d, J¼8.0, 1 H); 5.26 (d, J¼12.0, 1 H); 5.11–4.94
(m, 4 H); 4.54 (d, J¼12.0, 1 H); 4.44–4.33 (m, 2 H); 4.22 (t, J¼8.0, 1 H); 1.38 (d, J¼8.0, 3 H).
13C-NMR (100 MHz, CDCl3): 169.7; 156.9; 144.1; 143.7; 141.4; 135.74; 135.68; 135.1; 128.8; 128.7; 128.0;
127.9; 127.3; 125.3; 120.1; 75.6; 69.5; 68.1; 67.6; 58.7; 47.2; 18.7. HR-ESI-MS: 600.1782 ([MꢀH]ꢀ
,
C33H31NO8Pꢀ ; calc. 600.1787).
Benzyl O-[{[(2,2-Dimethylpropanoyl)oxy]methoxy}(hydroxy)phosphoryl]-N-{[(9H-fluoren-9-yl)-
methoxy]carbonyl}-l-threoninate (3). Compound 2 (0.3 g, 0. 5 mmol) in DMF (5.0 ml) under Ar was
treated with EtNiPr2 (0.174 ml, 1.00 mmol), followed by iodomethyl pivalate (POMI [18]; 0.241 g,
1.00 mmol), and the mixture was stirred at r.t. (overnight). The mixture was partitioned (H2O/AcOEt),
and the org. layer was washed with H2O and brine, dried (MgSO4), and concentrated, and the residue was
purified by CC (SiO2; AcOEt/hexanes 1:2!2 :1) to afford 3 (mixture of two diastereoisomers (ratio 1:1
as determined by 1H-NMR); 0.21 g, 60%). White semi-solid.
O-[{[(2,2-Dimethylpropanoyl)oxy]methoxy}(hydroxy)phosphoryl]-N-{[(9H-fluoren-9-yl)methoxy]-
carbonyl}-l-threonine (¼ [Fmoc-Thr[PO(OH)(OPOM)]-OH]; 1). A soln. of 3 (45 mg, 0.063 mmol) in
MeOH (1.25 ml) was hydrogenated over 10% Pd/C (5 mg, 1 h), then filtered, and concentrated, and the
residue was purified by CC (SiO2; CH2Cl2/MeOH 10 :1!4 :1) to afford 1 (23 mg, 68% ). Colorless oil.
[a]D ¼4.98 (c¼0.34, MeOH). UV (DAD, MeCN/H2O): 264, 289, 299 (Fmoc). 1H-NMR (400 MHz,
CD3OD): 7.80 (d, J¼8.0, 2 H); 7.70 (t, J¼8.0, 2 H); 7.39 (t, J¼8.0, 2 H); 7.31 (tt, J¼8.0, 4.0, 2 H); 5.58 (d,
J¼16.0, 2 H); 5.04–4.96 (m, 1 H); 4.43–4.35 (m, 3 H); 4.26 (t, J¼4.0, 1 H); 1.39 (d, J¼8.0, 3 H); 1.23 (s,
9 H). 13C-NMR (100 MHz, CD3OD): 178.4; 172.7; 159.1; 145.4; 142.7; 128.9; 128.3; 126.4; 121.1; 84.0;
76.4; 68.4; 60.1; 60.0; 39.9; 27.4; 19.0. HR-ESI-MS: 534.1524 ([MꢀH]ꢀ , C25H29NO10Pꢀ ; calc. 534.1529).
Synthesis of Ac-Pro-Leu-His-Ser-Thr(PO3(POM)2)-amide (¼1-Acetyl-l-prolyl-l-leucyl-l-histidyl-
l-seryl-O-(bis{[(2,2-dimethylpropanoyl)oxy]methoxy}phosphoryl)-l-threoninamide; 4). Peptide 4 was
synthesized on NovaSynꢄTG Sieber resin (Novabiochem, cat. No. 01-64-0092) using 1 according to
standard Fmoc-based protocols employing N-methylpyrrolidin-2-one (NMP) as solvent, and O-1H-
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU; 5.0 equiv.), 1-hydroxy-
benzotriazole (HOBt; 5.0 equiv.), and (10.0 equiv.) as coupling reagents. Terminal amine acetylation was
achieved using 1-acetyl-1H-imidazole. Following acetylation, the resin (0.1 mmol) was treated during 4 h
with POMI (242 mg, 1.0 mmol) and EtNiPr2 (0.174 ml, 1.0 ml) in DMF (3 ml). The resulting resin
(0.1 mmol) was washed with DMF, MeOH, CH2Cl2, and Et2O, and then dried under vacuum (overnight).
The peptide was cleaved from the resin using 1% TFA in CH2Cl2 (10 min; repeated five times). Resin
was removed by filtration, the filtrate was concentrated under vacuum, precipitated with Et2O, and the
precipitate was washed with cold Et2O. The resulting solid was dissolved in 50% aq. MeCN (5 ml) and
purified by RP prep. HPLC with a Phenomenex C18 column (250ꢁ21 mm) with a linear gradient from
20% aq. MeCN (0.1% TFA) to 90% MeCN (0.1% TFA) over 30 min at a flow rate of 10.0 ml/min.
Lyophilization provided 4 as white powder (4.8 mg; >99% pure by anal. HPLC). HR-ESI-MS: 903.4227
([MþH]þ , C38H64N8O15Pþ ; calc. 903.4229. By-product 6 was also isolated as white powder (7.0 mg; >
99% purity by anal.). HR-ESI-MS: 903.4227 ([MþH]þ , C38H64N8O15Pþ ; calc. 903.4229).
MS/MS Analysis of Peptides 4 and 6. Collision-induced dissociation (CID) product-ion mass spectra
for the [MþH]þ ion (m/z 903.4, 3.0-Da selection window) of 4 and 6 were generated by fragmentation in
the LTQ at an energy of 30 and analysis in the Orbitrap at a resolution of 15,000 (FWHM) over the
maximum allowable mass range. Each spectrum shown in the Figure was the average of ten or more scans
and resulted from an independent LC/MS/MS analysis of individual, HPLC-purified pentapeptide. A
shallow linear MeCN/H2O gradient (2–47% MeCN in 20 min), followed by a steeper linear gradient
(47–90% in 10 min), was employed on a 3-mm, 150ꢁ2.0 mm Cadenza CD-C18 HPLC column (Imtakt,
USA) at a flow rate of 250 ml/min for the separation and elution of these POM-peptide derivatives.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Received June 21, 2013