The synthesis of phosphopeptides via the Bpoc-based approach

Article abstract of DOI:10.1039/b617699m

The 2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc) group was examined as an N^α-protecting group in the stepwise assembly of the MAP Kinase ERK2 [178-188; Thr(P)¹⁸³, Tyr(P)¹⁸⁵] peptide. The mild acid deprotection of the Bpoc group permitted (i) incorporation of a fully protected phosphothreonyl derivative and (ii) a TFA-based final cleavage step. The first five C-terminal residues (184-188) were incorporated in the Fmoc mode of peptide synthesis, with all subsequent amino acids coupled as their Bpoc-Xxx-OH derivatives. The target product was obtained in high purity and yield, indicating that a Bpoc-based approach to phosphopeptide synthesis was compatible with both the acid-labile side chain protecting groups employed and Hmp-Wang resin. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b617699m

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
The synthesis of phosphopeptides via the Bpoc-based approach
Troy J. Attard,*†a Eric C. Reynoldsb and John W. Pericha
Received 4th December 2006, Accepted 6th December 2006
First published as an Advance Article on the web 12th January 2007
DOI: 10.1039/b617699m
The 2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc) group was examined as an Na-protecting group in the
stepwise assembly of the MAP Kinase ERK2 [178–188; Thr(P)183, Tyr(P)185] peptide. The mild acid
deprotection of the Bpoc group permitted (i) incorporation of a fully protected phosphothreonyl
derivative and (ii) a TFA-based final cleavage step. The first five C-terminal residues (184–188) were
incorporated in the Fmoc mode of peptide synthesis, with all subsequent amino acids coupled as their
Bpoc–Xxx–OH derivatives. The target product was obtained in high purity and yield, indicating that a
Bpoc-based approach to phosphopeptide synthesis was compatible with both the acid-labile side chain
protecting groups employed and Hmp–Wang resin.
Bpoc group for Na-protection. Since this method enables mild
Introduction
acidolytic Na-deprotection, it was envisaged that (a) Hmp–Wang
resin could be employed without hydrolysis of the peptide-resin
linkage, and (b) fully protected phosphothreonyl derivatives could
be incorporated into the growing peptide.
Protein phosphorylation is the most prevalent form of post-
translational modification in eukaryotic organisms and plays
important roles in processes such as protein trafficking,1 enzyme
activation2 and extracellular signal transmission.3 The study of
systems involving protein phosphorylation/dephosphorylation
has benefited significantly from the availability of synthetic phos-
phopeptides with which to probe consensus sequences or substrate
specificities.4,5 Recent developments in synthetic strategies for
the preparation of biologically significant phosphopeptides have
proved to be effective for simple or monophosphorylated peptides,
though synthetic difficulties have been encountered in cases of
more complex or multiphosphorylated peptides.6–8
Two general strategies are currently employed for solid phase
phosphopeptide synthesis: (a) the post-synthetic (‘global’) phos-
phorylation of the peptidyl target site, or (b) the incorporation
of a pre-phosphorylated amino acid derivative into the growing
peptide chain. A particular limitation of the latter approach
is the preclusion of fully protected phosphothreonyl and -seryl
derivatives from Fmoc-based syntheses due to the propensity
of the phosphorylated side chains to undergo base-catalysed
b-elimination during peptide assembly. While the use of Fmoc–
Xxx(PO3Bzl,H)–OH (Xxx = Ser, Thr) derivatives prevents
b-elimination by ionization of the phosphoryl oxygen, this method
is complicated by reports of incomplete acylation during the
coupling step, particularly in the case of Fmoc–Thr(PO3Bzl,H)–
OH.9
The synthesis of MAP Kinase ERK2 [178–188; Thr(P)183
,
Tyr(P)185], HTGFLT(P)EY(P)VAT (Scheme 4) (14) was chosen
to examine this method since the generation of phosphothreonyl
peptides have proven more difficult than for the corresponding
seryl or tyrosyl substrates.10 Furthermore, the application of
standard protocols for the assembly and subsequent global
phosphorylation of this particular sequence was reported to
be problematic by Johnson et al.,8 therefore providing an ideal
substrate to evaluate the efficacy of the Bpoc-based approach to
phosphopeptide preparation.
Results
Synthesis of MAP Kinase ERK2 [178–184; Thr(P)183],
HTGFLT(P)E (Scheme 2) (4)
The first stage of this study was to determine the compatibility of
the Bpoc method with the building block approach to phospho-
peptide synthesis. The synthesis of the shorter MAP Kinase [178–
184; Thr(P)183] peptide, HTGFLT(P)E (4), was selected to examine
the incorporation of the Bpoc–Thr(PO3Bzl2)–OH derivative in
order to ascertain the coupling efficiency and subsequent stability
of the phosphothreonyl residue under these synthetic conditions.
The requisite Bpoc–Thr(PO3Bzl2)–OH derivative was prepared
via the initial synthesis of Bpoc–Thr–OH followed by its ‘one-pot’
phosphorylation,11 according to Scheme 1. Phosphorylation was
effected using temporary t-butyldimethylsilyl (TBDMS) protec-
tion of the Bpoc–Thr–OH carboxyl group followed by phosphity-
lation with (BzlO)2PNiPr2–1H-tetrazole and subsequent oxidation
of the dibenzyl phosphite triester with tBuOOH. Deprotection of
the carboxyl group was achieved by treatment with 10% Na2S2O5
In view of these limitations, we have examined an alternative
approach to phosphopeptide synthesis using the highly acid-labile
aDept of Chemistry, University of Melbourne, Victoria, Australia 3010;
Fax: +61 (03) 8344 5104; Tel: +61 (03) 8344 4000
bCooperative Research Centre for Oral Health Science, School of Dental
Science and the Bio21 Institute of Molecular Science and Biotechnology,
The University of Melbourne, 30 Flemington Rd, Melbourne, Australia,
3010; Fax: +61 (03) 83442545; Tel: +61 (03) 83442562
† Current address: Cooperative Research Centre for Oral Health Science,
School of Dental Science and the Bio21 Institute of Molecular Science
and Biotechnology, The University of Melbourne, 30 Flemington Rd,
Melbourne, Australia 3010; Fax: +61 (03) 83442545; Tel: +61 (03)
83442562; tjattard@unimelb.edu.au
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resin (2) was cleaved by the addition of TFA–TES–H2O (95 : 2.5 :
2.5, v/v/v) for 6 h and the crude peptide (3) isolated by ethereal
precipitation.
The RP-HPLC profile of the crude peptide (3) (Fig 1a) showed
one major signal (peak 1) at 29.4 min, which gave a single peak
by MALDI-MS corresponding to the target phosphopeptide (4)
at m/z 882.7 [M–H]− (Fig 1b). Importantly, the mass spectrum of
the crude peptide (3) showed no evidence of a phosphothreonyl
deletion at m/z 700.8 [M–H]−, indicating quantitative incorpora-
tion of the Bpoc–Thr(PO3Bzl2)–OH derivative. As expected, the
high purity of the crude product also indicates that the benzyl
phosphate protecting group is stable to the mild acidic conditions
employed for Bpoc group removal.
Scheme 1 Reagents and conditions: (i) TBDMSCl (0.9 eq.)–NMM (1 eq.)
in THF (3 min), (ii) (BzlO)2PNiPr2 (1.2 eq.)–1H-tetrazole (3 eq.) (2 h), (iii)
14% tBuOOH (30 min), (iv) 10% Na2S2O5 (30 min).
(pH 4.0) and the product extracted using 5% NaHCO3 (pH 9.0)
to give the pure Bpoc–Thr(PO3Bzl2)–ONa salt in 63% yield.
The 13C NMR profile of the final product gave signals for Thr(P)
Ca, Thr(P) Cb and benzyl CH2 as broad singlets at 59.9, 69.4
and 80.2 ppm respectively. The aromatic C1-carbon of the benzyl
phosphate group was detected as a phosphorous-coupled doublet
at 135.7 ppm with a coupling constant of JP,C 6.5 Hz. The spectrum
showed no evidence of any Bpoc–Thr–ONa compound in the final
product since the characteristic peaks for the Thr–Ca and Thr–
Cb carbons at 58.9 and 67.7 ppm, respectively, were absent. The
phosphorylation of the protected threonyl synthon was confirmed
by its 31P NMR spectra which gave a major signal at −2.10 ppm
in ∼98% purity. The remaining Bpoc-protected amino acids were
synthesized according to the procedure outlined by Carey et al.12
in 51–74% yields.
The synthesis of HTGFLT(P)E (Scheme 2) (4) was accom-
plished by peptide extension from H–Glu(OtBu)–Wang with
all Bpoc amino acids and Boc–His(Trt)–OH incorporated as
the free acids, using a 1% TFA–DCM solution for N-terminal
deprotection. Upon completion of chain assembly, the peptide-
Fig. 1 Crude MAP Kinase ERK2 [178–184; Thr(P)183] (3), (a) RP-HPLC,
(b) MALDI-MS (peak 1).
Examination of Bpoc group acid lability
The next stage of this study was the determination of the minimum
acid requirement for the removal of the Bpoc group. While
Kemp et al.13 has shown that 0.5% TFA–DCM is sufficient for
deprotection within 10 min, Bpoc removal by an acid with a lower
Scheme 2 Reagents and conditions: (i) 25% piperidine–DMF (20 min),
(ii) Bpoc–amino acid (3 eq.)–HBTU (3 eq.)–NMM (9 eq.) (1 h) [Fi-
nal coupling: Boc–His(Trt)–OH], (iii) 1% TFA–DCM (20 min), (iv)
TFA–TES–H2O (95 : 2.5 : 2.5, v/v/v, 6 h).
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pKa is preferable for the stability of the resin linkage and side-chain
protecting groups. Formic acid–DCM was examined for suitability
as a deprotection solution in the synthesis of the MAP Kinase
ERK2 (182–184) peptide, H–Leu–Thr–Glu–OH (Scheme 3) (9),
in which the Thr(183) residue was coupled as the Bpoc–Thr–
OH derivative. Fmoc–Glu(OtBu)–Wang resin (1) was treated with
20% piperidine–DMF for 20 min and coupled to Bpoc–Thr–OH
for 1 h. Deprotection trials were then performed on the Bpoc–
Thr–Glu(OtBu)–Wang peptide-resin (5) using 5%, 10%, 15% and
20% formic acid–DCM solutions (20 min). Bpoc–Leu–OH was
subsequently coupled to each of the four deprotected dipeptides
and the protected peptide-resins (6)‡ cleaved with TFA–TES–
H2O (95 : 2.5 : 2.5, v/v/v) for 1 h. RP-HPLC and MALDI-
MS analysis of the resultant crude peptide (7)‡ from each trial
showed that the H–Thr–Glu–OH dipeptide (8) eluted at 8.6 min
{peak 1 (m/z 247.3, [M–H]−)} followed by the target tripeptide
(9) at 19.3 min {peak 2 (m/z 360.6 [M–H]−)} (Fig 2). Notably, the
dipeptide (8) resulting from incomplete Bpoc removal is absent in
the 15% and 20% formic acid trials, indicating that formic acid
concentrations of 15% or greater are sufficient for the removal of
the Bpoc protecting group within 20 min.
Fig. 2 Crude MAP Kinase ERK2 [182–184] (7) RP-HPLC profile from
formic acid–DCM deprotections of Bpoc–Thr–Glu(OtBu)–Wang (5): (a)
5% formic acid–DCM, (b) 10% formic acid–DCM, (c) 15% formic
acid–DCM, (d) 20% formic acid–DCM.
The Fmoc–Tyr(PO3Bzl2)–OH synthon was prepared using
temporary phenacyl carboxyl protection as described by Vale-
rio et al.14 Fmoc–Tyr–OPac was phosphorylated by treatment
with (BzlO)2PNiPr2–1H-tetrazole followed by oxidation with
mCPBA. The phenacyl group was removed by addition of Fmoc–
Tyr(PO3Bzl2)–OPac to a solution of Zn–EtOAc–AcOH–H2O (0.3 :
2 : 5 : 1, w/v/v/v) for 2 h and the Fmoc–Tyr(PO3Bzl2)–ONa
salt extracted with 5% NaHCO3 (pH 8.5). The combined base
extracts were acidified to pH 2.5 using solid citric acid, the product
extracted with DCM, and subsequent solvent evaporation under
reduced pressure gave Fmoc–Tyr(PO3Bzl2)–OH as a crispy white
foam in 73% yield.
The MAP Kinase ERK2 [178–188; Thr(P)183, Tyr(P)185] pep-
tide (Scheme 4) (14) was prepared by extension from Fmoc–
Thr(tBu)–Wang Resin (10) in the Fmoc mode of peptide syn-
thesis for the first five C-terminal residues. Following coupling
of Bpoc–Thr(PO3Bzl2)–OH to H–Glu(OtBu)–Tyr(PO3Bzl2)–Val–
Ala–Thr(tBu)–Wang, the deprotection of all intermediate N-
terminal residues was achieved using 20% formic acid–DCM for
20 min. At the completion of peptide synthesis, peptide-resin (12)
was cleaved by the addition of TFA–TES (95 : 5, v/v) for 6.5 h
and the crude peptide (13) isolated by ethereal precipitation.
Analysis by RP-HPLC gave a crude peptide spectrum of high
purity with a major peak at 32.3 min (Fig 3a). MALDI-MS
analysis of the main signal showed a single peak corresponding
to the target bis-phosphorylated peptide (14) (m/z 1397.4, [M–
H]−) (Fig 3b). Analysis of the crude peptide by MALDI-MS
showed no signal for either Thr(P) or Tyr(P) deletions, suggesting
full incorporation of the phosphorylated derivatives. Interestingly,
examination of both the crude peptide (13) and the major peak
from its RP-HPLC profile showed no signal for any contaminating
H-phosphonate peptide. This byproduct has been observed for
various global phosphorylations,6,15–18 and arises from the acid-
catalysed rearrangement of the intermediate phosphite triester un-
der concentrated reaction conditions during the phosphitylation
step. The absence of this undesired byproduct in the current study
was attributed to the lower concentration of 1H-tetrazole in the
Scheme 3 Reagents and conditions: (i) 25% piperidine–DMF (20 min),
(ii) Bpoc–Thr–OH (3 eq.)–HBTU (3 eq.)–NMM (9 eq.) (1 h), (iii) formic
acid–DCM [(a) 5%, (b) 10%, (c) 15% or (d) 20%; 20 min], (iv) Boc–Leu–OH
(3 eq.)–HBTU (3 eq.)–NMM (9 eq.) (1 h), (v) TFA–TES–H2O (95 : 2.5 :
2.5, v/v/v; 1 h).
Synthesis of MAP Kinase ERK2 [178–188; Thr(P)183, Tyr(P)185],
HTGFLT(P)EY(P)VAT (Scheme 4) (14)
Following the successful synthesis of HTGFLT(P)E (4) via Bpoc–
Thr(PO3Bzl2)–OH incorporation, this method was applied to
the extended bis-phosphorylated peptide from the MAP Kinase
ERK2 protein, HTGFLT(P)EY(P)VAT (14) for all residues
subsequent to the glutamyl residue. Since fully protected phos-
photyrosyl derivatives are not susceptible to b-elimination by
piperidine, the first five residues in the sequence were incorporated
as their Fmoc derivatives.
‡ Fully assembled peptide-resins resulting from each of the four formic
acid trials were all designated (6) for simplicity of discussion. Similarly, the
corresponding crude peptides were all designated (7).
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Discussion
An important feature of the Bpoc approach is that fully protected
phosphorylated threonyl derivatives may be directly incorporated
into the growing peptide chain, with Na-deprotections effected
using a 20% formic acid–DCM solution. This deprotection
strategy circumvents complications associated with base-catalysed
b-elimination of the protected phosphate group. Another major
advantage of this deprotection method is that TFA-labile side
chain protecting groups and resin linkages may be employed,
allowing the same mild acidic conditions that are utilized in Fmoc
solid phase syntheses for the final cleavage step. Formic acid was
chosen for Bpoc removal due to its lower pKa compared with TFA,
and is therefore expected to be more compatible for syntheses of
longer peptide chains.
For single Ser(P)- or Thr(P)-containing peptides, the global
phosphorylation method provides a convenient route to the
preparation of the monophosphorylated peptide, however long or
‘difficult’ peptide sequences may render this approach ineffective
due to the inaccessibility of phosphorylation reagents to the target
site. It has also been established that increasing phosphorylation
reagent concentrations to overcome this limitation is directly
related to an increase in H-phosphonate generation.18,19 In such
cases, a Bpoc-based approach would improve the purity of the final
product with respect to non-phosphorylated or H-phosphonylated
peptide byproducts and allow easier purification of the crude
sample in the absence of these contaminants.
Scheme 4 Reagents and conditions: (i) 25% piperidine–DMF (20 min),
(ii) Fmoc–aa (3 eq.)–HBTU (3 eq.)–NMM (9 eq.) (1 h), (iii) Bpoc–aa (3
eq.)–HBTU (3 eq.)–NMM (9 eq.) (1 h) [final coupling: Boc–His(Trt)–OH],
(iv) 20% formic acid–DCM (20 min), (v) TFA–TES (95 : 5, v/v, 6.5 h).
phosphitylation step during synthesis of the Bpoc–Thr(PO3Bzl2)–
OH and Fmoc–Tyr(PO3Bzl2)–OH derivatives, in comparison with
that required for solid phase phosphorylations.
The high purity of the crude MAP Kinase ERK2 [178–188;
Thr(P)183, Tyr(P)185] peptide (13) (Fig 3a) indicates that the Bpoc
methodology is also compatible with an Fmoc strategy when
used in combination for the same synthesis. In this case, the
phosphotyrosyl residue is located to the C-terminal side of the
phosphothreonyl residue, enabling the Fmoc approach for the
first five residues. While Fmoc–Tyr(PO3R2)–OH derivatives have
been successfully used in the Fmoc mode of synthesis,14,20–23
a
Bpoc protected phosphotyrosyl synthon may also conceivably be
utilized if the phosphotyrosyl residue occurs on the N-terminal
side of a phosphothreonyl or -seryl residue.
We anticipate that a Bpoc-based approach to phosphopeptide
synthesis would also facilitate the coupling of phosphorylated
derivatives in multiphosphorylated peptide chains. While the
development of Fmoc–Xxx(PO3Bzl,H)–OH (Xxx = Tyr, Thr, Ser)
derivatives has allowed the stepwise incorporation of phospho-
threonyl and -seryl derivatives in the Fmoc mode, this approach is
limited by the incomplete incorporation of the partially protected
derivative, caused by ionization of the phosphorylated moiety
during the coupling step.9 This problem may therefore be avoided
through the use of Bpoc–Xxx(PO3Bzl2)–OH synthons in the
Bpoc mode of peptide synthesis using 20% formic acid for Na-
deprotection on account of the stability of the benzyl phosphate
groups to such mild acid treatment. In regard to storage, it is
recommended that the phosphorylated Bpoc derivatives be stored
as their sodium salts at −20 ◦C or used immediately to avoid loss
of the highly labile Bpoc group over extended periods of time.
Conclusions
Fig. 3 Crude MAP Kinase ERK2 [178–188; Thr(P)183, Tyr(P)185] (13), (a)
RP-HPLC, (b) MALDI-MS (peak 1).
The Bpoc approach to phosphopeptide synthesis provides an effi-
cient method for the generation of phosphopeptides that are free
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from non-phosphorylated or H-phosphonylated contaminants.
The mild acidolytic Na-deprotection conditions permit the use
of TFA-labile side chain protection and resin linkages, allowing a
final cleavage step using TFA solutions.
with the following exceptions: a solution of Bpoc–OPh (5.0 mmol),
L-amino acid (5.0 mmol) and tetramethylguanidine (15 mmol) in
DMF (3.5 ml) was stirred at 50 ◦C for 48 h. The solvent was
evaporated under reduced pressure and the resultant oil dissolved
in 5% NaHCO3 (15 ml), transferred to a separation funnel and
washed with ether (2 × 30 ml). The aqueous layer was collected
and acidified to pH 6.5 (20% NaHSO4), washed with ether (2 ×
40 ml), acidified to pH 2.5 and the product extracted with DCM
(3 × 35 ml). Evaporation of the solvent gave the Bpoc–Xxx–OH
products in 51%–74% yields.
Experimental
General
Fmoc- and Boc-amino acids, HBTU, NMM and TFA were pur-
chased from Auspep and stored at 4 ◦C. Solvents were of AnalaR
grade with THF distilled from the potassium ketyl of ben-
zophenone prior to use. DMF, DCM, dibenzyl N,N-diisopropyl-
phosphoramidite, 1H-tetrazole, triethylsilane and phenacyl bro-
mide were obtained from Sigma–Aldrich Inc.
Solid phase peptide synthesis was performed manually on Hmp–
Wang resin using a solid phase reaction vessel attached to a
rotation instrument. The resin was suspended in a DMF–DCM (1 :
1, v/v) solution for 30 min prior to the commencement of peptide
synthesis. Acylation was achieved by the addition of a mixture of
amino acid (3 eq.), HBTU (3 eq.) and NMM (9 eq.) in a minimum
volume§ of DMF–DCM (1 : 1, v/v) to the Na-deprotected peptide-
resin and rotated for 1 h (see below for specific Na-deprotection
protocols). The peptide-resin was then filtered by suction and
rinsed with DCM (2 ml, 3 × 2 min). Cleavage of peptide-resins was
performed in a solid phase reaction vessel, the combined cleavage
filtrates evaporated to dryness by rotary evaporation and the crude
product isolated by ethereal precipitation (4 × 30 ml).
Synthesis of Bpoc-Thr(PO3Bzl2)–OH
N-Methylmorpholine (101 mg, 1.0 mmol) in anhydrous THF
(0.8 ml) and t-butyldimethylsilyl chloride (136 mg, 0.9 mmol) in
THF (0.8 ml) were added successively to a solution of Bpoc–Thr–
OH (356 mg, 1.0 mmol) in THF (1 ml) at room temperature. After
3 min, 1H-tetrazole (105 mg, 1.5 mmol) was added to the mixture,
followed by the addition of (BzlO)2PNiPr2 (415 mg, 1.2 mmol).
◦
t
After 2 h, the mixture was cooled to −20 C and 14% BuOOH
(0.9 ml) added for 30 min. The THF was evaporated under reduced
pressure and the reaction mixture transferred to a separation
funnel by the addition of ether (8 ml). An aqueous solution of
10% Na2S2O5 (pH 4.5, 6 ml) was added at 10–15 ◦C and the
mixture stirred rapidly for 20 min. The organic layer was collected,
washed with 10% Na2SO4 (2 × 6 ml) and the product extracted
with 5% NaHCO3 (pH 8.5; 3 × 4 ml). The combined aqueous
extract was evaporated under reduced pressure and the resultant
oil dissolved in DCM, filtered and the solvent evaporated to
give Bpoc–Thr(PO3Bzl2)–ONa in 402 mg (62.9% yield). The final
product, Bpoc–Thr(PO3Bzl2)–OH was generated prior to peptide
synthesis by dissolution in EtOAc (6 ml), washing with 10% citric
acid (10 ml) and solvent evaporation. 13C NMR (CDCl3): d 18.8,
27.9, 30.2, 59.9 (br s, Thr Ca), 69.4 (br s, Thr Cb), 80.2 (br s, Bzl
CH2), 124.7, 126.7, 127.0, 127.6, 127.9, 128.1, 128.4, 128.5, 135.7
Analytical RP-HPLC was performed using a Waters liquid
chromatograph instrument (model 510) equipped with a UV
detector (model 420.AC) and a Zorbax 300 SB-C18 reversed phase
column (4.6 mm × 25 cm). Peptide separation was achieved
using a linear acetonitrile gradient in 0.1% TFA (aq) at a flow
rate of 1 ml min−1. Semi-preparative RP-HPLC was performed
on a Perkin Elmer 200 Series liquid chromatograph instrument
with a Zorbax 300 SB–C18 reversed phase column (9.4 mm ×
25 cm) linked to a fluorescence detector (model ABI 783A)
and a chart recorder (model SE 120). Peptide separation was
accomplished at a flow rate of 2 ml min−1. MALDITOF-MS
data were obtained on a Voyager-DE MALDI bench top linear
mass spectrometer. Samples were prepared in a saturated a-cyano
hydroxycinnamic acid solution [solvent: MeCN–H2O–CHOOH
(33 : 66 : 1, v/v/v)]. 13C NMR spectra were recorded on a Unity-
300 fourier transform instrument operating at 300 and 75 MHz
respectively with chemical shifts in ppm relative to internal CDCl3
adjusted to 77.0 ppm (central peak). 31P NMR data were obtained
relative to internal H3PO4 (85%) adjusted to 0.0 ppm.
(d, JP,C 6.5 Hz, Bzl C1), 139.2, 140.9, 145.9, 155.7, 176.0 ppm. 31
NMR (85% H3PO4): d −2.1 ppm.
P
Preparation of MAP Kinase ERK2 [178–184; Thr(P)183];
H–His–Thr–Gly–Phe–Leu–Thr(P)–Glu–OH (4)
Fmoc-Glu(OtBu)–Wang (1) (36 mg, 0.02 mmol) was suspended
in 25% piperidine–DMF (1 ml) for 20 min and rinsed with DCM
(5 × 1 ml). Subsequent amino acid couplings were performed by
addition of Bpoc-amino acid (3 eq.)–HBTU (3 eq.)–NMM (9 eq.)
in DCM–DMF (1 : 1, v/v) (1 h) with deprotection of the Bpoc
Na-protecting group achieved by suspension of the peptide-resin
in 1% TFA–DCM (1 ml) for 20 min (N.B. the phosphothreonyl
residue was coupled as the Bpoc–Thr(PO3Bzl2)–OH acid). The N-
terminal amino acid was incorporated as the Boc–His(Trt)–OH
derivative. The resultant peptide-resin [Boc–His(Trt)–Thr(tBu)–
Gly–Phe–Leu–Thr(PO3Bzl2)–Glu(OtBu)–Wang] (2) was treated
with TFA–TES–H2O (95 : 2.5 : 2.5, v/v/v; 1 ml) for 6 h and
the crude peptide (3) (18 mg) isolated as described under General
procedure (above). Analytical RP-HPLC [gradient: 0–50% MeCN
in 50 min] gave one major peak (∼90%) at 29.4 min. MALDI-MS
(negative mode): m/z 882.7 [M–H]− (4).
General procedure for the synthesis of Bpoc–Xxx–OH [Xxx =
Thr(tBu), Gly, Phe, Leu]
(p-Biphenylyl)-2-propyloxycarbonylphenyl carbonate (BpocOPh)
was prepared in 98.3% yield using the procedure described by
Kemp et al.13 The preparation of all Bpoc-amino acids was
performed according to the procedure outlined by Carey et al.12
§ Minimum volume is defined as the minimum amount of solvent required
for adequate peptide-resin solvation during the coupling step. An increase
in the minimum volume required was observed during progression of
peptide assembly.
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Preparation of MAP Kinase ERK2 [182–184];
H–Leu–Thr–Glu–OH (9)
66.9, 70.2 (d, JP,C 6.0 Hz, Bzl CH2), 119.9 (d, JP,C 5.0 Hz, Tyr Ar
C3), 125.0, 127.0, 127.6, 127.9, 128.0, 128.5, 128.6, 130.7, 133.1,
135.0 (d, JP,C 7.1 Hz, Bzl C1), 141.2, 143.6, 143.7, 149.2 (d, JP,C
7.0 Hz, Tyr Ar C4), 155.7, 173.2 ppm. 31P NMR (85% H3PO4):
d −7.6 ppm.
Fmoc–Glu(OtBu)–Wang (1) (144 mg, 0.08 mmol) was suspended
in 25% piperidine–DMF (1 ml) for 20 min and rinsed with DCM
(5 × 1 ml). A mixture containing Bpoc–Thr–OH (3 eq.)–HBTU
(3 eq.) and NMM (9 eq.) in DCM–DMF (1 : 1, v/v) was added
to the H–Glu(OtBu)–Wang peptide-resin for 1 h. The resultant
Bpoc–Thr–Glu(OtBu)–Wang peptide-resin (5) was divided into
four equal portions (0.02 mmol each), and one of four solutions
containing formic acid–DCM (5%, 10%, 15% or 20%, v/v; 1 ml)
was added to each for 20 min. Each peptide-resin was rinsed
with DCM (3 × 1 ml) and Boc–Leu–OH coupled as above. The
resultant peptide-resins (6)‡ were suspended in a TFA–TES–H2O
(95 : 2.5 : 2.5, v/v/v, 1 ml) solution (1 h) and the corresponding
crude peptides (7)‡ isolated according to the general procedure
(above). Analytical RP-HPLC gave peaks for H–Leu–Thr–Glu–
OH (9) at 19.3 min and H–Thr–Glu–OH (8) at 8.6 min. MALDI-
MS (negative mode): m/z 360.6, [M–H]− (9), m/z 247.3 [M–H]−
(8).
Preparation of MAP Kinase ERK2 [178–188; Thr(P)183
,
Tyr(P)185]; H–His–Thr–Gly–Phe–Leu–Thr(P)–Glu–Tyr(P)–Val–
Ala–Thr–OH (14)
Peptide-resin [Boc–His(Trt)–Thr(tBu)–Gly–Phe–Leu–Thr(PO3-
Bzl2)–Glu(OtBu)–Tyr(PO3Bzl2)–Val–Ala–Thr(tBu)–Wang] (12)
was assembled by (a) standard Fmoc–tBu protocols for
residues 184–188 from Fmoc–Thr(tBu)–Wang resin (10) (38 mg,
0.025 mmol) including incorporation of Fmoc–Tyr(PO3Bzl2)–OH
and (b) the procedure outlined for the synthesis of peptide (4) for
residues 178–183 including incorporation of Bpoc–Thr(PO3Bzl2)–
OH and Boc–His(Trt)–OH as the N-terminal residue. All
couplings were effected as described under General procedure
(above); Fmoc deprotection was performed by addition of 25%
piperidine–DMF (1 ml) to the peptide-resin, while Bpoc removal
was achieved by suspension in 20% formic acid–DCM (1 ml).
Following completion of peptide assembly, the peptide-resin (12)
was suspended in TFA–TES (95 : 5, v/v; 1 ml) for 6.5 h and
the crude peptide (13) (32 mg) isolated according to general
procedure (above). Semi-preparative RP–HPLC purification
(gradient: 0–50% MeCN in 50 min) of crude peptide (13) (10 ×
2 mg) and lyophilization of the major eluting fraction gave MAP
Kinase ERK2 [178–188; Thr(P)183, Tyr(P)185]; H–His–Thr–Gly–
Phe–Leu–Thr(P)–Glu–Tyr(P)–Val–Ala–Thr–OH (14) as a fluffy
white powder (13 mg, 60% yield). MALDI-MS: (negative mode)
(m/z 1397.4 [M–H]−).
Synthesis of Fmoc–Tyr(PO3Bzl2)–OH
Triethylamine (203 mg, 2.0 mmol) in anhydrous THF (3 ml)
and phenacyl bromide (398 mg, 2.0 mmol) in THF (3 ml) were
added successively to a solution containing Fmoc–Tyr(tBu)–OH
(965 mg, 2.1 mmol) in THF (3 ml) and the mixture stirred for
8 h. The solvent was evaporated, the resultant foam redissolved
in EtOAc (20 ml) and the organic phase washed with NaCl (sat)
(2 × 20 ml), 10% citric acid (2 × 20 ml) and 0.1 M NaOH (2 ×
20 ml). The organic phase was collected, filtered and evaporated
under reduced pressure to give Fmoc–Tyr(tBu)–OPac as a white
powder (930 mg, 80.5% yield). Fmoc–Tyr(tBu)–OPac (930 mg,
1.61 mmol) was then treated with TFA–H2O (95 : 5, 3 ml) at 20 ◦C
for 1 h. Following evaporation of TFA, the resultant residue was
triturated with EtOEt–petroleum spirits (1 : 1, 50 ml) and the
Fmoc–Tyr–OPac powder filtered and dried (8 h) under reduced
pressure. Fmoc–Tyr–OPac (800 mg, 1.53 mmol) was dissolved in
anhydrous THF (2 ml) and (BzlO)2PNiPr2 (795 mg, 2.3 mmol)
in THF (2 ml) and 1H-tetrazole (193 mg, 2.76 mmol) added
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