4-Phosphopyrazol-2-yl alanine: A non-hydrolysable analogue of phosphohistidine

Article abstract of DOI:10.1039/c5cc01811k

We report the synthesis of a stable analogue of τ-phosphohistidine: 4-phosphopyrazol-2-yl alanine (pPza). Polyclonal antibodies generated against the mimic show high reactivity and selectivity for τ-phosphohistidine, with minor or no cross-reactivity towards non-phosphorylated histidine or O-phosphoamino acids, including phosphotyrosine.

Full text of DOI:10.1039/c5cc01811k

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4-Phosphopyrazol-2-yl alanine: a non-hydrolysable
analogue of phosphohistidine†
Cite this: Chem. Commun., 2015,
51, 7305
Matthew Lilley,ab Bezaleel Mambwe,b Mark J. Thompson,a Richard F. W. Jackson*a
and Richmond Muimo*b
Received 3rd March 2015,
Accepted 18th March 2015
DOI: 10.1039/c5cc01811k
www.rsc.org/chemcomm
We report the synthesis of a stable analogue of s-phosphohistidine:
4-phosphopyrazol-2-yl alanine (pPza). Polyclonal antibodies generated
against the mimic show high reactivity and selectivity for s-phospho-
histidine, with minor or no cross-reactivity towards non-phosphorylated
histidine or O-phosphoamino acids, including phosphotyrosine.
Fig. 1 Isomeric forms of phosphohistidine and the stable mimic,
4-phosphopyrazol-2-yl alanine (pPza).
Protein phosphorylation occurs on several amino acid residues:
serine, threonine, tyrosine, histidine, lysine, arginine, aspartate,
glutamate and cysteine.1 It regulates most cellular processes and
its dysregulation is linked to a number of diseases including have played a key role in elucidating the importance of O-phos-
cancer, diabetes, and hypertension amongst others.2 The O-phos- phorylation in cell signalling. For example, the generation of
phorylated amino acids (phosphoserine, phosphothreonine and phosphotyrosine specific antibodies in the early 1980s led to a
phosphotyrosine) are extensively reported but only account for massive upsurge in phosphotyrosine research and elucidation
approximately 50% of phosphorylation events in mammalian of the role of this posttranslational modification in many key
cells, indicating alternative phosphoamino acids (histidine, cellular processes. t-Phosphohistidine specific antibodies
lysine, arginine, aspartate, glutamate and cysteine) are abundant would likewise be pivotal in understanding the precise roles
but understudied.3
of histidine phosphorylation, but phosphohistidine itself is a
Phosphorylation of histidine was first identified 50 years ago by poor immunogen because of its high susceptibility to hydrolysis
Boyer and co-workers,4 and occurs on either of the two nitrogens (DG1 of hydrolysis À50 to À59 kJ molÀ1), and cannot be used to
located on its imidazole ring; accordingly, phosphorylated histidine generate specific antibodies.
exists either as p (pros)-phosphohistidine 1 or t (tele)-phospho-
An alternative strategy is to produce ‘phosphohistidine-like’
histidine 2 (Fig. 1), which is the dominant isomer found in proteins. antibodies by using stable unnatural amino acid analogues or
It is now well established that phosphorylation of histidine regulates mimics that are immunogenic. Since the discovery of phospho-
important cellular functions via the two-component phospho-relay histidine, only a relatively small number of potential analogues
signalling pathways in bacteria, fungi, and plants. Increasing have been developed including thio-phosphohistidine,7 phos-
evidence suggests phosphohistidine is also involved in regula- phofurylalanine,8 phosphopyrrole,5 phosphoryltriazolylalanine
tion of key cellular processes in mammalian cells including ion isomers,9–11 and most recently phosphoryltriazolylethylamine.12
channels, apoptosis, cell proliferation/differentiation, inflammation, A sulphonamide mimic of the transition state of phosphohistidine
chromatin biology, and cell signalling.5,6
phosphatase has been reported,13 as has the synthesis of a
Phospho-specific antibodies, with no cross-reactivity to complementary triazole.14 In most of the stable mimics, the labile
other phosphoamino acids or the non-phosphorylated residue, P–N bond is replaced with a hydrolytically stable P–C bond.
Introduction of a P–C bond was very successful in the case of
a Department of Chemistry, The University of Sheffield, Dainton Building, Sheffield,
phosphotyrosine,15 which is also relatively unstable. In one early
report of a phosphotyrosine antibody generated using azobenzene
phosphonate as immunogen, Frackelton et al. observed cross-
reactivity towards phosphohistidine 3,16 and thus provided the first
indication that it might be possible to raise antibodies specific to
phosphohistidine. However, antibodies subsequently raised against
S3 7HF, UK. E-mail: r.f.w.jackson@shef.ac.uk
b Department of Infection and Immunity, Academic Unit of Respiratory Medicine,
The University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2TH,
UK. E-mail: r.muimo@shef.ac.uk
† Electronic supplementary information (ESI) available: Experimental procedures
and NMR spectra for 2, 3, 5, 6, 7 and 8. See DOI: 10.1039/c5cc01811k
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the various analogues outlined above either cross-reacted signifi-
cantly with other phosphorylated amino acids; did not universally
recognise phosphohistidine in proteins; or did not recognise it at
all. Consequently, the study of N-phosphorylation in cell biology
has remained in its infancy, especially in eukaryotes, despite the
fact that phosphohistidine was discovered nearly 20 years prior to
phosphotyrosine and is reported to be much more abundant than
phosphotyrosine in certain tissues and organisms.17,18
Motivated by a previous report that suggested phosphofurylal-
anine8 might be a suitable non-hydrolysable and non-isomerisable
analogue of t-phosphohistidine 2, we considered that the simplest
modification would involve transposition of the nitrogen atom
bound to the labile phosphoryl group and the carbon bound to
the alanine residue. Pyrazole 3 therefore became our initial target.19
The synthesis of 3 was carried out by ring-opening of the Vederas
b-lactone 420–22 with 4-iodopyrazole to give the acid 5. Formation of
the methyl ester 6 and subsequent Hirao cross-coupling23 with
diethyl phosphite gave the fully protected amino acid 7. Finally two-
stage deprotection was carried out by hydrolysis of the methyl ester
to give the acid 8, followed by treatment with TMSBr to give the
target, 4-phosphopyrazol-2-yl alanine (pPza) 3 (Scheme 1).
Fig. 2 Dot-blots obtained using pPza rat antisera: (A) crude polyclonal
antisera. (B) Antibody affinity depleted using a sepharose histidine column.
(C) Antibody affinity depleted with sepharose bound glutaraldehyde linked
histidine–KLH column. (i) Histidine, (ii) t-phosphohistidine, (iii) phospho-
serine, (iv) phosphothreonine, (v) phosphotyrosine, (vi) 4-phosphopyrazol-
2-yl alanine (pPza).
To test the suitability of pPza 3 as an analogue of t-phos-
phohistidine, it was coupled to keyhole limpet hemocyanin (KLH)
via glutaraldehyde linkage and used as a hapten to generate rat and acid standards (Fig. 2; iii–v), linked via succinylated BSA, were
rabbit polyclonal antibodies. The resulting antisera was tested commercially sourced. We have already shown that these standards
for specificity using dot blot analysis (Fig. 2) against amino acid are detected by an antibody generated to a glutaraldehyde-linked
standards conjugated to bovine serum albumin (BSA), also via antigen, thus demonstrating that the absence of cross-reactivity
glutaraldehyde linkage. The polyclonal antibodies generated towards O-phosphoamino acids was not a false negative.24
from pPza 3 showed strong reactivity towards t-phosphohistidine,
Consequently, a two stage strategy was adopted to purify the
minor reactivity to histidine, and no cross-reactivity towards phos- rabbit pHis antibody: firstly, antisera was affinity depleted using an
photyrosine or other O-phosphoamino acids (Fig. 2, entry A). Cross- agarose column containing histidine conjugated to BSA via glutar-
reactivity towards histidine was removable by affinity depletion aldehyde linker (see ESI,† Fig. S1); secondly, the flow through from
using a histidine–KLH glutaraldehyde conjugated sepharose the first column was then further purified on an affinity column
column (Fig. 2, entry C) but not a histidine sepharose column containing immobilized BSA–t-phosphohistidine conjugate. ELISA
(Fig. 2, entry B) as previously described.24 The O-phosphoamino analysis showed the purified antibody had high specificity for pHis
with little or no cross-reactivity for pTyr or other phosphoamino
acids tested (Fig. 3).
Western blots were performed with t-phosphohistidine–BSA
conjugate to establish the utility of the antibody in a standard
biological technique (see ESI,† Fig. S3). Further western blot
analysis was also performed against an extract of cytosolic
proteins from ovine airway epithelia treated with either acid,
base or hydroxylamine buffers as previously described.25 Protein
bands (as yet unidentified) detected by the pPza rabbit antibody
are stable under alkaline conditions but labile in the presence of
hydroxylamine or acidic conditions (Fig. 4A), indicative of a
phosphoramidate bond. Lysates of human bronchial epithelial
cells (16HBE14o-) showed a considerable increase in phospho-
tyrosine content in several protein bands following treatment
with human epidermal growth factor (EGF, 15 nM) for 60 min at
37 1C in full growth medium (Fig. 4B, left panel), but this greatly
increased level of phosphotyrosine in the EGF treated cells
remained undetected by the pPza antibody (Fig. 4B, right panel).
These data provide evidence that in a complex protein sample,
the pPza antibody specifically detects phosphoramidate or N-phos-
Scheme 1 Synthesis of 4-phosphopyrazol-2-yl alanine 3.
7306 | Chem. Commun., 2015, 51, 7305--7308
phorylation and not acyl or O-phosphoamino acids.
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Fig. 3 Extract from the affinity purification of pPza rabbit antisera. Rabbit
polyclonal antibody affinity depleted using an agarose–BSA–His affinity column
was purified on an agarose–BSA–pHis affinity column: ELISA analysis showing
binding intenstity of pPza antibody to phosphoamino acids in various purification
fractions measured at 450 nm – input, flow through (FT), washes and eluates.
The ELISA plate was coated with BSA, BSA-His, BSA-pHis, BSA-pThr, BSA-pSer,
BSA-pTyr or BSA-pPZa. The full purification profile is included in the ESI,† Fig. S2.
Fig. 5 Detection of endogenous phosphohistidine in known phosphohistidine
containing proteins (NDPK-A or B, and protein Gb from airway epithelia).
(A) Immunoblot (IB) of NDPK-A and NDPK-B; (B) western blot detection of
phosphohistidine and NDPK-A in immunoprecipitates (IP) of NDPK-A, NDPK-B
and phosphohistidine (pHis) from cytosol of ovine airway epithelia or (C) western
blot detection of phosphohistidine in immunoprecipitates (IP) of protein Gb
(antibodies R = rabbit polyclonal; M = mouse monoclonal) in lysates of human
bronchial epithelial cells (16HBE14o-) probed with pPza rabbit antibody.
of many histidine-phosphorylated proteins and be useful in inter-
rogation of proteomes. We therefore sought to provide further
evidence that the pPza antibody recognises phosphohistidine inde-
pendently of the amino acid sequences flanking the histidine
phosphorylation sites. Fig. 5 shows immunoblot detection of endo-
genous phosphohistidine in different proteins – NDPK-A, NDPK-B,
and protein Gb26,27 – immunoprecipitated from lysates of human
bronchial epithelial cells (16HBE14o-) and further demonstrates the
potential and wide-ranging utility of the pPza generated antibody to
detect phosphohistidine in a range of proteins. As expected, heat
treatment of the immunoprecipitates at 90 1C for 5 min in Laemmli
buffer resulted in loss of pHis signal (data not shown).
Significantly, the pPza generated antibody selectively recognizes
free t-phosphohistidine, detects phosphorylated histidine residues
in several proteins and shows little or no cross-reactivity with
O-phosphoamino acids.
In conclusion, we report successful synthesis of 4-phosphopyrazol-
2-ylalanine 3 and demonstrate its potential as a stable analogue of
t-phosphohistidine.‡ Thus, both pPza and resulting antibodies are
likely to be important tools to explore and characterize the role of
phosphohistidine in various cell types and cellular processes.
We thank the EPSRC Life Sciences Interface DTC for support
(ML), and B. Marsh for experimental assistance. The 16HBE14o-
cells were provided by Dr D. Gruenert (California Pacific Medical
Center, CA).
Fig. 4 Western blot detection of phosphohistidine (anti-pHis) and phos-
photyrosine (anti-pTyr) following EGF, acid or base treatments. (A) Western
blot of cytosolic proteins from ovine airway epithelia treated with buffer
control, 0.1 M HCl, 0.1 M NaOH, 0.4 M acetic acid/0.1 M hydroxylamine, for
30 min at 60 1C, and probed for phosphohistidine (pPza rabbit antibody,
Rb). (B) Lysates of human bronchial epithelial cell lines (16HBE14o-) treated
with or without human epidermal growth factor (EGF, 15 nM) for 1 hour
and probed for phosphotyrosine staining (clone 4G10 antibody) or the
rabbit antibody generated with pPza (right panel), (control, untreated).
Notes and references
‡ During the preparation of this manuscript, the synthesis of a closely
related pyrazolylethylamine derivative, and its use to generate antibodies,
was reported.28 These antibodies showed significantly higher selectivity
towards phosphohistidine (compared to other O-phosphoamino acids) than
A pan-pHis specific antibody that recognises pHis epitopes
independently of sequence context should allow the detection antibodies generated from the previously reported triazole analogues.
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