COMMUNICATION
Fmoc-chemistry of a stable phosphohistidine analoguew
Tom E. McAllister,ab Michael G. Nixa and Michael E. Webb*ab
Received 5th October 2010, Accepted 27th October 2010
DOI: 10.1039/c0cc04238b
We report the synthesis of the phosphohistidine analogue,
Fmoc-4-diethylphosphonotriazolylalanine 5 and its incorporation
into peptides. Our synthesis of 5 has enabled us to demonstrate
that the analogue is compatible with Fmoc-solid phase peptide
synthesis (SPPS) conditions. Standard cleavage conditions yield
the diethyl phosphonate-protected peptide, however this can be
subsequently deprotected using trimethylsilyl bromide to yield
the free phosphonic acid-containing peptides.
Phosphorylation of amino acids is central to the post-
translational regulation of proteins1 and the role of the acid-stable
Fig. 1 Structure of tele-phosphohistidine 1 and analogues designed to
phosphate monoesters, phosphoserine, phosphothreonine and
phosphotyrosine in cell physiology and biochemistry is well
established. Phosphorylation of several other amino acids
including histidine, arginine, lysine and aspartate is also
observed.2,3 In these cases the phosphorylated amino acid
residues are less stable due to the inherent instability of the
phosphoramidate and mixed phospho-anhydride bond.
Because of this relative lability, phosphorylated histidine and
aspartate both act to mediate phosphate transfer in pathways
of bacterial two-component signal transduction4 and
phosphohistidine is observed as a reactive intermediate in
numerous enzymatic reaction mechanisms. Histidine
phosphorylation has also been observed in mammalian systems,
including on histones I and IV,5 on heterotrimeric G-proteins,6
on P-selectin7 and on annexin I.8 Corresponding histidine
kinases9 and histidine phosphatases10 have been described.
This suggests that regulated phosphorylation of histidine may
be important for eukaryotic cell biochemistry. Further study
of the importance of histidine phosphorylation is currently
hampered by the lack of available biochemical and chemical
tools. Mass spectrometric methods to detect the modification
have recently been described11 but these are not currently
readily applicable to whole proteome studies and until
the recent publication by Kee et al.12 there were no
available methods to detect the modification selectively using
antibodies.
mimic it including those of Schenkels et al. 2 and Kee et al. 4. We
report the use of the Fmoc-protected derivative 5.
stable analogues of phosphohistidine have previously been
reported: the furanyl and pyrrolylalanine derivatives 2 & 3
(Fig. 1).2 The synthesis of the furanyl derivative was reported
by Schenkels et al.15 and takes ten steps leading to a
compound still requiring further derivatisation before being
suitable for incorporation into peptides using Fmoc chemistry.
We therefore chose to synthesize the triazolylalanine
phosphonates 4 & 5. In these molecules the labile P–N bond
is replaced with
a
hydrolytically stable P–C bond;
dephosphonylation of these molecules would first require
protonation at the carbon adjacent to the phosphorus atom
leading to loss of aromaticity suggesting that these molecules
will not be readily hydrolysed in acid. This work complements
that of Kee et al.12 who recently reported the synthesis of
Boc-4-diethylphosphonotriazolylalanine 4 and its application
to the biochemistry of phosphohistidine.
Our synthetic route (Scheme 1) to the triazole derivative is
based upon the cycloaddition of diethyl acetylenylphosphonate 6
with azidoalanine derivatives. Several routes to 6 are described
in the literature,16 however we prepared it by direct addition of
acetylenyl magnesium bromide to diethyl chlorophosphate
in THF to give the desired alkyne 6 in 24% yield.w
N-Boc-azidoalanine 8 was generated from commercially available
N-a-Boc-diaminopropionic acid 7 via Cu-catalysed diazo-transfer
using the method of Link et al.,17 Cu(I)-catalysed [3+2]
cycloaddition of 8 with the alkyne 6 then generated the
Boc-protected triazolylalanine 4 in 88% yield. This could be
converted to the corresponding Fmoc-protected triazolylalanine
5 in 60% overall yield. Alternatively, this compound has
subsequently been prepared by direct addition of commercially
available Fmoc-azidoalanine 10 to the alkyne 6 to generate 5
in 96% yield.
The tele-regioisomer (t-pHis, 1) of phosphohistidine is the
dominant form observed in proteins.2 The chemical instability
of both this and the pros-regioisomer (p-pHis) is due to
protonation of the non-phosphorylated imidazole nitrogen
(pKa E 6.4 in t-pHis).2 Methods to chemically phosphorylate13
and thiophosphorylate14 histidine in a protein context have
been described, however we wished to develop an effective
stable mimic of the residue amenable to Fmoc-SPPS. Two
a School of Chemistry, University of Leeds, Leeds, UK
b Astbury Centre for Structural Molecular Biology,
University of Leeds, Leeds, UK. E-mail: m.e.webb@leeds.ac.uk;
Tel: +44 (0)113 343 6423
w Electronic supplementary information (ESI) available: Full experi-
mental and DFT analysis. See DOI: 10.1039/c0cc04238b
The protected phosphonotriazole 5 is readily incorporated
into peptides using standard Fmoc-SPPS methodology. We
synthesized the test peptide sequence, Ac-GMTSTzAA-NH2
11, corresponding to the phosphocarrier domain of pyruvate,
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 1297–1299 1297
McAllister, Tom E.
