Development of Stable Phosphohistidine Analogues

Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 09/2010; 132(41):14327-9. DOI: 10.1021/ja104393t
Source: PubMed


Protein phosphorylation is one of the most common and extensively studied posttranslational modifications (PTMs). Compared to the O-phosphorylation of Ser, Thr, and Tyr residues, our understanding of histidine phosphorylation is relatively limited, particularly in higher eukaryotes, due to technical difficulties stemming from the intrinsic instability and isomerism of phosphohistidine (pHis). We report the design and synthesis of stable and nonisomerizable pHis analogues. These pHis analogues were successfully utilized in solid-phase peptide synthesis and semi-synthesis of histone H4. Significantly, the first antibody that specifically recognizes pHis was obtained using the synthetic peptide as the immunogen.

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Available from: Jung-Min Kee, Nov 27, 2014
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    • "Recently, sequence-specific pHis polyclonal antibodies toward pHis18 in histone H4 have been generated (Kee et al., 2010). First-and second-generation ''pan-pHis'' polyclonal antibodies against 3-pHis have also been reported (Kee et al., 2013, 2015). "
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    ABSTRACT: Summary Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
    Cell 07/2015; 162(1):198-210. DOI:10.1016/j.cell.2015.05.046 · 32.24 Impact Factor
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    • "c Determined by comparison with the specific rotations reported by Muir and co-workers (Kee et al. 2010) d Determined by comparison with the specific rotation of the same material synthesised by protonolysis of the benzyl esters 11e/12e e Determined from the relative integrals of the peaks corresponding to the enantiomers by enantioselective HPLC "
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    ABSTRACT: Histidine-phosphorylated proteins and the corresponding kinases are important components of bacterial and eukaryotic cell-signalling pathways, and are therefore potential drug targets. The study of these biomolecules has been hampered by the lability of the phosphoramidate functional group in the phosphohistidines and the lack of generic antibodies. Herein, the design and concise synthesis of stable triazolylphosphonate analogues of N1- and N3-phosphohistidine, and derivatives suitable for bioconjugation, are described.
    Amino Acids 11/2011; 43(2):857-74. DOI:10.1007/s00726-011-1145-2 · 3.29 Impact Factor
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    ABSTRACT: 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.
    Chemical Communications 01/2011; 47(4):1297-9. DOI:10.1039/c0cc04238b · 6.83 Impact Factor
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