ChemComm
Communication
In conclusion, the Ln complexes reported here function
as efficient, selective and chemically stable tags for cysteine-
containing peptides and proteins. Moreover, we have devised a
new method of protein attachment,17 based on displacement of
the nitro group in a p-substituted pyridine moiety that creates a
short link between the tag and protein, compared to previous
methods with electron poor alkene or maleimide electrophiles.
The tagging reaction is thiol-specific and proceeds rapidly in
water at room temperature, demonstrated by the facile ligation
of the single, exposed Cys residue of BSA. Using [Ln.L2], tagged
peptides can be further constrained by coordination of proximal
anionic amino acid residues (Glu and pSer) to the Ln centre.
These examples highlight the advantages of the nucleophilic
substitution of an activated aromatic nitro group over existing
protocols for Cys binding. The range of applications could
embrace introduction of a heavy atom label to aid crystallo-
graphic analysis, may include examples in NMR analysis created
by the introduction of one (or more) Ln ions of differing shift
and relaxation ability and could extend to protein labelling with
radioisotopes or luminescent species, e.g. a Eu(III) complex
analogue of the recent, very bright series of complexes.12,15,16
We thank Dr Jackie Mosely for analysis of the MALDI MS
data and the ERC for support (FCC 266804).
Fig. 3 Emission spectra of: (upper) [Eu.L2]; (centre) [Eu.L2] in 1 mM NH4OAc
solution and; (lower) [Eu.L2] in 1 mM Na2HPO4 solution (293 K, H2O, pH 6, 10 mM
complex).
Notes and references
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3 P. H. J. Keizers, A. Saragliadis, Y. Hiruma, M. Overhand and
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Fig. 4 (upper) Emission spectra of: [Eu.L2] in water (green); [Eu.L2] in 1 mM
NH4OAc solution (blue) and; [Eu.L2-peptide 1] (pink) in water. (lower) Overlaid
emission spectra of: [Eu.L2] in water (green); [Eu.L2-peptide 2] (orange) and;
[Eu.L2-peptide 3] (blue) in water.
¨
4 D. Haussinger, J.-r. Huang and S. Grzesiek, J. Am. Chem. Soc., 2009,
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the emission fingerprint spectrum recorded for [Eu.L2] in
acetate solution (Fig. 4), and differs clearly from that of
[Eu.L2] in water alone, indicating that the terminal Glu residue
is directly interacting with the Eu metal. Likewise, the Eu
emission spectral form of [Eu.L2-peptide 2] was very similar
to that recorded for [Eu.L2] in phosphate solution, consistent
with ligation of the terminal pSer residue of peptide 2 to the Eu
centre. Additionally, the 31P NMR spectrum of Yb.L2-peptide 2
showed two signals at 3.4 and À50.6 ppm in the ratio 2 : 1,
¨
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¨
¨
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12 S. J. Butler, B. K. McMahon, R. Pal, D. Parker and J. W. Walton,
at 3.4 ppm was assigned to the species in which the pSer moiety
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was unbound (free peptide dp = 0.2 ppm). The broad signal at
13 H. E. Stanger and S. H. Gellman, J. Am. Chem. Soc., 1998, 120, 4236.
À50.6 ppm corresponds to the pSer-bound species, confirmed 14 S. Aime, A. S. Batsanov, A. Beeby, M. Botta, R. S. Dickins,
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variation accords with an Yb-P separation, r, of 3.50(0.07) Å,
consistent with structural data for related Yb-phosphinate
49, 1600.
complexes, where r = 3.39(0.02) Å.14 Finally, the europium 16 B. K. McMahon, R. Pal and D. Parker, Chem. Commun., 2013,
emission spectrum of [Eu.L2-peptide 3] was identical to that
49, 5763.
17 Reactions of 4-nitro-2-hydroxymethyl pyridine with cysteine or glutathione
of [Eu.L2] in water, indicating that in this case binding of the
(pH 7, 20 1C, 40 min) went to completion, suggesting a role in tagging an
terminal residue to the lanthanide ion was not occurring.
unbound ligand for antibody/peptide radiolabelling.
c
9106 Chem. Commun., 2013, 49, 9104--9106
This journal is The Royal Society of Chemistry 2013