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. 2011 Jul 20;22(7):1402-9.
doi: 10.1021/bc200131k. Epub 2011 Jun 7.

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

D Rajasekhar Reddy  1 , Laura E Pedró Rosa, Lawrence W Miller
Affiliations

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

D Rajasekhar Reddy et al. Bioconjug Chem. .

Abstract

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.

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Figures

Figure 1
Figure 1
Variants of carbostyril 124-polyaminocarboxylates and their modes of attachment to biomolecules.
Figure 2
Figure 2
UV absorption spectra of representative trimethoprim-polyaminocarboxylate Eu3+ complexes and the reference compound (DTPA-cs124) described in this study.
Figure 3
Figure 3
Normalized emission spectra (λex = 330 nm) of Eu3+ complexes of (A) DTPA-cs124CF3− TMP (6); (B) TTHA-cs124CF3− TMP (7); (C) cs124-TTHA-TMP (10); and (D) Tb3+ complex of 10.
Figure 4
Figure 4
Relative emission intensity over time in the presence of EDTA for trimethoprim-polyaminocarboxylate conjugates and DTPA-cs124. (A) Eu3+ complexes. (B) Tb3+ complexes.
Figure 5
Figure 5
Intramolecular, luminescence resonance energy transfer (LRET) between eDHFR-bound Tb3+ complexes of TTHA-cs124-TMP (9) or cs124-TTHA-TMP (10) and GFP. Increasing concentrations of either purified eDHFR-GFP or a bacterial lysate containing the protein were titrated against a constant concentration (20 nM) of each compound. Sensitized GFP emission (520 nm) and Tb3+ emission (615 nm) was detected after a time delay of 100 μs, upon pulsed excitation with near-UV light (~340 nm). The y-axis represents the percent change in the 520/615 emission ratio. Lines represent nonlinear least squares fit to the data.
Scheme 1
Scheme 1
Synthesis of TMP conjugated to (A) cs124-CF3 (3) and (B) cs124 (5).
Scheme 2
Scheme 2
Synthesis of sensitized TMP-polyaminocarboxylate conjugates.
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References

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