Journal of the American Chemical Society
ARTICLE
simultaneously to Gly-Phe-Gly (20), the result would be
AMPhe-Gly-Gly (22), which has an aminomethyl side chain,
an N-terminal ammonium group, and a peptide tail. One may
expect, therefore, that 22 would bind to Q7 ∼2.8 kcal/mol more
stably than 1 (the sum of the two energies). Just like the above
example, however, we find that 22 binds to Q7 5.0À5.1 kcal/mol
more stably than 20. Therefore, the aminomethyl side chain, the
N-terminal ammonium group, and the peptide tail work syner-
gistically to stabilize the binding of 22 to Q7.
thus there is a good chance that it can be metabolically
incorporated into proteins as an affinity tag in vivo.17
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details, isother-
b
1
mal titration calorimetry data, H NMR spectra and limiting
chemical shifts for the modified phenylalanine side chains, mass
spectra, and a derivation of the competitive binding analysis.
This material is available free of charge via the Internet at
Enthalpy and Entropy. Figure 6 shows a plot of the entropic
vs enthalpic contributions to the free energy of binding to Q7 for
all eight compounds in Table 1. What is immediately apparent is
the straight line fit, which was applied purposely to only the five
data points plotted as filled circles. This extraordinarily linear
(R2 > 0.999) correlation shows a consistent compensation of
enthalpic gain with entropic loss among the five compounds with
very similar affinities in the 0.21À0.46 μM range. Of the remaining
three compounds, two have lower affinity (1 and 20) and lie above
the line with less favorable enthalpy and entropy, while one has
much higher affinity (22) and lies below the line with more
favorable enthalpy and entropy. We do not claim to understand
this phenomenon, but it is interesting to observe that the two
compounds lying above the line, Phe (1) and Gly-Phe-Gly (20),
have the least number of stabilizing interactions as discussed in the
section above and in Figure 5. Similarly, Phe-Gly-Gly (19), AMPhe
(3), and Gly-AMPhe-Gly (23), which lie on the line, have one
additional stabilizing interaction. Finally, AMPhe-Gly-Gly
(22), which lies below the line, has two additional stabilizing
interactions. Collectively, the thermodynamic and spectroscopic
data presented here emphasize the importance of four groups for the
binding of Q7 to AMPhe-Gly-Gly (22): the aromatic side chain, the
side chain aminomethyl group, the peptide backbone, and the
N-terminal ammonium group. This complex provides a rare
example of cooperative interaction among multiple functional groups
to achieve extraordinary stability.
’ AUTHOR INFORMATION
Corresponding Author
’ ACKNOWLEDGMENT
We thank Frank Truong for helpful discussions. Financial
support from the National Science Foundation (CHE-0748483),
the Welch Foundation (W-1640), and the Henry Dreyfus
Teacher-Scholar Awards Program is gratefully acknowledged.
’ REFERENCES
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’ CONCLUSIONS
This is the first example of high-affinity, site-specific recogni-
tion of a peptide containing a noncanonical amino acid by a
synthetic receptor. We find that adding an aminomethyl group to
N-terminal Phe yields a 500-fold increase in binding affinity for
Q7, a Kd value of 0.95 nM, and selectivity versus other sites and
canonical residues in excess of 3.5 kcal/mol. This combination of
affinity and selectivity makes it worthwhile to consider whether
binding to N-terminal AMPhe would be selective in a proteomic
context and thus useful as an affinity tag for protein purification.
In this capacity, AMPhe should have certain advantages com-
pared to other protein tags, either genetically encoded (e.g., His,
FLAG, GST)15 or chemically introduced (e.g., biotin or bis-
(dimethylaminomethyl)ferrocene derivatives4c) because it re-
places existing Phe residues (thus requiring less genetic
modification), it is a much smaller addition to the protein, it
enables elution from the affinity matrix under a wide range of
conditions (native and denaturing) using a competitive guest,
and it does not require the introduction of other proteins such as
antibodies or streptavidin. Higher-affinity guests for Q7 exist,
including several ferrocene and adamantyl derivatives,4b,11b and
others have shown that proteinÀferrocene conjugates can be
selectively isolated from cell extracts.16 Compared to such
conjugates, AMPhe has the chief advantage that, with only two
additional heavy atoms, it still closely resembles native Phe, and
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(9) Shaikh, M.; Mohanty, J.; Singh, P. K.; Nau, W. M.; Pal, H.
Photochem. Photobiol. Sci. 2008, 7, 408–414.
(10) % quenching = (Ianalyte À Imin)/(Imax À Imin), where Ianalyte is
the intensity of 20 μM Phe derivative + 2 μM Q7 + 2 μM AO, Imax is the
intensity of 2 μM Q7 + 2 μM AO, and Imin is the intensity of 2 μM AO.
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dx.doi.org/10.1021/ja207825y |J. Am. Chem. Soc. 2011, 133, 17087–17092