Clickable Pyrrolysine Mimics
tonated in standard E. coli media (pH 7.4), the amino group
of (R)-8 would be chiefly protonated which would hinder its
ability to serve as a hydrogen-bond acceptor. However, at
slightly higher pH, the amine should be deprotonated and,
consequently, the readthrough efficiency of (R)-8 would be
expected to increase. Indeed, when 2 mm (R)-8 was used,
the production of full-length mCherry was higher at more
basic pH of the growth medium (Figure 5). The total
amount of the produced protein peaks at pH 8.5, with a
yield five times greater than that at pH 6.5.
Figure 3. Concentration-dependent readthrough efficiency of pyrrolysine
analogs (R)-8, (S)-8, and their equimolar mixture 8 at pH 8.5. Error bars
indicate standard deviations calculated from three independent runs.
that (R)-8 is markedly more efficient on its own than in the
presence of (S)-8. One possible explanation is that the sig-
nificantly less readily incorporable isomer (S)-8 serves none-
theless as an effective competitive inhibitor of incorporation
of (R)-8.
The observation that the d-Pra derivative (R)-8 exhibits a
much higher readthrough efficiency than its l-Pra counter-
part (S)-8 is notable. It was previously established by Poly-
carpo et al.[10] and Li et al.[7] that in amide-type analogs pos-
sessing cyclic acyl substituents at the lysine N-6, a heteroa-
tom needs to be placed within the ring at a position strictly
corresponding to that of the imine nitrogen in 1 (carbamate-
type analogs do not require an additional heteroatom).[11]
Therefore, while 2 and (R)-18 (Figure 4) are competent pyr-
rolysine mimics, (S)-18, (S)-19, and (R)-19 are not.[7,10]
Figure 5. pH-dependent readthrough efficiency of 2 mm (R)-8. Fluores-
cence intensity of cell culture grown in media at various pH was normal-
ized to that recorded at pH 6.5. Error bars indicate standard deviations
calculated from three independent runs.
Although these data appeared to support our initial hy-
pothesis, the results of other studies did not. Specifically,
readthrough efficiency experiments with other pyrrolysine
analogs, particularly 2 and 3, yielded similar pH-dependent
profiles (Figure S4 in the Supporting Information). These
latter findings suggest that the hydrogen-bonding interaction
which we invoked in our initial hypothesis (vide supra) is
presumably not the major contributor to the observed pH
effect. Instead, it is most likely that the a-amino group of
the lysine residue, which is an invariant structural feature in
all the pyrrolysine analogs tested, is responsible for the ob-
served pH effect. Another possibility is that the intracellular
concentration of the pyrrolysine analogs is also pH depen-
dent consistent with the fact that this parameter is also
known to affect transport and metabolism of amino acids in
E. coli resulting in changes to their cellular concentration.[20]
Finally, the ability of PylS to recognize its substrates could
be influenced by pH via changes in the protonation state of
key residues such as Tyr384 (the hydrogen-bond donor in
our original hypothesis). Ultimately, whatever the actual
reason, higher pH levels appear to be favorable for read-
through regardless of the structural and electronic proper-
ties of our pyrrolysine analogs.
Figure 4. Potential pyrrolysine analogs studied by Polycarpo et al. (18)[10]
and Li et al. (19).[7]
Having identified (R)-8 as a strong outperformer of 3 in
both the ease of synthesis and incorporation efficiency, we
then focused on optimizing the expression conditions by ad-
justing the pH of the E. coli medium. Our working rationale
for this study was based on the previous structural analysis
of M. mazei PylS bound to adenylated 1 and pyrophosphate
which suggested that a conserved tyrosine (Tyr384) on a
flexible loop moves into the substrate-binding pocket to
form, via its hydroxyl group, a hydrogen bond with the
imine nitrogen of 1.[18] This interaction might be important
for pyrrolysine recognition and subsequent charging onto
PylT. We speculated that, given its analogous position, the
amine in (R)-8 could participate in a similar hydrogen
bond.[19] While the imine group of 1 would be fully depro-
Following the demonstration that (R)-8 can be efficiently
incorporated into mCherry, we expanded our studies to de-
termine whether the developed protocol could be applied to
other proteins. Therefore, calmodulin (CaM),[21] a small 17-
kDa model protein widely used in biochemical and structur-
al studies, was chosen as the second target for the incorpora-
tion of (R)-8. Rattus norvegicus CaM cDNA bearing a UAG
Chem. Asian J. 2010, 5, 1765 – 1769
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