New charge-bearing amino acid residues that promote β-sheet secondary structure

Article abstract of DOI:10.1021/ja510265e

Proteinogenic amino acid residues that promote β-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display β-sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also bear an ionizable group in the side chain.

Full text of DOI:10.1021/ja510265e

Article
pubs.acs.org/JACS
New Charge-Bearing Amino Acid Residues That Promote β‑Sheet
Secondary Structure
Stacy J. Maynard, Aaron M. Almeida, Yasuharu Yoshimi,† and Samuel H. Gellman*
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: Proteinogenic amino acid residues that promote β-
sheet secondary structure are hydrophobic (e.g., Ile or Val) or only
moderately polar (e.g., Thr). The design of peptides intended to
display β-sheet secondary structure in water typically requires one set
of residues to ensure conformational stability and an orthogonal set,
with charged side chains, to ensure aqueous solubility and discourage
self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also
bear an ionizable group in the side chain.
INTRODUCTION
RESULTS AND DISCUSSION
Initial Design: Ether Derivative of Thr. Our initial design
hypothesis focused on derivatives of threonine in which the side
chain hydroxyl is used to form an ether linkage to a unit bearing
an ionizable group. Scheme 1 shows the synthesis of protected
■
■
β-Sheet is a very common motif within folded proteins. Small,
autonomously folding peptides that display β-sheet secondary
structure in the absence of a specific tertiary context are useful
tools for exploring intrinsic relationships between sequence and
β-sheet stability1 and for establishing characteristic spectro-
scopic signatures of this motif.2 In addition, autonomously
folding systems have been valuable for probing noncovalent
interactions involving post-translationally introduced units.3
Engineered β-sheets have been used to explore preferred modes
of intermolecular β-strand associations, phenomena that
underlie amyloid formation;4 such β-sheet designs may
ultimately lead to diagnostic or therapeutic tools for amyloid
diseases.5
Scheme 1. Synthesis of Amino Acids
Design of β-sheet-forming peptides is challenging because
the natural residues that promote this secondary structure are
hydrophobic (Ile, Val, Phe, Trp, Tyr) or only moderately polar
(Thr).6 Therefore, sequences intended to adopt β-sheet
conformations but not self-associate in aqueous solution must
be rich both in β-promoting residues and in solubility-
promoting residues, the latter usually bearing charged side
chains. The necessity of using distinct sets of residues for
conformational propensity and solubility represents a signifi-
cant design constraint. Here we explore the hypothesis that β-
sheet propensity and peripheral charge can be combined in a
single α-amino acid residue.
Side chain branching adjacent to the backbone (“β-
branching”), as in Ile, Val and Thr, is correlated with high β-
sheet propensity;7 therefore, we have synthesized and evaluated
new amino acids that contain both a β-branch point and an
ionizable group in the side chain (Figure 1).
amino acid 3 from aziridine 1, which was generated from L-
threonine via well-precedented methods.8a Vederas et al. have
shown that nucleophiles can open closely related Thr-derived
aziridines in the presence of BF3 with high stereoselectivity,8
and this approach was useful in our case. The residue derived
from 3, which we designate TO+, was readily incorporated into
synthetic peptides via Fmoc-based solid-phase synthesis. TO+
can be viewed as an analogue of Lys that contains a β-branch
point in the side chain.
We used 12-mer peptide I as a benchmark for evaluating the
β-sheet propensity of TO+ and other new residues described
below (Figure 1). The conformational behavior of I was
previously characterized via NMR.9 In aqueous solution this
peptide adopts an antiparallel two-stranded β-sheet conforma-
tion (“β-hairpin”) with a reverse turn at the DPro-Gly segment.
Received: October 6, 2014
Published: November 13, 2014
© 2014 American Chemical Society
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Figure 1. Sequences of hairpin peptides.
D-Proline was employed to promote a type II′ reverse turn,
which favors β-sheet interactions between flanking strands
composed of L-residues.10 NMR data indicate ∼68% population
of the β-hairpin conformation at 4 °C in aqueous buffer (pH
3.8).9b
Peptide II is the analogue of I in which Lys-9 has been
replaced with TO+. 2D NMR analysis suggested significant
population of the β-hairpin conformation at 4 °C in aqueous
buffer (pH 3.8).11 The NOEs observed for II are consistent
with adoption of the expected β-hairpin conformation. For
additional insight, we compared chemical shifts for α protons
(δCαH) of residues within I and II. δCαH values are sensitive to
secondary structure, with residues that participate in α-helix
displaying upfield shifts and residues that participate in β-sheet
showing downfield shifts relative to random coil values.12
Figure 2a shows ΔδCαH data, the deviation from the random
coil chemical shift at each residue, for peptides I and II. The
LPro-6 diastereomers of I and II were used to provide the
random coil δCαH values because we have previously shown
that replacing DPro with LPro abolishes β-hairpin folding in I
and comparable designs.9 For both I and II, ΔδCαH ≥ 0.2 ppm
for the segments Tyr-2 to Val-5 and Orn-8 to Ile-10, which is
consistent with the expected locations of the two β-strand
segments. The consistent negative ΔδCαH values for Leu-11
presumably arise from the proximity of the Tyr side chain to
Leu-11 CαH in the β-hairpin conformation. The near-zero
values at DPro-6 and Gly-7 are consistent with the expected
turn, and the near-zero values at Arg-1 and Gln-12 are
consistent with “fraying” at the termini.
Figure 2. Comparison of ΔδCαH values for the (A) parent, TS+, and
TO+ peptides and (B) parent and TS− peptides. All peptides were
referenced relative to unfolded controls of the same sequence. The
ΔδCαH values at or adjacent to the substitution cannot be directly
compared as the substitution changes the dynamic range. ΔδCαH
values at hydrogen bonding positions in the core of the peptide have
been shown to most accurately reflect the population of the β-
hairpin.2b
1; the key step was use of trityl thiol to open the Thr-derived
aziridine.14 Protected amino acid 5a allowed solid-phase
synthesis of peptide III, which contains TS+ in place of Lys-9
of I.
2D NMR analysis of III revealed interstrand NOEs
consistent with significant population of the expected β-hairpin
conformation in aqueous buffer at 4 °C. Resonance overlap
hindered identification of NOEs involving the Tyr-2 side chain
at this temperature, but at 10 °C such NOEs could be detected
(Figure 3). All NOEs are consistent with the expected β-sheet
structure.
ΔδCαH data for III (Figure 2a) suggest that the extent of β-
hairpin formation is higher for this peptide than for II, which
contains the TO+ residue, because the absolute value of ΔδCαH
is larger at each strand residue (positions 2−5 and 8−11) for
III than for II. Moreover, the ΔδCαH values are larger at most
strand positions for III relative to parent peptide I, which
suggests that the TS+ residue stabilizes the β-hairpin
conformation relative to Lys.
The ΔδCαH comparison between I and II suggests that the
new TO+ residue may have a slightly lower β-sheet propensity
than does Lys. This surprising conclusion emerges because the
absolute ΔδCαH values for II are smaller than those for I at
most strand residues common to the two peptides.
Redesign: Thioether Derivatives of Thr. The unexpected
behavior of TO+ led us to re-evaluate our design hypothesis,
which focused exclusively on β-branching in the side chain. The
side chain oxygen of Thr can form an H-bond with a nearby
backbone N−H, which leads to Thr backbone torsion angles
(ϕ and ψ) more compatible with the α-helical than β-sheet
secondary structure.7a,b An ether is a better H-bond acceptor
than a comparable alcohol.13a If the H-bond acceptor ability of
the ether oxygen in TO+ works against β-sheet propensity, then
we hypothesized that the desired conformational properties
should be achieved by replacing this oxygen atom with sulfur,
to generate TS+. Thioethers are much poorer H-bond acceptors
than are ethers.13 A protected α-amino acid that could be used
to incorporate TS+ residues was prepared as shown in Scheme
We explored the scope of this design strategy by evaluating
the residue TS−, an analogue of TS+ that bears an acidic rather
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Figure 3. Cross-strand NOEs observed for peptide III at 10 °C in pH
3.8 aqueous buffer.
Figure 4. Reference peptides for thermodynamic analysis. Differences
from the peptide of interest are highlighted in blue.
than a basic side chain along with a thioether-based β-branch
point. Peptide IV, prepared using 5b, is an analogue of I in
which Glu-4 is replaced by TS−. The NOEs observed at 4 °C
are consistent with the formation of the expected β hairpin.
ΔδCαH data for IV (Figure 2b) suggest that this peptide forms
a more stable β-hairpin than that formed by I. Circular
dichroism spectra of peptides I, III, and IV (phosphate buffer,
pH = 7.0) are consistent with the formation of a β hairpin and
the stability trends observed by NMR.11
Table 1. Population and Thermodynamic Analysis for the
a
Reporter Positions in Hairpins I−IV
Val 3
(%)
Val 5
(%)
Orn 8
(%)
Ile 10
(%)
ΔΔGfold
peptide
(kcal/mol)
b
I
70
62
85
93
76
77
93
96
65
63
76
84
59
51
71
77
c
c
II (TO+)
III (TS+)
IV (TS−)
+0.1 0.2
−0.5 0.4
−0.6 0.3
d
ΔδCαH-based population analysis was undertaken in order
to estimate the thermodynamic impact of replacing Lys-9 with
either TO+ or TS+ or replacing Glu-4 with TS−. Residues 3, 5,
8, and 10 occupy hydrogen bonded positions within the β-
hairpin conformation adopted by I−IV, and we previously
found that these positions are optimal “indicators” for
population analysis in I and related peptides.2b,9b Such peptides
are presumed to equilibrate rapidly between β-hairpin and
unfolded states on the NMR time scale. The ΔδCαH value
measured at each indicator residue represents a populated-
weighted average of the contributions from the fully unfolded
state and the fully folded (i.e., β-hairpin) state. For each
sequence, we use the LPro-6 diastereomer to estimate ΔδCαH
for each indicator residue in the fully unfolded state, and we use
a macrocyclized analogue with DPro−Gly turns at both ends to
estimate δCαH in the fully folded state.11 (The reference
peptides for III are shown in Figure 4.) Table 1 shows the β-
hairpin population deduced in this way at each of the four
indicator positions in peptides I−IV. The variation among the
four values for each molecule is a measure of the intrinsic
uncertainty associated with this quantification strategy, which
involves independent analysis at four distinct sites within each
peptide. Despite this uncertainty, the data support the
qualitative conclusion that replacing Lys-9 of I with TO+ (II)
has little effect on β-hairpin population, while replacing Lys-9
with TS+ (III) or Glu-4 with TS− (IV) leads to enhanced β-
hairpin population.
a
The ΔΔG was calculated for each reporter position in the hairpin and
b
c
then averaged. Data from Syud et al. (ref 9a). Replacement for Lys-9.
For II relative to III ΔΔG = −0.8 0.5 kcal/mol. Replacement for
Glu-4.
d
not cleanly distinguished given the uncertainties in Table 1,
direct comparison of III vs II shows unambiguously that TS+
enhances β-hairpin stability relative to TO+. The magnitude of
the β-sheet stabilization provided by TS+ or TS− relative to
proteinogenic residues with unbranched side chains, Lys and
Glu, respectively, is significant given that the natively folded
state of a typical globular protein, containing a few hundred
residues, is only ∼5 kcal/mol more stable than the unfolded
state.15
It should be noted that differences between ΔδCαH values
(bar heights) from Figure 2a and % folded values from Table 1
are not directly comparable. Each ΔδCαH value in Figure 2a is
determined by the difference between the chemical shift
(δCαH) for a given residue in one of three peptides (I, II or
III) and the analogous chemical shift from a reference peptide
that has L-proline in place of D-proline. The L-proline peptide
represents the fully unfolded state. The % folded values in
Table 1 are generated from an algebraic expression based on
three chemical shifts, the two mentioned above and the δCαH
value for the analogous cyclic peptide, which represents the
fully folded state. Since this last value can vary among
macrocyclic peptides with different sequences, quantitative
correlations between ΔδCαH values (Figure 2a) and % folded
values (Table 1) may not be evident at specific residues,
particularly at or adjacent to substitution positions. In addition,
another factor may prevent such quantitative correlations: the
relationship between changes in ΔδCαH and changes in %
folding is not linear. For these reasons, and to account for local
factors that can influence δCαH values at particular residues, we
Table 1 shows ΔΔGfold calculated for peptides II−IV relative
to parent I, and for III relative to II, based on the residue-
specific population. This thermodynamic analysis shows that
replacing Lys with TO+, the basic Thr derivative with an ether
linkage at the branch point, fails to enhance β-hairpin stability,
which invalidates our original design hypothesis. In contrast,
the analogous basic residue containing a thioether linkage at the
branch point, TS+, stabilizes the β-hairpin conformation.
Although the ΔΔGfold values for II vs I and for III vs I are
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Figure 5. CD data for peptides (A) I, (B) III, and (C) IV in phosphate buffer (pH 7.0) or acetate buffer (pH 3.8). CD data for peptides (D) I, (E)
III, and (F) IV in phosphate buffer at 4, 20, and 37 °C.
draw conclusions based on measurements at multiple sequence
positions.
Circular Dichroism: Effects of pH and Temperature.
Circular dichroism (CD) provides information on peptide
folding that is intrinsically of lower structural resolution than
the insights available from 2D NMR; however, CD can be very
useful for qualitative comparisons. CD data in the far-UV
region (190−250 nm) arise largely from the backbone amide
groups and therefore report on secondary structure. The far-
UV signatures of peptides I, III, and IV obtained in the buffer
used for NMR studies, 100 mM acetate buffer, pH 3.8, all
manifest a minimum at 215 nm (Figure 5), which is
characteristic of β-sheet secondary structure and therefore
consistent with NMR data for these peptides.
For each peptide, the far-UV CD spectrum obtained in the
NMR buffer is not significantly different from the spectrum
Figure 6. Sequences of hairpin peptides.
obtained in 100 mM phosphate buffer, pH 7.0 (Figure 5a−c),
which indicates that β-hairpin folding is not pH-dependent over
The ΔδCαH data indicate that replacing Lys-15 with TO+
does not lead to a significant change in β-hairpin population,
this range. This observation is important because the Glu or
TS− side chain carboxyl group is likely to be largely protonated
because the ΔδCαH values for VI are within error of those for
V at strand positions Gln-2 to Ser-7 (Figure 7). In contrast,
replacing Lys-15 with TS+ results in consistently higher ΔδCαH
values among the N-terminal strand residues (Figure 7). These
conclusions are similar to those derived from comparisons
among I-III, which also differ at a single residue (Lys vs TO+ vs
TS+). To evaluate TS− in this β-hairpin system, we evaluated
VIII, the derivative of V in which Ile-3 has been replaced by
Glu. ΔδCαH analysis indicates that this change causes a
significant decline in Δδ-hairpin population,11 which may
reflect two factors, the loss of β-branching and the loss of a
hydrophobic side chain. Replacing Glu-3 of VIII with TS−, to
generate IX, leads to an increase in β-hairpin population, as
judged by ΔδCαH data;11 however, these data indicate that the
TS− residue in IX is not as effective as the Ile residue of V in
terms of stabilizing the β-hairpin conformation. Despite the
apparently diminished β-sheet propensity of the TS− residue
relative to the more hydrophobic Ile residue, TS− represents a
at pH 3.8, but fully deprotonated at pH 7.0. Varying
temperature from 4 to 20 to 37 °C does not significantly
alter the far UV CD of I, III or IV (Figure 5d−f), which
suggests that conclusions drawn from NMR analysis at low
temperature are relevant to room or physiological temper-
atures.
Evaluation of the New Residues in a Different β-
Hairpin Context. We turned to a different β-hairpin system to
explore the generality of the behavior observed in derivatives of
peptide I for new residues TO+, TS+ and TS− (Figure 6). The
design of peptide V was based on the sequence of a β-hairpin
that occurs at the N-terminus of ubiquitin; a central DPro−Gly
segment was used to promote autonomous β-hairpin folding.
Previously reported NMR data suggest that the expected β-
hairpin conformation is significantly populated in aqueous
solution.10 We prepared analogues of V in which Lys-15 is
replaced with TO+ (VI) or TS+ (VII).
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while Thr is similar to Gly. The data clearly show that TS+ and
TS− are significantly more hydrophilic that Thr or Ile.
CONCLUSIONS
■
We have identified a new family of unnatural α-amino acid
residues featuring two properties, intrinsic conformational
propensity and side chain charge, that have proven to be
valuable for design of peptides that fold autonomously to β-
sheet secondary structure in aqueous solution. These two
properties are not paired in any proteinogenic amino acid
residue, which has made it challenging to design small peptides
that adopt β-sheet conformations but do not aggregate. High β-
sheet propensity is generally associated with β-branching in a
residue’s side chain. Our initial attempt to build charge and side
chain branching into a new residue, involving an ether linkage
at the branch point, was not successful. Thioether-based branch
points, on the other hand, lead to the desired properties. We
have illustrated this approach with two new residues, TS+ and
TS−, which feature basic and acidic side chains, respectively.
The versatility of the synthetic route will enable preparation of
many related thioether-containing α-amino acids.
Figure 7. Comparison of ΔδCαH values for the parent, TS+, and TO+
peptides. All peptides were referenced relative to unfolded controls of
the parent. The ΔδCαH values at or adjacent to the substitution
cannot be directly compared as the substitution changes the dynamic
range. The ΔδCαH values at hydrogen bonding positions in the core
of the peptide have been shown to most accurately reflect the
population of the β-hairpin.2b
ASSOCIATED CONTENT
* Supporting Information
Experimental details including NMR spectra, NMR structure
calculations, and chemical shift deviation analysis. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
useful design tool because one can match the charge provided
by Glu while enhancing the folding tendency.
Hydrophilicity Assessements. Our design strategy is
based on the assumption that new amino acid residues with a
side chain that contains both a β-branch point and an ionizable
group will be more hydrophilic than the proteinogenic β-
branched residues (Thr, Ile and Val). To test this hypothesis,
we evaluated the hydrophilicities of TS+ and TS− along with
selected proteinogenic residues, Lys, Glu, Gly, Thr and Ile,
using a previously described system.16 This method employs
the N-4-nitrobenzoyl derivatives of the amino acids. Distribu-
tion coefficients are determined (at equilibrium) between equal
volumes of octanol and aqueous buffer (100 mM phosphate,
pH 7.0). The parameter of comparison, Π, is normalized: the
logarithm of the distribution coefficient of glycine, log(DGlycine),
is subtracted from the logarithm of distribution coefficient of
the amino acid under consideration, log(Damino acid) to calculate
Π for that amino acid. Table 2 shows Π values measured for
TS+ and TS− and the selected proteinogenic residue.
AUTHOR INFORMATION
Corresponding Author
gellman@chem.wisc.edu
■
Present Address
†Department of Applied Chemistry and Biotechnology,
Graduate School of Engineering, University of Fukui, 3-9-1
Bunkyo, Fukui 910-8507, Japan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH (GM-061238). NMR
spectrometers were purchased with partial support from NIH
(Grant No. 1 S10 RR13866-01) and NSF. Y.Y. thanks the
University of Fukui, Japan, for research sabbatical support.
Table 2. Normalized Octanol/Water Distribution
Coefficients of Selected 4-Nitrobenzoyl Amino Acid
Derivatives
REFERENCES
■
(1) (a) Searle, M. S.; Ciani, B. Curr. Opin. Struct. Biol. 2004, 14, 458.
(b) Hughes, R. M.; Waters, M. L. Curr. Opin. Struct. Biol. 2006, 16,
514. (c) Gao, J. M.; Bosco, D. A.; Powers, E. T.; Kelly, J. W. Nat.
Struct. Mol. Biol. 2009, 16, 684. (d) Kier, B. L.; Shu, I.; Eidenschink, L.
A.; Andersen, N. H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10466.
(e) Freire, F.; Almeida, A. M.; Fisk, J. D.; Steinkruger, J. D.; Gellman,
S. H. Angew. Chem., Int. Ed. 2011, 50, 8735. (f) Cheng, P. N.; Pham, J.
D.; Nowick, J. S. J. Am. Chem. Soc. 2013, 135, 5477.
(2) (a) Woys, A. M.; Almeida, A. M.; Wang, L.; Chiu, C. C.;
McGovern, M.; Pablo, J. J.; Skinner, J. L.; Gellman, S. H.; Zanni, M. T.
J. Am. Chem. Soc. 2012, 134, 19118. (b) Syud, F. A.; Espinosa, J. F.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 11577.
amino acid
Π
Lys
TS+
Glu
TS−
Gly
Thr
Ile
−2.44 0.02
−1.47 0.04
−1.85 0.4
−1.75 0.2
0
0.07 0.01
1.59 0.08
(3) Riemen, A. J.; Waters, M. L. J. Am. Chem. Soc. 2010, 132, 9007.
(4) Liu, C.; Sawaya, M. R.; Cheng, P. N.; Zheng, J.; Nowick, J. S.;
Eisenberg, D. J. Am. Chem. Soc. 2011, 133, 6736.
(5) (a) Cheng, P. N.; Liu, C.; Zhao, M.; Eisenberg, D.; Nowick, J. S.
Nat. Chem. 2012, 4, 927. (b) Sievers, S. A.; Karanicolas, J.; Chang, H.
As we predicted, TS+ and TS− cluster with Lys and Glu, all
significantly preferring aqueous buffer relative to octanol. It is
interesting to note that TS− and Glu are very similar on this
scale, while TS+ is somewhat less hydrophilic than Lys. In
contrast, Ile significantly prefers octanol to aqueous buffer,
W.; Zhao, A.; Jiang, L.; Zirafi, O.; Stevens, J. T.; Munch, J.; Baker, D.;
̈
Eisenberg, D. Nature 2011, 475, 96.
16687
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Journal of the American Chemical Society
Article
(6) (a) Chou, P. Y.; Fasman, G. D. Trends Biochem. Sci. 1977, 2, 128.
(b) Smith, C. K.; Withka, J. M.; Regan, L. Biochemistry 1994, 33, 5510.
(c) Minor, D. L.; Kim, P. S. Nature 1994, 371, 264.
(7) (a) Swindells, M. B.; MacArthur, M. W.; Thornton, J. M. Nat.
Struct. Biol. 1995, 2, 596. (b) Munoz, V.; Serrano, L. Proteins: Struct.,
Funct., Genet. 1994, 20, 301. (c) Bai, Y.; Englander, S. W. Proteins:
Struct., Funct. Genet. 1994, 18, 262.
(8) (a) Liu, H.; Pattabiraman, V. R.; Vederas, J. C. Org. Lett. 2007, 9,
4211. (b) Liu, W.; Chan, A. S. H.; Liu, H.; Cochrane, S. A.; Vederas, J.
C. J. Am. Chem. 2011, 133, 14216. (c) Tarrade, A.; Dauban, P.; Dodd,
R. H. J. Org. Chem. 2003, 68, 9521.
(9) (a) Syud, F. A.; Stanger, H. E.; Gellman, S. H. J. Am. Chem. Soc.
2001, 123, 8667. (b) Stanger, H. E.; Gellman, S. H. J. Am. Chem. Soc.
1998, 120, 4236.
(10) Haque, T. S.; Gellman, S. H. J. Am. Chem. Soc. 1997, 119, 2303.
(11) See Supporting Information for details.
(12) (a) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry
1992, 31, 1647.
(13) (a) Kebarle, P. Annu. Rev. Phys. Chem. 1977, 28, 445. (b) Arnett,
E. M.; Mitchell, E. J.; Murty, T. S. S. R. J. Am. Chem. Soc. 1974, 96,
3875.
(14) (a) Liu, W.; Chan, A. S. H.; Liu, H.; Cochrane, S. A.; Vederas, J.
C. J. Am. Chem. Soc. 2011, 133, 14216. (b) Narayan, R. S.;
VanNieuwenhze, M. S. Org. Lett. 2005, 7, 2655. (c) Carrillo, A. K.;
VanNieuwenhze, M. S. Org. Lett. 2012, 14, 1034.
(15) Maxwell, K. L.; Wildes, D.; Zarrine-Afsar, A.; De Los Rios, M.
A.; Brown, A. G.; Friel, C. T.; Hedberg, L.; Horng, J.; Bona, D.; Miller,
E. J.; Vallee-Belisle, A.; Main, E. R. G.; Bemporad, F.; Qiu, L.; Teilum,
́ ́
K.; Vu, N.; Edwards, A. M.; Ruczinski, I.; Poulsen, F. M.; Kragelund, B.
B.; Michnick, S. W.; Chiti, F.; Bai, Y.; Hagen, S. J.; Serrano, L.;
Oliveberg, M.; Raleigh, D. P.; Wattung-Stafshede, P.; Radford, S. E.;
Jackson, S. E.; Sosnick, T. R.; Marqusee, S.; Davidson, A. R.; Plaxco, K.
W. Protein Sci. 2005, 14, 602.
(16) (a) Woll, M. G.; Hadley, E. B.; Mecozzi, S.; Gellman, S. H. J.
Am. Chem. Soc. 2006, 128, 15932. (b) Wilce, M. C. J.; Aguilar, M.;
Hearn, T. W. Anal. Chem. 1995, 67, 1210.
16688
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