Side-chain-to-tail cyclization of ribosomally derived peptides promoted by aryl and alkyl amino-functionalized unnatural amino acids

Article abstract of DOI:10.1039/c6ob00192k

A strategy for the production of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward promoting the formation of medium to large-sized peptide macrocycles via intein-mediated side-chain-to-C-terminus cyclization. While only partial cyclization was observed with precursor proteins containing para-amino-phenylalanine, efficient peptide macrocyclization could be achieved using O-2-aminoethyl-tyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, post-translational reduction of the azido-containing counterpart, O-2-azidoethyl-tyrosine, directly in E. coli cells. This methodology could be successfully applied for the production of a 12 mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin as compared to the acyclic counterpart (K_D: 5.1 μM vs. 22.4 μM), thus demonstrating its utility toward the creation and investigation of novel, functional macrocyclic peptides.

Full text of DOI:10.1039/c6ob00192k

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. Fasan, J. Frost,
Z. Wu, Y. Chong Lam and A. Owens, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB00192K.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Organic & Biomolecular Chemistry
Page 1 of 8Journal Name
Dynamic Article Links ►
View Article Online
DOI: 10.1039/C6OB00192K
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Side-chain-to-tail cyclization of ribosomally derived peptides promoted
by aryl and alkyl amino-functionalized unnatural amino acids
John R. Frost§, Zhijie Wu§, Yick Chong Lam§, Andrew E. Owens and Rudi Fasan*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A strategy for the production of sideꢀchainꢀtoꢀtail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two
genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward
promoting the formation of medium to largeꢀsized peptide macrocycles via inteinꢀmediated sideꢀchainꢀtoꢀCꢀterminus cyclization. While
only partial cyclization was observed with precursor proteins containing paraꢀaminoꢀphenylalanine, efficient peptide macrocyclization
10 could be achieved using Oꢀ2ꢀaminoethylꢀtyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, postꢀ
translational reduction of the azidoꢀcontaining counterpart, Oꢀ2ꢀazidoethylꢀtyrosine, directly in E. coli cells. This methodology could be
successfully applied for the production of a 12mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin
as compared to the acyclic counterpart (KD: 5.1 ꢁM vs. 22.4 ꢁM), thus demonstrating its utility toward the creation and investigation of
novel, functional macrocyclic peptides.
15
Introduction
Owing to their peculiar structural and conformational
properties, macrocyclic peptides provide attractive scaffolds for
20 targeting complex biomolecular interfaces.1ꢀ5 The occurrence of
macrocyclic backbones amongst biologically active peptides6
found in nature further motivates the increasing interest toward
this structural class.1ꢀ5 As illustrated by a growing body of
studies, macrocyclization can help overcome important
25 limitations of linear peptides such as limited proteolytic stability7ꢀ
9
and membrane permeability.10ꢀ14 The conformational
rigidification imparted by macrocyclization on the peptide
structure can also aid in preꢀorganizing the molecule into a
bioactive conformation, resulting in enhanced selectivity and
30 binding affinity for the target biomolecule.15ꢀ18
While a variety of synthetic methods have been
investigated for the synthesis of cyclic peptides,19 the ability to
cyclize ribosomally derived polypeptides can offer key
advantages toward the synthesis, combinatorial diversification,
35 and functional evaluation of collections of peptide
macrocycles.20,21 Indeed, the application of these strategies has
enabled the successful identification of cyclopeptide inhibitors
for a variety of target proteins and enzymes.22ꢀ29
Our group has reported methods to rapidly generate
40 structurally diverse Macrocyclic Organo Peptide Hybrids
(MOrPHs), via a dual ligation strategy involving arbitrary nonꢀ
peptidic linkers and ribosomally produced inteinꢀfusion proteins
bearing an unnatural amino acid equipped with a bioorthogonal
functional group.9,30ꢀ33 More recently, complementary
45 methodologies has been developed to enable the biosynthesis of
cyclic and bicyclic peptides of arbitrary sequence in living
bacterial cells.34ꢀ36 As illustrated in these and other studies,18,37
55 Scheme 1. Synthesis of macrocyclic peptides via thiolꢀcatalyzed
cyclization of biosynthetic precursor proteins containing aminoꢀ
the type of backbone connectivity and intramolecular linkage can
functionalized unnatural amino acids. UAA: Unnatural amino
play a significant role in affecting the functional properties (e.g.,
acid, GyrA: GyrA miniꢀintein from Mycobacterium xenopi. Nu1
corresponds to pꢀaminoꢀphenylalanine (pAmF) and Nu2
60 corresponds to Oꢀ2ꢀaminoethylꢀtyrosine (O2ameY).
50 protein binding affinity) of the resulting macrocyclic peptide.
Accordingly, we have been interested in exploring alternative
approaches for creating peptide macrocycles through the postꢀ
translational processing of ribosomally derived polypeptides.
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Organic & Biomolecular Chemistry
Page 2 of 8
View Article Online
DOI: 10.1039/C6OB00192K
Here, we report the development of a new strategy to
achieve this goal, which involves the sideꢀchainꢀtoꢀtail
cyclization of inteinꢀfused polypeptides by means of aminoꢀ
functionalized unnatural amino acids. We also demonstrate how
this cyclization strategy led to the development of a cyclic
peptide with improved binding affinity toward a model target
protein.
Table 1. Biosynthetic precursors investigated in this study. CBD:
Chitin Binding Domain, OpgY: Oꢀpropargylꢀtyrosine. MeaF: 3ꢀ
(2ꢀmercaptoꢀethyl)aminoꢀphenylalanine.
5
Entry
Construct Name
UAA
Target Sequence
1
2
3
4
5
6
7
8
9
CBDꢀ5mer(pAmF)
CBDꢀ6mer(pAmF)
CBDꢀ7mer(pAmF)
CBDꢀ8mer(pAmF)
CBDꢀ10mer(pAmF)
CBDꢀ10mer(OpgY)
5mer(O2ameY)
pAmF
pAmF
pAmF
pAmF
pAmF
OpgY
OameY
OameY
OameY
OameY
OpgY
TGSGT
TGSYGT
TGSEYGT
TGSAEYGT
TGSKLAEYGT
TGSKLAEYGT
TGSGT
Results and Discussion
10
Peptide macrocyclization strategy
Scheme 1 illustrates the general strategy investigated in
6mer(O2ameY)
8mer(O2ameY)
TGSYGT
the present study. This approach involves the use of
a
TGSAEYGT
FTNVHPQFANA
FTNVHPQFANA
FTNVHPQFANA
15 recombinant biosynthetic precursor in which an arbitrary target
sequence is framed between an aminoꢀfunctionalized unnatural
amino acid and a Cꢀterminal intein. In the latter, the conserved Cꢀ
terminal asparagine residue (Asn198 in GyrA intein) and cysteine
residue at the intein+1 position is mutated (N198A) and removed,
20 respectively, to allow for the formation of a transient thioester
linkage at the junction between the peptide target sequence and
intein while preventing splicing of the intein itself. We
envisioned that thiolꢀinduced cleavage of the intein moiety on
this precursor polypeptide would form a reactive Cꢀterminal
25 thioester intermediate, which would then undergo aminolysis by
action of the sideꢀchain amino group from the unnatural amino
acid upstream of the target sequence to yield the desired sideꢀ
chainꢀtoꢀtail cyclopeptide product (Scheme 1). Extending upon
our previous studies on the cyclization of inteinꢀfused
30 polypeptides with aminoꢀthiol containing amino acids,35 this
strategy would enable the formation of macrocyclic peptides
featuring alternative sideꢀchainꢀtoꢀtail connectivities, thus
broadening the repertoire of peptide macrocycles accessible via
10 Strepꢀ11mer(O2ameY)
11
12
Strepꢀ11mer(OpgY)
Strepꢀ11mer(MeaF)
MeaF
70
Entry 1). This protein construct was expressed in BL21(DE3) E.
coli cells and purified by nickelꢀaffinity chromatography using a
polyhistidine tag fused to the Cꢀterminus of the GyrA intein. The
purified protein was then incubated with either benzylmercaptan
75 or thiophenol in phosphate buffer at pH 7.0. Tris(2ꢀ
carboxyethyl)phosphine (TCEP) was also added to the reaction
mixture to maintain the thiols in the protein and reagents in the
reduced form. As shown in Figure 1, MALDIꢀTOF mass
spectrometry (MS) analysis of the reaction in the presence of
80 benzyl mercaptan showed the formation of only the acyclic
thioester intermediate ('a') together with a smaller amount of the
hydrolysis byproduct ('h'). In contrast, the reaction in the presence
of thiophenol was found to result in formation of the desired
posttranslational
35 polypeptides.
elaboration
of ribosomally produced
Para-amino-phenylalanine-mediated cyclization
The realization of the reaction scheme outlined above
40 was anticipated to require the choice of an aminoꢀfunctionalized
unnatural amino acid with suitable structural and reactivity
properties. First, it should exhibit the desired nucleophilic
reactivity in aqueous media and under conditions in which the
cyclization through other nucleophilic amino acids (e.g., lysine)
45 is not favorable. Secondly, it should be amenable to the ribosomal
incorporation into the precursor polypeptide through convenient
methods such as amber stop codon suppression38 with orthogonal
aminoacylꢀtRNA synthetases. In light of these considerations, we
selected pꢀaminoꢀphenylalanine (pAmF) (Scheme 1, Nu1) as a
50 promising candidate for mediating the peptide cyclization
process. Because of its significantly lower pKa compared to
lysine (4.5 vs. 10.5 for Lys εꢀNH2), the aryl amino group in
pAmF is expected to remain unprotonated and to possess higher
nucleophilic reactivity in aqueous buffer at nearꢀneutral pH.
55 Indeed, aniline and derivatives thereof have been successfully
applied as nucleophilic catalysts for promoting condensation
reactions under physiological conditions.39,40 In addition, an
orthogonal tRNA / tRNA amino acyl synthetase (AARS) pair was
previously made available to enable the incorporation of pAmF
60 into recombinant proteins in response to an amber stop codon.41
To investigate the feasibility of pAmFꢀmediated
85
Figure 1. pAmFꢀmediated cyclization of 5mer peptide sequence.
AꢀB) MALDIꢀTOF MS spectra of small MW products from
reaction of CBDꢀ5mer(pAmF) (100 ꢀM) with 10 mM thiol
benzylmercaptan (A) or thiophenol (B), and TCEP (10 mM) after
90 24 hours. 'm' : macrocycle, 'h': hydrolysis product. [m+H]+ calc:
8345.2, obs: 8345.4; [h+H]+ calc: 8363.2, obs: 8363.6; [a+H]+
calc: 8469.4 , obs: 8469.0 C) Schematic structures of the
macrocyclic product (m), thioester intermediate (a), and
hydrolysis (h) byproduct.
peptide cyclization, a model precursor polypeptide was initially
tested, which comprises a Nꢀterminal chitinꢀbinding domain
(CBD) followed by pAmF, a 5mer target sequence (TGSGT), and
65 the GyrA miniꢀintein from Myobacterium xenopi42 (Table 1,
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 8
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C6OB00192K
macrocyclic peptide product ('m', Figure 1), along with the linear
byproduct resulting from hydrolysis of the thiophenol thioester
('h'). Due to the presence of a large Nꢀterminal tag (CBD protein)
lysine does not mediate the cyclization process under the applied
conditions.
Although the results above supported the feasibility of
and identical target sequence, it is reasonable to assume that the 50 the general strategy outlined in Scheme 1, they also showed that
5
cyclic and acyclic species generated in these experiments have
similar ionization properties. Accordingly, the ratio of
macrocycle : hydrolysis product in the thiophenol reaction was
estimated to be approximately 1:5. Furthermore, no significant
the desired thioester aminolysis reaction mediated by pAmF does
not effectively outcompete hydrolysis of the thioester
intermediate. This phenomenon can be attributed to the limited
nucleophilicity of aryl amino group of pAmF in the context of the
changes in product distribution were observed over a 24 hour 55 present reaction. The noticeable increase in the pAmFꢀmediated
10 period.
macrocyclization efficiency observed with the longer, 7mer to
10mer target sequences (Figures 1-2) also suggests that
unfavourable conformational constraints could be another factor
contributing to the limited cyclization efficiency of the pAmFꢀ
To gain insight into the effect of the length of the target
peptide sequence on the efficiency of the macrocyclization
reaction, four additional biosynthetic precursors with 6 to 10
amino acidꢀlong target sequences were generated (Table 1, 60 containing proteins in the context of smaller rings (i.e., for 5mer
15 entries 2ꢀ5). As for the 5mer peptide, these sequences of arbitrary
composition were previously found to have no particular
propensity either toward cyclization or against it,31,32 thus
providing ideal model structures for testing the functionality of
the present method. After purification, the corresponding proteins
20 were incubated with thiophenol and TCEP as described above.
Importantly, MALDIꢀTOF MS analysis showed the formation of
the desired macrocyclic peptide for all the reactions, thus
demonstrating that pAmFꢀmediated macrocyclization can be
to 6mer target sequences).
Identification of O-2-aminoethyl-tyrosine-tRNA synthetase
65
The considerations above prompted the design of an
alternative aminoꢀfunctionalized unnatural amino acid capable of
mediating macrocyclization with higher efficiency. To this end,
we identified Oꢀ2ꢀaminoethylꢀtyrosine (O2ameY, Scheme 1) as a
promising candidate. Compared to pAmF, the sideꢀchain alkyl
achieved across target peptide sequences of variable length (5ꢀ 70 amino functionality in O2ameY was expected to be more
25 10mer) and composition (Figure 2). As observed for the 5mer
target sequence, however, these reactions also led to the
accumulation of the hydrolysis byproduct in approximately 3:1
(6mer) to 2:1 (7mer to 10mer) excess over the macrocycle. Since
sterically accessible to nucleophilic attack to the Cꢀterminal
thioester group (Scheme 1), thus favouring cyclization over
hydrolysis of the thioester intermediate. This hypothesis was also
supported by our previous observations in the context of peptide
the 10mer sequence also contains a lysine residue, this construct 75 cyclization promoted by aminoꢀthiol unnatural amino acids.35
30 also provided an opportunity to probe the chemoselectivity of the
cyclization reaction. Accordingly, a control protein construct
(Table 1, Entry 6) was prepared by incorporating Oꢀpropargylꢀ
tyrosine (OpgY)43 instead of pAmF upstream of the 10mer target
Furthermore, the estimated pKa of the amino group in O2ameY
(~9) was deemed suitable for mediating the desired cyclization
process under nearꢀneutral conditions.
Although
a
wide variety of tyrosineꢀ and
sequence. OpgY lacks a nucleophilic sideꢀchain and is thus not 80 phenylalanineꢀderivatives have been successfully incorporated
35 expected to react with the Cꢀterminal thioester. Incubation of the
CBDꢀ10mer(OpgY) construct with thiophenol resulted in the
formation of hydrolysis product (Figure S1), thus confirming that
into proteins using engineered variants of the tyrosylꢀtRNA
synthetase from Methanococcus jannaschii or the pyrrolysylꢀ
tRNA synthetase from Methanosarcina sp.,38,44,45 the
incorporation of unnatural amino acids containing polar sideꢀ
85 chains have been very challenging due to the hydrophobic nature
of the active sites in these enzymes.46 Accordingly, finding a
AARS variant for the efficient recognition and incorporation of
O2ameY into the precursor protein was expected to be very
difficult. To overcome this problem, we envisioned an alternative
90 approach that entails the incorporation of the unnatural amino
acid Oꢀ2ꢀazidoethylꢀtyrosine (O2azeY), followed by unmasking
40 Figure 2. pAmFꢀmediated cyclization of 6mer to 10mer peptide
sequences. MALDIꢀTOF MS spectra for the reactions with the
indicated precursor proteins after incubation with 10 mM
thiophenol and 10 mM TCEP (24 hours). The calculated and
observed m/z values for the macrocyclic (m) and hydrolyzed (h)
45 products are shown.
Figure 3. Screening of engineered aminoacylꢀtRNA synthetases
for ribosomal incorporation of O2azeY. The graph reports the
95 relative fluorescence measured in the YFP reporter assay for the
panel of AARSs in the presence of O2azeY, O2ameY, and no
unnatural amino acid. Data are normalized to the fluorescence
value obtained with pAcFꢀRS in the presence of pAcF.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Organic & Biomolecular Chemistry
Page 4 of 8
View Article Online
DOI: 10.1039/C6OB00192K
of the desired alkyl amino functionality via a Staudinger 40 Since pAcFꢀRS is unable to incorporate O2ameY directly
reduction of the azide group by means of the phosphineꢀbased
reducing agent (i.e., TCEP) added during the cyclization reaction.
To identify a suitable AARS for incorporation of
(Figure 3), it can be derived that the 'in vivo' conversion of
O2azeY to O2ameY must occur at the postꢀtranslational level,
that is after O2azeY is charged onto the suppressor tRNA and
incorporated into the protein by ribosomal synthesis. Partial
5
O2azeY, we screened a panel of M. jannaschii tyrosylꢀtRNA
synthetase variants previously engineered for the incorporation of 45 reduction of azidoꢀcontaining groups in living cells has been
paraꢀsubstituted tyrosine and phenylalanine derivatives such as
3ꢀaminotyrosine (3AmY)47, pꢀazidophenylalanine (AzF)48, pꢀ
aminophenylalanine (pAmF)49, Oꢀpropargyltyrosine (OpgY)43,
previously reported53 but not in the context of compounds like
OazeY.54 Importantly, our serendipitous discovery that O2azeY is
efficiently converted to O2ameY during protein expression in E.
coli cells provides a streamlined approach to obtain O2ameYꢀ
10 Oꢀ(2ꢀbromoethyl)ꢀtyrosine (O2beY)36, and pꢀacetylphenylalanine
(pAcF)50 (Figure 3). The ability of these AARSs to incorporate 50 containing recombinant proteins for the present and other
O2azeY in response to an amber stop codon was measured by
means of a Yellow Fluorescent Protein (YFP) reporter protein
containing a TAG codon within the Nꢀterminal region of the
15 protein sequence.35 In this assay, the suppression efficiency and
fidelity of each synthetase is measured based on the relative
expression levels of the reporter YFP protein in the presence and
in the absence of O2azeY, respectively. As shown in Figure 3,
both O2beYꢀRS and pAcFꢀRS were found to efficiently
20 incorporate O2azeY into the reporter YFP protein. Notably, the
applications.
O2ameY-mediated macrocyclization
55
Having acquired the capability of producing O2ameYꢀ
containing proteins in an efficient manner, we proceeded to
investigate the feasibility and functionality of the envisioned
O2ameYꢀmediated macrocyclization strategy (Scheme 1). To this
end, we expressed and isolated the model 5mer construct CBDꢀ
protein yield obtained with pAcFꢀRS in the presence of O2azeY 60 5mer(O2ameY), in which O2ameY is placed upstream of the
is comparable to that observed in the presence of pAcF, for which
this enzyme variant was selected. Despite the structural
differences between pAcF and O2azeY, this result is in line with
25 the 'polyspecificity' often exhibited by engineered AARS
target sequence TGSGT. To identify optimal conditions for
macrocyclization, this precursor protein was incubated under
reducing (TCEP) conditions in the presence of either
benzylmercaptan or thiophenol and at varying pH (7.5, 8.2, and
enzymes toward unnatural amino acids other than those involved 65 9.0), (Figure 4). The products of these reactions were analysed
in the functional selection process.51 As expected, none of the
tested AARS variants were able to efficiently incorporate
O2ameY (Figure 3), likely reflecting the incompatibility of its
30 polar sideꢀchain with the hydrophobic environment surrounding
the para position of the tyrosine substrate in the M. jannaschii
tyrosylꢀtRNA synthetase structure.52 Because of its superior
performance in the YFP assay, pAcFꢀRS was selected for
preparation of the O2azeYꢀcontaining proteins.
by MALDIꢀTOF MS and the extent of intein cleavage was
measured by SDSꢀPAGE followed by densitometric analysis of
the gel bands corresponding to fullꢀlength protein and cleaved
GyrA intein (Figures S2 and S3).35
As summarized in Figure 4A, a high degree of intein
splicing (75ꢀ90%) was observed across most of the conditions
tested, consistent with the ability of both thiol nucleophiles to
induce intein cleavage via transthioesterification. Despite the
generally higher levels of intein cleavage in the presence of
70
35
To our surprise, the purified O2azeYꢀcontaining YFP
protein did not show the expected molecular weight but rather a 75 benzyl mercaptan, in particular at pH 7.5, these reactions mainly
mass that is consistent with the quantitative reduction of the sideꢀ
chain azido group of O2azeY to the corresponding amine
derivative, to effectively yield a O2ameYꢀcontaining protein.
resulted in the formation of the acyclic thioester intermediate ('a')
along with small quantities of the hydrolysis byproduct ('h'). At
higher pH (9.0) however, the acyclic intermediate is fully
Figure 4. O2ameYꢀmediated peptide macrocyclization. A) Extent of intein splicing as measured by SDSꢀPAGE gel densitometry for
CBDꢀ5mer(O2ameY) after 24 h with benzylmercaptan (BnSH) or thiophenol (PhSH) at varying pH. b) Representative structures of 'm' ,
'h' , and 'a'. MALDIꢀTOF mass spectra of CBDꢀ5mer(O2ameY) after 24 h with c) benzyl mercaptan and d) thiophenol at pH 7.5, 8.2,
and 9.0. 'm' : macrocycle, 'h' : acyclic hydrolysis product, 'a' : acyclic thioester product.
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 8
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C6OB00192K
established that macrocyclization of the OameYꢀcontaining
30 constructs occurs most efficiently at pH 8.2 with the addition of
thiophenol as the catalyst, as these conditions provide an optimal
combination of high intein cleavage (75%) with high conversion
to the desired macrocyclic product (70ꢀ80%). These results also
clearly demonstrated the superiority of the O2ameYꢀbased
35 cyclization strategy over that based on pAmFꢀcontaining
constructs described earlier.
converted to desired macrocyclic product ('m') along with the
hydrolysis byproduct (Figure 4C) in an approximately 1:4 ratio.
In stark contrast, the thiophenolꢀcatalyzed reactions
were found to yield the desired macrocyclic peptide as the major
product across all the tested pH values (Figure 4D). As the pH is
raised from pH 7.5 to 9.0, macrocyclization becomes more
favourable over hydrolysis, as reflected by an increase of the
macrocycle ('m') : hydrolysis product ('h') ratio from 3:2 to 7:3.
Interestingly, the thiophenol thioester intermediate is not
5
To assess the value of the O2ameYꢀbased approach
toward generating cyclic peptides of low to medium MW (600ꢀ
1,000 Da), 5mer to 8mer target peptide sequences lacking a Nꢀ
40 terminal tail were investigated (Table 1, entries 7ꢀ9). Following
expression and purification, the corresponding precursor proteins
were subjected to the optimized reaction conditions (10 mM
PhSH, 10 mM TCEP, pH 8.2), followed by LCꢀMS analysis.
Notably, all of these reactions led to the desired smallꢀMW
45 peptide macrocycle as the only product, whose cyclic structure
was further corroborated by the characteristic MS/MS
fragmentation profile (Figure 5). Based on the measured extent
of intein cleavage and the lack of observation of the thioester
intermediate or hydrolysis byproduct, the macrocycle conversion
50 yield of these reactions was estimated to be around 65ꢀ80%. To
gain further insight into the kinetics of these reactions, the
cyclization of the 5mer(O2ameY) construct was monitored over
time (Figure 6). These studies releaved that the precursor protein
had undergone 50% intein cleavage after 6 hours and reached a
55 maximum of 80% cleavage after 18ꢀ24 hours (Figure S4). The
incomplete cleavage even at extended reaction times suggests that
a fraction of the precursor protein carries a nonꢀfunctional GyrA
intein, which is unable to undergo the thiolꢀinduced
transthioesterification, possibly due to partial misfolding during
60 the expression and/or purification step. In concomitance with the
cleavage of the precursor protein, the macrocyclic product was
found to progressively accumulate over time. Interestingly, no
accumulation of the thioester intermediate was observed at any of
the time points. Altogether, these results indicate that the rateꢀ
65 limiting step of the overall reaction is the thiophenolꢀinduced
transthioesterification step and that all of the precursor protein
capable of reacting with the thiol catalyst undergoes O2ameYꢀ
mediated cyclization. As shown by the plot of Figure 6, about
50% and 80% of the total macrocyclic product is formed after 4.5
70 and 12 hours, respectively.
10 observed in these reactions, even at earlier time points (data not
shown), indicating that, once formed, this species is rapidly
converted to the macrocyclic product via O2ameYꢀmediated
aminolysis or is hydrolyzed. The higher efficiency of the
macrocyclization process at higher pH can be rationalized
15 considering that a larger fraction of O2ameY sideꢀchain amine is
deprotonated at more alkaline conditions and thus become
available for nucleophilic attack to the thiophenol thioester group.
On the other hand, the better results obtained with PhSH vs.
BnSH is consistent with the higher reactivity of thiophenol
20 thioesters as compared to the benzyl mercaptan thioester
counterparts.55 On the basis of these these experiments, we
Figure 6. Time course experiment for cyclization of
5mer(OameY) construct. The graph plots the % of intein cleavage
75 (♦) and the relative amount of macrocyclic product (■), thioester
intermediate (▲), and hydrolysis byproduct (X) over time as
measured by LCꢀMS (Figure S4).
Synthesis of streptavidin-binding cyclic peptide
To demonstrate the utility of this approach toward
Figure 5. Chemical structures, LCꢀMS ion extract
25 chromatograms (inserts), and MS/MS spectra for macrocyclic
peptide products obtained via O2ameYꢀmediated cyclization of
5mer(OameY), 6mer(OameY), and 8mer(OameY) precursor
proteins.
80
generating functional peptide macrocycles,
a biosynthetic
precursor encompassing a histidineꢀprolineꢀglutamine (HPQ)
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Organic & Biomolecular Chemistry
Page 6 of 8
View Article Online
DOI: 10.1039/C6OB00192K
motif known to bind streptavidin56ꢀ58 was generated (Table 1,
entry 10). Specifically, this protein construct contained an Nꢀ
terminal FLAG affinity tag (MDYKDDDYK), followed by a
linker sequence (GSSG), and
a 11mer target sequence
5
(FTNVHPQFANA) flanked by O2ameY and GyrA. Subjecting
the precursor protein Strepꢀ11mer(O2ameY) to the standard
reaction conditions led to the production of the desired
macrocyclic peptide 1, as confirmed by LCꢀMS and MALDIꢀ
TOF MS (Figure 7A). In the reaction only a minimal fraction
10 (<5%) of the hydrolysis byproduct was observed ('h', Figure 7A),
demonstrating the efficiency of the macrocyclization reaction also
in context of a 11mer target sequence and in the presence of
alternative residues (i.e., Ala instead of Thr) at the 'Iꢀ1' position
preceding the intein. Based on the observed extent of intein
15 cleavage (63%), the macrocycle yield for this transformation was
estimated to be ~60%. In contrast, sufficient amounts of the
pAmFꢀcyclized peptide for streptavidin binding studies could not
be isolated, primarily due to inefficient cyclization in the
presence of this unnatural amino acid as observed in the context
20 of the model peptide sequences (Figure 2).
In order to evaluate the effect of cyclization on the
streptavidin binding affinity of the peptide, the control linear
peptide FLAGꢀ(OpgY)ꢀFTNVHPQFANAꢀCO2H (2) was also
prepared. The latter carries the nonꢀnucleophilic Oꢀpropargylꢀ
25 tyrosine (OpgY) in place of O2ameY and was produced by
thiophenolꢀinduced cleavage of the corresponding precursor
protein (Table 1, entry 11) followed by thioester hydrolysis under
alkaline conditions (pH 8.0). The successful isolation of this
peptide in the linear form further demonstrated the excellent
30 chemoselectivity of the O2ameYꢀmediated cyclization process, as
none of the several nucleophilic residues present in the target
sequence and FLAG tag caused cyclization of the peptide under
the applied reaction conditions.
The dissociation constants (KD) for the cyclic and linear
35 peptide were determined by means of an immunoassay, in which
the FLAGꢀtagged peptides were incubated on streptavidinꢀcoated
plates at varying concentrations between 0.05ꢀ40 ꢁM. The
amount of the streptavidinꢀbound peptide was then measured
based on the corresponding colorimetric signal (450 nm), after
40 incubation with a horseradish peroxidase (HRP) conjugated antiꢀ
FLAG antibody followed by addition of the HRP substrate oꢀ
phenylinediamine. In this assay, the O2ameYꢀcontaining
macrocycle exhibited a KD of 5.1 ꢁM, whereas the linear peptide
exhibited a KD of 22.4 ꢁM (Figure 7B). The 4ꢀfold lower KD for
45 the cyclic peptide compared to its linear counterpart clearly
evidenced the beneficial effect of the macrocyclic backbone
toward improving the binding affinity for the target protein. A
direct comparison was also made with the cyclopeptide 3 (Figure
Figure 7. Streptavidinꢀbinding peptides. A) Chemical structure,
65 LCMS ion extract chromatogram (insert), and MALDIꢀTOF MS
spectrum of macrocycle 1 obtained via cyclization of Strepꢀ
11mer(O2ameY). [m+H]+ calc: 2849.1; obs: 2849.8. BꢀC)
Streptavidin binding curves and schematic structure for O2ameYꢀ
cyclized peptide 1 (KD: 5.1 ꢁM ± 0.4), MeaFꢀcyclized peptide 3
70 (KD = 7.7 ꢁM ± 0.6), and the linear peptide 2 (KD = 22.4 ꢁM ±
1.8).
7C), which comprises an identical target sequence as
1
Conclusion
50 (FTNVHPQFANA) but features a different sideꢀchainꢀtoꢀtail
linkage as obtained via cyclization by means of 3ꢀ(2ꢀmercaptoꢀ
ethyl)aminoꢀphenylalanine (MeaF)35. In this assay, the MeaFꢀ
cyclized peptide 3 exhibited an approximately 1.5ꢀfold higher KD
(7.7 ꢁM) than the O2ameYꢀcyclized peptide 1 (Figure 7B). Since
55 the peptideꢀstreptavidin interaction is primarily driven by the
HPQ motif35 and is not expected to directly involve the
75
We have developed a new methodology to achieve the
formation of macrocyclic peptides via an intramolecular ligation
of aminoꢀfunctionalized unnatural amino acids to the Cꢀterminus
of a genetically encoded peptide. Using this approach, 5ꢀ to 11ꢀ
amino acidꢀlong peptides could be efficiently cyclized to generate
intramolecular linkage, the differential binding affinity of these 80 mediumꢀ to largeꢀmembered rings (23ꢀ44 atoms). In these
compounds nicely illustrates the subtle effects of the sideꢀchainꢀ
toꢀtail linkage in modulating the protein binding properties of
60 these macrocyclic peptides.
compounds, the Nꢀterminal tail can be readily altered to include
either a single residue or various affinity tags to facilitate the
immobilization, isolation, and/or detection of these macrocyles
for characterization purposes. Importantly, this strategy could be
85 applied to generate a cyclic peptide with improved streptavidinꢀ
binding affinity, highlighting the effect of macrocyclization and
the type of sideꢀchainꢀtoꢀtail connectivity toward modulating this
functional property.
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 8
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C6OB00192K
Another important result of this study concerns the
discovery that the azidoꢀcontaining unnatural amino acid O2azeY
is consistently and quantitatively reduced to the amineꢀcontaining
residue O2ameY in living E. coli cells. While the exact
mM) and lysed by sonication. Protein was purified from the cell
lysate by NiꢀNTA affinity chromatography using Tris buffer (50
mM; pH 7.4; NaCl 150 mM) with 20 mM imidazole for washing
and 300 mM imidazole for elution. The recovered protein was
5
mechanism of this process was not investigated, it is most likely 70 exchanged into potassium phosphate buffer (50 mM, NaCl 150
dependent upon the reducing intracellular environment provided
by the bacterial host. As exemplified by the present study, this in
vivo azide reduction process could be exploited to produce
proteins containing O2ameY as well as other aminoꢀ
mM, pH 7.4), concentrated, and stored at ꢀ80C.
Macrocyclization reactions
10 functionalized unnatural amino acids, a currently challenging task
using commonly adopted aminoacylꢀtRNA synthetases for amber
stop codon suppression. These results are also insightful in that
they provide a cautionary note about the often assumed but
clearly not universal, bioorthogonal nature of azidoꢀbased
15 functionalities in biological systems.
75
Reactions were carried out incubating the precursor
protein (100 ꢁM) in potassium phosphate buffer (50 mM, NaCl
150 mM) in the presence of TCEP (10 mM) and thiophenol or
benzyl mercaptan (10 mM). The pH of the reaction was adjusted
to the specified value, and the reaction was gently mixed for up to
80 24 hours at room temperature. Protein splicing of the CBDꢀfused
substrates was monitored and quantified by SDSꢀPAGE and
densitometric analysis of the gel bands corresponding to the fullꢀ
length protein (24ꢀ31 kDa) and spliced GyrA intein (~22 kDa)
using ImageJ software. The low MW products (2ꢀ9 kDa) were
85 analyzed by MALDIꢀTOF MS. The products of the reactions with
the biosynthetic precursors without Nꢀterminal CBD were
analysed by LCꢀMS. The extent of intein cleavage was
determined based on the deconvoluted MS spectra for the fullꢀ
length protein and spliced GyrA intein, whereas the relative
90 amount of the macrocyclic product, hydrolysis product, and
thiophenol thioester intermediate were estimated based on the
corresponding extractedꢀion chromatograms. Each reaction was
carried out at least in duplicate.
Experimental Procedures
General
20
Chemical reagents and solvents were purchased from
Sigma–Aldrich, Acros Organics, and Fluka. Silica gel
chromatography purifications were carried out by using AMD
Silica Gel 60 230–400 mesh. 1H and 13C NMR spectra were
25 recorded on Bruker Avance spectrometers by using solvent peaks
as reference. LCꢀMS analyses were performed on a Thermo
Scientific LTQ Velos ESI/ionꢀtrap mass spectrometer coupled to
an Accela UꢀHPLC. MALDIꢀTOF spectra were acquired on a
Bruker Autoflex III MALDIꢀTOF spectrometer by using a
30 stainless steel MALDI plate and sinapinic acid as matrix.
95 Fluorescence assay for AARS screening
E. coli BL21(DE3) cells were coꢀtransformed with a
pET22ꢀbased plasmid encoding for MetGly(amber stop)YFPꢀHis6
and a pEVOLꢀbased plasmid encoding for an appropriate
100 aminoacylꢀtRNA synthetase and cognate amber suppressor
tRNA. Cells were then grown in LB media containing ampicillin
(50 mg/L) and chloramphenicol (26 mg/L) at 37°C overnight.
The overnight cultures were used to inoculate 96ꢀdeep well plates
containing M9 minimal media. At an OD600 of 0.6, cell cultures
105 were induced by adding arabinose (0.06%), IPTG (0.2 mM), and
the appropriate unnatural amino acid (final concentration of 1
mM for Lꢀisomer). After overnight growth at 27°C, the cell
cultures were diluted (1:1) with phosphate buffer (50 mM, 150
mM NaCl, pH 7.5) and fluorescence intensity (λex = 514 nm; λem
110 = 527 nm) was determined using a Tecan Infinite 1000 plate
reader. Cell cultures containing no unnatural amino acid were
included as negative controls. Each sample was measured in
triplicate. The fluorescence values were normalized to that
obtained for cells expressing pAcFꢀRS in the presence of pAcF.
115
Synthesis of unnatural amino acids
Synthetic procedures and spectral data relative to the
35 preparation and characterization of O2azeY and O2ameY are
reported in the Supplementary Information. OpgY was
synthesized as described previously.32
Plasmid constructs
40
The biosynthetic precursors described in Table 1 were
expressed using pET22ꢀbased plasmids and the cloning
procedures for preparation of these constructs were reported
previously.32,36 The plasmids for expression of the amber stop
45 codon suppression system were derived from pEVOL59 and their
preparation was described previously.35,36
Protein Expression and Purification
50
Proteins were expressed in E. coli BL21(DE3) cells
cotransformed with the pET22ꢀbased plasmid for expression of
the protein precursor and the pEVOLꢀbased vector for expression
of the appropriate AARS (pAmFꢀRS, pAzFꢀRS, 3AmYꢀRS,
pAcFꢀRS, OpgYꢀRS, O2beYꢀRS). After overnight growth, cells
55 were used to inoculate M9 media supplemented with ampicillin
(50 mg Lꢀ1), chloramphenicol (34 mg Lꢀ1) and glycerol (1%). At
an OD600 of 0.6, the culture was supplemented with the
appropriate unnatural amino acid (pAmF, O2azeY, or OpgY) at a
final concentration of 2 mM and expression of the AARS was
60 induced by addition of Lꢀarabinose (0.06%). After one hour of
shaking at 30ᵒC, protein expression was induced by addition of
0.25mM isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG). Cultures
were grown for an additional 12 hours at 27ᵒC and harvested by
centrifugation at 3400 g. Cells were resuspended in Tris buffer
65 (50 mM; pH 7.4) containing NaCl (150 mM) and imidazole (20
Streptavidin binding studies
Cyclic peptide 1 was prepared according to the macrocyclization
conditions described above. Linear peptide 2 was produced by
120 thiophenolꢀinduced cleavage of the corresponding precursor
protein (Table 1, entry 11) followed by thioester hydrolysis under
alkaline conditions (pH 9.0). Following completion of the
reactions (24 hours), the pH was adjusted to 7.0 and the solution
was dialyzed against water overnight with a 500 Da cutꢀoff
125 membrane. The reaction was then filtered through a 10 kDa cutꢀ
off centrifugal membrane (Millipore) at 3600 g. The filtrate was
lyophilized and the peptide was resuspended in Tris buffer (50
mM, 150 mM NaCl, pH 7.4). A solution of each peptide (100
uL), was added to NeutrAvidinꢀcoated 96ꢀwell plates (Pierce) at
130 various concentrations (0.1 – 40 ꢀM) in triplicate. After
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

Organic & Biomolecular Chemistry
Page 8 of 8
View Article Online
DOI: 10.1039/C6OB00192K
23 Wrighton, N. C.; Farrell, F. X.; Chang, R.; Kashyap, A. K.; Barbone,
F. P.; Mulcahy, L. S.; Johnson, D. L.; Barrett, R. W.; Jolliffe, L. K.;
Dower, W. J., Science, 1996, 458.
24 Tavassoli, A.; Benkovic, S. J., Angew. Chem. Int. Ed. Engl., 2005,
44, 2760.
25 Tavassoli, A.; Lu, Q.; Gam, J.; Pan, H.; Benkovic, S. J.; Cohen, S.
N., ACS Chem. Biol., 2008, 3, 757.
26 Morimoto, J.; Hayashi, Y.; Suga, H., Angew. Chem. Int. Ed. Engl.,
2012, 51, 3423.
75 27 Heinis, C.; Rutherford, T.; Freund, S.; Winter, G., Nat. Chem. Biol.,
2009, 5, 502.
28 Kawakami, T.; Ishizawa, T.; Fujino, T.; Reid, P. C.; Suga, H.;
Murakami, H., ACS Chem Biol, 2013.
incubation at room temperature for 2 hours, the wells were then
washed three times with 200 ꢀL of Tris buffer (50 mM, 150 mM
NaCl, pH 7.4) containing 0.5% Tween 20. A solution of antiꢀ
FLAGꢀantibodyꢀHRP conjugate (100 uL, 1:2500 dilution in Tris
buffer) was added to each well and incubated for 1 hour. The
wells were washed three times with 200 ꢀL Tris buffer containing
0.5% Tween 20. Finally, 100 ꢀL of SigmaFast OPD solution was
added to each well, and the absorbance at 450 nm was measured
after 20 minutes. KD values were calculated with SigmaPlot via
70
5
10 fitting of the doseꢀresponse curves using a 1:1 binding model.
Acknowledgements
29 Baeriswyl, V.; Rapley, H.; Pollaro, L.; Stace, C.; Teufel, D.; Walker,
80
85
E.; Chen, S.; Winter, G.; Tite, J.; Heinis, C., ChemMedChem, 2012,
7, 1173.
30 Smith, J. M.; Vitali, F.; Archer, S. A.; Fasan, R., Angew. Chem. Int.
Ed. Engl., 2011, 50, 5075.
31 Satyanarayana, M.; Vitali, F.; Frost, J. R.; Fasan, R., Chem.
Commun. (Camb.), 2012, 48, 1461.
32 Frost, J. R.; Vitali, F.; Jacob, N. T.; Brown, M. D.; Fasan, R.,
Chembiochem, 2013, 14, 147.
33 Smith, J. M.; Hill, N. C.; Krasniak, P. J.; Fasan, R., Org. Biomol.
Chem. , 2014, 12, 1135.
90 34 Bionda, N.; Fasan, R., Chembiochem, 2015, 16, 2011.
35 Frost, J. R.; Jacob, N. T.; Papa, L. J.; Owens, A. E.; Fasan, R., ACS
Chem. Biol., 2015, 10, 1805.
This work was supported by the U.S. National Institutes of Health
15 (grant R21 CA187502). MS instrumentation was supported by
the U.S. National Science Foundation (grants CHEꢀ0840410 and
CHEꢀ0946653).
Notes and references
*
Department of Chemistry, University of Rochester, Rochester, NY
20
14627, USA. E-mail: rfasan@ur.rochester.edu
§ These authors contributed equally to this work.
36 Bionda, N.; Cryan, A. L.; Fasan, R., ACS Chem. Biol., 2014, 9, 2008.
37 Chen, S.; MoralesꢀSanfrutos, J.; Angelini, A.; Cutting, B.; Heinis, C.,
†
Electronic Supplementary Information (ESI) available: Synthetic
procedures and spectral data for O2azeY and O2ameY. See
DOI: 10.1039/b000000x/
95
Chembiochem, 2012, 13, 1032.
25
30
35
40
45
38 Liu, C. C.; Schultz, P. G., Annu. Rev. Biochem., 2010, 79, 413.
39 Dirksen, A.; Hackeng, T. M.; Dawson, P. E., Angew. Chem. Int. Ed.
Engl., 2006, 45, 7581.
1
2
3
Marsault, E.; Peterson, M. L., J. Med. Chem. , 2011, 54, 1961.
Cardote, T. A.; Ciulli, A., ChemMedChem, 2015.
Villar, E. A.; Beglov, D.; Chennamadhavuni, S.; Porco, J. A., Jr.;
Kozakov, D.; Vajda, S.; Whitty, A., Nat. Chem. Biol., 2014, 10, 723.
Bionda, N.; Fasan, R., 2015, 431.
40 Crisalli, P.; Kool, E. T., J. Org. Chem., 2013, 78, 1184.
100 41 Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A.
B.; King, D. S.; Horn, D. M.; Schultz, P. G., J. Am. Chem. Soc., 2003,
125, 935.
42 Telenti, A.; Southworth, M.; Alcaide, F.; Daugelat, S.; Jacobs, W.
R.; Perler, F. B., J. Bacteriol., 1997, 179, 6378.
105 43 Deiters, A.; Schultz, P. G., Bioorg. Med. Chem. Lett., 2005, 15,
1521.
4
5
6
7
Yudin, A. K., Chem. Sci., 2015, 6, 30.
Nolan, E. M.; Walsh, C. T., Chembiochem, 2009, 10, 34.
Fairlie, D. P.; Tyndall, J. D.; Reid, R. C.; Wong, A. K.; Abbenante,
G.; Scanlon, M. J.; March, D. R.; Bergman, D. A.; Chai, C. L.;
Burkett, B. A., J. Med. Chem., 2000, 43, 1271.
Wang, D.; Liao, W.; Arora, P. S., Angew. Chem. Int. Ed. Engl., 2005,
44, 6525.
44 Lang, K.; Chin, J. W., Chem. Rev., 2014, 114, 4764.
45 Dumas, A.; Lercher, L.; Spicer, C. D.; Davis, B. G., Chem. Sci.,
2015, 6, 50.
8
9
Smith, J. M.; Frost, J. R.; Fasan, R., Chem. Commun. (Camb.), 2014,
50, 5027.
110 46 Nguyen, D. P.; Elliott, T.; Holt, M.; Muir, T. W.; Chin, J. W., J. Am.
Chem. Soc., 2011, 133, 11418.
10 Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.;
Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J., Science,
2004, 305, 1466.
11 Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lokey, R. S.;
Jacobson, M. P., J. Am. Chem. Soc., 2006, 128, 14073.
12 Rezai, T.; Yu, B.; Millhauser, G. L.; Jacobson, M. P.; Lokey, R. S.,
J. Am. Chem. Soc., 2006, 128, 2510.
13 Bock, J. E.; Gavenonis, J.; Kritzer, J. A., ACS Chem. Biol., 2013, 8,
488.
50 14 Qian, Z.; Liu, T.; Liu, Y. Y.; Briesewitz, R.; Barrios, A. M.; Jhiang,
S. M.; Pei, D., ACS Chem. Biol., 2013, 8, 423.
15 Rizo, J.; Gierasch, L. M., Annu. Rev. Biochem., 1992, 61, 387.
16 Dias, R. L.; Fasan, R.; Moehle, K.; Renard, A.; Obrecht, D.;
Robinson, J. A., J. Am. Chem. Soc., 2006, 128, 2726.
55 17 Diana, D.; Basile, A.; De Rosa, L.; Di Stasi, R.; Auriemma, S.; Arra,
C.; Pedone, C.; Turco, M. C.; Fattorusso, R.; D'Andrea, L. D., J. Biol.
Chem., 2011, 286, 41680.
18 Quartararo, J. S.; Wu, P.; Kritzer, J. A., Chembiochem, 2012, 13,
1490.
60 19 White, C. J.; Yudin, A. K., Nat. Chem., 2011, 3, 509.
20 Smith, J. M.; Frost, J. R.; Fasan, R., J. Org. Chem., 2013, 78, 3525.
21 Frost, J. R.; Smith, J. M.; Fasan, R., Curr. Opin. Struct. Biol., 2013,
23, 571.
47 Seyedsayamdost, M. R.; Xie, J.; Chan, C. T.; Schultz, P. G.; Stubbe,
J., J. Am. Chem. Soc., 2007, 129, 15060.
48 Chin, J. W.; Santoro, S. W.; Martin, A. B.; King, D. S.; Wang, L.;
Schultz, P. G., J. Am. Chem. Soc., 2002, 124, 9026.
49 Santoro, S. W.; Wang, L.; Herberich, B.; King, D. S.; Schultz, P. G.,
Nat. Biotechnol., 2002, 20, 1044.
50 Wang, L.; Zhang, Z.; Brock, A.; Schultz, P. G., Proc. Natl. Acad.
Sci. USA, 2003, 100, 56.
115
120 51 Young, D. D.; Young, T. S.; Jahnz, M.; Ahmad, I.; Spraggon, G.;
Schultz, P. G., Biochemistry, 2011, 50, 1894.
52 Kobayashi, T.; Nureki, O.; Ishitani, R.; Yaremchuk, A.; Tukalo, M.;
Cusack, S.; Sakamoto, K.; Yokoyama, S., Nat. Struct. Biol., 2003, 10,
425.
125 53 Chatterjee, A.; Sun, S. B.; Furman, J. L.; Xiao, H.; Schultz, P. G.,
Biochemistry, 2013, 52, 1828.
54 Tuley, A.; Wang, Y. S.; Fang, X.; Kurra, Y.; Rezenom, Y. H.; Liu,
W. R., Chem. Commun. (Camb.), 2014, 50, 2673.
55 Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H., Science,
130
135
1994, 266, 776.
56 Bayer, E. A.; BenꢀHur, H.; Wilchek, M., Methods Enzymol., 1990,
184, 80.
57 Katz, B. A., Biochemistry, 1995, 34, 15421.
58 Giebel, L. B.; Cass, R. T.; Milligan, D. L.; Young, D. C.; Arze, R.;
Johnson, C. R., Biochemistry, 1995, 34, 15430.
59 Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G., J. Mol. Biol.,
2010, 395, 361.
22 O'Neil, K. T.; Hoess, R. H.; Jackson, S. A.; Ramachandran, N. S.;
65
Mousa, S. A.; DeGrado, W. F., Proteins, 1992, 14, 509.
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]