Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids

Article abstract of DOI:10.1021/acschembio.5b00119

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids be

Full text of DOI:10.1021/acschembio.5b00119

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Article
Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo
Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids
John R Frost, Nicholas T. Jacob, Louis J Papa, Andrew E. Owens, and Rudi Fasan
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00119 • Publication Date (Web): 01 May 2015
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ACS Chemical Biology
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Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in
Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural
Amino Acids
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John R. Frost, Nicholas T. Jacob, Louis J. Papa, Andrew E. Owens, and Rudi Fasan*
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester NY, 14627.
KEYWORDS
Peptide macrocyclization, Cyclic peptides, Intein, Protein splicing, Unnatural amino acid.
ABSTRACT: A versatile method for orchestrating the formation of sideꢀchainꢀtoꢀtail cyclic peptides from ribosomally derived
polypeptide precursors is reported. Upon ribosomal incorporation into inteinꢀcontaining precursor proteins, designer unnatural amiꢀ
no acids bearing sideꢀchain 1,3ꢀ or 1,2ꢀaminothiol functionalities are able to promote the cyclization of a downstream target peptide
sequence via a Cꢀterminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and comꢀ
position could be generated in a pHꢀtriggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a
new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy
was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 ꢀM) from a library of cyclic peptides produced
in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles.
arbitrary sequence within a cell, thereby providing the capability
of coupling library production with a selection system or phenoꢀ
typic screen.42ꢀ48 In this regard, a viable method is the soꢀcalled
splitꢀintein mediated circular ligation of peptides and proteins
(SICLOPPS) introduced by Benkovic and coworkers,49 which
enables the intracellular formation of headꢀtoꢀtail cyclic peptides
by exploiting the trans splicing reactivity of the natural split intein
DnaE. More recently, a strategy useful for the generation in vitro
and in vivo of macrocylic peptides constrained by an interꢀsideꢀ
chain thioether bond was reported by our group.50 While these
methods provide a route to obtain headꢀtoꢀtail or sideꢀchainꢀtoꢀ
sideꢀchain cyclic peptides, complementary strategies to access
alternative peptide macrocycle architectures would be highly deꢀ
sirable. For example, neither of these approaches allows the forꢀ
mation of sideꢀchainꢀtoꢀtail cyclic peptides, a molecular topology
that is found in many bioactive peptide natural products (e.g. baciꢀ
tracin A, polymixin D).
INTRODUCTION
Macrocyclic peptides occupy a most relevant region of the chemiꢀ
cal space and have attracted increasing attention as molecular
scaffolds for addressing challenging pharmacological targets.1, 2
Important benefits arising from the cyclization of peptide seꢀ
quences include preꢀorganization into a bioactive conformation
and reduced entropic costs upon complex formation, which can
result in enhanced binding selectivity and affinity, respectively,
toward a target biomolecule.3ꢀ10 In addition, macrocyclization has
proven to be beneficial toward improving the proteolytic
stability11ꢀ13 and cell penetration ability14ꢀ16 of peptideꢀbased molꢀ
ecules. These advantageous features are well reflected by the
widespread occurrence of ring topologies amongst natural biologꢀ
ically active peptides, such as those produced by nonꢀribosomal
peptide synthetases17,
(e.g. cyclosporin, polymyxin) or those
18
belonging to the class of ribosomally synthesized and postꢀ
translationally modified peptides (RiPPs)19 (e.g., lanthipeptides20,
cyclotides21, cyanobactins22).
As part of our efforts toward the development of methꢀ
ods for the synthesis of macrocyclic organoꢀpeptide hybrids,51ꢀ54
we recently described the ability of bifunctional oxyamine/1,3ꢀ
aminoꢀthiol aryl reagents to induce the efficient cyclization of
polypeptide precursors containing a target peptide sequence
flanked by a sideꢀchain keto group and a Cꢀterminal intein moieꢀ
ty.52 Albeit two possible routes for macrocyclization are available
in this system (e.g. sideꢀchain → Cꢀend versus Cꢀend → sideꢀ
chain ligation), mechanistic studies revealed that this process is
largely driven by the intermolecular attack of the 1,3ꢀaminoꢀthiol
aryl moiety onto the inteinꢀcatalyzed thioester bond within the
polypeptide precursor.53 These mechanistic insights suggested to
us the possibility of directing the biosynthesis of sideꢀchainꢀtoꢀtail
macrocyclic peptides through the ribosomal expression of a preꢀ
cursor polypeptide in which an appropriately designed aminoꢀ
thiolꢀbased unnatural amino acid is placed upstream of an intein
(Figure 1). Given the expected rate enhancement in moving from
an intermolecular to an intramolecular setting, we envisioned this
construct could undergo a spontaneous postꢀtranslational 'selfꢀ
Because of the attractive properties of macrocyclic pepꢀ
tides, the development of methods for the preparation of this class
of compounds has attracted significant interest.23ꢀ27 Among these,
strategies that rely on the cyclization of genetically encoded pepꢀ
tide sequences offer several advantageous features, which include
the opportunity to create and evaluate large combinatorial librarꢀ
ies of these molecules by combining genetic randomization with
highꢀthroughput display/screening platforms.24ꢀ26 For example,
biopanning of phage displayed peptide libraries cyclized via disulꢀ
fide bridges28ꢀ32 or exogenous crossꢀlinking agents33ꢀ35 has enaꢀ
bled the discovery of potent inhibitors of enzymes and proteins.
Coupling in vitro translation methods with mRNA display, other
groups have successfully isolated potent macrocyclic peptide
inhibitors for a variety of other therapeutically relevant protein
targets.36ꢀ41
Despite this progress, there are currently only a few
methods that enable the production of macrocyclic peptides of
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Scheme 1. Synthetic routes for the preparation of (A) 3ꢀaminoꢀ4ꢀ
mercaptomethylꢀphenylalanine (AmmF), and (B) 3ꢀ(2ꢀmercaptoꢀ
ethyl)aminoꢀphenylalanine (MeaF).
processing' reaction through (a) capturing of the transient inteinꢀ
catalyzed thioester linkage by the sideꢀchain thiol functionality,
followed by (b) irreversible acyl shift to the neighboring amino
group to give the desired macrocyclic peptide. Here, we describe
the successful implementation of this design strategy and its funcꢀ
tionality toward enabling the ribosomal synthesis of macrocyclic
peptides both in vitro and in living cells. Furthermore, it is shown
how this approach could be successfully applied to evolve and
isolate a cyclopeptide with improved binding affinity toward a
model target protein (streptavidin).
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is capable of charging a cognate tRNA molecule with multiple
UAAs while maintaining discriminating selectivity against the
natural amino acids.56, 57 We reasoned this feature could be exꢀ
ploited to find a suitable AARS for recognition of AmmF. To this
end, we generated a panel of six previously reported AARSs deꢀ
rived from Methanocaldococcus jannaschii tyrosylꢀtRNA syntheꢀ
tase (MjTyrꢀRS) in combination with the cognate amber stop coꢀ
don suppressor tRNA (MjtRNATyrCUA). This panel included
MjTyrꢀRS variants selected for recognition of 3ꢀaminotyrosine
Figure 1. Overview of the strategy for generating sideꢀchainꢀtoꢀtail macꢀ
rocyclic peptides via cyclization of ribosomally derived, inteinꢀfused
precursor proteins by means of aminoꢀthiol unnatural amino acids. 'Nu'
refers to the nucleophilic 1,3ꢀ or 1,2ꢀaminothiol moiety.
(3AmY)58,
pꢀaminophenylalanine
(pAmF)59,
pꢀ
RESULTS AND DISCUSSION
azidophenylalanine (AzF)60, pꢀacetylphenylalanine (pAcF)61, Oꢀ
propargyltyrosine (OpgY)62, and Oꢀ(2ꢀbromoethyl)ꢀtyrosine
(O2beY)50(Figure 2A).
Synthesis of 1,3-aminothiol amino acid, AmmF. In previous
work, we recognized the peculiar ability of an oꢀaminoꢀ
mercaptomethylꢀaryl (AMA) group to undergo efficient Cꢀ
terminal ligation at GyrA intein thioesters under catalystꢀfree
conditions.53 Based on this knowledge, an amino acid incorporatꢀ
ing
this
reactive
moiety,
3ꢀaminoꢀ4ꢀmercaptomethylꢀ
phenylalanine (AmmF, Scheme 1A), was designed in order to test
its ability to mediate peptide cyclization according to the strategy
outlined in Figure 1. As outlined in Scheme 1A, gramꢀscale synꢀ
thesis of AmmF was carried out starting from commercially availꢀ
able methyl 3ꢀaminoꢀ4ꢀmethylꢀbenzoate, followed by NꢀBoc proꢀ
tection, benzylic bromination and benzylic substitution with
tritylmercaptan to afford intermediate 3. Lithium aluminum hyꢀ
dride reduction of the methyl ester group in 3, followed by mesylꢀ
ation, and substitution of the mesyl group with diethylacetamꢀ
idomalonate, yielded intermediate 5. Following deprotection, the
desired product, AmmF, was obtained in 41% overall yield over 8
steps.
Figure 2. Orthogonal aminoacylꢀtRNA synthetases for ribosomal incorpoꢀ
ration of the aminoꢀthiol amino acids. (A) Structures of the target amino
acids, AmmF and MeaF, and other UAAs mentioned in the text. (B) Relaꢀ
tive fluorescence measured in the YFP reporter assay for the panel of
AARSs in the presence of AmmF or MeaF. Data are normalized to the
highest value within each series. (C) SDSꢀPAGE analysis of purified
wildꢀtype YFP (lane 1) and YFP variants incorporating AmmF (lane 2)
and MeaF (lane 3).
Aminoacyl-tRNA synthetase for ribosomal incorporation of
AmmF. With AmmF in hand, we sought to identify an orthogonal
aminoacylꢀtRNA synthetase (AARS) / tRNA pair for the riboso
mal incorporation of this unnatural amino acid (UAA) via amberꢀ
stop codon (TAG) suppression.55 Interestingly, a number of engi
neered AARS enzymes have been shown to be 'polyspecific', that
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Figure 3. Precursor polypeptides, reaction intermediates and final macrocyclic peptide products for (A) AmmFꢀmediated and (B) MeaFꢀmediated peptide
cyclization. (A) Expression of the AmmFꢀcontaining precursor protein (a') in E. coli results in the formation of the benzothiazine adduct (b'), which upon
pHꢀinduced deprotection in vitro leads to the target macrocyclic peptide (d'). (B) Expression of the MeaFꢀcontaining precursor protein (a'') in E. coli results
in spontaneous, postꢀtranslational peptide cyclization to give the thiolactone intermediate (b'') and then the final macrolactam product (c'') via an S→N acyl
shift rearrangement.
Among these, 3AmYꢀRS was considered a particularly promising
candidate owing to the structural similarity between 3AmY and
AmmF. The relative ability of the AARSs to incorporate AmmF
in response to a TAG codon was established via a fluorescence
assay with a Yellow Fluorescent Protein (YFP) variant containing
an amber stop codon at the Nꢀterminus. In this assay, the suppresꢀ
sion 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 AmmF, respectively (Figure 2B).
Gratifyingly, the 3AmYꢀRS synthetase was found capable of inꢀ
corporating AmmF with good efficiency as compared to most of
the other AARSs. Interestingly, the AzFꢀRS synthetase exhibited
even better performance in that respect, as judged by the higher
fluorescence signal (+15%) measured in the assay. This result is
consistent with previous observations by Schultz and coworkers
regarding the ability of AzFꢀRS to recognize various meta, paraꢀ
disubstituted phenylalanine derivatives, in addition to AzF.57 Furꢀ
ther experiments were then carried out to quantitatively assess the
suppression efficiency of AzFꢀRS with AmmF. For this purpose,
YFP(AmmF) and wildꢀtype YFP were expressed under identical
conditions and purified by Niꢀ affinity chromatography. Direct
comparison of the expression yield (16 vs. 45 mg/L culture, reꢀ
spectively) indicated a suppression efficiency of 36% (Figure
2C), a value comparable to that reported for AARSs specifically
evolved for the ribosomal incorporation of other UAAs.57
would allow us to probe the formation of peptide macrocycles of
varying ring size. In addition, the Nꢀterminal chitin binding doꢀ
main (CBD) would serve as a large (~8 kDa) affinity tag to faciliꢀ
tate both the isolation of the products formed in cells and measꢀ
urement of the extent of intein cleavage via SDSꢀPAGE densiꢀ
tometry. Accordingly, the precursor proteins were produced in E.
coli cells coꢀtransformed with a pETꢀbased plasmid encoding for
the precursor protein and a pEVOL64ꢀbased plasmid for the coꢀ
expression of the AzFꢀRS/tRNACUA pair for the siteꢀselective
incorporation of AmmF.
Contrary to our expectations, only low levels of intein
cleavage (20ꢀ25%) were observed for all the constructs under the
applied expression conditions (12 hrs, 27oC), as determined by
SDSꢀPAGE analysis. These values were comparable to those
obtained with control constructs in which AmmF was substituted
for a nonꢀnucleophilic UAA (= pAcF). Moreover, no traces of the
desired macrocyclic compounds were detected after processing
the cell lysates with chitin beads.
These results were surprising, given that compounds
sharing the oꢀaminoꢀmercaptomethylꢀaryl moiety of AmmF can
induce nearly quantitative clevage of GyrAꢀfusion proteins (80ꢀ
90%) in less than 10ꢀ12 hours.53 To investigate the basis of this
unexpected lack of reactivity, the AmmFꢀcontaining constructs
were isolated in fullꢀlength form using the Cꢀterminal His tag and
then incubated with thiophenol to cleave the intein and release the
Nꢀterminal CBDꢀcontaining fragment. MALDIꢀTOF analysis of
these reactions showed the accumulation of a single product corꢀ
responding to the linear peptide with an additional mass of +26
Da for each of the constructs (Figure S1). In contrast, under idenꢀ
tical conditions, control proteins containing pAcF instead of
AmmF produced a species corresponding to the expected linear
peptide with no modifications. These results pointed at a postꢀ
translational modification occurring specifically at the level of the
unnatural amino acid in the AmmFꢀcontaining constructs. The
Characterization of AmmF-containing precursor proteins. Havꢀ
ing identified a viable AARS for AmmF incorporation, we tested
the ability of this amino acid to promote peptide macrocyclization
according to the scheme outlined in Figure 1. To this end, we
investigated a panel of six constructs encompassing target peptide
sequences of variable length (4 to 12 amino acids) framed in beꢀ
tween AmmF and the N198A variant of GyrA miniꢀintein from
Myobacterium xenopi63 (Entries 1ꢀ6, Table 1). These constructs
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observed mass difference was found to be consistent with the
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formation of a 2,4ꢀdihydroꢀbenzo[1,3]thiazine adduct (Figure 3A,
species b') resulting from a condensation/decarboxylation reacꢀ
tion of the AMA moiety in AmmF with pyruvate, an abundant
metabolite in E. coli.65 Importantly, a similar reaction, leading to a
thiazolidine adduct, was reported for polypeptides carrying an
exposed Nꢀterminal cysteine upon expression in this organism.66
To confirm the assignment, a model reaction was carried out by
incubating a smallꢀmolecule surrogate of AmmF, methyl 3ꢀ
aminoꢀ4ꢀ(mercaptomethyl)benzoate, with pyruvate under physioꢀ
logical conditions (50 mM KPi, 150 mM NaCl, pH 7.5). LCꢀMS
analysis revealed quantitative conversion of the compound to the
pyruvate condensation adduct within a short time (Figure S2).
Altogether, these experiments indicated that rapid and quantitative
intracellular modification of AmmF by pyruvate impedes the
desired in vivo peptide cyclization process. Attempts to minimize
this modification by altering the expression conditions66 failed to
result in a detectable increase of the unmodified AmmFꢀ
containing protein.
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Table 1. Precursor protein constructs investigated in this study. CBD =
Chitin Binding Domain; UAA = unnatural amino acid (i.e. AmmF or
MeaF); GyrA = Mxa GyrA intein (N198A variant).
Construct
Entry
Peptide sequence
name
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2
CBDꢀ4T
CBDꢀ5T
CBDꢀ6T
CBDꢀ8T
CBDꢀ10T
CBDꢀ12T
MGꢀ8T
CBDꢀ(UAA)TGSTꢀGyrAꢀHis6
CBDꢀ(UAA)TGSGTꢀGyrAꢀHis6
3
CBDꢀ(UAA)TGSYGTꢀGyrAꢀHis6
4
CBDꢀ(UAA)TGSAEYGTꢀGyrAꢀHis6
CBDꢀ(UAA)TGSKLAEYGTꢀGyrAꢀHis6
CBDꢀ(UAA)TGSWGKLAEYGTꢀGyrAꢀHis6
MG(UAA)TGSAEYGTꢀGyrAꢀHis6
5
6
7
8
CBDꢀ7T
Strep1
CBDꢀ(UAA)TGSAYGTꢀGyrAꢀHis6
9
MG(UAA)FTNVHPQFANAꢀGyrAꢀHis6
FLAGꢀGSSG(UAA)FTNVHPQFANAꢀGyrAꢀHis6
FLAGꢀGSSG(UAA)FTNVHPQSANAꢀGyrAꢀHis6
FLAGꢀGSSG(UAA)FTNYHPQDANAꢀGyrAꢀHis6
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12
FlagꢀStrep1
FlagꢀStrep2
FlagꢀStrep3
In vitro macrocyclization of AmmF-containing constructs. Theꢀ
se findings prompted us to explore the possibility to execute the
desired AmmFꢀmediated peptide macrocyclization upon chemical
unmasking of its modified 1,3ꢀaminoꢀthiol functionality. While
not directly applicable for intracellular synthesis of cyclic pepꢀ
tides, this approach would provide a means to trigger the forꢀ
mation of peptide macrocycles in a timeꢀcontrolled manner in
vitro. Although deprotection of cysteineꢀderived thiazolidines has
been achieved under rather harsh, protein denaturing conditions
(0.2 M methoxyamine, 6 M guanidinium chloride),67, 68 we disꢀ
covered that the AmmF dihydrobenzothiazine adduct can be readꢀ
ily converted to the free 1,3ꢀaminothiol counterpart by simple
exposure of the protein to slightly acidic pH, under which condiꢀ
tions GyrA intein remains functional. Gratifyingly, incubation of
the AmmFꢀcontaining constructs at pH 5.0 and in the presence of
sodium 2ꢀsulfanylethanesulfonate (MESNA) resulted in the forꢀ
mation of small molecular weight products with masses correꢀ
sponding to the expected CBDꢀfused peptide macrocycle in each
case, as determined by MALDIꢀTOF MS (Figure S3). Notably,
identical results could be achieved in the absence of any thiol
catalyst, resulting in the clean formation of the desired macrocyꢀ
clic lactams as the only product (Figure 4A).
Figure 4. pHꢀinduced cyclization of AmmFꢀcontaining constructs in vitro.
A) MALDIꢀTOF MS spectra of the macrocyclic peptides generated under
catalystꢀfree conditions. The calculated ("c") and observed ("o") m/z valꢀ
ues corresponding to the [M+H]+ adduct of the macrocylic product are
indicated. R' = chitin binding domain. B) Extent of intein cleavage for
constructs CBDꢀ4T(AmmF) through CBDꢀ12T(AmmF) at pH 5.0 after 24
hours as determined by SDSꢀPAGE. C) Chemical structure, extractedꢀion
chromatogram (insert), and MS/MS spectrum of the macrocyclic peptide
obtained from pHꢀinduced cyclization of the MGꢀ8T(AmmF) construct.
Calculated m/z values for the [MꢀH2O+H]+ and [Mꢀ2H2O+H]+ species are
1014.07 and 996.05, respectively.
(+57, Figure S4), demonstrating the occurrence of the desired
S→N acyl transfer rearrangement (c' → d' step, Figure 2A) to
give rise to the hydrolytically stable, sideꢀchainꢀtoꢀCꢀterminus
amide linkage. Although the extent of intein cleavage in these
reactions was not elevated (15ꢀ28%, Figure 4B), AmmFꢀ
mediated cyclization was found to be largely independent of the
Exposure of these macrocyclic products to iodoacetamꢀ
ide resulted in complete conversion to the Sꢀalkylated adduct
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target peptide length, enabling the formation of peptide macrocyꢀ
an about 16% higher efficiency than that achieved with AmmF
and AzFꢀRS (Figure 2C).
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cles spanning from 4 to 12 amino acid residues with equal effiꢀ
ciency. Suspecting that steric clashes between the CBD and intein
protein may disfavor attack of AmmF thiol group onto the downꢀ
stream thioester (Figure 3A), an 8mer construct in which the
CBD was replaced with a smaller Nꢀterminal tail (MetꢀGly) was
also tested (Entry 7, Table 1). Incubation of the corresponding
protein precursor at pH 5.0 resulted in the formation of the desired
peptide macrocycle, whose cyclic structure was further confirmed
by MS/MS analysis (Figure 4C). Importantly, the construct was
determined to undergo 64% intein cleavage (vs. 28% for the
CBDꢀfused counterpart) with the macrocycle as the only detectaꢀ
ble product, thereby demonstrating the possibility to dramatically
increase the yields of these macrocyclization reactions via the use
of shorter (or more flexible) Nꢀterminal tails.
In vivo cyclization of MeaF-containing precursor proteins. To
compare sideꢀbyꢀside the performance of MeaF vs. AmmF toward
promoting peptide macrocyclization in vivo, the same six conꢀ
structs with a 4mer to 12mer target sequence (Entries 1ꢀ6, Table
1) were expressed in E. coli in the presence of MeaF and the
O2beYꢀRSꢀbased suppressor system. Notably, SDSꢀPAGE analyꢀ
sis of these proteins revealed a significantly larger degree of intein
cleavage (75ꢀ85%, Figure 5) as compared to the related AmmFꢀ
containing counterparts (20ꢀ25%) under identical expression conꢀ
ditions. Since the two sets of constructs differ only by the sideꢀ
chain aminothiol functionality in the installed UAA, these results
strongly suggested that the ribosomally incorporated MeaF had
remained available for nucleophilic attack on the intein thioester.
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Design and synthesis of 1,2-aminothiol unnatural amino acid
for in vivo macrocycle formation. Having demonstrated the posꢀ
sibility to generate macrocyclic peptides in vitro by means of
AmmF, we next sought an alternative aminothiol UAA to enable
this process to take place in vivo. To this end, a second phenylalaꢀ
nine derivative was designed, namely 3ꢀ(2ꢀmercaptoꢀethyl)aminoꢀ
Phe (MeaF, Figure 1), which was expected to exhibit superior
performance than AmmF in that respect based on the following
arguments. First, the secondary amino group of MeaF was exꢀ
pected to disfavor condensation with pyruvate in cells, thereby
preserving the sideꢀchain aminothiol functionality required for
cyclization. Second, the more acidic and less sterically hindered
sulphydryl group in MeaF compared to that in AmmF (i.e. pKa of
~8.569 vs. ~9.570) would favor nucleophilic attack onto the downꢀ
stream intein thioester, in particular at the nearꢀneutral conditions
of the intracellular milieu. Third, the thiolactone formed during
the initial transthiosterification step was envisioned to be less
strained in the presence of MeaF vs. AmmF (b'' vs. c', Figure 3)
Finally, MeaF 1,2ꢀaminothiol moiety was expected to remain
capable of undergoing an S→N acyl shift rearrangement to yield
the desired Nꢀalkyl macrolactam, as suggested by peptide ligation
studies with other aminothiol compounds71.
Figure 5. MeaF mediated intein cleavage. A) SDSꢀPAGE analysis of the
precursor proteins CBDꢀ4T(MeaF) through CBDꢀ12T(MeaF) after expresꢀ
sion for 12 hours in E. coli. Bands corresponding to fullꢀlength protein and
spliced GyrA intein are labeled. B) The extent of intein cleavage as measꢀ
ured by gel band densitometric analysis.
To examine the outcome of these in vivo reactions, the
cleaved Nꢀterminal fragments were isolated from the cell lysates
using chitinꢀcoated resin beads. To our delight, MALDIꢀTOF MS
analysis of these samples revealed the occurrence of a macrocyꢀ
clic product ("cyc", Figure 6) corresponding to either the thioꢀ
lactone or the isobaric lactam (species b'' and c'', respectively, in
Figure 3B), as the only or largely predominant product for the
constructs with 6 to 12 amino acidꢀlong target sequences. In conꢀ
trast, the 4mer and 5mer constructs showed only MS signals conꢀ
sistent with the acyclic, hydrolyzed product (Figure S5). Altꢀ
hough this species could arise from hydrolysis of the fullꢀlength
inteinꢀcontaining precursor protein directly, the higher degree of
intein cleavage observed with the MeaFꢀcontaining vs. AmmFꢀ
containing constructs (Figure 5) suggests that it more likely deꢀ
rives from hydrolysis of the thiolactone intermediate formed after
MeaFꢀmediated transthioesterification. Importantly, no adducts
were observed for any of the constructs, indicating that the sideꢀ
chain functionality of MeaF did not react with pyruvate or other
intracellular metabolites.
As summarized in Scheme 1B, MeaF was prepared
starting from 3ꢀnitroꢀbenzyl bromide via alkylation with diethylaꢀ
cetamidomalonate and nitro group reduction by hydrogenation
with Pd/C catalyst. Reductive amination of intermediate 8 with αꢀ
chloroacetaldehyde, followed by substitution with triphenylmeꢀ
thylmercaptan resulted in the protected intermediate 10. Deprotecꢀ
tion with TFA followed by decarboxylation with concentrated
HCl resulted in the desired amino acid MeaF in 38% isolated
yield over six steps. This route could be scaled up to the gram
scale (1.5 g final product) with no significant reduction in the
overall yield.
To assess the occurrence and extent of S→N acyl shift
AARS for ribosomal incorporation of MeaF. The same approach
described above for AmmF was applied to identify a suitable
AARS for amber stop codon suppression with MeaF. Initial
screening of the panel of engineered Mj AARSs showed that both
pAmFꢀRS and AzFꢀRS can recognize and incorporate MeaF into
the reporter YFP protein (Figure 2B). At the same time, O2beYꢀ
RS emerged as the most promising enzyme for this purpose based
on the approximately 20% higher fluorescent signal in the assay
(Figure 2B). This synthetase, which carries a Ala32Gly mutation
in the OpgYꢀRS background, was engineered by our group to
accommodate tyrosine derivatives containing larger alkyl substitꢀ
uents at the para position.50 Considering the much lower incorpoꢀ
ration of MeaF obtained with OpgYꢀRS, this substitution is clearꢀ
ly beneficial also toward recognition of metaꢀsubstituted aromatic
UAAs such as MeaF. Comparison of the expression yield of this
protein (23 mg/L culture) with that of wildꢀtype YFP (45 mg/L
culture) indicated a suppression efficiency as high as 52%, that is
rearrangement in the 6mer to 12mer macrocyclic products, the
corresponding cell lysate samples were first treated with iodoaꢀ
cetamide under alkaline conditions (pH 8) prior to chitinꢀaffinity
purification. Through this procedure, the macrolactam is convertꢀ
ed to its corresponding Sꢀcarboxyamidomethyl adduct ('m', Fig-
ure S6) whereas the thiolactone is hydrolysed and alkylated, thus
appearing as the Sꢀalkylated adduct of the linear acyclic peptide
('a'). These experiments showed that the 6mer, 10mer, and 12merꢀ
based macrocycles were present exclusively or mostly (~90% for
12mer) in the thiolactone form. As an exception, the 8merꢀbased
macrocycle was found to have undergone partial rearrangement,
being present in about 40% as the desired lactam product and the
remaining 60% as thiolactone as estimated based on the intensity
of the corresponding MS signals (Figure S6C) and assuming the
two species share similar ionization properties. Collectively, these
results demonstrated the ability of MeaF to efficiently mediate the
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first step (i.e. intramolecular transthioesterification) of the enviꢀ
byꢀproduct, which would be also captured by this procedure as
determined via control experiments, indicated that MeaFꢀ
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sioned macrocyclization process within 12 hours. The partial rearꢀ
rangement of the 8merꢀbased construct also suggested that the
second step (S,N acyl shift) is comparatively much slower. This
finding is consistent with the slow kinetics of the S,N acyl transfer
observed for 1,2ꢀaminothiol moieties containing secondary
amines in the context of peptide ligation studies71.
Given the remarkable stability in vivo of the thiolactone
intermediate for the 6mer to 12mer constructs, we reasoned that
longer culture times could result in the intracellular formation of
the desired macrolactam product. Accordingly, cell cultures were
grown for 24 hours prior to isolation and analysis of the spliced
products using the procedure described above. Gratifyingly, these
experiments showed a dramatic increase in the S,N acyl transfer
rearrangement for all the constructs. Indeed, the 6merꢀbased macꢀ
rocycle was found to consist for the most part of the desired lacꢀ
tam product (~60%, Figure 6A). Even better results were
achieved with the 8mer, 10mer, and 12merꢀbased macrocycles,
which were isolated exclusively in the lactam form (Figure 6, C-
E). These results were corroborated by parallel tests in which the
peptides were isolated via chitin affinity immediately after cell
lysis under alkaline conditions (pH 8.0). Under these conditions,
MS analysis revealed a macrocycle : hydrolyzed product ratio
(corresponding to the lactam : thiolactone ratio) comparable or
identical to the 'm' : 'a' ratio obtained after incubation with iodoaꢀ
cetamide, thus further confirming the occurrence of the S,N acyl
transfer step inside the cells.
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An intriguing aspect emerging from these studies conꢀ
cerns the effect of the target peptide length on the outcome of
MeaFꢀmediation peptide cyclization in vivo. Our results indicate
that 4mer and 5mer sequences undergo MeaFꢀinduced transꢀ
thioesterification, but the resulting thiolactones are apparently
susceptible to hydrolysis before the irreversible S,N acyl transfer
process can take place. In contrast, both of these steps occur effiꢀ
ciently in the context of longer peptide sequences (6ꢀ12 amino
acids). Possibly, a higher ring strain in the thiolactone intermediꢀ
ate and/or slower kinetics for the subsequent ring contraction step
may be at the basis of the higher susceptibility of the shorter conꢀ
structs toward hydrolysis. In the future, it will be interesting to
determine whether alternative aminothiol UAAs and/or alterations
within the peptide sequence (e.g. at the 'Inteinꢀ1' site) can provide
access also to these smaller rings. Another implication of the reꢀ
sults above is that 7mer peptide sequences should also be amenaꢀ
ble to cyclization. To test this hypothesis, the construct correꢀ
sponding to Entry 8 in Table 1 was prepared and expressed. As
anticipated, this construct underwent extensive cyclization in vivo
(~75ꢀ80% intein cleavage), resulting in the formation of the deꢀ
sired CBDꢀfused peptide macrocycle as the major product
(~60:40 lactam:thiolactone ratio, Figure 6B). Altogether, these
studies demonstrated the functionality of the methodology outꢀ
lined in Figure 3B to orchestrate the spontaneous, intracellular
formation of sideꢀchainꢀtoꢀtail peptide macrocycles in E. coli.
Figure 6. MALDIꢀTOF spectra of the macrocyclization products obtained
from the MeaFꢀcontaining constructs as isolated from E. coli cells after 12
hours (left panel) and 24 hours (right panel), using pH 5.0 buffer and pH
8.0 buffer containing iodoacetamide, respectively. (A) CBDꢀ6T(MeaF);
(B) CBDꢀ7T(MeaF); (C) CBDꢀ8T(MeaF); (D) CBDꢀ10T(MeaF); and (E)
CBDꢀ12T(MeaF). See also Figures S5ꢀS6. "cyc" = macrocyclic product in
either thiolactone or lactam form; "h" = hydrolyzed product, "m" = macroꢀ
lactam (Sꢀcarboxyamidomethyl adduct), "a" = acyclic side product (Sꢀ
carboxyamidomethyl adduct), * = acetylated "a". The calculated ("c") and
observed ("o") m/z values corresponding to the [M+H]+ adducts are indiꢀ
cated and color coded.
Generation of a streptavidin-binding macrocyclic peptide in
living cells. To demonstrate the production of a bioactive macroꢀ
cyclic peptide in living cells, an additional construct (Entry 9,
Table 1) was designed based on a streptavidinꢀbinding headꢀtoꢀ
tail cyclopeptide reported by Benkovic and coworkers.72 In this
case, the precursor protein consists of a short Nꢀterminal tail
(MetꢀGly), followed by MeaF and a 11mer peptide sequence enꢀ
compassing a histidineꢀprolineꢀglutamine (HPQ) motif known to
interact with streptavidin.73 74 After expression of this construct in
E. coli, cell lysates were passed over streptavidinꢀcoated beads to
isolate any streptavidinꢀbound material. Remarkably, analysis of
the eluate revealed the presence of the desired macrocyclic pepꢀ
tide (cyclo(Strep1)) as the only product, as shown in the MS specꢀ
trum in Figure 7A. The lack of detectable amounts of the acyclic
mediated cyclization had occurred with high efficiency, that is
outcompeting hydrolysis of the fullꢀlength construct and/or of the
thiolactone intermediate. Furthermore, the precursor protein was
determined to have undergone >75% cleavage (Figure 7C), a
value comparable to that observed for target sequences of compaꢀ
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rable length in the CBDꢀfused format. Based on the expression
level of the isolated GyrA protein and extent of in vivo cyclization
variants, named FlagꢀStrep2 and FlagꢀStrep3, which exhibited >2ꢀ
1
fold higher signal in the immunoassay over that of the parent cyꢀ
clic peptide (Figure S8). Upon sequencing, FlagꢀStrep2 was
found to carry a single mutation (F22S), whereas both of the HRP
flanking positions were mutated in FlagꢀStrep3 (V18Y, F22D)
(Entries 11ꢀ12, Table 1). Interestingly, many of the other imꢀ
proved variants contain a Tyr or Phe residue in position 18 (4/10)
and a polar residue (H, D, S, R) in position 22 (6/10), thus definꢀ
ing a consensus for these sites. Efficient production in vitro of the
two best cyclic peptides, Flagꢀcyclo(Strep2) and Flagꢀ
cyclo(Strep3), confirmed that both sequences undergo MeaFꢀ
mediated cyclization, leading to the expected macrocycles as the
only product (Figure S9). These cyclic peptides, along with the
parent cyclic peptide Flagꢀcyclo(Strep1), were then tested in the
assay to measure their relative affinity for streptavidin (Figure
8C). From the resulting doseꢀresponse curve, the parent cyclic
peptide was found to bind streptavidin with an equilibrium dissoꢀ
ciation constant (KD) of 7.7 ꢀM. Importantly, both of the evolved
macrocyclic peptides showed significantly (2ꢀ to 7ꢀfold) improved
affinity for this protein, with measured KD values of 4.2 ꢀM
(Flagꢀcyclo(Strep2)) and 1.1 ꢀM (Flagꢀcyclo(Strep3). Altogether,
these studies provides a proofꢀofꢀprinciple demonstration of the
utility of the present methodology in aiding the discovery of macꢀ
rocyclic binders against a target protein of interest.
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Figure 7. Isolation of streptavidinꢀbinding peptide macrocycle from E.
coli. (A) MALDIꢀTOF spectrum and (B) chemical structure of the cyꢀ
clo(Strep1) as isolated from E. coli cells using streptavidinꢀcoated beads.
The HPQ motif is highlighted in green. (C) LCꢀMS spectrum of the Niꢀ
affinity purified fullꢀlength precursor protein ('FL') and spliced GyrA
intein (GyrA) illustrating the extent of intein cleavage occurred in vivo.
, the yield of the Strep1ꢀderived cyclopeptide was estimated to be
around 0.85 mg Lꢀ1.
Isolation of improved streptavidin-binding peptide from macro-
cyclic peptide library. To begin to explore the potential utility of
this methodology toward the discovery of macrocyclic peptides
with novel or improved function, cyclo(Strep1) was subjected to
mutagenesis in search for cyclopeptide variants with enhanced
affinity toward streptavidin. To this end, we availed of insights
from the available crystal structure of streptavidin bound to a
HPQꢀcontaining peptide isolated by phage display.75 Inspection of
this complex revealed that whereas the key HPQ motif inserts into
the biotin binding site of streptavidin, the amino acid residues
flanking this region also lie within short distance from the protein
surface (< 6 Å, Figure S7). In addition, these residues are likely
to influence the βꢀturn conformation of the HPQ sequence, thereꢀ
by affecting the affinity of the peptide ligand for the protein. Acꢀ
cordingly, a library of cyclic peptides was generated by randomizꢀ
ing the positions flanking the HPQ tripeptide in the Strep1 target
sequence (V18, F22) using the degenerate codon NDC (12 coꢀ
dons/12 amino acids). In the corresponding precursor proteins, a
FLAG peptide (sequence: DYKDDDDK) was placed upstream of
the UAA and the semiꢀrandomized target sequence in order to
obtain macrocyclic peptides with an Nꢀterminal affinity tag useful
for detection purposes. As a reference, a construct encoding for a
FLAGꢀtagged version of cyclo(Strep1) was also prepared (Entry
10, Table 1).
Figure 8. Selection of improved streptavidinꢀtargeting cyclic peptides. (A)
Schematic representation of the in vitro assay used to screen the Strep1ꢀ
derived library and measure KD for peptide binding to streptavidin. HRP:
horseradish peroxidase. (B) Results from the primary screening of the
FlagꢀStrep1(V18NDC/F22NDC) library in 96ꢀwell format. The absorbꢀ
ance values are normalized to that of the reference cyclic peptide Flagꢀ
cyclo(Strep1). (C) Streptavidin binding curves for isolated Flagꢀ
cyclo(Strep1) (KD = 7.7 ± 0.6 ꢁM), Flagꢀcyclo(Strep2) (KD = 4.2 ± 0.1
ꢁM), and Flagꢀcyclo(Strep3) (KD = 1.1 ± 0.2 ꢁM)
CONCLUSIONS
To screen the library, an ELISAꢀlike assay was impleꢀ
mented and validated using a synthetic biotinylated FLAG pepꢀ
tide. As schematically illustrated in Figure 8A, this assay relies
on capturing the streptavidinꢀbinding peptide on a streptavidinꢀ
coated plate, followed by colorimetric detection and quantificaꢀ
tion of the amount of bound peptide by means of an antiꢀFLAG
antibodyꢀhorseradish peroxidase (HRP) conjugate. After expresꢀ
sion and cell lysis in a 96ꢀwell plate format, 480 recombinants
from the FlagꢀStrep1(V18NDC/F22NDC) library were screened
using this assay. This experiment led to the identification of ten
variants displaying a significantly enhanced response (>1.5 fold)
as compared to the reference Flagꢀcyclo(Strep1) peptide (Figure
8B). Reꢀscreening of these ‘hits’ yielded two most promising
In summary, we have developed a novel and versatile methodoloꢀ
gy useful for generating peptide macrocycles from ribosomally
produced polypeptides in vitro and in vivo. This approach leverꢀ
ages the ability of two genetically encodable aminothiol amino
acids, AmmF and MeaF, to induce a sideꢀchain→Cꢀend peptide
cyclization via an inteinꢀmediated intramolecular transthioesterifiꢀ
cation followed by S,N acyl shiftꢀinduced ring contraction mechꢀ
anism. Whereas the AmmFꢀbased strategy (Figure 3A) provides a
means to generate macrocyclic peptides in vitro in a pHꢀ
controlled manner, the MeaFꢀbased strategy (Figure 3B) offers
the opportunity to achieve the spontaneous cyclization of peptide
sequences of variable length (6mer to 12mer) and composition
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directly in living bacterial cells. As such, it provides a new apꢀ
acid were included as controls. Each sample was measured in
triplicate.
proach, complementary in scope to those previously reported,49, 50
to direct the biosynthesis of cyclic peptides of completely arbiꢀ
trary sequence. Compared to SICLOPPS, for example, the present
method offers the versatility to access the desired peptide macroꢀ
cycle in a broader range of molecular arrangements (i.e. cyclic,
lariat, or Cꢀterminally fused to a carrier protein) according to the
nature of the Nꢀterminal tail. In this study, this structural feature
was leveraged to couple inꢀcell library generation with a high
throughput in vitro assay to achieve the affinity maturation of a
streptavidinꢀtargeting cyclic peptide. Beyond that, we expect this
methodology to make possible the screening of genetically encodꢀ
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8
Protein expression and purification. Proteins were expressed in
E. coli BL21(DE3) cells coꢀtransformed with the plasmid encodꢀ
ing for the biosynthetic precursor and pEVOL_AzF or
pEVOL_O2beY vectors. After overnight growth, cells were used
to inoculate M9 medium (0.4 L) supplemented with ampicillin (50
mgꢂL−1), chloramphenicol (34 mgꢂL−1), and 1ꢂ% glycerol. At an
OD600 of 0.6, protein expression was induced by adding Lꢀ
arabinose (0.05%), IPTG (0.25 mM), and AmmF or MeaF (2
mM). Cultures were grown for an additional 12ꢀ24 h at 27ꢂ°C and
harvested by centrifugation at 3,400ꢂg. Frozen cells were resusꢀ
pended in Tris buffer (50 mM; pH 7.4) containing 300 mM NaCl
and 20 mM imidazole and lysed by sonication. Protein purificaꢀ
tion by NiꢀNTA affinity chromatography was carried out using a
Tris buffer (50 mM, pH 7.4, NaCl 150 mM) containing 50 mM
and 300 mM imidazole for protein loading and elution, respecꢀ
tively. Protein samples were concentrated in potassium phosphate
buffer (50 mM, NaCl 150 mM, pH 7.5), and stored at −80ꢂ°C.
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ed cyclopeptide libraries in a wide range of other formats, which
48
include not only intracellular selection42ꢀ44,
or reporter sysꢀ
tems45, but also twoꢀhybrid76 or surface display77 systems. Whereꢀ
as the molecular size of the macrocycles currently accessible with
this method (i.e. 900ꢀ1,600 Da) seems appropriate for targeting
extended biomolecular interfaces characterizing proteinꢀprotein
interactions,28ꢀ31, 78 future studies will explore whether this strateꢀ
gy can be further evolved to provide access to smaller peptide ring
structures.
In vitro macrocyclization reactions. Reactions were carried out
incubating the AmmFꢀcontaining precursor protein (50 ꢁM) in
potassium phosphate buffer (50 mM, NaCl 150 mM, pH 5.0) in
the presence of TCEP (10 mM). For the thiolꢀcatalyzed reactions,
10 mM MESNA was also added. After 1.5 h, the pH of the soluꢀ
tion was adjusted to 5.0. Intein cleavage was monitored and quanꢀ
tified by SDSꢀPAGE and densitometry analysis of the gel bands
using ImageJ software. The low MW products (8–10 kDa) of the
reactions were analyzed by MALDIꢀTOF using a Brüker Autoflex
II mass spectrometer.
EXPERIMENTAL SECTION
Cloning and plasmid construction. Genes encoding for the preꢀ
cursor proteins were placed under an ITPGꢀinducible T7 promoter
in pET22 vectors (Novagen). Construction of the plasmids for
expression of constructs CBDꢀ4T through CBDꢀ12T was deꢀ
scribed previously.52 Construct 9 of Table 1 was prepared followꢀ
ing an identical procedure. Construct 10 of Table 1 was prepared
by stepwise assembly PCR using FLAGꢀHPQꢀGyrA_for1/3,
FLAGꢀHPQꢀGyrA_for2/3, and FLAGꢀHPQꢀGyrA_for3/3 (Table
S1) as forward primers, T7_Term_long_rev as reverse primer, and
pET22b_CBDꢀ12T as template. The PCR product (0.68 Kbp)
was cloned into the Nde I / Xho I cassette of pET22b(+) vector.
The FLAGꢀStrep1ꢀderived library was prepared through randomiꢀ
zation of position V18 and F22 in pET22b_FLAGꢀStrep1 with
NDC degenete codons (= 12 codons encoding for C, D, F, G, H, I,
L, N, R, S, V, Y). The corresponding plasmid library was preꢀ
pared by overlap extension PCR using primers HPQ_SOE(A)_for,
In vivo macrocyclization reactions. The AmmFꢀcontaining preꢀ
cursor proteins were expressed in E. coli BL21(DE3) cells (50 mL
culture) as described above. After 12 and 24 hours after induction,
cells were harvested by centrifugation (3,400ꢂg), reꢀsuspended in
800 ꢀL of potassium phosphate buffer (50 mM, NaCl 150 mM) at
either pH 5.0 or 8.0, followed by lysis via sonication. The samples
at pH 5.0 were used for detection of the thioester intermediate and
they were processed immediately. The samples at pH 8.0 were
incubated with iodoacetamide (50 mM) for 3 hours at room temꢀ
perature prior to further processing. For protein isolation, the cell
lysate samples were clarified by centrifugation (20,100 g) and
passed over chitinꢀcoated beads (New England Biolabs), the beads
were washed with the lysis buffer, and the chitinꢀbound proteins
were eluted with 70% acetonitrile in water (1 mL) followed by
MALDIꢀTOF MS analysis.
HPQ_SOE(A)_rev,
HPQ(NDC)_SOE(B)_for,
and
HPQ(NDC)_SOE(B)_rev for fragment generation and then priꢀ
mers HPQ_SOE(A)_for and T7m _Term_long_rev for amplificaꢀ
tion of the final gene (1.35 Kbp), which was then cloned into the
Xba I / Xho I cassette of pET22b(+) vector. The library was transꢀ
formed into E. coli DH5α cells and a plasmid stock was generated
by pooling > 1,000 c.f.u.
Synthesis of AmmF and MeaF. Detailed synthetic procedures
and characterization data for AmmF and MeaF as provided as
Supplemental Information.
Isolation of streptavidin binding peptide. The Strep1(MeaF)
protein construct was expressed in E. coli BL21(DE3) cells (50
mL culture) for 24 hours at 27ꢂ°C as described above. After harꢀ
vesting, cells were reꢀsuspended in 800 ꢀL of potassium phosꢀ
phate buffer (50 mM, NaCl 150 mM, 7.5), lysed via sonication,
followed by centrifugation at 20,100 g. The clarified cell lysate
was passed over streptavidin beads (Pierce) and the immobilized
peptide was eluted with 70% acetonitrile in water and analyzed by
MALDIꢀTOF MS.
Fluorescence-based assay. E. coli BL21(DE3) cells were coꢀ
transformed with a pET22_YFP(stop) plasmid50 encoding for
MetGly(amber stop)YFPꢀHis6 and a pEVOL plasmid encoding
for the appropriate aminoacylꢀtRNA synthetase. Cells were then
grown in LB media containing ampicillin (50 mg/L) and chloramꢀ
phenicol (26 mg/L) at 37°C overnight. The overnight cultures
were used to inoculate 96ꢀdeep well plates containing minimal
M9 media. At an OD600 of 0.6, cell cultures were induced by addꢀ
ing arabinose (0.06%), IPTG (0.2 mM), and either AmmF or
MeaF at a final concentration of 2 mM. After overnight growth at
27°C, the cell cultures were diluted (1:1) with phosphate buffer
Library Screening. Recombinants from the FLAGꢀ
Strep1(V18NDC/F22NDC) library were expressed in E. coli
BL21(DE3) cells in 96 deep well plates (1 mL culture) for 24
hours at 27ꢂ°C. After harvesting, cells were reꢀsuspended in 400
ꢁL of TBS (Tris 50 mM, NaCl 150 mM, 7.4), and incubated for 1
h at 37ꢂ°C in the presence of lysosyme, DNase, and MgCl2 to lyse
the cells, followed by centrifugation at 3,400 g. The clarified cell
(50 mM, 150 mM NaCl, pH 7.5) and fluorescence intensity (λex
=
514 nm; λem = 527 nm) was determined using a Tecan Infinite
1000 plate reader. Cell cultures containing no unnatural amino
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lysate (200 ꢁL) was transferred to NeutrAvidin coated 96ꢀwell
active and selective alpha(V)beta(3) integrin antagonists, J.
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plates (Pierce) and incubated for 2 h at room temperature. The
plates were washed three times with 200 ꢁL TBS + 0.5% Tween
20. Next, a solution of antiꢀFLAGꢀantibody HRP conjugate (100
ꢁL, 1:2500 dilution) was added to each well and incubated for 1
h. The plates were washed three times with 200 ꢁL TBS + 0.5%
Tween 20. 100 ꢁL of SigmaFast OPD solution was added to each
well and the absorbance at 450 nm was measured after 20
minutes. The signals were normalized to the signal corresponding
to the cells containing the FLAGꢀStrep1 cyclic peptide. The ten
most active variants (>1.5 rel. act.) were reꢀexpressed and
screened again in triplicate using the same procedure (Figure S7).
Med. Chem. 42, 3033ꢀ3040.
(6) Graciani, N. R., Tsang, K. Y., McCutchen, S. L., and Kelly,
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KD determination for streptavidin binding. The cyclic peptides
Flagꢀcyclo(Strep1), Flagꢀcyclo(Strep2), and Flagꢀcyclo(Strep3)
were obtained via cyclization of the corresponding fullꢀlength
precursor proteins in TBS in the presence of TCEP (20 mM) and
thiophenol (10 mM). The fullꢀlength precursor proteins were obꢀ
tained via expression in E. coli BL21(DE3) cells for 7 h at 22ᵒC,
followed by Niꢀaffinity purification as described above. In each
case, the macrocyclization reaction proceeded quantitatively
yielding the cyclic peptide as the only product as determined by
SDSꢀPAGE and MS (Figure S9). A solution of each of the macꢀ
rocyclic peptide (50 ꢁL) was added to NeutrAvidin coated plates
at varying concentration (0.156 ꢀ 20 ꢁM) and assayed in duplicate
as described above. KD values were calculated with SigmaPlot via
fitting of the doseꢀresponse curves using a 1:1 binding model.
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, additional MS
spectra and SDSꢀPAGE gels. This material is available free of
charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT
This work was supported in part by the National Science Foundaꢀ
tion grant CHEꢀ1112342 and in part by National Institute of
Health grant R21 CA187502. N.T.J. and L.J.P. acknowledges the
NSF REU program for financial support. MS instrumentation was
supported by the NSF grants CHEꢀ0840410 and CHEꢀ0946653.
We also thank Dr. Kestutis Bendinskas (SUNY Oswego) for
providing access to Bruker Autoflex II MALDIꢀTOF mass specꢀ
trometer.
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45
46
47
48
49
50
51
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53
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55
56
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60
80x55mm (300 x 300 DPI)
ACS Paragon Plus Environment