Modular assembly of macrocyclic organo-peptide hybrids using synthetic and genetically encoded precursors

Article abstract of DOI:10.1002/anie.201101331

Mix, click, cyclize: Conformationally constrained organo-peptide hybrids can be constructed by a tandem chemoselective reaction between a synthetic molecule and a recombinant protein. Diverse macrocyclic structures were obtained in cyclic, lariat, and protein-tethered configurations by varying the nature of the synthetic and biosynthetic precursors. Copyright

Full text of DOI:10.1002/anie.201101331

DOI: 10.1002/anie.201101331
Protein Chemistry
Modular Assembly of Macrocyclic Organo–Peptide Hybrids Using
Synthetic and Genetically Encoded Precursors**
Jessica M. Smith, Francesca Vitali, Steven A. Archer, and Rudi Fasan*
Macrocyclic peptides and peptide-containing molecules are
attractive molecular scaffolds for the development of bioac-
tive compounds to modulate biomolecular interactions. These
structures combine a high degree of functional complexity
with restricted conformational flexibility, which make them
well-suited to achieve selective and tight binding to extended
biomolecular interfaces, such as those mediating protein–
protein and protein–nucleic acid complex formation.[1] Com-
pared to linear peptides, conformationally constrained pep-
tide-based ligands often exhibit higher proteolytic stability,[2]
enhanced cell permeability,[3] and higher affinity towards the
target biomolecule,[4] which render them valuable as probes
and potential pharmacological agents. Indeed, many cyclic
and lariat peptides isolated from natural sources[5] exhibit
potent biological activities and have provided a source of
viable drugs.[1d]
the size and composition of which can be readily diversified
by varying the nature of the synthetic and biosynthetic
precursors (Scheme 1).
Our approach takes advantage of the reactivity of intein
proteins[12] and the opportunity to introduce bioorthogonal
functionalities into proteins by amber stop codon suppres-
sion.[13] We envisioned that incorporating an alkyne-bearing
non-natural amino acid within the N-terminal portion of an
intein-fused polypeptide would yield a recombinant protein
carrying two functional groups with orthogonal reactivity,
namely the alkyne moiety and the thioester bond transiently
formed at the junction with the intein by reversible N!S acyl
transfer. A tandem chemoselective reaction could thus be
exploited to mediate coupling of this biosynthetic precursor
(BP) to an azide/hydrazide-containing synthetic precursor
(SP) and promote the formation of an organo–peptide
macrocycle (Scheme 1).
To test our design, we first constructed a plasmid
(pBP_MG6) encoding for a 6mer target sequence (TS6:
TGSYGT) preceded by Met, Gly, and the amber stop codon
TAG and fused at the C terminus to the N-terminal cysteine
of intein GyrA from Mycobacterium xenopi.[14] This construct
was expressed in E. coli in the presence of O-propargyltyr-
osine (OpgY) and a previously described mutant tRNACUA
(MjtRNACUA)/tyrosyl-tRNA synthetase (MjTyrRS) pair[15]
encoded by a second vector (pEVOL[16]). The latter allow
the site-selective incorporation of OpgY at the N-terminal
end of the target sequence in the biosynthetic precursor by
stop codon suppression. The resulting protein, called MG6,
was purified by nickel-affinity chromatography and its
identity confirmed by MALDI-TOF (Figure 1a). To test the
macrocyclization reaction, the bifunctional synthetic precur-
sor 1 was synthesized and coupled to MG6 by CuI-catalyzed
azide–alkyne 1,3-dipolar cycloaddition[17] (CuAAC; 20 min)
followed by removal of the copper catalyst and excess 1 by
fast buffer exchange (2 min). Formation of the MG6-1 adduct
occurred quantitatively and was followed by complete splic-
ing of the GyrA intein after 16 h as indicated by MALDI-
TOF analysis (Figure 1a). This process was accompanied by
the accumulation of a product with molecular mass (m/z
1016.3) corresponding to the desired organo–peptide macro-
cycle 7, as revealed by LC-MS (Figure 1b). Along with the
major macrocyclic product, the formation of a small amount
(ca. 20%) of the acyclic peptide H2N-G(OpgY-1)TGSYGT-
COOH (8; m/z 1034.3) was also observed, indicating that
hydrolysis of the MG6-1 adduct competes to a minor extent
with the macrocyclization process. The cyclic backbone of the
predominant product (7) was further evidenced by MS/MS
analysis (Figure 1c), which showed few fragments as a result
of multiple ring-opening pathways leading to acylium ions of
Both biosynthetic[6] and synthetic[7] methods have been
implemented to afford peptides in cyclic or conformationally
constrained configurations. Genetic encoding offers the
unrivalled advantage that vast molecular libraries (108–1010)
can be rapidly created by combinatorial mutagenesis and
readily explored using genetic selection or ultrahigh-through-
put screening methods.[6,8] However, the pool of building
blocks available for construction of biological peptide libra-
ries remains restricted, limiting the degree of ligand diversity
achievable through these approaches. In contrast, synthetic
methods can draw upon a much broader spectrum of
precursor structures, including non-natural amino acids,[9]
peptoids,[10] and amino acid unrelated scaffolds,[11] which can
be exploited to confer improved or novel conformational and
target-binding properties to peptide-based ligands.
Integrating the advantages of biological and synthetic
approaches would open unprecedented opportunities for
ligand diversification and molecular discovery. Towards this
goal, we have developed a method that allows the embedding
of non-proteogenic synthetic moieties into genetically en-
coded peptidic frameworks. This strategy enables the modular
assembly of macrocyclic organo–peptide hybrids (MOrPHs),
[*] J. M. Smith,[+] Dr. F. Vitali,[+] S. A. Archer, Prof. Dr. R. Fasan
Department of Chemistry, RC Box 270216, University of Rochester
Rochester, NY 14627 (USA)
E-mail: fasan@chem.rochester.edu
[+] These authors contributed equally to this work.
[**] This work was supported by startup funds from the University of
Rochester and a NSF Graduate Fellowship to J.M.S. The authors
thank Prof. Peter Schultz for kindly providing the pEVOL vector
encoding for the mutant MjtRNA and MjTyrRS.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201101331.
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fusion protein (ca. 15%, 16 h), the latter deriving from
background hydrolysis of the protein as indicated by obser-
vation of the linear peptide H2N-G(OpgY)TGSYGT-COOH
by LC-MS (Supporting Information, Figure S1). The reaction
was then carried out using an analogue of 1 (compound 2),
which carries a methyl ester in place of the hydrazide. After
coupling 2 to MG6, only background splicing of the MG6-2
adduct (ca. 20%, 16 h) and accumulation of the hydrolysis
product H2N-G(OpgY-2)TGSYGT-COOH were observed
(Supporting Information, Figure S2), which confirmed the
direct involvement of the hydrazide in 1 in macrocyclization.
Finally, the regioselectivity of the CuAAC was investigated by
coupling 1 (and the other SPs described later) to N-Boc-
protected O-propargyltyrosine methyl ester under identical
conditions used for MOrPH synthesis (50 mm KPi, pH 7.5).
This reaction afforded the disubstituted 1,4-triazole product
1
as single regioisomer, as determined by H NMR and NOE
experiments (Supporting Information), confirming the excel-
lent regioselectivity of this reaction.[17] Altogether, these
studies demonstrate that the assembly of the hybrid macro-
cycle occurred with the expected regiochemistry and accord-
ing to the envisioned route; that is, by intramolecular attack
of the nucleophilic hydrazide on the thioester linkage after
formation of the BP-SP adduct.
Encouraged by these results, we investigated the viability
of this strategy to assemble diverse MOrPHs by varying the
genetically encoded portion of the macrocycle. To this end,
biosynthetic precursors comprising shorter (TS4, TS5) and
longer (TS8, TS10, TS12) target sequences (Scheme 1) were
prepared (named MG4, MG5, MG8, MG10, and MG12,
respectively). As for TS6, these target sequences were
randomly chosen with the exception of the I-1 position
(preceding the intein), where a threonine was introduced as
this substitution was reported to induce minimal self-splicing
of GyrA fusion proteins during expression in E. coli.[19] After
coupling with 1, the desired macrocycle formed as the almost
exclusive product (95–100%) from all the reactions except
that involving MG4, which yielded an approximately 1:1 ratio
of macrocycle and acyclic product (Figure 2a) as estimated
from the corresponding LC-MS extracted-ion chromato-
grams. Tandem mass spectrometry further confirmed the
cyclic structure of the produced MOrPHs (Supporting
Information, Figure S3). Importantly, these results indicated
that macrocyclization is strongly favored over the competing
thioester hydrolysis (leading to the acyclic product) across
target sequences from five up to twelve amino acids, allowing
for the efficient assembly of MOrPHs of variable ring size
(Supporting Information, Figure S3). In these reactions,
splicing of the BP-SP adduct was found to be higher (90–
100%) in the context of MG4, MG5, and MG6 compared to
MG8, MG10, and MG12 (50–75%) after overnight incuba-
tion at room temperature (Figure 2b). This trend can be
rationalized considering that in the BPs with shorter target
sequences, the protein-bound SP is positioned closer to the
intein, favoring the nucleophilic attack of the hydrazide on
the thioester linkage. As observed with MG6, CuAAC
coupling proceeded quantitatively with the various biosyn-
thetic precursors, as judged from MALDI-TOF spectra
acquired immediately after the coupling reaction. Based on
Scheme 1. Strategy for the modular synthesis of macrocyclic organo–
peptide hybrids (MOrPHs). The vector pBP encodes for a linear
polypeptide (biosynthetic precursor) comprising an N-terminal tail
(green), O-propargyl tyrosine (OpgY), a target sequence TS (yellow),
and GyrA intein (purple). Upon coupling of this protein to a synthetic
precursor (blue) by CuI-catalyzed alkyne–azide cycloaddition (CuAAC),
the thioester bond at the intein junction is intercepted by the
nucleophilic hydrazide to yield an organo–peptide macrocycle. The
various N-terminal tails, target sequences, and synthetic precursors
described in this study are indicated.
the same m/z as observed in cyclic peptides.[18] In comparison,
the minor acyclic product (8) exhibited a fragmentation
pattern typical for a linear peptide (Figure 1d).
Control experiments were carried out to confirm the
mechanism and specificity of the reaction. Omitting the
copper catalyst from the reaction with 1 resulted in no
macrocycle formation and much reduced splicing of the intein
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Figure 1. a) MALDI-TOF spectrum of biosynthetic precursor MG6 after purification (left), after coupling to 1 by CuAAC (middle), and after
overnight incubation at room temperature (right). The observed (o) and calculated (c) m/z values ([M+H]+ species) are indicated.
NaAsc=sodium ascorbate, TCEP=tris(2-carboxyethyl)phosphine. b) Extracted-ion chromatograms for m/z corresponding to the macrocycle (left)
and the acyclic product (right) as obtained from LC-MS analysis of the reaction mixture after 16 h. Light gray peak: unrelated multicharged ions
from spliced GyrA intein. c) MS/MS spectrum of the MOrPH product 7 (precursor ion: m/z 1016.3). d) MS/MS spectrum of the acyclic product 8
(precursor ion: m/z 1034.3) with assignment of the fragment ions.
Next, we investigated the possibility of diversifying
MOrPH architecture by varying the structure of the synthetic
precursor. To this end, compounds 3–6 (Scheme 1) were
synthesized, which incorporate phenyl, biphenyl, and
diphenyl scaffolds. Bi- and diaryl structures are, among
others, recurring motifs (“privileged structures”) in small
molecules with biological activity,[20] including those found to
inhibit protein–protein interactions.[21] These SPs were also
designed to evaluate the effect of the distance between the
azide and the hydrazide on the efficiency of macrocyclization.
Such distance increases in the order 3 (5.5 ꢀ), 4 (6 ꢀ), 1 (7 ꢀ),
5 (9–11 ꢀ), 6 (12–15 ꢀ), as calculated based on energy-
minimized conformations of these molecules (MM2 force-
field). Reactions of 3–6 with the BPs containing target
sequences of varying length were performed as described
for 1 and analyzed by LC-MS. Notably, the desired macro-
Figure 2. Reactions between SP 1, 3, 4, 5, and 6 and the MG
biosynthetic precursors. a) Fraction of MOrPH formed ([MOrPH]/
cyclic product was obtained for all 30 combinations tested
(Supporting Information, Figure S4), demonstrating the func-
tionality of the method across widely different SP structures
and its versatility to afford diverse MOrPHs by varying the
synthetic and peptidic portion of these structures.
([MOrPH]+[acyclic product]) and b) percentage of splicing of the
corresponding BP-SP adducts ([GyrA]/([GyrA]+[unspliced BP-SP])) as
determined by LC-MS (16 h).
this, the percentage of MOrPH produced, and the extent of
protein splicing (Figure 2), the overall yield for MOrPH
formation in the reaction of 1 with the MG constructs was
estimated to range from about 50% (MG4, MG8, MG10,
MG12) to more than 80% (MG5, MG6).
The ratio of macrocycle versus acyclic product produced
in these reactions was analyzed to assess the relative
efficiency of macrocyclization in the context of the various
SP/BP pairs (Figure 2a). Interestingly, MOrPH assembly was
found to occur with highest efficiency (> 85%) with the 5mer
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target sequence and all the SPs and with the 6mer, 8mer,
large rate acceleration in the hydrazide-induced intein splic-
ing reaction for the intramolecular versus intermolecular
mechanism. An important consequence of this rate difference
is that hydrazide-dependent splicing occurs exclusively after
tethering of the synthetic precursor to the protein, providing
an excellent control over the undesired intermolecular
reaction.
To determine whether milligram amounts of pure MOrPH
product could be obtained using the described method, a
scaled-up reaction was carried out using 1 and about 50 mg of
purified MG6 protein. After the reaction, the low-molecular-
weight products could be isolated from the reaction mixture
by filtration followed by solid-phase extraction, yielding
about 2 mg of a mixture of 7 and 8 in about 80:20 ratio and
90% purity. MOrPH 7 was then successfully isolated in more
than 95% purity by further purification using C18 reverse-
phase HPLC (Supporting Information, Figure S5).
10mer, and 12mer target sequences and 1, 5, and 6. Synthetic
precursors with closely spaced azide/hydrazide (less than 6 ꢀ
as in 3 and 4) were suboptimal for cyclization with target
sequences longer than six amino acid residues, while the
shortest target sequence tested (4mer) yielded an approx-
imately 1:1 mixture of macrocycle and acylic product regard-
less of the SP structure. With respect to the extent of BP-SP
adduct splicing, the trend observed in the reactions with 1 was
reproduced in the reactions with the other four SPs as well
(Figure 2b), supporting our conclusions regarding the higher
reactivity of BPs with shorter target sequences.
To investigate the kinetics of MOrPH formation, splicing
of MG5-1 and MG10-1 adducts, which produce MOrPH
almost exclusively, was monitored over time. These studies
revealed that adduct splicing (and thus MOrPH formation)
occurs in large part within the first two hours from the
coupling reaction (Figure 3). In contrast, a high concentration
Various bioactive peptides and depsipeptides found in
nature display a lariat backbone, where an N- or C-terminal
tail is connected to a cyclized portion of the peptide
sequence.[7e,h,22] To explore the scope of the method to
prepare MOrPHs in lariat configuration, the five synthetic
precursors (1, 3–6) were reacted with the biosynthetic
precursor Lar5, which consists of a pentamer N-terminal tail
(MGYTA) and a pentamer target sequence (ADWGT).
Macrocyclization was found to proceed efficiently in all cases,
as indicated by the extent of splicing of the BP-SP adducts
(50–60%) and the observation of the desired lariat macro-
cycles as the sole product by MALDI-TOF (Figure 4).
These results suggested that modification of the N-
terminal portion in the biosynthetic precursor was compatible
with MOrPH formation. To further investigate this aspect, the
six target sequences TS4 to TS12 were fused to a larger N-
terminal tail consisting of the 71 amino acid chitin binding
domain (CBD) of chitinase A1 from Bacillus circulans. The
resulting CBD fusion biosynthetic precursors were tested in
reactions with 1 and 3–6. As illustrated by the MALDI-TOF
spectra (Figure 5; Supporting Information, Figures S6–S9),
the desired CBD-tethered MOrPHs were obtained as the sole
Figure 3. Time course measurement of GyrA splicing for MG5-1
^
*
adduct ( ), MG10-1 adduct ( ), MG5 in the presence of 1 at 50 mm
~
( ), and MG5 alone (&) as determined by LC-MS analysis. Error bars
are calculated from experiments carried out in duplicate.
of unbound 1 (50 mm) caused a negligible amount of protein
splicing over background hydrolysis, even over extended
periods of time (12 h), which is consistent with the slow
kinetics observed for intein-mediated ligations using nucleo-
philes other than thiols.[12b] Overall, these results denoted the
Figure 4. MALDI-TOF spectra of the lariat MOrPHs. Calculated (c) and observed (o) m/z values for the sodium adduct are indicated along with
the peaks corresponding to the proton and potassium adducts. SRC=standard reaction conditions (see the Supporting Information).
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support or a cellular/viral structure for screening purposes. In
particular, we envision that coupling MOrPH synthesis with a
display method will provide a powerful tool to discover
valuable MOrPH compounds with tailored protein-binding
properties.
Received: February 22, 2011
Published online: April 19, 2011
Keywords: click chemistry · intein · macrocycles · mutagenesis ·
.
non-natural amino acids
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