Sequence-Selective Decapeptide Synthesis by the Parallel Operation of Two Artificial Molecular Machines

Article abstract of DOI:10.1021/jacs.1c01234

We report on the preparation of a decapeptide through the parallel operation of two rotaxane-based molecular machines. The synthesis proceeds in four stages: (1) simultaneous operation of two molecular peptide synthesizers in the same reaction vessel; (2) selective residue activation of short-oligomer intermediates; (3) ligation; (4) product release. Key features of the machine design include the following: (a) selective transformation of a thioproline building block to a cysteine (once it has been incorporated into a hexapeptide intermediate by one molecular machine); (b) a macrocycle-peptide hydrazine linkage (as part of the second machine) to differentiate the intermediates and enable their directional ligation; and (c) incorporation of a Glu residue in the assembly module of one machine to enable release of the final product while simultaneously removing part of the assembly machinery from the product. The two molecular machines participate in the synthesis of a product that is beyond the capability of individual small-molecule machines, in a manner reminiscent of the ligation and post-translational modification of proteins in biology.

Full text of DOI:10.1021/jacs.1c01234

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Article
Sequence-Selective Decapeptide Synthesis by the Parallel
Operation of Two Artificial Molecular Machines
Javier Echavarren,† Malcolm A. Y. Gall, Adrian Haertsch,† David A. Leigh,* Justin T. J. Spence,†
Daniel J. Tetlow, and Chong Tian†
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ABSTRACT: We report on the preparation of a decapeptide
through the parallel operation of two rotaxane-based molecular
machines. The synthesis proceeds in four stages: (1) simultaneous
operation of two molecular peptide synthesizers in the same
reaction vessel; (2) selective residue activation of short-oligomer
intermediates; (3) ligation; (4) product release. Key features of the
machine design include the following: (a) selective transformation
of a thioproline building block to a cysteine (once it has been
incorporated into a hexapeptide intermediate by one molecular machine); (b) a macrocycle-peptide hydrazine linkage (as part of the
second machine) to differentiate the intermediates and enable their directional ligation; and (c) incorporation of a Glu residue in the
assembly module of one machine to enable release of the final product while simultaneously removing part of the assembly
machinery from the product. The two molecular machines participate in the synthesis of a product that is beyond the capability of
individual small-molecule machines, in a manner reminiscent of the ligation and post-translational modification of proteins in
biology.
chain-length limitations of rotaxane-based molecular synthe-
sizers might be to operate two different artificial molecular
machines in one pot and then ligate their products in situ
(Figure 1). In this way track-sequence information could be
processed by multiple machines and then added together to
generate a product not obtainable through the use of a single
machine alone.
Molecular Machine Designs and Operation Pathway.
The strategy behind such a molecular machine-driven synthesis
is outlined in Figure 1. A decapeptide product of specific
sequence is produced by a series of functional modules that
make up two molecular machines and their tracks. The
synthesis occurs in four consecutive stages: (1) Parallel
operation of the two rotaxane-based molecular machines; (2)
selective residue activation by release of the C-terminus
hydrazide and N-terminus cysteine groups of the machine
products; (3) ligation of the resulting short-sequence
intermediate peptides; and (4) final product release through
the activation of a glutamate residue installed in the machine I
assembly module.
INTRODUCTION
■
Chemists are adopting principles from the natural world to
help develop artificial molecular machines capable of
increasingly sophisticated operations.1 Most examples of
functional synthetic molecular machines2−8 to date feature a
single type working alone to achieve a task.9−11 This contrasts
with the complex and tightly regulated functional networks
common in biology.12 An example of the latter is
ubiquitination, in which ubiquitin and a substrate peptide are
made independently by ribosomes before being subsequently
ligated by the cooperative action of enzymes.13,14 This
produces a protein product that is beyond the scope of
synthesis by a single ribosome alone.
Rotaxane-based artificial molecular machines have previously
been developed that produce short sequence-specific
oligomers, most commonly peptides, in an approach inspired
by aspects of the way the ribosome builds proteins.15−32
However, a significant limitation of current track-based
molecular synthesizers is that when each amino acid is
extracted from the strand, the resulting cyclic transition state
that transfers the amino acid to the terminus of the growing
chain increases in size.15 Every addition slows the transfer of
further amino acids until eventually other processes (inter-
molecular reactions, hydrolysis, etc.) compete with the
machine operation and the machine stops working. This
effectively limits the size of product that can be made through
such an approach. Inspired by the strategy evolution has
selected for ubiquitination, we considered that a solution to the
Received: February 1, 2021
Published: March 25, 2021
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© 2021 American Chemical Society
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of the cysteine abstracting a building block from the
track to form a thioester, followed by transfer of the acyl
group to the amino terminus of the elongation site,
simultaneously forming a new peptidic bond and
regenerating the catalytic thiol group for further
barrier-removing reactions.
(vi) The lack of strong binding interactions between the ring
and track, often a residual feature in rotaxanes not
prepared by active template synthesis, ensures the ring
movement between the track barriers is as unrestricted
as possible.
(vii) Rigid spacers between the building blocks on the track
inhibit track folding, preventing reactions out of
sequence.
The following new features were introduced for the
cooperative synthesis shown in Figure 1:
(viii) The machines were operated simultaneously in the same
reaction vessel, but at high dilution to disfavor
intermolecular reactions.
(ix) The assembly modules are connected to machine I and
II by different functional groups (amide and hydrazone,
respectively) to allow the chemoselective revealing of the
C-terminus of one short-sequence intermediate.
(x) Track I contains a thioproline (Thi) residue that can be
unmasked48 following machine operation to provide a
Cys residue that enables subsequent ligation49 of the
short-sequence intermediates.
(xi) Assembly module I contains a Glu as the second amino
acid, enabling the first two amino acids arising from
assembly module I to be removed from the final product
during its liberation from the machine. Like many of our
design solutions for artificial machines that work at the
molecular level, this feature is inspired by considering
how biology solves a related problem. In eukaryotic
ribosomal protein synthesis, each sequence is initiated
with a methionine residue codified by the start codon
AUG. Following the ribosome operation the now-
superfluous N-terminal methionine is cleaved by
methionine aminopeptidase (MAP) to yield the final
peptide product.50
Figure 1. Decapeptide synthesis by a pair of modular artificial
molecular machines: 1. Parallel operation of two molecular
synthesizers with different assembly modules, assembly module-
macrocycle linkages and track sequences; 2. Selective residue
activation; 3. Ligation of short-sequence intermediates; 4. Release of
the decapeptide product.
The molecular machine designs are related to previous
rotaxane-based peptide synthesizers15,19−21 and incorporate
the following features:
(i) The rotaxane architecture confers processivity (the ring
cannot dethread or exchange with others in the bulk
until all the barriers have been removed).
In C-to-N sequence, the decapeptide product formed in
Figure 1 consists of the following: one amino acid from
assembly module I (AA1: Gly), AA2−4 incorporated in
sequence from track I (AA2: Tyr(Me); AA3: Leu; AA4:
transformed from Thi to Cys), AA5−7 from assembly module
II (AA5: Cys; AA6: Gly; AA7: Gly), AA8−10 incorporated in
sequence from track II (AA8: Phe; AA9: Leu; AA10: Ala).
(ii) The unreactive bulky “stopper” at the left-hand terminus
of the axle provides the directionality necessary for
sequence-specific synthesis (the component dynamics
are not ratcheted, so the ring moves incessantly in both
directions along the available section of track, but only
movements away from the stoppered end of the track
bring the ring-bound assembly module close enough to
an axle-bound building block for a reaction to occur).
(iii) The reactive barriers are amino acyl derivatives, and the
sequence they appear on the track is the sequence they
are incorporated in the machine-made short-oligomer
intermediate.
(iv) The macrocycle contains both a pyridine group,
necessary for rotaxane formation of the machine-track
conjugate by active template synthesis,33−46 and an
assembly module featuring a cysteine residue at one end
of a tripeptide and an elongation site for the growing
oligomeric chain at the other.
RESULTS AND DISCUSSION
■
Synthesis of Molecular Machine-Track Conjugates 1
and 2. Molecular machine-track conjugates 1 and 2 were
prepared in a rotaxane elongation strategy19 via successive
Cu(I)-catalyzed alkyne−azide cycloaddition (CuAAC) reac-
tions of various synthons (Scheme 1). Active template CuAAC
synthesis33 generated one-barrier [2]rotaxanes 3 and 4 (see
Supporting Information for synthesis and characterization,
sections 3.1 and 3.2). The mechanically interlocked structure
1
of each species was confirmed by mass spectrometry and H
NMR (see Supporting Information). Machine 1 was
synthesized by CuAAC coupling of rotaxane 3 and the two-
barrier fragment 5 (Scheme 1, route a). The assembly module
for this molecular machine was attached to the macrocycle
(v) The machines operate through multiple successive
native chemical ligation (NCL) reactions,47 the thiol
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a
Scheme 1. Synthesis of 1 and 2 by Elongation of the Corresponding One-Barrier Rotaxane Synthons
a
Reagents and conditions: Route a: (i) 3 (1.0 equiv), 5 (1.5 equiv), Cu(MeCN)4PF6 (1.5 equiv), Tentagel TBTA resin (3.0 equiv), CH2Cl2, t-
BuOH (5:1), rt, 48 h, 63%. Route b: (i) 4 (1.0 equiv), 6 (1.0 equiv), Cu(MeCN)4PF6 (0.45 equiv), Tentagel TBTA resin (0.5 equiv), CH2Cl2, t-
BuOH (8:1), rt, 48 h, 86%. (ii) BocGlyGlyCys(Trt)NHNH2, aniline, CH2Cl2, rt, 48 h, 50%.
prior to rotaxane formation (see Supporting Information
section 3.1 and 3.2 for synthesis). Molecular machine-track
conjugate 2 was assembled by the CuAAC reaction of 4 with
the two-barrier extension unit 6,19 followed by condensation
with the Boc-Gly-Gly-Cys(Trt)-NHNH2 assembly module II
(Scheme 1, route b).
ually, we explored their contemporaneous operation within the
same reaction vessel. The two machines were combined,
deprotected, and operated under the standard conditions
(Figure 2C, Supporting Information section 3.5). Pleasingly,
the mass spectrum of the one-pot operation of 1 and 2 is
virtually a superimposition of the spectra from the individual
operation of each machine (Figure 2C, right), with both of the
desired hexapeptide products (7 and 8) formed. Crucially, no
byproducts were observed corresponding to intermolecular
reactions (neither out of sequence nor higher order peptides),
confirming that the two machines operate in parallel without
effecting the outcome from the other’s.
Selective Residue Activation of Sequences 7 and 8.
Next we looked at coupling the two hexapeptide products of
operation by a hydrazide ligation protocol. To do so the
thioproline of 7 and hydrazone of 8 needed to be converted to
the corresponding terminal cysteine, 9, and hydrazide 10,
respectively (Scheme 2 and Supporting Information, section
3.7). Conveniently, the deprotection of thioproline is reported
to proceed under mild conditions with methoxyamine
hydrochloride,51 while the same conditions should cleave the
hydrazone in 8 via an exchange reaction. Accordingly, the
mixture of 7 and 8 was treated with methoxyamine
hydrochloride in MeOH at 50 °C. Pleasingly, hydrazide 10
was liberated from the macrocycle accompanied by depro-
tection of the thioproline to generate the N-terminal cysteine
residue on the amide macrocycle (9).
Individual Operation of Machine-Track Conjugates 1
and 2. With both machines in hand, we investigated their
operations individually (Figure 2A and 2B, and Supporting
Information). Each machine-track conjugate was separately
subjected to global deprotection with trifluoroacetic acid and
triisopropylsilane (removing the Boc and trityl groups). The
resulting protecting-group-free machines were then subjected
to a standard set of operation conditions: Each machine was
heated in a microwave in the presence of diisopropylethyl-
amine (i-Pr2NEt) and 4 Å molecular sieves in a mixture of
acetonitrile/dimethylformamide at 75 °C for 15 h. The
operation was monitored by mass spectrometry (Figure 2A
and 2B; see Supporting Information, sections 3.3 and 3.4 for
details) which in each case confirmed the formation of the
desired peptide products (7 and 8, respectively). The sequence
fidelity of these hexapeptide intermediates was confirmed by
tandem mass spectrometry (Figure 2 A and 2B, bottom). In
accordance with previous observations on small-molecule
peptide synthesizers,19,20 we also detected some truncated
products arising from the hydrolysis of barriers, whose
intensity in the mass spectrum does not necessarily correlate
with their relative abundance in the product mixture.
Ligation of Intermediates 9 and 10. Following selective
residue activation the resulting mixture was subjected to
hydrazide ligation conditions.52 Initially, the hydrazide is
Parallel Operation of Machines 1 and 2. Having
confirmed the successful operation of each machine individ-
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Figure 2. (A) Top: Mass spectrum of the reaction mixture following the operation of machine 1. Signals in gray correspond to adducts with
dimethylformamide (DMF; e.g. 1015 = 942 + DMF; 1244 = 1171 + DMF) and minor unidentified species. Bottom: Tandem mass-spectrometry
sequencing of 1. (B) Top: Mass spectrum of the reaction mixture following the operation of machine 2. Bottom: Tandem mass-spectrometry
sequencing of 2. (C) Left: Parallel operation of both molecular machines, 1 and 2, in the same reaction vessel (“one-pot” synthesis). Right: Mass
spectrum of the reaction mixture following the one-pot operation of 1 and 2. Peaks in blue correspond to products arising from the operation of
machine 1; peaks in red correspond to products arising from the operation of machine 2. Reagents and conditions: (i) (i-Pr)3SiH, CF3CO2H,
CH2Cl2, rt, 30 min. (ii) i-Pr2NEt, MeCN/DMF (3:1), 4 Å molecular sieves, 75 °C, MW, 15 h.
oxidized at pH 3 by NaNO2 to the corresponding acyl azide
(Scheme 2B, hydrazide oxidation),53 after which a large excess
of 4-mercaptophenylacetic acid (MPAA) is added and the pH
is adjusted to 7 by the use of buffer. The added MPAA has a
multipurpose role: (i) It forms a thioester by reaction with the
acyl azide (Scheme 2 B, thiolysis), (ii) it reduces any oxidized
cysteine species back to the thiol, and (iii) it reacts with, thus
removing, the residual NaNO2.49 Next, transthioesterification
of the thioester and the terminal cysteine of 9 takes place to
join the fragments (Scheme 2 B, transthioesterification).
Finally, a 1,5-S,N-acyl shift completes the reaction (Scheme
2B, S to N acyl shift), rendering dodecapeptide 11.
After 3 h, tris(2-carboxyethyl)phosphine (TCEP) was added
to the reaction mixture to reduce any oxidized thiols formed
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Scheme 2. (A) Ligation of Short Oligomer Intermediates to Form a Single Peptide; (B) Mechanism of Ligation; (C)
a
Mechanism of Glutamic Acid Cleavage Mediated by OGT and (D) Direct Glutamic Ester Cleavage
a
Reagents and conditions: (i) MeONH2·HCl, MeOH, 50 °C, 3 h. (ii) pH = 3, NaNO2, 6 M guanidinium chloride, 0.2 M Na2HPO4, HFIP
(hexafluoro-2-propanol), −10 °C, 20 min, then pH = 7, 4-mercaptophenylacetic acid, rt, 3 h, 25% over 2 steps. (iii) LiOH, THF/H2O, rt, 30 min.
The double-headed arrows are used as shorthand to indicate the formation and collapse of tetrahedral intermediates in the mechanism.
1
Figure 3. Partial H NMR (600 MHz, DMSO-d6, 298 K) of (A) hydrazide 10 (unpurified reaction product); (B) ligated peptide 11; and (C)
amide 7 (unpurified reaction product). Dashed lines indicate analogous proton resonances in the different molecules. Letters correspond to the
labeling shown in Scheme 2. Peaks shaded gray correspond to residual solvent.
during the reaction. The ligation product was purified by
precipitation in methanol and isolated in 25% yield. The
identity of 11 was confirmed by HR-ESI(+) mass spectrometry
and NMR (see Supporting Information, section 3.7).
of diagnostic peaks from both fragments, such as signals from
the amide-linked macrocycle (Ha−Hf), glutamic ester (Hg−
Hh), and methyl tyrosine (Hi, Hj, and Hk), as well as from the
phenyl alanine (Ho, Hp, and Hq) and alanine (Hu and Hv)
originating from the hydrazide fragment, together with signals
from both leucine residues (protons Hr−Ht and Hl−Hn).
1
Qualitative comparison of the H NMR spectrum of 11 with
the intermediates 7 and 10 (Figure 3)54 showed incorporation
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Release of Decapeptide 12 by Glutamic Ester
Cyclization. Finally, we investigated removal of the ligated
peptide from the remaining macrocycle and cysteine residue of
assembly module I by selective glutamic acid cleavage.55 We
originally envisaged an approach based on the naturally
occurring backbone protein cleavage of human cell factor 1
(HCF-1), where a glutamic acid is activated as the glycosyl
glutamate ester by O-GlcNAc transferase (OGT) (Scheme 2
C, OGT-activation). This ester is then converted to a transient
pyroglutamate intermediate (Scheme 2C, cyclization and loss
of GluNAc) that spontaneously undergoes hydrolysis to the
carboxylic acid and lactam (Scheme 2C, hydrolysis).55 Since
the Glu residue in 11 is an O-Bzl ester, we reasoned it might
undergo a similar transformation. Accordingly, 11 was reacted
with LiOH for 30 min at room temperature (Scheme 2 D and
Supporting Information, section 3.8) and we were pleased to
observe direct formation of the desired decapeptide acid 12
together with the corresponding lactam 13. Presumably the
mechanism is analogous to HCF-1 cleavage, via an imide (14)
formed by direct attack of the amide on the benzyl ester
(benzyl alcohol as the leaving group, Scheme 2D, cyclization).
Imide 14 is then cleaved under the basic aqueous conditions to
give decapeptide 12 (Scheme 2D, hydrolysis), with N-to-C
sequence PivAlaLeuPheGlyGlyCysCysLeuTyr(Me)Gly.56−58
Justin T. J. Spence − Department of Chemistry, University of
Manchester, Manchester M13 9PL, U.K.; orcid.org/0000-
0003-1386-2673
Daniel J. Tetlow − Department of Chemistry, University of
Manchester, Manchester M13 9PL, U.K.
Chong Tian − Department of Chemistry, University of
Manchester, Manchester M13 9PL, U.K.; orcid.org/0000-
0001-7264-9042
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c01234
Author Contributions
†J.E., A.H., J.T.J.S., and C.T. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Engineering and Physical Sciences Research
Council (EPSRC; EP/P027067/1) and the European Union
(European Research Council (ERC) Advanced Grant no.
786630 and H2020 project Euro-Sequences, H2020-MSCA-
ITN-2014, Grant no. 642083) for funding and the University
of Manchester Mass Spectrometry Service Centre for high-
resolution mass spectrometry. D.A.L. is a Royal Society
Research Professor.
CONCLUSIONS
■
Two artificial molecular machines, each composed of several
functional modules, can work in parallel to carry out the
sequence-selective synthesis of peptides in one pot without
crossover or scrambling. The machine designs incorporate
fragments that can be activated to allow intermediates to be
ligated and released from the machine, the latter in a process
reminiscent of the cleavage of a “starting codon”. The overall
synthesis generates a decapeptide of programmed sequence, a
product length unreachable with the current generation of
artificial molecular synthesizers acting alone. Nature’s solutions
to the challenges posed by operating machinery at the
nanoscale continue to serve as useful examples to guide the
design of increasingly complex artificial molecular machines
capable of performing increasingly demanding tasks.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c01234.
Experimental procedures, characterization of new
compounds, and spectroscopic data (PDF)
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