Sequence-specific peptide synthesis by an artificial small-molecule machine

Article abstract of DOI:10.1126/science.1229753

The ribosome builds proteins by joining together amino acids in an order determined by messenger RNA. Here, we report on the design, synthesis, and operation of an artificial small-molecule machine that travels along a molecular strand, picking up amino acids that block its path, to synthesize a peptide in a sequence-specific manner. The chemical structure is based on a rotaxane, a molecular ring threaded onto a molecular axle. The ring carries a thiolate group that iteratively removes amino acids in order from the strand and transfers them to a peptide-elongation site through native chemical ligation. The synthesis is demonstrated with ～10¹⁸ molecular machines acting in parallel; this process generates milligram quantities of a peptide with a single sequence confirmed by tandem mass spectrometry.

Full text of DOI:10.1126/science.1229753

Sequence-Specific Peptide Synthesis by an Artificial Small-Molecule
Machine
Bartosz Lewandowski et al.
Science 339, 189 (2013);
DOI: 10.1126/science.1229753
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ing one expansion/contraction cycle. fad is the (Fig. 4C). Within 7 min of charging, the voltage of
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and moist substrates. Given a certain E, R, and This was lower than the peak output voltage of the
fad, Eqs. 1 and 2 roughly define a theoretical max- generator, possibly due to voltage drop across
imum and minimum limit on the required thick- the rectifying diodes and/or current leakage of
ness of the actuator to perform fast locomotion. the capacitor.
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This PEE-PPy polymer composite system fea-
for PEE-PPy actuators on a moist paper was tures an interpenetrating network of a rigid poly-
roughly 15 to 40 mm. Actuators thinner than 15 mm mer with a soft, hydrolytically sensitive polymer
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water gradients to mechanical work. Besides me-
Because this PEE-PPy actuator could contin- chanical vibration energy, the generator based
uously extract chemical potential energy out of on this powerful actuator can use ubiquitous low-
ambient water gradients to perform mechanical temperature water gradients as its energy source,
work, it should be able to drive a piezoelectric in contrast to state-of-the-art piezoelectric energy
element to convert the mechanical energy into scavengers that rely solely on mechanical vibration
electrical energy. A 9-mm-thick piezoelectric poly- energy (26). Thus, the water-gradient–driven actu-
vinylidene difluoride (PVDF) film was metallized, ator and generator demonstrated potential applica-
wired, and insulated on both faces (Fig. 4A). A tions as sensors, switches, and power sources for
27-mm-thick PEE-PPy actuator was attached to ultralow-power devices.
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Acknowledgments: M.M. and R.L. conceived the idea and
designed the experiments. M.M. and L.G. performed the
experiments. M.M., L.G., D.G.A., and R.L. contributed materials
and/or tools, analyzed the data, and wrote the paper.
This research was supported in part by a National Heart, Lung,
and Blood Institute Program of Excellence in Nanotechnology
(PEN) Award, contract no. HHSN268201000045C; National
Cancer Institute grant CA151884; and Armed Forces Institute
of Regenerative Medicine Award no. W81XWH-08-2-0034.
Experimental procedures, additional data for materials
characterization, and the device test are presented in the
supplementary materials. We thank D. Bong for thoughtful
discussion and N. Zhang for help in the preparation of figures.
one face of the PVDF element. When placed on a
moist substrate with the actuator facing down, the
actuator bent and stretched the PVDF element
References and Notes:
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repeatedly (movie S5), generating an open-circuit
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analysis of this background noise giving an av-
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Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6116/186/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S16
Tables S1 and S2
Eqs. S1 to S3
Movies S1 to S5
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17 September 2012; accepted 9 November 2012
10.1126/science.1230262
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(bacterial) to ~4.3-MD (eukaryotic) molecular
machine found in all living cells that assembles
amino acids from tRNA building blocks into a
peptide chain with an order defined by the
sequence of the mRNA strand that it moves
along. Artificial small-molecule machines (5)
have previously been used to store information
(6, 7) and do mechanical work (8–11); others
have been employed in synthesis to processive-
Sequence-Specific Peptide Synthesis by
an Artificial Small-Molecule Machine
Bartosz Lewandowski,1 Guillaume De Bo,1 John W. Ward,1 Marcus Papmeyer,1 Sonja Kuschel,1
María J. Aldegunde,2 Philipp M. E. Gramlich,2 Dominik Heckmann,2 Stephen M. Goldup,2
Daniel M. D’Souza,2 Antony E. Fernandes,2 David A. Leigh1,2
*
The ribosome builds proteins by joining together amino acids in an order determined by messenger ly epoxidize an unsaturated polymer (12, 13),
RNA. Here, we report on the design, synthesis, and operation of an artificial small-molecule
machine that travels along a molecular strand, picking up amino acids that block its path, to
switch “on” and “off” catalytic activity (14–17),
and change the handedness of a reaction product
synthesize a peptide in a sequence-specific manner. The chemical structure is based on a rotaxane, (18). Large synthetic DNA molecules have been
a molecular ring threaded onto a molecular axle. The ring carries a thiolate group that iteratively used to guide the formation of bonds between
removes amino acids in order from the strand and transfers them to a peptide-elongation site
through native chemical ligation. The synthesis is demonstrated with ~1018 molecular machines
acting in parallel; this process generates milligram quantities of a peptide with a single sequence
confirmed by tandem mass spectrometry.
unnatural building blocks (19–22) and assemble
1School of Chemistry, University of Manchester, Oxford Road,
Manchester M13 9PL, UK. 2School of Chemistry, University of
Edinburgh, The King’s Buildings, West Mains Road, Edinburgh
EH9 3JJ, UK.
ells achieve the sequence-specific synthe- molecular machines that transcribe information
sis of information-rich oligomers and poly- from the genetic code (1). The most extraordi-
*To whom correspondence should be addressed. E-mail:
C
mers through the operation of complex nary of these is the ribosome (2–4), a ~2.6-MD david.leigh@manchester.ac.uk
www.sciencemag.org SCIENCE VOL 339 11 JANUARY 2013
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REPORTS
gold nanoparticles in particular sequences (23). tificial molecular machine is based on several cessivity during the machine’s operation (remi-
Here, we report on the design, synthesis, and elements that have analogs in either ribosomal niscent of the way that subunits of the ribosome
operation of a rotaxane-based small-molecule (2–4) or nonribosomal (24) protein synthesis: clamp the mRNA strand) and bears a catalyst—a
machine in which a functionalized macrocycle Reactive building blocks (the role played by tethered thiol group—that detaches the amino
operates on a thread containing building blocks tRNA-bound amino acids) are delivered in a se- acid building blocks from the strand and passes
in a predetermined order to achieve sequence- quence determined by a molecular strand (the role them on to another site at which the resulting
specific peptide synthesis. The design of the ar- played by mRNA). A macrocycle ensures pro- peptide oligomer is elongated in a single specific
Fig. 1. Synthesis of rotaxane-based molecular machine 1, incorporating
a strand bearing amino acid building blocks (2), a macrocycle (3) with a
site for attachment of the reactive arm, and a terminal blocking group (5)
that prevents the threaded macrocycle from coming off the strand until all
of the amino acid groups have been cleaved. Structure 1 is shown as the
major diastereomer; a small amount of epimerization (<5%) of some of the acyl amino units occurs during incorporation into the track. Boc, CO2C(CH3)3; Piv, COC(CH3)3;
Trt, CPh3; Ph, phenyl. Reaction conditions: (i) Cu(CH3CN)4PF6 in dichloromethane:t-butanol (2:1), room temperature, 4 days, 30%. (ii) PhNH2 (catalyst),
BocGlyGlyCys(S-Trt)NHN=CHC6H4OCH3 in 3:1 dimethylsulfoxide: aqueous 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0), 60°C, 2 days, 90%. The italicized
letters indicate key signals in the 1H NMR spectrum shown in Fig. 2B. For the full lettering scheme and assignments, see the supplementary materials, page S28 (26).
Fig. 2. Proton NMR spectrum of (A) the noninterlocked
thread and (B) rotaxane 1, in d6-dimethylsulfoxide
(500 MHz, 298 K). Rotaxane 1 exists in both E- and
Z-hydrazone forms. The assignments correspond to
the lettering shown in Fig. 1. C′, S-Trt-cysteine; G′,
N-Boc-glycine; F′, N-Boc-phenylalanine; L′, N-Boc-
leucine; A′, N-Piv-alanine. ppm, parts per million.
190
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REPORTS
sequence, through chemistry related to non- threading of strand 2 during the Cu(I)-catalyzed conjugate 4, we used reversible hydrazone ex-
ribosomal peptide synthesis (24). cycloaddition of the terminal alkyne with the change to introduce a cysteine derivative bearing
The chemical structure of the artificial mo- azide-bearing stopper group 5, leading to the the reactive arm [a trityl (Trt)–protected thiol
lecular machine, 1, is shown in Fig. 1. Strand 2 assembly of rotaxane 4 in 30% yield (Fig. 1, i). group] and the site for peptide elongation [a
bears three amino acids attached to the track by This active template (27, 28) strategy ensured tert-butoxycarbonyl carbamate (Boc)–protected
weak phenolic ester linkages (25) and separated that the resulting threaded structure did not have amine at the end of a glycylglycine residue]
from each other by rigid spacers that minimize residual attractive intercomponent interactions (Fig. 1, ii). The fully assembled machine 1 is
the possibility of the reactive arm of the machine that would tend to localize the position of the stable in its protected form, with upfield shifts
coming into contact and reacting with a building ring rather than allow it to move freely up and of the HP1 triazole and nearby HS12-S14 proton
block out of sequence (26). Macrocycle 3 con- down the strand between blocking groups. Once signals evident in the 1H nuclear magnetic res-
tains an endotopic pyridine group that directs the we assembled the macrocycle-strand-stopper onance (NMR) spectrum on account of shielding
from the phenyl rings of the macrocycle, con-
firming that the ring is trapped in the region of
the strand between the terminal stopper and the
Boc-phenylalanine ester (Fig. 2).
We used acid-catalyzed cleavage of the Boc
and trityl protecting groups (Fig. 3, i) to activate
the molecular machine and then allowed it to oper-
ate (Fig. 3, ii) at 60°C under microwave heating
in a 3:1 acetonitrile:dimethylformamide solution in
the presence of N,N-diisopropylethylamine (a non-
nucleophilic base) and tris(2-carboxyethyl)phosphine
(a reducing agent that cleaves any disulfide bonds
formed through thiol oxidation). The design of the
machine is such that once the thiolate residue of
the cysteine group (6a) is deprotected, it is poised
to undergo a transacylation reaction with the first
amino acid phenolic ester that blocks the macro-
cycle’s path on the track (6b). We hypothesized that
the subsequently formed phenylalanine thioester
(6c) would be able to react further, transferring
the amino acid by native chemical ligation (29) to
the glycylglycine amine group by an 11-membered-
ring transition state [the dipeptide spacer between
the cysteine residue and the amine of the peptide-
elongation site was introduced because native
chemical ligation is reported to be very slow via
8-membered-ring transition states (30)]. This se-
quence simultaneously transfers the amino acid
to the end of the growing peptide (6d) and re-
generates the catalytic thiolate group, ready for
the cleavage and transfer of further building blocks.
S-N acyl transfer is a key feature of nonribosomal
peptide synthesis (24).
Once the covalent bond connecting an amino
acid to the strand is broken, the macrocycle is
able to move further along the track until its path
is blocked by the next amino acid group (6d).
The O-S acyl transfer/S-N acyl transfer/catalyst
regeneration/ring movement process continues
(6e to 6h) until the last amino acid on the track
is cleaved (6h) and the macrocycle detaches from
the strand (8) with the newly formed, full length,
peptide attached (7). The artificial molecular ma-
chine synthesizes the peptide from the C terminus
to the N terminus, the opposite direction of ribo-
somal translation (2–4).
After a 36-hour operation of 6a at 60°C, no
starting material remained, as shown by high-
Fig. 3. Proposed mechanism for sequence-specific peptide
synthesis by molecular machine 1. After activation of the ma-
chine by acidic cleavage of the Boc and Trt protecting groups,
under basic conditions successive native chemical ligation re-
performance liquid chromatography (HPLC), and
actions transfer the amino acid building blocks to the peptide-elongation site on the macrocycle in the
two major products were isolated from the re-
action mixture (26). We used 1H NMR spectrosco-
py and mass spectrometry to identify one product
as the completely deacylated thread, 8. The other
order they appear on the thread. Once the final amino acid is cleaved, the macrocycle bearing the
synthesized oligopeptide 7 dethreads from the strand. The hydrazide peptide 9 is subsequently released
from the macrocycle by hydrolysis. Reaction conditions: (i) 20% CF3CO2H in dichloromethane, room tem-
perature, 2 hours, 100%. (ii) ((CH3)2CH)2NEt, (HO2CCH2CH2)3P in 3:1 acetonitrile:dimethylformamide,
60°C, 36 hours. Et, ethyl. (iii) 30% CF3CO2H in 3:1 dichloromethane:water, room temperature, 18 hours. product had a 1H NMR spectrum and molecular
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REPORTS
weight (Fig. 4) consistent with the macrocycle apparent in Fig. 4C (725.73 for the LeuGlyGlyCys- subsequently be cleaved from the macrocycle by
bearing the hydrazone linked to the hexapeptide macrocycle, 742.92 for the PheGlyGlyCys- hydrolysis (Fig. 3, iii).
(Piv)AlaLeuPheGlyGlyCys, 7 [Piv, COC(CH3)3]. macrocycle), and product 7 was confirmed as
On a scale of tens of milligrams, we performed
To confirm that the product of the molecular corresponding to the intended sequence isomer. the synthesis of the small peptide through auton-
machine’s operation had the amino acids as-
With the use of HPLC-MS analysis of the omous multistep production by artificial small-
sembled in the correct order, we used tandem reaction mixture from the operation of 1, we did molecule machine 1, corresponding to parallel
mass spectrometry (MS/MS) to determine the not detect any products corresponding to other synthesis by ~1018 machines. Once operation is
peptide sequence. We then validated the se- peptide compositions (neither different sequences initiated, the synthetic tasks performed by 1 pro-
quence by comparing it with an authentic sample nor peptides with more or less than one Phe, Leu, ceed automatically, requiring no further inter-
and an isomer in which the order of the Phe and or Ala residue), indicating that the peptide syn- vention. As the catalytic thiolate is constrained
Leu residues was reversed, each prepared un- thesis occurs overwhelmingly within the confines by the threaded architecture of the machine from
ambiguously by conventional peptide synthesis of the molecular machine. In contrast, a control reacting with building blocks out of sequence,
(26). Figure 4A shows the 1H NMR spectrum of reaction carried out under identical conditions the act of balancing the rate of reactions with
the molecular machine product 7, and Fig. 4B but using the nonthreaded strand and macrocycle the speed that templates rearrange (19–22) is un-
shows the MS/MS spectrum of one isotope of a yielded several products, including strands with necessary for a rotaxane-based machine.
2+ ion of 7 derivatized through the cysteine as one or more amino acid groups cleaved, but there
Rotaxane 1 is a (very) primitive analog of
an S,N-acetal, a species that gave a sufficient was no evidence for the formation of 7 under the ribosome. Limitations of the first-generation
signal-to-noise ratio for the MS/MS experiment. these conditions. Thus, the threaded architecture artificial system include slow kinetics (1 takes
Figure 4C shows the superimposition of the of the molecular machine—encompassing the ~12 hours to make each amide bond, compared
MS/MS spectra of a similar isotope and ion catalytic site, elongation site, and the building to the 15 to 20 amide bonds synthesized per sec-
from the authentic samples of the macrocycle block strand—is essential for the sequential pep- ond by a ribosome) and loss of the sequence
bearing the sequence (Piv)AlaPheLeuGlyGlyCys tide synthesis, and the mode of operation of the information on the strand as it is translated into
(red peaks) and (Piv)AlaLeuPheGlyGlyCys molecular machine is consistent with the mech- the product. Furthermore, the size of oligopeptide
(blue peaks). The difference in the fragmenta- anism shown in Fig. 3. The peptide (9), still bear- that can be produced may ultimately be restricted
tion masses of the two sequence isomers is ing the GlyGlyCys unit at the C terminus, could by the size of the cyclic transition states involved
Fig. 4. (A) Proton NMR spectrum of molecular machine operation product derivatized macrocycles bearing the peptide sequences (Piv)AlaPheLeuGlyGlyCys
7 in d6-dimethylsulfoxide (500 MHz, 298 K). (B) Tandem mass spectrum of (red; 2+ ion isotope-selected m/z = 876.73) and (Piv)AlaLeuPheGlyGlyCys (blue;
a single isotope of the 2+ ion [mass/charge ratio (m/z) = 876.64] of S,N-acetal– 2+ ion isotope-selected m/z = 876.92), each prepared unambiguously by con-
derivatized 7. (C) Superimposed tandem mass spectra of 2+ ions of S,N-acetal– ventional peptide synthesis.
192
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research was funded by the Engineering and Physical Sciences
Research Council (UK). We are grateful to the following
organizations for postdoctoral fellowships: Fundacja na Rzecz
Nauki Polskiej (to B.L.), Fonds de la Recherche Scientifique
and Wallonie-Bruxelles International (to G.D.B.), the European
Union 7th Framework Marie Curie Intra European Fellowship
Program (to M.J.A.), Deutscher Akademischer Austausch
Dienst (to P.M.E.G. and D.H.), and Deutsche Akademia der
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reducing the sequence integrity of the peptide synthesis.
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materials on Science Online.
Supplementary Materials
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Materials and Methods
Supplementary Text
Figs. S1 to S37
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Yoko Sakata,1,2 Shuhei Furukawa,1,2* Mio Kondo,1,2 Kenji Hirai,3 Nao Horike,2
Yohei Takashima,2 Hiromitsu Uehara,2 Nicolas Louvain,1,2 Mikhail Meilikhov,2
Takaaki Tsuruoka,2,5 Seiji Isoda,1 Wataru Kosaka,2 Osami Sakata,4 Susumu Kitagawa1,2,3
*
We fabricated MSME nanopores by crystal
downsizing, which regulated the flexibility of the
framework, and demonstrated the switchable sorp-
tion events based on the presence of two intercon-
vertible pore shapes (Fig. 1). Among the variety of
flexible PCPs, we chose a PCP with a twofold
interpenetrated framework (10–12)—namely,
[Cu2(bdc)2(bpy)]n (1, bdc = 1,4-benzenedicarboxylate,
bpy = 4,4′-bipyridine) (13)—that exhibits a co-
operative structural transformation from the
Flexible porous coordination polymers change their structure in response to molecular
incorporation but recover their original configuration after the guest has been removed.
We demonstrated that the crystal downsizing of twofold interpenetrated frameworks of
[Cu2(dicarboxylate)2(amine)]n regulates the structural flexibility and induces a shape-memory
effect in the coordination frameworks. In addition to the two structures that contribute to the
sorption process (that is, a nonporous closed phase and a guest-included open phase), we
isolated an unusual, metastable open dried phase when downsizing the crystals to the
mesoscale, and the closed phase was recovered by thermal treatment. Crystal downsizing
suppressed the structural mobility and stabilized the open dried phase. The successful
isolation of two interconvertible empty phases, the closed phase and the open dried phase,
provided switchable sorption properties with or without gate-opening behavior.
1World Premier International Research Initiative–Institute for
Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,
Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. 2Exploratory Re-
search for Advanced Technology (ERATO) Kitagawa Integrated
hape-memory materials alter their mor- framework materials in which the application
phological appearance in response to an of an adsorption stress deforms the original shape
S
external stimulus (for example, mechanical of the nanopore into a temporary shape, which Pores Project, Japan Science and Technology Agency (JST), Kyoto
Research Park Building #3, Shimogyo-ku, Kyoto 600-8815,
stress created by macroscopic structural deforma- is maintained even after desorption, and thermal
tion), hold their new temporary shape after the treatment then recovers the original shape.
Japan. 3Department of Synthetic Chemistry and Biological Chem-
istry, Graduate School of Engineering, Kyoto University, Katsura,
Nishikyo-ku, Kyoto 615-8510, Japan. 4Synchrotron X-ray Station
at SPring-8, National Institute for Materials Science (NIMS),
Kouto, Sayo, Hyogo 679-5148, Japan. 5Frontiers of Innovative
Research in Science and Technology (FIRST), Konan University,
7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047,
Japan.
stimulus has been removed, and return to their
Our design takes advantage of the flexibility
original morphology in the presence of another of crystalline porous coordination polymers (PCPs)
external stimulus (1, 2). For instance, a metal alloy (3–8), which are assembled from organic spokes
can exhibit shape-memory effect if it has two and inorganic joints. These flexible PCPs coop-
phases that can interconvert reversibly; that is, eratively reconfigure their framework structures
without requiring atoms to diffuse through the in response to the incorporation of molecules into
structure. Here, we describe a molecular-scale the nanopores; this adsorption process triggers
*To whom correspondence should be addressed. E-mail:
shuhei.furukawa@icems.kyoto-u.ac.jp (S.F.); kitagawa@
shape-memory effect (MSME) in nanoporous the deformation of the pore shape. Most flexible icems.kyoto-u.ac.jp (S.K.)
www.sciencemag.org SCIENCE VOL 339 11 JANUARY 2013
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