University of Groningen
Novel peptide replicators from dynamic combinatorial libraries
Altay, Yigit
DOI:
10.33612/diss.90041906
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Publication date: 2019
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Altay, Y. (2019). Novel peptide replicators from dynamic combinatorial libraries. University of Groningen. https://doi.org/10.33612/diss.90041906
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Y. Altay, M. Altay and S. Otto – “Existing Self-Replicators Can Direct the Emergence of New Ones”, Eur. J. Chem., 2018, 24, 11911-11915.
Chapter 4
Existing Self-Replicators Can Direct the
Emergence of New Ones
Science is the most reliable guide in life.
Mustafa Kemal Atat¨urk
Abstract
The study of the interplay between different self-replicating molecules consti-tutes an important new phase in the synthesis of life and in unravelling the origin of life. Here we show how existing replicators can direct the nature of a newly formed replicator. Starting from the same building block, 6-ring tors formed when the mixture was exposed to pre-existing 6-membered replica-tors, while pre-formed 8-membered replicators funneled the building block into 8-ring replicators. Not only ring size, but also the mode of assembly of the rings into stacks was inherited from the pre-existing replicators. These results show that the nature of self-replicating molecules can be strongly influenced by the interplay between different self-replicators, overriding preferences innate to the structure of the building block.
164 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.1
Introduction
T
he process through which chemistry can transition into biology remains shrouded in mystery, yet represents one of the grand challenges in contemporary sci-ence.1–3 At some stage in the origin of life and in the process of synthesizing life de novo, the ability to replicate needs to emerge. A good understanding of the re-quirements for self-replication now exists4–9and several chemical self-replicating sys-tems have been reported.4–15 Also the emergence of self-replicators from relatively complex mixtures (in particular dynamic combinatorial libraries, DCLs16–21) has been described by Philp22–25and by our group.26–33Our group previously developed DLCs using building blocks equipped with two thiol groups that can oxidize to form a disulfide macrocycles which continuously exchange building blocks through reversible disulfide exchange reactions. These building blocks were equipped with short peptide chains, containing alternating hydrophilic and hydrophobic amino acids, predisposing them to the formation of β-sheet assemblies. Beyond a critical macrocycle size (which depends on the peptide sequence)29 macrocycles can assemble into stacks, held to-gether by β- sheets. This assembly process stabilizes the macrocycles that assemble and leads to the autocatalytic formation of more of these assembling macrocycles (i.e. self-replication) through a nucleation-growth mechanism. Exponential replica-tion can be enabled by mechanical agitareplica-tion that causes growing fibers to fragment, thereby increases the number of fiber ends from which the fibers grow.27Until now, most efforts have focused on systems in which only a single replicator emerges and persists.The next phase in the development of such inanimate systems towards life involves the evolution of replicators5,34,35 and, subsequently, replicator communities.34–40 In this new phase new questions arise, including: How do replicators interact and what are the consequences of such interactions? And how do existing replicators affect the emergence of new ones? While answers to the former question can be informed by knowledge from contemporary biology, where much is known about how species interact, the latter question has no known counterpart in biology. In current life, all new species derive from existing ones and no new life seems to emerge from scratch. Yet in the early stages of biogenesis replicator emergence is likely to have been much more common.
In this chapter, we describe that existing self-replicators can steer the emergence of new ones, resulting in self-replicators that are different from those that would have emerged in the absence of pre-existing replicators. Thus, replicator composition is not merely dictated by the availability of specific precursors, but becomes dependent on the history of the sample and interactions with pre-existing replicators.
4.1. Introduction 165
Figure 4.1: a) Structures of the dithiol building blocks. b) Oxidation of the tyrosine con-taining building block leads to formation of a dynamic combinatorial library of differently sized macrocyclic disulfides. c) Selective formation of 16or 18upon cross-seeding with other
pre-formed replicators made from building blocks 2-6 and schematic representation of a ten-tative mechanism through which these pre-existing replicators direct the formation of the new replicator 16 and 18.
164 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.1
Introduction
T
he process through which chemistry can transition into biology remains shrouded in mystery, yet represents one of the grand challenges in contemporary sci-ence.1–3 At some stage in the origin of life and in the process of synthesizing life de novo, the ability to replicate needs to emerge. A good understanding of the re-quirements for self-replication now exists4–9and several chemical self-replicating sys-tems have been reported.4–15 Also the emergence of self-replicators from relatively complex mixtures (in particular dynamic combinatorial libraries, DCLs16–21) has been described by Philp22–25and by our group.26–33Our group previously developed DLCs using building blocks equipped with two thiol groups that can oxidize to form a disulfide macrocycles which continuously exchange building blocks through reversible disulfide exchange reactions. These building blocks were equipped with short peptide chains, containing alternating hydrophilic and hydrophobic amino acids, predisposing them to the formation of β-sheet assemblies. Beyond a critical macrocycle size (which depends on the peptide sequence)29 macrocycles can assemble into stacks, held to-gether by β- sheets. This assembly process stabilizes the macrocycles that assemble and leads to the autocatalytic formation of more of these assembling macrocycles (i.e. self-replication) through a nucleation-growth mechanism. Exponential replica-tion can be enabled by mechanical agitareplica-tion that causes growing fibers to fragment, thereby increases the number of fiber ends from which the fibers grow.27Until now, most efforts have focused on systems in which only a single replicator emerges and persists.The next phase in the development of such inanimate systems towards life involves the evolution of replicators5,34,35 and, subsequently, replicator communities.34–40In this new phase new questions arise, including: How do replicators interact and what are the consequences of such interactions? And how do existing replicators affect the emergence of new ones? While answers to the former question can be informed by knowledge from contemporary biology, where much is known about how species interact, the latter question has no known counterpart in biology. In current life, all new species derive from existing ones and no new life seems to emerge from scratch. Yet in the early stages of biogenesis replicator emergence is likely to have been much more common.
In this chapter, we describe that existing self-replicators can steer the emergence of new ones, resulting in self-replicators that are different from those that would have emerged in the absence of pre-existing replicators. Thus, replicator composition is not merely dictated by the availability of specific precursors, but becomes dependent on the history of the sample and interactions with pre-existing replicators.
4.1. Introduction 165
Figure 4.1: a) Structures of the dithiol building blocks. b) Oxidation of the tyrosine con-taining building block leads to formation of a dynamic combinatorial library of differently sized macrocyclic disulfides. c) Selective formation of 16or 18upon cross-seeding with other
pre-formed replicators made from building blocks 2-6 and schematic representation of a ten-tative mechanism through which these pre-existing replicators direct the formation of the new replicator 16 and 18.
166 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.2
Results and Discussion
In the course of our work on the emergence of replicators from DCLs26–33 we
pre-pared building block 1 (Figure 4.1), differing from our previously reported building blocks by featuring a tyrosine residue. We prepared DCLs by oxidizing 1 at 3.8 mM concentration in borate buffer (50 mM, pH 8.2). Depending on the speed of oxidation and the mode of agitation the product distribution differs markedly. When slowly oxidizing a stirred solution by exposing it to oxygen from the air, the cyclic trimer 13 is the dominant product (Figure 4.2a). Repeating this experiment in the absence
of agitation produced a mixture of trimer and tetramer macrocycles (Figure 4.2b). Oxidizing the solution rapidly to 80% using perborate and subsequently placing it under an inert atmosphere also produced a mixture of 13and 14(Figure 4.2c).
How-ever, when this experiment was repeated but now the sample was exposed to air after being oxidized with perborate, cyclic octamer was formed (Figure 4.2d). This behavior was found to be qualitatively reproducible (see Figure 4.8).
Figure 4.2: Kinetic profiles41(monitored at 254 nm) of libraries made from peptide 1 (3.8
mM in 50 mM borate buffer, pH 8.2) a) stirred at 1200 rpm in air, b) left non-agitated and exposed to air, c) oxidized to 80% by perborate and stirred at 1200 rpm under inert atmosphere, d) oxidized to 80% by perborate and stirred at 1200 rpm while exposed to air. Data for the repeats of these experiments are shown in Figure 4.8.
4.2. Results and Discussion 167
For the samples that produced mixtures of 13and 14 we did not detect any
self-assembled structures by TEM analysis. However, for the experiments of Figure 4.2a and d, producing mainly 13or 18, respectively, TEM analysis revealed the presence
of fibers (Figure S72). Seeding experiments confirmed that 18is able to self-replicate
(vide supra). Similar experiments on 13 were inconclusive, as the emerge of 13 is
limited by the rate of oxidation of 1 (cf. Figure 4.2a).
The results above show that the behavior of DCLs made from 1 is unusually sen-sitive to small changes in the experimental conditions and that the different products that are formed are separated by relatively high activation energy barriers. The rel-atively high plasticity of this system (even some hexamer can be formed transiently - cf. Figure 4.2a) makes it an ideal candidate to probe the extent to which replicator emergence can directed by introducing other replicators.
Figure 4.3: Kinetic profiles (monitored at 254 nm) of replicator growth for libraries of peptide 1 (3.8 mM in 50 mM borate buffer, pH 8.2. Samples were oxidized to 80 mol% with respect to monomer (with a 80 mM perborate solution) and then exposed to 10 mol% of a) 26, b) 36, c) 46, d) 56, e) 26, f) 68.
168 4. Existing Self-Replicators Can Direct the Emergence of New Ones Thus, we exposed freshly prepared DLCs made from building block 1 to a series of different replicators: 26, 36, 46, 56, 58 and 68 (10 mol%) which we prepared fol-lowing previously described procedures.29 Remarkably, all DCLs to which hexamer replicators (26, 36, 46and 56) were added exhibited product distributions dominated by hexamer 16 (Figure 4.3a-d). Furthermore, all libraries seeded with octamer repli-cators (58and 68) formed 18dominated libraries (Figure 4.3e-f). In some cases also mixed macrocycles were observed (1n26-n in the presence of 26 and 1n58-n in the presence of 58). Thus, pre-existing replicators control the ring size of the new repli-cators made from building block 1, in some cases overriding the inherent preference of 1 to produce octamer replicator.
Figure 4.4: Kinetic profiles (monitored at 254 nm) of replicator growth for libraries made from building block 1 (3.8 mM in 50 mM borate buffer, pH 8.2). Samples are pre-oxidized to 80 mol% with respect to monomer (with a 80 mM perborate solution), non-seeded (blue) or seeded (red) on day 0 with 10 mol% of (a) 18 obtained from the DCL corresponding to
Figure 2f and (b) 16 obtained from the DCL corresponding to Figure 4.3a. For clarity only
the data of the relevant macrocycle size is shown.
In order to confirm that 16 and 18 are self-replicators, seeding experiment were performed using the seeds obtained from the libraries corresponding to Figure 4.3a and 4.3f. When a freshly prepared library of peptide 1 is seeded with 10 mol% seed of 18or 16, we observed rapid growth of the corresponding macrocycles (Figure 4.4), relative to the non-seeded control (blue lines), confirming that 18and 16are indeed able to self-replicate.
4.2. Results and Discussion 169
Figure 4.5: CD spectra of samples dominated by a) 16and b) 18and c) normalized
max-imum thioflavin T fluorescence emission intensity (at 492 nm) of non-seeded or seeded (10 mol %) DCLs made from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): i, non-agitated; ii, stirred at 1200 rpm and kept under a nitrogen atmosphere; iii, stirred at 1200 rpm in the presence of air; iv, seeded with 46; v, seeded with 56; vi, seeded with 36; vii, seeded with
26; viii, oxidized to 80% using perborate and stirred at 1200 rpm; ix, seeded with 58; x,
seeded with 68.
The structures of the newly formed 16 and 18 replicators were characterized by circular dichroism (CD) spectroscopy, thioflavin T fluorescence assays, TEM and IR spectroscopy. The CD spectra of the samples dominated by 16 showed a pos-itive helicity at around 190 nm and a negative helicity at 210 nm (Figure 4.5a). These bands appear at wavelengths that are somewhat smaller than those typical for β-sheets. According to Pribic et al.42 such shifted signals may arise in tyrosine con-taining peptides due to π-π* transitions that complicate the far UV region of the CD spectrum and are still in agreement with a β-sheet structure. The three libraries that
168 4. Existing Self-Replicators Can Direct the Emergence of New Ones Thus, we exposed freshly prepared DLCs made from building block 1 to a series of different replicators: 26, 36, 46, 56, 58 and 68 (10 mol%) which we prepared fol-lowing previously described procedures.29 Remarkably, all DCLs to which hexamer replicators (26, 36, 46and 56) were added exhibited product distributions dominated by hexamer 16(Figure 4.3a-d). Furthermore, all libraries seeded with octamer repli-cators (58 and 68) formed 18dominated libraries (Figure 4.3e-f). In some cases also mixed macrocycles were observed (1n26-n in the presence of 26 and 1n58-n in the presence of 58). Thus, pre-existing replicators control the ring size of the new repli-cators made from building block 1, in some cases overriding the inherent preference of 1 to produce octamer replicator.
Figure 4.4: Kinetic profiles (monitored at 254 nm) of replicator growth for libraries made from building block 1 (3.8 mM in 50 mM borate buffer, pH 8.2). Samples are pre-oxidized to 80 mol% with respect to monomer (with a 80 mM perborate solution), non-seeded (blue) or seeded (red) on day 0 with 10 mol% of (a) 18 obtained from the DCL corresponding to
Figure 2f and (b) 16 obtained from the DCL corresponding to Figure 4.3a. For clarity only
the data of the relevant macrocycle size is shown.
In order to confirm that 16 and 18 are self-replicators, seeding experiment were performed using the seeds obtained from the libraries corresponding to Figure 4.3a and 4.3f. When a freshly prepared library of peptide 1 is seeded with 10 mol% seed of 18or 16, we observed rapid growth of the corresponding macrocycles (Figure 4.4), relative to the non-seeded control (blue lines), confirming that 18and 16 are indeed able to self-replicate.
4.2. Results and Discussion 169
Figure 4.5: CD spectra of samples dominated by a) 16 and b) 18and c) normalized
max-imum thioflavin T fluorescence emission intensity (at 492 nm) of non-seeded or seeded (10 mol %) DCLs made from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): i, non-agitated; ii, stirred at 1200 rpm and kept under a nitrogen atmosphere; iii, stirred at 1200 rpm in the presence of air; iv, seeded with 46; v, seeded with 56; vi, seeded with 36; vii, seeded with
26; viii, oxidized to 80% using perborate and stirred at 1200 rpm; ix, seeded with 58; x,
seeded with 68.
The structures of the newly formed 16 and 18 replicators were characterized by circular dichroism (CD) spectroscopy, thioflavin T fluorescence assays, TEM and IR spectroscopy. The CD spectra of the samples dominated by 16 showed a pos-itive helicity at around 190 nm and a negative helicity at 210 nm (Figure 4.5a). These bands appear at wavelengths that are somewhat smaller than those typical for β-sheets. According to Pribic et al.42such shifted signals may arise in tyrosine con-taining peptides due to π-π* transitions that complicate the far UV region of the CD spectrum and are still in agreement with a β-sheet structure. The three libraries that
170 4. Existing Self-Replicators Can Direct the Emergence of New Ones produced 18 replicators showed three rather different signatures in their CD spec-tra (Figure 4.5b) which we tentatively assigned42to parallel β-sheet (for the sample in which 18 emerged autonomously), anti-parallel β-sheet (when the formation of 18 was triggered by 58) and mixed (parallel and anti-parallel) β-sheet structures (when the formation of 18 was triggered by 68). Thioflavin T assays showed an at least 40 times increase in emission for the 16 containing sample and an at least 20 times increase for the 18 containing samples, compared to samples in the absence of replicator, which supports amyloid type β-sheet structures for all samples.
Negative staining transmission electron microscopy (TEM) revealed bundles of fibers having a right-handed helicity for all samples dominated by 16 (Figure 4.6a-d and Figure 4.80). For the samples of 18 that showed parallel β-sheets we observed single fibers with a width of„4.5 nm. In contrast, for samples 18that showed anti-parallel β-sheets we observed a high degree of lateral association of the fibers. In the sample of 18 that showed mixed β-sheets we observed laterally associated fibers along with single fibers. Thus, it appears that the lateral association of the fibers occurs through antiparallel β-sheet formation.
Figure 4.6: Transmission electron microscopy images of DCLs made from 1 seeded with (a) 26; (b) 36; (c) 46; (d) 56; (e) 58 and (f) 68.
4.2. Results and Discussion 171
We further characterized the different assemblies of 16 and 18 by IR spectroscopy (Figure 4.7). The frequency of the C=O bands are in the range expected for β-sheet assemblies.43 Only for the sample of 1
8 where we suspect the formation of antiparallel β-sheets and which exhibited extensive lateral association of the fibers, we observed an additional band at 1615 cm-1, which is associated with the phenyl ring of tyrosine. The fact that this band is only observed for the 18 sample that shows extensive laterally associated fibers suggests that in this sample those phenols are in a an environment that differs from the one in a non-associated fiber, which would be in agreement with the postulated occurrence of parallel and antiparallel β-sheet assemblies.
Figure 4.7: ATR-IR spectra of different replicators obtained from DCLs made from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): vii, seeded with 26; viii, oxidized to 80% using
perborate and stirred at 1200 rpm in the presence of air; ix, seeded with 58. Assignment:
TFA at 1675 cm-1; amide I band (C=O) at 1631 cm-1; C-C ring stretching at 1615 cm-1;
amide II band (C-N) at 1537 cm-1; C-C ring stretching at 1515 cm-1.
Taken together these results suggest that replicator 18 can exhibit different modes of assembly: one in which its fibers show lateral association through anti-parallel β-sheet formation and one in which such interactions are absent. Remarkably, this mode of assembly is dictated by the replicator that triggered its emergence. Thus, not only the information regarding ring size is transferred, but also information regarding the mode of assembly of these rings. Seeding experiments showed that both forms of information are to some extent hereditable: seeding a DCL made from 1 with a sample of 18 that showed laterally associated fibers induced the formation of more 18 that also showed fiber bundles, while seeding a similar DCL with a sample of 18
170 4. Existing Self-Replicators Can Direct the Emergence of New Ones produced 18 replicators showed three rather different signatures in their CD spec-tra (Figure 4.5b) which we tentatively assigned42 to parallel β-sheet (for the sample in which 18 emerged autonomously), anti-parallel β-sheet (when the formation of 18 was triggered by 58) and mixed (parallel and anti-parallel) β-sheet structures (when the formation of 18 was triggered by 68). Thioflavin T assays showed an at least 40 times increase in emission for the 16 containing sample and an at least 20 times increase for the 18 containing samples, compared to samples in the absence of replicator, which supports amyloid type β-sheet structures for all samples.
Negative staining transmission electron microscopy (TEM) revealed bundles of fibers having a right-handed helicity for all samples dominated by 16 (Figure 4.6a-d and Figure 4.80). For the samples of 18 that showed parallel β-sheets we observed single fibers with a width of„4.5 nm. In contrast, for samples 18 that showed anti-parallel β-sheets we observed a high degree of lateral association of the fibers. In the sample of 18 that showed mixed β-sheets we observed laterally associated fibers along with single fibers. Thus, it appears that the lateral association of the fibers occurs through antiparallel β-sheet formation.
Figure 4.6: Transmission electron microscopy images of DCLs made from 1 seeded with (a) 26; (b) 36; (c) 46; (d) 56; (e) 58 and (f) 68.
4.2. Results and Discussion 171
We further characterized the different assemblies of 16 and 18 by IR spectroscopy (Figure 4.7). The frequency of the C=O bands are in the range expected for β-sheet assemblies.43 Only for the sample of 1
8 where we suspect the formation of antiparallel β-sheets and which exhibited extensive lateral association of the fibers, we observed an additional band at 1615 cm-1, which is associated with the phenyl ring of tyrosine. The fact that this band is only observed for the 18 sample that shows extensive laterally associated fibers suggests that in this sample those phenols are in a an environment that differs from the one in a non-associated fiber, which would be in agreement with the postulated occurrence of parallel and antiparallel β-sheet assemblies.
Figure 4.7: ATR-IR spectra of different replicators obtained from DCLs made from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): vii, seeded with 26; viii, oxidized to 80% using
perborate and stirred at 1200 rpm in the presence of air; ix, seeded with 58. Assignment:
TFA at 1675 cm-1; amide I band (C=O) at 1631 cm-1; C-C ring stretching at 1615 cm-1;
amide II band (C-N) at 1537 cm-1; C-C ring stretching at 1515 cm-1.
Taken together these results suggest that replicator 18 can exhibit different modes of assembly: one in which its fibers show lateral association through anti-parallel β-sheet formation and one in which such interactions are absent. Remarkably, this mode of assembly is dictated by the replicator that triggered its emergence. Thus, not only the information regarding ring size is transferred, but also information regarding the mode of assembly of these rings. Seeding experiments showed that both forms of information are to some extent hereditable: seeding a DCL made from 1 with a sample of 18 that showed laterally associated fibers induced the formation of more 18 that also showed fiber bundles, while seeding a similar DCL with a sample of 18
172 4. Existing Self-Replicators Can Direct the Emergence of New Ones that showed no fiber bundling induced more 18 that existed as non-associated fibers (See Figure 4.81).
Finally, we investigated the influence of the sample history on replicator com-position. We prepared DCLs having the same building block compositions as those shown in Figures 4.3a-f, but now the building blocks were mixed prior to oxidation. All of the DCLs produced 13 and 14 along with mixed trimers and tetramers (See Figure 4.52-4.75), but no significant amount of any replicator. These data show that the history of the sample is an essential factor determining not only the nature, but also the presence or absence of replicators.
4.3
Conclusions
In summary, our results show that both the molecular structure (ring size) and the mode of assembly (parallel or antiparallel β- sheets and degree of fiber bundling) of newly emerging replicators can be controlled by pre-existing replicators. The in-teractions between replicators can override the preference for a particular structure and ring size innate to the structure of the building blocks of the replicator. While involving similar (but not identical) replicators to the ones reported in a previous study,26 our current system show behavior that is exactly opposite to that reported previously. In the previous work we showed how one specific pre-existing replica-tors can help the formation of one other specific replicator, that does not readily form by itself. This behavior was only observed for these two specific replicators and the structure of the newly formed replicator appeared to be invariable. Our current results shows that replicators, rather than having an apparently predeter-mined structure, can also be highly plastic and respond to the presence of any of a range of pre-existing replicators by adopting the ring size of the specific replicator it was exposed to. As a consequence the composition and nature of the system of replicators reflects sample history and inter-replicator interactions, which are both key prerequisites for Darwinian evolution.
4.4
Acknowledgements
M. Altay is gratefully acknowledged for discussions on the results, proof-reading this chapter and for giving precious feedback.
4.5. Materials and Methods 173
4.5
Materials and Methods
4.5.1
Materials
Doubly distilled water was used in all experiments. Boric anhydride (Sigma-Aldrich) and sodium hydroxide (Merck Chemicals) were utilized for buffer preparation and pH adjustments. For UPLC measurements, UPLC grade acetonitrile, water and trifluoroacetic acid were purchased from Biosolve BV.
Solvents used in the peptide synthesis were reagent grade. Dichloromethane, dimethylformamide, oxyma and N,N-diisopropylcarbodiimide were used without fur-ther purification. Reagents were purchased from Sigma-Aldrich, Merck, Fluka, or Acros and used as received. Preloaded resins and Fmoc-protected amino acids were purchased from Merck Chemicals and Sigma Aldrich.
4.5.2
Peptide Synthesis
Peptides 1 and 4 were synthesized by conventional peptide synthesis using pre-loaded Wang resins. The peptides were purified by preparative HPLC using a Phenomenex Jupiter (10µm, C5, 300 ˚A, 250ˆ21.2mm) column.
Peptides 2, 3, 5 and 6 were synthesized by Cambridge Peptides Ltd. (Birming-ham, U.K.) by coupling 3,5-bis(tritylthio)-benzoic acid, which was synthesized via a previously reported procedure,44 at the N-termini. All peptides had purity higher than 95%. Impurities were mainly resulting from the formation of disulfides (i.e. dimers, trimers etc.).
4.5.3
Library Preparation and Sampling
Building blocks were dissolved in borate buffer (50 mM, pH 8.2) to prepare a library of peptides at 3.8 mM concentration. The volume of each library was at least 500µL. All libraries were equilibrated in an HPLC vial (12ˆ32 mm) with a Teflon-coated screw cap. Except for the non-stirred samples, all HPLC vials were equipped with a cylindrical stirrer bar (2ˆ5 mm, Teflon coated, purchased from VWR) and were stirred at 1200 rpm using an IKA RCT basic hot plate stirrer. All experiments were performed at ambient conditions except for those set up in a glovebox under an inert atmosphere (corresponding to Figure 4.27). For sampling, 10µL of each sample was transferred to another vial containing 10µL DMF and diluted to 200 µL with UPLC grade water prior to UPLC or LC-MS analysis.
172 4. Existing Self-Replicators Can Direct the Emergence of New Ones that showed no fiber bundling induced more 18that existed as non-associated fibers (See Figure 4.81).
Finally, we investigated the influence of the sample history on replicator com-position. We prepared DCLs having the same building block compositions as those shown in Figures 4.3a-f, but now the building blocks were mixed prior to oxidation. All of the DCLs produced 13 and 14 along with mixed trimers and tetramers (See Figure 4.52-4.75), but no significant amount of any replicator. These data show that the history of the sample is an essential factor determining not only the nature, but also the presence or absence of replicators.
4.3
Conclusions
In summary, our results show that both the molecular structure (ring size) and the mode of assembly (parallel or antiparallel β- sheets and degree of fiber bundling) of newly emerging replicators can be controlled by pre-existing replicators. The in-teractions between replicators can override the preference for a particular structure and ring size innate to the structure of the building blocks of the replicator. While involving similar (but not identical) replicators to the ones reported in a previous study,26 our current system show behavior that is exactly opposite to that reported previously. In the previous work we showed how one specific pre-existing replica-tors can help the formation of one other specific replicator, that does not readily form by itself. This behavior was only observed for these two specific replicators and the structure of the newly formed replicator appeared to be invariable. Our current results shows that replicators, rather than having an apparently predeter-mined structure, can also be highly plastic and respond to the presence of any of a range of pre-existing replicators by adopting the ring size of the specific replicator it was exposed to. As a consequence the composition and nature of the system of replicators reflects sample history and inter-replicator interactions, which are both key prerequisites for Darwinian evolution.
4.4
Acknowledgements
M. Altay is gratefully acknowledged for discussions on the results, proof-reading this chapter and for giving precious feedback.
4.5. Materials and Methods 173
4.5
Materials and Methods
4.5.1
Materials
Doubly distilled water was used in all experiments. Boric anhydride (Sigma-Aldrich) and sodium hydroxide (Merck Chemicals) were utilized for buffer preparation and pH adjustments. For UPLC measurements, UPLC grade acetonitrile, water and trifluoroacetic acid were purchased from Biosolve BV.
Solvents used in the peptide synthesis were reagent grade. Dichloromethane, dimethylformamide, oxyma and N,N-diisopropylcarbodiimide were used without fur-ther purification. Reagents were purchased from Sigma-Aldrich, Merck, Fluka, or Acros and used as received. Preloaded resins and Fmoc-protected amino acids were purchased from Merck Chemicals and Sigma Aldrich.
4.5.2
Peptide Synthesis
Peptides 1 and 4 were synthesized by conventional peptide synthesis using pre-loaded Wang resins. The peptides were purified by preparative HPLC using a Phenomenex Jupiter (10µm, C5, 300 ˚A, 250ˆ21.2mm) column.
Peptides 2, 3, 5 and 6 were synthesized by Cambridge Peptides Ltd. (Birming-ham, U.K.) by coupling 3,5-bis(tritylthio)-benzoic acid, which was synthesized via a previously reported procedure,44 at the N-termini. All peptides had purity higher than 95%. Impurities were mainly resulting from the formation of disulfides (i.e. dimers, trimers etc.).
4.5.3
Library Preparation and Sampling
Building blocks were dissolved in borate buffer (50 mM, pH 8.2) to prepare a library of peptides at 3.8 mM concentration. The volume of each library was at least 500µL. All libraries were equilibrated in an HPLC vial (12ˆ32 mm) with a Teflon-coated screw cap. Except for the non-stirred samples, all HPLC vials were equipped with a cylindrical stirrer bar (2ˆ5 mm, Teflon coated, purchased from VWR) and were stirred at 1200 rpm using an IKA RCT basic hot plate stirrer. All experiments were performed at ambient conditions except for those set up in a glovebox under an inert atmosphere (corresponding to Figure 4.27). For sampling, 10µL of each sample was transferred to another vial containing 10µL DMF and diluted to 200 µL with UPLC grade water prior to UPLC or LC-MS analysis.
174 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.5.4
Seeding Experiments
A freshly prepared library of peptide 1 (3.8 mM) was oxidized to 80% with freshly prepared sodium perborate solution (80 mM) such that it contained mostly monomer, trimer and tetramer. After 15-30 min, a previously prepared solution containing predominantly hexamer, 16or octamer, 18 (10 mol %) was added. All libraries were monitored with UPLC and UPLC-MS.
4.5.5
Cross Seeding Experiments
For libraries seeded with pre-existing replicators, solutions containing mostly 11, 13 and 14 were prepared according to the procedure described above. Pre-formed seed (10 mol %) 26, 36, 46, 56, 58or 68was added. All libraries were stirred at 1200 rpm and monitored by UPLC and UPLC-MS.
4.5.6
Circular Dichroism (CD)
Samples were prepared by diluting libraries to 0.24 mM (with respect to building block concentration). All spectra were collected in a Jasco J-715 CD spectropo-larimeter using HELMA quartz cuvettes with path length of 1.0 mm. Continuous spectra were obtained from 190 nm to 300 nm at room temperature. All reported spectra were an average of three scans recorded at a scan rate of 200 nm/min with 2-nm step interval and measured in millidegrees. Solvent spectra were subtracted from all spectra.
4.5.7
Thioflavin T (ThT) Fluorescence Assay
A ThT stock solution (2.2 mM) was prepared in 10 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) and filtered through a 0.2µm syringe filter. On the day of analysis, 50µL of the stock solution was diluted into 5 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) to generate the working solution of 22µm. The fluorescence intensity of 450µL ThT solution was measured by excitation at 440 nm (slit width 5 nm) and emission between 480-700 nm (slit width 5 nm), averaging 3 accumulations. An aliquot of 80µL of peptide solution (100 µL in borate buffer) was added to the HELMA 10ˆ2 mm quartz cuvette, incubated for 2 min, and the intensity was measured over 3 accumulations. All fluorescence measurements were performed on a JASCO FP6200 fluorimeter equipped with a 480 nm high pass cut-off filter on the emission channel to avoid high order diffractions coming from the excitation.
4.5. Materials and Methods 175
4.5.8
Fourier Transform Infrared Spectroscopy
FTIR measurements were carried out using a FTIR spectrometer (PerkinElmer Spec-trum Series 400) equipped with a Ge ATR device. Measurements were carried out in the spectral range 450-4000 cm-1 with a resolution of 4 cm-1. Each spectrum was an average of 64 scans to increase the signal to noise ratio. Freeze dried samples were directly placed on the Ge ATR crystal.
4.5.9
Negative Staining Transmission Electron Microscopy
A small drop (5µL) of sample was deposited on a 400 mesh copper grid covered with a thin carbon film (Agar Scientific). After 30 s, the droplet was blotted on filter paper. The sample was then stained twice (4µL each time) with a solution of 2% uranyl acetate deposited on the grid and blotted on the filter paper after 30 s each time. The grids were observed in a Philips CM120 cryo-electron microscope operating at 120 kV. Images were recorded on a slow scan CCD camera.
174 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.5.4
Seeding Experiments
A freshly prepared library of peptide 1 (3.8 mM) was oxidized to 80% with freshly prepared sodium perborate solution (80 mM) such that it contained mostly monomer, trimer and tetramer. After 15-30 min, a previously prepared solution containing predominantly hexamer, 16or octamer, 18 (10 mol %) was added. All libraries were monitored with UPLC and UPLC-MS.
4.5.5
Cross Seeding Experiments
For libraries seeded with pre-existing replicators, solutions containing mostly 11, 13 and 14 were prepared according to the procedure described above. Pre-formed seed (10 mol %) 26, 36, 46, 56, 58or 68was added. All libraries were stirred at 1200 rpm and monitored by UPLC and UPLC-MS.
4.5.6
Circular Dichroism (CD)
Samples were prepared by diluting libraries to 0.24 mM (with respect to building block concentration). All spectra were collected in a Jasco J-715 CD spectropo-larimeter using HELMA quartz cuvettes with path length of 1.0 mm. Continuous spectra were obtained from 190 nm to 300 nm at room temperature. All reported spectra were an average of three scans recorded at a scan rate of 200 nm/min with 2-nm step interval and measured in millidegrees. Solvent spectra were subtracted from all spectra.
4.5.7
Thioflavin T (ThT) Fluorescence Assay
A ThT stock solution (2.2 mM) was prepared in 10 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) and filtered through a 0.2µm syringe filter. On the day of analysis, 50µL of the stock solution was diluted into 5 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) to generate the working solution of 22µm. The fluorescence intensity of 450µL ThT solution was measured by excitation at 440 nm (slit width 5 nm) and emission between 480-700 nm (slit width 5 nm), averaging 3 accumulations. An aliquot of 80µL of peptide solution (100 µL in borate buffer) was added to the HELMA 10ˆ2 mm quartz cuvette, incubated for 2 min, and the intensity was measured over 3 accumulations. All fluorescence measurements were performed on a JASCO FP6200 fluorimeter equipped with a 480 nm high pass cut-off filter on the emission channel to avoid high order diffractions coming from the excitation.
4.5. Materials and Methods 175
4.5.8
Fourier Transform Infrared Spectroscopy
FTIR measurements were carried out using a FTIR spectrometer (PerkinElmer Spec-trum Series 400) equipped with a Ge ATR device. Measurements were carried out in the spectral range 450-4000 cm-1with a resolution of 4 cm-1. Each spectrum was an average of 64 scans to increase the signal to noise ratio. Freeze dried samples were directly placed on the Ge ATR crystal.
4.5.9
Negative Staining Transmission Electron Microscopy
A small drop (5µL) of sample was deposited on a 400 mesh copper grid covered with a thin carbon film (Agar Scientific). After 30 s, the droplet was blotted on filter paper. The sample was then stained twice (4µL each time) with a solution of 2% uranyl acetate deposited on the grid and blotted on the filter paper after 30 s each time. The grids were observed in a Philips CM120 cryo-electron microscope operating at 120 kV. Images were recorded on a slow scan CCD camera.
176 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.6
Kinetic Profiles
Figure 4.8: Kinetic profiles of libraries made from peptide 1 (3.8 mm in 50 mM borate buffer, pH 8.12) (a) stirred at 1200 rpm without any pre-oxidation, (b) left non-agitated, (c) 80% pre-oxidized with perborate solution (80 mM) and stirred at 1200 rpm under inert atmosphere, (d) 80% pre-oxidized with perborate solution (80 mM) and stirred at 1200 rpm. Plots shown on the same line represent repeats of the same experiment.
4.7. UPLC and LC-MS analyses 177
Figure 4.9: Kinetic profiles of libraries made from peptide 1 (3.8 mm in 50 mM borate buffer, pH 8.12), 80% pre-oxidized with perborate solution (80 mM) and seeded with 10 mol % (a) 26 (b) 36 (c) 46 (d) 56 (e) 58 (f) 68. All libraries stirred at 1200 rpm. Plots shown
on the same line represent repeats of the same experiment.
These systems, like similar replicating systems we investigated before, are prone to considerable quantitative variability that is mostly likely a result of the inherent sensitivity of the kinetics of exponentially growing systems to small perturbations.
4.7
UPLC and LC-MS analyses
UPLC analyses were performed on a Waters Acquity UPLC I-class system equipped with a PDA detector. A reversed-phase UPLC column (Aeris 1.7µm. XB-C18 150 ˆ 2.10 mm, purchased from Phenomenex) was used in the analyses of all samples, while UV absorbance was monitored at 254 nm. The column temperature was equilibrated at 35˝C prior to injections.
UPLC-MS analyses were performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF. The mass spectrometer was operated under the following conditions: electrospray ionization mode, positive; capillary voltage, 2.5 kV; sampling cone voltage, 30 V; extraction cone voltage, 4.0 V. Samples were prepared
176 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.6
Kinetic Profiles
Figure 4.8: Kinetic profiles of libraries made from peptide 1 (3.8 mm in 50 mM borate buffer, pH 8.12) (a) stirred at 1200 rpm without any pre-oxidation, (b) left non-agitated, (c) 80% pre-oxidized with perborate solution (80 mM) and stirred at 1200 rpm under inert atmosphere, (d) 80% pre-oxidized with perborate solution (80 mM) and stirred at 1200 rpm. Plots shown on the same line represent repeats of the same experiment.
4.7. UPLC and LC-MS analyses 177
Figure 4.9: Kinetic profiles of libraries made from peptide 1 (3.8 mm in 50 mM borate buffer, pH 8.12), 80% pre-oxidized with perborate solution (80 mM) and seeded with 10 mol % (a) 26 (b) 36 (c) 46 (d) 56 (e) 58 (f) 68. All libraries stirred at 1200 rpm. Plots shown
on the same line represent repeats of the same experiment.
These systems, like similar replicating systems we investigated before, are prone to considerable quantitative variability that is mostly likely a result of the inherent sensitivity of the kinetics of exponentially growing systems to small perturbations.
4.7
UPLC and LC-MS analyses
UPLC analyses were performed on a Waters Acquity UPLC I-class system equipped with a PDA detector. A reversed-phase UPLC column (Aeris 1.7µm. XB-C18 150 ˆ 2.10 mm, purchased from Phenomenex) was used in the analyses of all samples, while UV absorbance was monitored at 254 nm. The column temperature was equilibrated at 35˝C prior to injections.
UPLC-MS analyses were performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF. The mass spectrometer was operated under the following conditions: electrospray ionization mode, positive; capillary voltage, 2.5 kV; sampling cone voltage, 30 V; extraction cone voltage, 4.0 V. Samples were prepared
178 4. Existing Self-Replicators Can Direct the Emergence of New Ones
by dissolving 10µL of a 3.8 mM library in 10 µL DMF and then further diluting them by 180µL UPLC grade water. The operating parameters were the following: eluent flow rate 0.3 mL/min; eluent A, UPLC grade water with 0.1 v% trifluoroacetic acid; eluent B, UPLC grade acetonitrile with 0.1 v% trifluoroacetic acid.
During the analysis, all the peaks that have a peak area more than 1% on the 254 nm trace, is integrated and their contribution is considered for the kinetic plots. However, it is extremely difficult to characterize some peaks. All the building blocks we use have at least 95% purity. Especially due to the possible deletion products in the remaining 5%, they can form macrocycles using other deletion products and also with the actual building blocks. As their concentration do not increase or decrease significantly in the course of these experiments, they are generally omitted from the kinetic profiles and only the most relevant peaks are shown.
4.7.1
UPLC Methods
Libraries were analysed using the following method (linear gradient) and a Phe-nomenex Aeris Peptide column.
Solvent A: ULC/MS grade water (0.1% trifluoroacetic acid added) Solvent B: ULC/MS grade acetonitrile (0.1% trifluoroacetic acid added)
Time, min. A% B% 0.00 90 10 1.00 90 10 1.30 75 25 3.00 72 28 11.00 60 40 11.50 5 95 12.00 5 95 12.50 90 10 17.00 90 10
4.7. UPLC and LC-MS analyses 179
4.7.2
UPLC and LC-MS analysis of Peptide 1
Figure 4.10: UPLC trace (monitored at 254 nm) of monomer of the peptide 1 (3.8 mM in 50 mM borate buffer, pH 8.2) after purification.
Figure 4.11: Mass spectrum of monomer 11 from the LC-MS analysis of peptide 1
af-ter purification (corresponding to Figure 4.10). Calculated isotopic profile for [M+2H]2+
(species, abundance): 388.67 (M, 100%), 389.17 (M+1, 70.84%), 389.68 (M+2, 39.42%); m/z calculated: 776.35 [M+H]1+, 389.18 [M+2H]2+; m/z observed: 776.33 [M+H]1+,
180 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.3
UPLC and LC-MS analysis of Seed 2
6Figure 4.12: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 2 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 20 days. This hexamer sample was used as seed.
Figure 4.13: Mass spectrum of cyclic hexamer 26from the LC-MS analysis of a stirred
library made from peptide 2 (corresponding to Figure 4.12). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1138.21 (M, 100%), 1138.46 (M+1, 99.89%),
1138.69 (M+2, 85.93%), 1138.94 (M+3, 67.79%), 1139.19 (M+4, 46.22%); m/z calcu-lated: 1517.7 [M+3H]3+, 1138.53 [M+4H]4+; 911.02 [M+5H]5+; m/z observed: 1517.29
[M+3H]3+, 1138.21 [M+4H]4+; 910.96 [M+5H]5+.
4.7. UPLC and LC-MS analyses 181
4.7.4
UPLC and LC-MS analysis of Seed 3
6Figure 4.14: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 3 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 42 days. This hexamer sample was used as seed.
Figure 4.15: Mass spectrum of cyclic hexamer 36from the LC-MS analysis of a stirred
library made from peptide 3 (corresponding to Figure 4.14). Calculated isotopic profile for [M+4H]4+(species, abundance): 1147.72 (M, 100%), 1147.97 (M+1, 87.26%), 1148.23
(M+2, 62.93%), 1148.48 (M+3, 46.73%), 1148.73 (M+4, 34.80%); m/z calculated: 1529.80 [M+3H]3+, 1147.60 [M+4H]4+; 918.28 [M+5H]5+; m/z observed: 1529.66 [M+3H]3+,
180 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.3
UPLC and LC-MS analysis of Seed 2
6Figure 4.12: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 2 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 20 days. This hexamer sample was used as seed.
Figure 4.13: Mass spectrum of cyclic hexamer 26from the LC-MS analysis of a stirred
library made from peptide 2 (corresponding to Figure 4.12). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1138.21 (M, 100%), 1138.46 (M+1, 99.89%),
1138.69 (M+2, 85.93%), 1138.94 (M+3, 67.79%), 1139.19 (M+4, 46.22%); m/z calcu-lated: 1517.7 [M+3H]3+, 1138.53 [M+4H]4+; 911.02 [M+5H]5+; m/z observed: 1517.29
[M+3H]3+, 1138.21 [M+4H]4+; 910.96 [M+5H]5+.
4.7. UPLC and LC-MS analyses 181
4.7.4
UPLC and LC-MS analysis of Seed 3
6Figure 4.14: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 3 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 42 days. This hexamer sample was used as seed.
Figure 4.15: Mass spectrum of cyclic hexamer 36from the LC-MS analysis of a stirred
library made from peptide 3 (corresponding to Figure 4.14). Calculated isotopic profile for [M+4H]4+(species, abundance): 1147.72 (M, 100%), 1147.97 (M+1, 87.26%), 1148.23
(M+2, 62.93%), 1148.48 (M+3, 46.73%), 1148.73 (M+4, 34.80%); m/z calculated: 1529.80 [M+3H]3+, 1147.60 [M+4H]4+; 918.28 [M+5H]5+; m/z observed: 1529.66 [M+3H]3+,
182 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.5
UPLC and LC-MS analysis of Seed 4
6Figure 4.16: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 12 days. This hexamer sample was used as seed.
Figure 4.17: Mass spectrum of cyclic hexamer 46from the LC-MS analysis of a stirred
library made from peptide 4 (corresponding to Figure 4.16). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1069.24 (M, 100%), 1069.49 (M+1, 87.12%),
1069.74 (M+2, 68.62%), 1069.99 (M+3, 47.58%), 1070.23 (M+4, 28.03%); m/z calcu-lated: 1425.66 [M+3H]3+, 1069.50 [M+4H]4+; 855.80 [M+5H]5+; m/z observed: 1425.32
[M+3H]3+, 1069.24 [M+4H]4+; 855.58 [M+5H]5+.
4.7. UPLC and LC-MS analyses 183
4.7.6
UPLC and LC-MS analysis of Seed 5
6Figure 4.18: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 42 days. This hexamer sample was used as seed.
Figure 4.19: Mass spectrum of cyclic hexamer 56from the LC-MS analysis of a stirred
library made from peptide 5 (corresponding to Figure 4.18). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1048.55 (M, 100%), 1048.80 (M+1, 77.46%),
1049.01(M+2, 65.72%), 1049.26 (M+3, 48.41%), 1049.52 (M+4, 32.96%); m/z calcu-lated: 1397.64 [M+3H]3+, 1048.48 [M+4H]4+; 838.98 [M+5H]5+; m/z observed: 1397.41
182 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.5
UPLC and LC-MS analysis of Seed 4
6Figure 4.16: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 12 days. This hexamer sample was used as seed.
Figure 4.17: Mass spectrum of cyclic hexamer 46from the LC-MS analysis of a stirred
library made from peptide 4 (corresponding to Figure 4.16). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1069.24 (M, 100%), 1069.49 (M+1, 87.12%),
1069.74 (M+2, 68.62%), 1069.99 (M+3, 47.58%), 1070.23 (M+4, 28.03%); m/z calcu-lated: 1425.66 [M+3H]3+, 1069.50 [M+4H]4+; 855.80 [M+5H]5+; m/z observed: 1425.32
[M+3H]3+, 1069.24 [M+4H]4+; 855.58 [M+5H]5+.
4.7. UPLC and LC-MS analyses 183
4.7.6
UPLC and LC-MS analysis of Seed 5
6Figure 4.18: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 42 days. This hexamer sample was used as seed.
Figure 4.19: Mass spectrum of cyclic hexamer 56from the LC-MS analysis of a stirred
library made from peptide 5 (corresponding to Figure 4.18). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1048.55 (M, 100%), 1048.80 (M+1, 77.46%),
1049.01(M+2, 65.72%), 1049.26 (M+3, 48.41%), 1049.52 (M+4, 32.96%); m/z calcu-lated: 1397.64 [M+3H]3+, 1048.48 [M+4H]4+; 838.98 [M+5H]5+; m/z observed: 1397.41
184 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.7
UPLC and LC-MS analysis of Seed 5
8Figure 4.20: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 20 days. This octamer sample was used as seed.
Figure 4.21: Mass spectrum of cyclic octamer 58from the LC-MS analysis of a stirred
library made from peptide 5 (corresponding to Figure 4.20). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1397.15 (M, 100%), 1397.40 (M+1, 95.17%), 1397.65
(M+2, 78.53%), 1397.90 (M+3, 61.84%), 1398.15 (M+4, 42.69%); m/z calculated: 1863.19 [M+3H]3+, 1397.64 [M+4H]4+; 1118.31 [M+5H]5+; m/z observed: 1862.51 [M+3H]3+,
1397.15 [M+4H]4+; 1117.91 [M+5H]5+.
4.7. UPLC and LC-MS analyses 185
4.7.8
UPLC and LC-MS analysis of Seed 6
8Figure 4.22: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 6 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 14 days. This octamer sample was used as seed.
Figure 4.23: Mass spectrum of cyclic octamer 68from the LC-MS analysis of a stirred
library made from peptide 6 (corresponding to Figure 4.22). Calculated isotopic profile for [M+4H]4+(species, abundance): 1365.14 (M, 100%), 1365.39 (M+1, 96.43%), 1365.64 (M+2, 79.54%), 1365.89 (M+3, 62.12%), 1366.13 (M+4, 40.98%); m/z calculated: 1820.52 [M+3H]3+, 1365.64 [M+4H]4+; 1092.712 [M+5H]5+; m/z observed: 1819.84 [M+3H]3+, 1365.14 [M+4H]4+; 1092.51 [M+5H]5+.
184 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.7
UPLC and LC-MS analysis of Seed 5
8Figure 4.20: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 20 days. This octamer sample was used as seed.
Figure 4.21: Mass spectrum of cyclic octamer 58from the LC-MS analysis of a stirred
library made from peptide 5 (corresponding to Figure 4.20). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1397.15 (M, 100%), 1397.40 (M+1, 95.17%), 1397.65
(M+2, 78.53%), 1397.90 (M+3, 61.84%), 1398.15 (M+4, 42.69%); m/z calculated: 1863.19 [M+3H]3+, 1397.64 [M+4H]4+; 1118.31 [M+5H]5+; m/z observed: 1862.51 [M+3H]3+,
1397.15 [M+4H]4+; 1117.91 [M+5H]5+.
4.7. UPLC and LC-MS analyses 185
4.7.8
UPLC and LC-MS analysis of Seed 6
8Figure 4.22: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 6 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 14 days. This octamer sample was used as seed.
Figure 4.23: Mass spectrum of cyclic octamer 68from the LC-MS analysis of a stirred
library made from peptide 6 (corresponding to Figure 4.22). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1365.14 (M, 100%), 1365.39 (M+1, 96.43%), 1365.64 (M+2, 79.54%), 1365.89 (M+3, 62.12%), 1366.13 (M+4, 40.98%); m/z calculated: 1820.52 [M+3H]3+, 1365.64 [M+4H]4+; 1092.712 [M+5H]5+; m/z observed: 1819.84 [M+3H]3+, 1365.14 [M+4H]4+; 1092.51 [M+5H]5+.
186 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.9
UPLC and LC-MS analysis of a pre-oxidized DCL made
from 1
Figure 4.24: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 in a 80 mol % pre-oxidized library (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 40 days.
Figure 4.25: Mass spectrum of cyclic octamer 18from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.24). Calculated isotopic profile for [M+4H]4+(species, abundance): 1549.68 (M, 100%), 1549.93 (M+1, 90.55%), 1550.18 (M+2, 73.48%), 1550.43 (M+3, 53.24%), 1550.68 (M+4, 35.37%); m/z calculated: 2065.93 [M+3H]3+, 1549.7 [M+4H]4+; 1239.96 [M+5H]5+; m/z observed: 2065.50 [M+3H]3+, 1549.68 [M+4H]4+; 1239.94 [M+5H]5+.
4.7. UPLC and LC-MS analyses 187
4.7.10
UPLC and LC-MS analysis of a DCL made from 1
without pre-oxidation
Figure 4.26: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 in a stirred library (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 35 days.
4.7.11
UPLC and LC-MS analysis of a DCL made from 1
equilibrated under an inert atmosphere
Figure 4.27: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 in a library equilibrated under an inert atmosphere (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 40 days.
188 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.28: Mass spectrum of dimer 12from the LC-MS analysis of a stirred library made
from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+2H]2+
(species, abundance): 775.61(M, 100%), 776.11 (M+1, 78.70%), 776.61 (M+2, 58.33%); m/z calculated: 1548.7 [M+H]+, 775.35 [M+2H]2+; 517.23 [M+3H]3+; m/z observed:
1548.27 [M+H]+, 775.35 [M+2H]2+; 517.23 [M+3H]3+.
Figure 4.29: Mass spectrum of cyclic trimer 13 from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 775.02(M, 100%), 775.36 (M+1, 87.49%), 775.70 (M+2,
65.73%), 776.03 (M+3, 42.22%); m/z calculated: 1162.53 [M+2H]2+, 775.35 [M+3H]3+; 581.76 [M+4H]4+; m/z observed: 1062.00 [M+2H]2+, 775.02 [M+3H]3+; 581.26 [M+4H]4+.
4.7. UPLC and LC-MS analyses 189
Figure 4.30: Mass spectrum of cyclic tetramer 14 from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 1032.82 (M, 100%), 1033.16 (M+1, 93.27%), 1033.50 (M+2, 66.78%), 1033.83 (M+3, 39.73%); m/z calculated: 1549.70 [M+2H]2+, 1033.47 [M+3H]3+; 775.35 [M+4H]4+; m/z observed: 1548.69 [M+2H]2+, 1032.82 [M+3H]3+;
774.85 [M+4H]4+.
Figure 4.31: Mass spectrum of cyclic pentamer 15from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 1290.80 (M, 100%), 1291.13 (M+1, 80.89%), 1291.46
(M+2, 55.01%), 129180 (M+3, 35.37%); m/z calculated: 1936.88 [M+2H]2+, 1291.58
[M+3H]3+; 968.94 [M+4H]4+; m/z observed: 1935.65 [M+2H]2+, 1290.80 [M+3H]3+;
188 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.28: Mass spectrum of dimer 12from the LC-MS analysis of a stirred library made
from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+2H]2+
(species, abundance): 775.61(M, 100%), 776.11 (M+1, 78.70%), 776.61 (M+2, 58.33%); m/z calculated: 1548.7 [M+H]+, 775.35 [M+2H]2+; 517.23 [M+3H]3+; m/z observed:
1548.27 [M+H]+, 775.35 [M+2H]2+; 517.23 [M+3H]3+.
Figure 4.29: Mass spectrum of cyclic trimer 13 from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 775.02(M, 100%), 775.36 (M+1, 87.49%), 775.70 (M+2,
65.73%), 776.03 (M+3, 42.22%); m/z calculated: 1162.53 [M+2H]2+, 775.35 [M+3H]3+; 581.76 [M+4H]4+; m/z observed: 1062.00 [M+2H]2+, 775.02 [M+3H]3+; 581.26 [M+4H]4+.
4.7. UPLC and LC-MS analyses 189
Figure 4.30: Mass spectrum of cyclic tetramer 14 from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 1032.82 (M, 100%), 1033.16 (M+1, 93.27%), 1033.50 (M+2, 66.78%), 1033.83 (M+3, 39.73%); m/z calculated: 1549.70 [M+2H]2+, 1033.47 [M+3H]3+; 775.35 [M+4H]4+; m/z observed: 1548.69 [M+2H]2+, 1032.82 [M+3H]3+;
774.85 [M+4H]4+.
Figure 4.31: Mass spectrum of cyclic pentamer 15from the LC-MS analysis of a stirred
library made from peptide 1 (corresponding to Figure 4.27). Calculated isotopic profile for [M+3H]3+(species, abundance): 1290.80 (M, 100%), 1291.13 (M+1, 80.89%), 1291.46
(M+2, 55.01%), 129180 (M+3, 35.37%); m/z calculated: 1936.88 [M+2H]2+, 1291.58
[M+3H]3+; 968.94 [M+4H]4+; m/z observed: 1935.65 [M+2H]2+, 1290.80 [M+3H]3+;
190 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.12
UPLC and LC-MS analysis of a non-agitated DCL made
from 1
Figure 4.32: UPLC trace (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 in a non-agitated library (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring at 1200 rpm for 40 days.
4.7. UPLC and LC-MS analyses 191
4.7.13
UPLC and LC-MS analysis of a DCL made from 1
seeded with 2
6Figure 4.33: UPLC trace (monitored at 254 nm) of the product mixture obtained from a DCL made from 1 seeded with 26 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring
192 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.34: Mass spectrum of mixed cyclic hexamer 1521from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 26(corresponding to Figure 4.33).
Cal-culated isotopic profile for [M+4H]4+(species, abundance): 1157.90 (M, 100%), 1158.15 (M+1, 89.02%), 1158.39 (M+2, 69.94%), 1158.64 (M+3, 48.18%), 1158.88 (M+4, 31.96%); m/z calculated: 1543.34 [M+3H]3+, 1157.50 [M+4H]4+; 926.00 [M+5H]5+; m/z observed:
1543.22 [M+3H]3+, 1157.90 [M+4H]4+; 926.53 [M+5H]5+.
Figure 4.35: Mass spectrum of mixed cyclic hexamer 1422from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 26(corresponding to Figure 4.33).
Cal-culated isotopic profile for [M+4H]4+(species, abundance): 1153.91 (M, 100%), 1154.15
(M+1, 93.68%), 1154.39 (M+2, 68.82%), 1154.64 (M+3, 36.46%), 1154.89 (M+4, 31.79%); m/z calculated: 1538.00 [M+3H]3+, 1153.50 [M+4H]4+; 922.80 [M+5H]5+; m/z observed:
4.7. UPLC and LC-MS analyses 193
4.7.14
UPLC and LC-MS analysis of a DCL made from 1
seeded with 3
6Figure 4.36: UPLC trace (monitored at 254 nm) of the product mixture obtained from a DCL made from 1 seeded with 36 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring
194 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.37: Mass spectrum of mixed cyclic hexamer 1531from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 36(corresponding to Figure 4.36).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1159.42 (M, 100%), 1159.66 (M+1, 91.33%), 1159.91 (M+2, 70.36%), 1160.15 (M+3, 51.53%), 1160.40 (M+4, 33.08%); m/z calculated: 1545.35 [M+3H]3+, 1159.01 [M+4H]4+; 927.21 [M+5H]5+; m/z observed:
1545.24 [M+3H]3+, 1159.42 [M+4H]4+; 927.74 [M+5H]5+.
Figure 4.38: Mass spectrum of mixed cyclic hexamer 1432from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 36(corresponding to Figure 4.36).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1153.91 (M, 100%), 1154.15
(M+1, 93.68%), 1154.39 (M+2, 68.82%), 1154.64 (M+3, 36.46%), 1154.89 (M+4, 31.79%); m/z calculated: 1542.04 [M+3H]3+, 1156.53 [M+4H]4+; 925.22 [M+5H]5+; m/z observed:
1542.26 [M+3H]3+, 1156.93 [M+4H]4+; 925.95 [M+5H]5+.
4.7. UPLC and LC-MS analyses 195
Figure 4.39: Mass spectrum of an unknown macrocycle denoted as x1 from the LC-MS analysis of a stirred library made from peptide 1 seeded with 36(corresponding to Figure
4.36). Calculated isotopic profile for [M+4H]4+(species, abundance): 1168.91 (M, 100%), 1169.15 (M+1, 92.51%), 1169.40 (M+2, 71.53%), 1169.64 (M+3, 49.75%), 1169.89 (M+4, 33.52%); m/z observed: 1557.89 [M+3H]3+, 1168.91 [M+4H]4+; 935.33 [M+5H]5+.
Figure 4.40: Mass spectrum of an unknown macrocycle denoted as x2 from the LC-MS analysis of a stirred library made from peptide 1 seeded with 36(corresponding to Figure
4.36). Calculated isotopic profile for [M+4H]4+(species, abundance): 1166.42 (M, 100%),
1166.66 (M+1, 91.46%), 1166.90 (M+2, 68.07%), 1167.15 (M+3, 47.97%), 1167.40 (M+4, 31.23%); m/z observed: 1554.57 [M+3H]3+, 1166.42 [M+4H]4+; 933.34 [M+5H]5+.
194 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.37: Mass spectrum of mixed cyclic hexamer 1531from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 36(corresponding to Figure 4.36).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1159.42 (M, 100%), 1159.66 (M+1, 91.33%), 1159.91 (M+2, 70.36%), 1160.15 (M+3, 51.53%), 1160.40 (M+4, 33.08%); m/z calculated: 1545.35 [M+3H]3+, 1159.01 [M+4H]4+; 927.21 [M+5H]5+; m/z observed:
1545.24 [M+3H]3+, 1159.42 [M+4H]4+; 927.74 [M+5H]5+.
Figure 4.38: Mass spectrum of mixed cyclic hexamer 1432from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 36(corresponding to Figure 4.36).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1153.91 (M, 100%), 1154.15
(M+1, 93.68%), 1154.39 (M+2, 68.82%), 1154.64 (M+3, 36.46%), 1154.89 (M+4, 31.79%); m/z calculated: 1542.04 [M+3H]3+, 1156.53 [M+4H]4+; 925.22 [M+5H]5+; m/z observed:
1542.26 [M+3H]3+, 1156.93 [M+4H]4+; 925.95 [M+5H]5+.
4.7. UPLC and LC-MS analyses 195
Figure 4.39: Mass spectrum of an unknown macrocycle denoted as x1 from the LC-MS analysis of a stirred library made from peptide 1 seeded with 36(corresponding to Figure
4.36). Calculated isotopic profile for [M+4H]4+(species, abundance): 1168.91 (M, 100%), 1169.15 (M+1, 92.51%), 1169.40 (M+2, 71.53%), 1169.64 (M+3, 49.75%), 1169.89 (M+4, 33.52%); m/z observed: 1557.89 [M+3H]3+, 1168.91 [M+4H]4+; 935.33 [M+5H]5+.
Figure 4.40: Mass spectrum of an unknown macrocycle denoted as x2 from the LC-MS analysis of a stirred library made from peptide 1 seeded with 36(corresponding to Figure
4.36). Calculated isotopic profile for [M+4H]4+(species, abundance): 1166.42 (M, 100%),
1166.66 (M+1, 91.46%), 1166.90 (M+2, 68.07%), 1167.15 (M+3, 47.97%), 1167.40 (M+4, 31.23%); m/z observed: 1554.57 [M+3H]3+, 1166.42 [M+4H]4+; 933.34 [M+5H]5+.
196 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.15
UPLC and LC-MS analysis of a DCL made from 1
seeded with 4
6Figure 4.41: UPLC trace (monitored at 254 nm) of the product mixture obtained from a DCL made from 1 seeded with 46 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring
4.7. UPLC and LC-MS analyses 197
Figure 4.42: Mass spectrum of mixed cyclic hexamer 1541from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 46(corresponding to Figure 4.41).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1146.40 (M, 100%), 1146.65 (M+1, 82.41%), 1146.89 (M+2, 68.58%), 1147.14 (M+3, 44.08%), 1147.38 (M+4, 29.56%); m/z calculated: 1527.99 [M+3H]3+, 1146.00 [M+4H]4+; 916.80 [M+5H]5+; m/z observed:
198 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.16
UPLC and LC-MS analysis of a DCL made from 1
seeded with 5
6Figure 4.43: UPLC trace (monitored at 254 nm) of the product mixture obtained from a DCL made from 1 seeded with 56 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring
4.7. UPLC and LC-MS analyses 199
Figure 4.44: Mass spectrum of mixed cyclic hexamer 1551from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 56(corresponding to Figure 4.43).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1142.67 (M, 100%), 1142.90
(M+1, 98.98%), 1143.14 (M+2, 87.22%), 1143.39(M+3, 67.36%), 1143.64 (M+4, 47.11%); m/z calculated: 1523.52 [M+3H]3+, 1142.64 [M+4H]4+; 914.11 [M+5H]5+; m/z observed: 1523.22 [M+3H]3+, 1142.67 [M+4H]4+; 914.53 [M+5H]5+.
200 4. Existing Self-Replicators Can Direct the Emergence of New Ones
4.7.17
UPLC and LC-MS analysis of a DCL made from 1
seeded with 5
8Figure 4.45: UPLC trace (monitored at 254 nm) of the product mixture obtained from a DCL made from 1 seeded with 58 (3.8 mM in 50 mM borate buffer, pH 8.2) after stirring
4.7. UPLC and LC-MS analyses 201
Figure 4.46: Mass spectrum of cyclic mixed octamer 1751from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 58(corresponding to Figure 4.45).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1529.80 (M, 100%), 1530.05 (M+1, 92.48%), 1530.30 (M+2, 77.18%), 1530.55 (M+3, 56.60%), 1530.80 (M+4, 42.32%); m/z calculated: 2039.74 [M+3H]3+, 1529.80 [M+4H]4+; 1223.84 [M+5H]5+; m/z observed:
202 4. Existing Self-Replicators Can Direct the Emergence of New Ones
Figure 4.47: Mass spectrum of cyclic mixed octamer 1652from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 58(corresponding to Figure 4.45).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1510.79 (M, 100%), 1511.04 (M+1, 94.94%), 1511.29 (M+2, 76.93%), 1511.54 (M+3, 59.48%), 1511.79 (M+4, 40.37%); m/z calculated: 2014.58 [M+3H]3+, 1510.94 [M+4H]4+; 1208.75 [M+5H]5+; m/z observed:
2013.66 [M+3H]3+, 1510.79 [M+4H]4+; 1208.84 [M+5H]5+.
Figure 4.48: Mass spectrum of cyclic mixed octamer 1553 from the LC-MS analysis of a
stirred library made from peptide 1 seeded with 58(corresponding to Figure 4.45).
Cal-culated isotopic profile for [M+4H]4+ (species, abundance): 1491.53 (M, 100%), 1491.79
(M+1, 98.88%), 1492.03 (M+2, 96.50%), 1492.29 (M+3, 63.68%), 1492.53 (M+4, 59.42%); m/z calculated: 1989.43 [M+3H]3+, 1492.07 [M+4H]4+; 1193.66 [M+5H]5+; m/z observed:
