University of Groningen Novel peptide replicators from dynamic combinatorial libraries Altay, Yigit

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University of Groningen

Novel peptide replicators from dynamic combinatorial libraries

Altay, Yigit

DOI:

10.33612/diss.90041906

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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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118 2. A Structural Survey in the Search for Novel Self-Replicating Peptides [67] Bowerman, C. J.; Ryan, D. M.;

Nissan, D. A.; Nilsson, B. L. Mol. BioSyst. 2009, 5, 1058-1069. [68] Bowerman, C. J.; Nilsson, B. L. J.

Am. Chem. Soc. 2010, 132, 9526-9527. [69] Bowerman, C. J.; Liyanage, W.; Federation, A. J.; Nilsson, B. L. Biomacromolecules 2011, 12, 2735-2745. [70] Swanekamp, R. J.; DiMaio, J. T. M.; Bowerman, C. J.; Nilsson, B. L. J. Am. Chem. Soc. 2012, 134, 5556-5559.

[71] Vassar, P.; Culling, C. Arch. Pathol-ogy 1959, 68, 487-498.

[72] Levine, H. Protein Sci. 1993, 2, 404-410.

[73] Barth, A. Biochim. Biophys. Acta 2007, 1767, 1073-1101.

[74] Leonetti, G.; Otto, S. J. Am. Chem. Soc. 2015, 137, 2067-2072.

[75] Ugwu, S. O.; Apte, S. P. The Ef-fect of Buﬀers on Protein Confor-mational Stability. In Pharmaceuti-cal Technology; 2004.

[76] Cecil, R.; McPhee, J. R. Adv. Prot. Chem. 1959, 14, 255.

[77] Otto, S.; Furlan, R.; Sanders, J. Science 2002, 297, 590-593.

[78] Reif, M. M.; H¨unenberger, P. H.; Oostenbrink, C. J. Chem. Theory Comput. 2012, 8, 37053723.

[79] Reif, M. M.; Winger, M.; Oosten-brink, C. J. Chem. Theory Comput. 2013, 9, 12471264.

[80] Berendsen, H. J. C.; Postma, J. P. M.; Gunsteren, W. F. v.; Her-mans, J. Interaction Models for Wa-ter in Relation to Protein Hydration. In Intermolecular Forces; Pullman, B., Ed.; The Jerusalem Symposia on Quantum Chemistry and Biochem-istry; Springer: Netherlands, 1981. [81] Hess, B. J. Chem. Theory Comput.

2008, 4, 116-122.

[82] Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, 952962. [83] Bussi, G.; Donadio, D.;

Par-rinello, M. J. Chem. Phys. 2007, 126, 14101.

[84] Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Di-Nola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 36843690.

[85] Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem. Theory Comput. 2008, 4, 435447. [86] Komaromy, D.; Stuart, M. C. A.;

Monreal Santiago, G.; Tezcan, M.; Krasnikov, V. V.; Otto, S. J. Am. Chem. Soc. 2017, 139, 6234–6241.

Published as:

Y. Altay, M. Tezcan and S. Otto – “Emergence of a New Self-Replicator from a Dynamic Combinatorial Library Requires a Specific Pre-Existing Replicator”, J. Am. Chem. Soc., 2017, 139, 13612 - 13615.

Chapter 3 Emergence of a New Self-Replicator

Requires a Specific Pre-Existing Replicator

The essence of life is statistical improbability on a colossal scale.

Richard Dawkins

Abstract

Our knowledge regarding the early steps in the formation of evolvable life and what constitutes the minimal molecular basis of life remains far from complete. The recent emergence of systems chemistry reinvigorated the investigation of systems of self-replicating molecules to address these questions. Most of these studies focus on single replicators and the effects of replicators on the emer-gence of other replicators remains under-investigated. Here we show the cross-catalyzed emergence of a novel self-replicator from a dynamic combinatorial library made from a threonine containing peptide building block, which, by it-self, only forms trimers and tetramers that do not replicate. Upon seeding of this library with different replicators of different macrocycle size (hexamers and octamers), we observed the emergence of hexamer replicator consisting of six units of the threonine peptide only when it is seeded with an octamer replicator containing eight units of a serine building block. These results reveal for the first time how a new replicator can emerge in a process that relies critically on the assistance by another replicator through cross-catalysis and that replicator composition is history dependent.

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120 3. Emergence of a New Self-Replicator

3.1 Introduction

H

ow life originated and how life may be synthesized de novo are among the grandchallenges in contemporary science. Research in these areas has focused on the biomolecules essential to current life (proteins, RNA and DNA) or on the bottom-up construction of chemical systems that mimic the essential characteristics of life. Over the last decades, the fields of systems chemistry,1–3_{and dynamic combinatorial}

chem-istry4–9_{in particular, have developed synthetic systems that capture some of the}

es-sential characteristics of life: compartmentalization,10–13_{reaction networks}

(address-ing the issue of metabolism)14–17 _{and systems featuring self- and cross-replicating}

molecules.18–21

In this study, we will focus on self-replication. Until now, relatively few replicators have been reported. Following pioneering work by von Kiedrowski,22–26 _completely

synthetic replicators were developed by Rebek27,28 _{and Philp.}29–33 _{Joyce, Lehman}

and Szostak developed systems of replicating RNAs34–37_{while Chmielewski}38 _along

with Ghadiri,39–41_Ashkenasy42–46_{and us,}47–51_{focused on peptide-based replicators.}

While in the majority of these studies replicators were designed in full structural de-tail, we explored how replicators emerged from complex mixtures where the structure of the emerging replicator was not pre-determined. For this purpose, we developed dynamic combinatorial libraries (DCLs) made from building-blocks featuring two thiol groups for reversible disulfide chemistry48 and a peptide that is predisposed to β-sheet formation by virtue of alternating hydrophobic and hydrophilic amino-acid residues. In solution, reaction between these building blocks and oxygen from the air yields a DCL that consists of a mixture of disulfide macrocycles of diﬀerent ring sizes (Figure 3.1). If one of the macrocycles can stabilize itself through self-assembly, the product distribution shifts towards this compound at the expense of the other compounds in the library. Assembly occurs through a nucleation-growth mechanism which allows exponential replication to be achieved: growing fibers break into frag-ments by mechanical agitation (i.e. stirring or shaking), which increases the number of ends from where the fibers grow.48

In most of our previous studies,51 _{the emergence of replicators occurred}

spon-taneously. In an intriguing recent study,47 we showed that assembly-driven self-replication could also be triggered by a template that raises the concentration of the potential replicator above its critical aggregation concentration. However, until now, the role of existing replicators on the emergence of new ones has received little attention. Yet, cross-catalysis may be a powerful mechanism for the diversification and evolution of replicators and the development of replicator “ecosystems”.52

We now report a system in which autonomous replicator emergence is not ob-served, but where replicator emergence requires cross-catalysis by another pre-existing

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3.1. Introduction 121

Figure 3.1: (a) A dynamic combinatorial library of diﬀerently sized macrocyclic disulfides is formed upon oxidation of the threonine containing peptide functionalized dithiol. (b) Selective formation of replicator 16 upon cross-seeding. (c) Schematic representation of the

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122 3. Emergence of a New Self-Replicator replicator. Cross-catalyzed emergence was mediated only by a replicator with a specific ring size and peptide sequence. We also show that replication is strongly dependent on the sample history marking an important step in the direction of self-replicator evolution.

3.2 Results and Discussion

In the course of a systematic investigation of the eﬀect of the structure of the peptide building blocks with the general architecture shown in Figure 3.1 on the formation of replicators, we encountered unexpected behavior upon using building block 1. In agitated DCLs made from 2-5, individually, self-replicators emerged spontaneously in all cases. Yet in similar experiments starting from building block 1, we did not observe any replicators. Using our standard protocol we rapidly oxidized a solution of 1 (3.8 mM in 50 mM borate buﬀer pH 8.1) to 80% (conversion of thiols into disulfides) using sodium perborate solution (80 mM), followed by slower further oxidation mediated by oxygen present in the air. Solutions were stirred at 1200 rpm or left non-agitated. The kinetic profiles of these libraries, monitored by UPLC-MS, show the formation trimers (13) and tetramers (14), neither of which self-assembles or self-replicates (Figure 3.2a;

see Figure 3.5 for the non-agitated sample). The absence of any replicator might be explained with the library reaching complete oxidation before any replicator had the chance to emerge (disulfide exchange requires a catalytic amount of thiol). In order to prevent freezing the library by complete oxidation, we repeated the experiment at a constant oxidation level of 80% and monitored the composition of the small DCL over the course of one month under an inert atmosphere. Also in this experiment 13 and 14 dominated the library and no replicator emergence was observed (Figure

3.2b).

We then investigated whether the formation of replicators made from 1 could be induced by cross-seeding with pre-formed replicators made from building blocks 2, 3, 4 or 5. Thus, 10 mol % of 26, 36, 46, 48, 58, prepared following published

protocols,50,51 _{was added to stirred DCLs made from 1 and the compositions of the}

mixtures were monitored over time. These experiments failed to induce the formation of new macrocycles (see Figure 3.6) with one notable exception: only cross-seeding with 48induced the formation of a new macrocycle (16, Figure 3.2c). The sigmoidal

growth of 16is consistent with self-replication.

To confirm that 16 is a self-replicator, we added it as a seed to a DCL made from

1. Figure 3.2d shows that 16 grows rapidly upon seeding and accounts for 72% of

the library material within one day (see Figure 3.9 for results of seeding with smaller amounts of seed).53 _{Note that the lag phase, that was observed in the library to}

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3.2. Results and Discussion 123

Figure 3.2: Kinetic profile of a dynamic combinatorial library made from building block 1 (3.8 mM in 50 mM borate buﬀer, pH 8.1) stirred at 1200 rpm and (a) kept under ambient conditions, (b) 80% oxidized and kept under an inert atmosphere; kinetic profile of a dynamic combinatorial library made from an 80% oxidized solution of building block 1 (3.8 mM in 50 mM borate buﬀer, pH 8.1) seeded with (c) 48 (cross-seeding), (d) 16 (self-seeding), stirred

at 1200 rpm.

of this lag phase suggests that growth of 16 occurs on the ends of fibers of 48, but it

is a relatively rare event and that fragmentation of these nuclei of 16(induced by 48)

into secondary nuclei is required before replication of 16becomes eﬃcient (see Figure

3.2c). This interpretation was further supported by the fact that seeding a library made from 1 with 48 in the absence of mechanical agitation dramatically slowed

down the rate of replication of 16 (Figure 3.7). Detailed analysis of the early stage

of the growth of 16 seeded by 48 (Figure 3.8) did not reveal any mixed macrocycles

containing both building blocks, lending further support for the proposed mechanism. The structure of the assemblies formed by 16 was characterized by transmission

electron microscopy (TEM), circular dichroism (CD) spectroscopy and thioflavin T fluorescence assays (Figure 3.3). Negative staining TEM micrographs of a sample dominated by 16revealed laterally associated fibers that were approximately 100 nm

long (Figure 3.3c). In contrast, libraries made from 1 containing mostly cyclic trimers and tetramers did not show any ordered or disordered aggregates. The CD spectrum

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Figure 3.3: (a) CD spectra and (b) maximum thioflavin T fluorescence emission intensity (at 492 nm) of non-seeded and seeded (10 mol %) libraries made from building block 1 (3.8 mM in 50 mM borate buﬀer, pH 8.1): i, stirred; ii, inert atm.; iii, non-agitated; iv, seeded with 48; v, seeded with 58; vi, seeded with 26; vii, seeded with 36; viii, seeded with 46.(c)

TEM micrographs of the library corresponding to Figure 3.2c. Scale bars are 100 nm.

of samples dominated by 16 showed positive helicity at 196 nm and negative helicity

at 218 nm, indicative of a β-sheet structure (Figure 3.3a).54_{We observed only random}

coil secondary structure for all other libraries made from 1 that were dominated by trimers and tetramers. Thioflavin T fluorescence measurements55_{also confirmed a}

β-sheet amyloid-fibril-like56_{structure of 1}

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3.2. Results and Discussion 125 in emission intensity, while all libraries dominated by 13 and 14 showed a 3-fold

increase at most (Figure 3.3b).

In order to investigate whether cross-catalysis between the 16 and 48 replicators

is reciprocal, libraries were made from building block 4 and the eﬀect of seeding by 16 was probed. Mechanical agitation was not applied, as agitation induces the

autonomous formation of the 48 replicator. We observed the emergence of 48 and

46 alongside some mixed hexamers containing both building blocks 1 and 4 in the

library seeded with 16, whereas the non-seeded control only led to the formation of

non-assembling 43and 44macrocycles (Figure 3.4a-b). Thus, cross-catalysis appears

to be reciprocal, albeit not completely symmetrical: 48 induces the formation of 16

but not 18 while 46 does not give rise to any replicators based on 1, within the

time frame of our experiments. Conversely, 16induces the formation of both 46 and

48. These perhaps non-intuitive cross-catalytic eﬀects prompted us to explore their

origin. The fact that replicators based on the more hydrophobic building block 1 are hexamers, while those based on the more hydrophilic 4 are predominantly octamers fits with the general trend that is reported previously:51 more hydrophobic building blocks allow for self-assembly and concomitant replication already at a smaller ring size. What remains puzzling is why the replication by 16 is only triggered by 48

and not by any other of our established replicators. We suspect that this could be due to specific interactions involving the OH-groups of the serine residue in 4. This hypothesis is supported by the observation that octamers of alanine containing building block 5, thus lacking these OH groups, do not induce the formation of any replicators based on 1.

Finally, we investigated to what extent the replicator distribution is dependent on sample history. We prepared a DCL with the same overall building block composition as the one shown in Figure 3.4b (3.42 mM in 1 and 0.38 mM in 4) but now mixed these building blocks at the start of the experiment. We monitored the sample over a period of 40 days but did not detect the emergence of replicators 16or 48 (Figure

3.4c). This experiment shows that the history of the sample is a decisive factor in determining replicator presence or absence, just like the evolutionary history dictates the species composition in current life. While history-dependence is a wide-spread phenomenon in materials science57_{and protein folding,}58_{it had not yet been reported}

to dictate the nature of self-replicating molecules. Moreover, the history dependence observed here involves the interaction history between molecules in the mixture, and not merely the history in terms of physical properties, such as pH or temperature.

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Figure 3.4: Kinetic profile of a non-agitated dynamic combinatorial library made from building block 4 (3.8 mM in 50 mM borate buﬀer, pH 8.1) 80% oxidized (a) non-seeded (b) after addition of 10 mol percent of pre-formed 16 as seed. (c) Kinetic profile of a library

made by mixing peptide 1 and peptide 4 (3.42 mM in 1 and 0.38 mM in 4) in borate buﬀer (pH 8.1, 50 mM) to form 3.8 mM library. The library was then oxidized to 80% with freshly prepared perborate solution (80 mM) and stirred at 1200 rpm under an inert atmosphere.

3.3 Conclusions

In conclusion, we have shown how the emergence of a new replicator based on threonine-containing building block 1 relies on the presence of a specific pre-existing replicator containing serine residues. Cross-catalysis between replicators (and there-fore replicator mutation) in this system was found to be remarkably specific, as structurally closely related replicators failed to show the same eﬀect. Our results also constitute an important first step in the development of abiotic systems of repli-cators in the direction of primitive life. While in previous work the presence of the building blocks of a replicator was typically suﬃcient for replicators to emerge (af-ter some lag phase), in contemporary life, new species only derive from pre-existing

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3.4. Acknowledgements 127 ones (for as far as we know, no new life forms emerge solely from abiotic materials). Thus, in the current life, the species present at any given time point reflect not only the available resources, but also the evolutionary history of the various biological species. Our results represent a first step in the transition from a regime where repli-cator abundance is governed by building block availability to one where pre-existing replicators control the new replicator population. So, instead of replicator distri-butions being defined solely by the present conditions, the systems (evolutionary) history is now also becoming an essential controlling factor. Such history-dependent systems of self-replicators represent an important strategic direction in research on the origin of life and the development of de novo life.

3.4 Acknowledgements

M. Altay is gratefully acknowledged for discussions on the results, proof-reading this chapter and for giving precious feedback.

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3.5 Materials and Methods

Peptide No. Peptide Sequence Replicator Replicator Macrocycle Size

1 XGLKTK-OH 16 Hexamer 2 XGLKFK-OH 26 Hexamer 3 XGLKChaK-OH 36 Hexamer 4 XGLKSK-OH 46 Hexamer 48 Octamer 5 XGLKAK-OH 58 Octamer

Table 3.1: List of peptides, replicators and libraries set up in the study. X denotes the 3,5-dimercaptobenzoyl group.

3.5.1 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.

3.5.2 Peptide Synthesis

Peptide 1 was synthesized by conventional peptide synthesis using pre-loaded Wang resins. It was purified by preparative HPLC using a Phenomenex, Jupiter (10µm, C5, 300 ˚A, 250ˆ21.2mm) column.

Peptides 2, 3, 4 and 5 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,60 _{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.)

3.5. Materials and Methods 129

3.5.3 Library Preparation and Sampling

Building blocks were dissolved in borate buﬀer (50 mM, pH 8.12) 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 (12_{ˆ32 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 the DCL shown in Figure 3.2b which was set up in a glovebox under an inert atmosphere. 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.

3.5.4 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, 16 (10 mol%) was added. All libraries were monitored with

UPLC and UPLC-MS.

3.5.5 Cross Seeding Experiments

Solutions containing mostly 11, 13and 14 were prepared according to the procedure

described above. After 5 minutes, pre-formed seed (10 mol %) 26, 36, 46, 48, or

58 was added. All libraries were stirred at 1200 rpm and monitored by UPLC and

UPLC-MS.

3.5.6 Reverse Cross Seeding Experiments

A solution containing mostly 41, 43 and 44was prepared according to the procedure

described above. This library was split into two, and 10 mol % of pre-formed 16was

added to one of the solutions while the other one was left as control. The libraries were not agitated and monitored by UPLC and UPLC-MS.

3.5.7 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 spectropho-tometer 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

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3.5 Materials and Methods

Peptide No. Peptide Sequence Replicator Replicator Macrocycle Size

1 XGLKTK-OH 16 Hexamer 2 XGLKFK-OH 26 Hexamer 3 XGLKChaK-OH 36 Hexamer 4 XGLKSK-OH 46 Hexamer 48 Octamer 5 XGLKAK-OH 58 Octamer

Table 3.1: List of peptides, replicators and libraries set up in the study. X denotes the 3,5-dimercaptobenzoyl group.

3.5.1 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.

3.5.2 Peptide Synthesis

Peptide 1 was synthesized by conventional peptide synthesis using pre-loaded Wang resins. It was purified by preparative HPLC using a Phenomenex, Jupiter (10µm, C5, 300 ˚A, 250ˆ21.2mm) column.

Peptides 2, 3, 4 and 5 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,60 _{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.)

3.5. Materials and Methods 129

3.5.3 Library Preparation and Sampling

Building blocks were dissolved in borate buﬀer (50 mM, pH 8.12) 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 (12_{ˆ32 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 the DCL shown in Figure 3.2b which was set up in a glovebox under an inert atmosphere. 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.

3.5.4 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, 16(10 mol%) was added. All libraries were monitored with

UPLC and UPLC-MS.

3.5.5 Cross Seeding Experiments

Solutions containing mostly 11, 13and 14 were prepared according to the procedure

described above. After 5 minutes, pre-formed seed (10 mol %) 26, 36, 46, 48, or

58 was added. All libraries were stirred at 1200 rpm and monitored by UPLC and

UPLC-MS.

3.5.6 Reverse Cross Seeding Experiments

A solution containing mostly 41, 43and 44was prepared according to the procedure

described above. This library was split into two, and 10 mol % of pre-formed 16was

added to one of the solutions while the other one was left as control. The libraries were not agitated and monitored by UPLC and UPLC-MS.

3.5.7 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 spectropho-tometer 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

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130 3. Emergence of a New Self-Replicator 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.

3.5.8 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 8.12) 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 8.12) 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 µm in borate buffer) was added to the HELMA 10_{ˆ2 mm quartz cuvette, incubated for 2 min, and the} intensity was measured over 3 accumulations. All fluorescence measurements were recorded on a JASCO FP6200 fluorimeter equipped with a 480 nm cut-off filter on the emission channel.

3.5.9 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 immediately after first deposition and 30 s after the second deposition. The grids were observed in a Philips CM12 electron microscope operating at 120 kV. Images were recorded on a slow scan CCD camera.

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3.6. Kinetic Profiles 131

3.6 Kinetic Profiles

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.

Figure 3.5: Kinetic profile of a non-agitated library of peptide 1 (no pre-oxidation, 3.8 mM in borate buﬀer, pH 8.12).

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Figure 3.6: Kinetic profiles of DCLs of peptide 1 (80% pre-oxidation, 3.8 mM in borate buﬀer, pH 8.12) seeded with 10 mol % (a) 48 (b) 58(c) 26 (d) 36(e) 46. Plots shown on the

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3.6. Kinetic Profiles 133

Figure 3.7: Kinetic profile of a non-agitated library of peptide 1 seeded with 48on day 0.

Figure 3.8: Detailed kinetic profile of a stirred library of peptide 1 seeded with 48 in the

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Figure 3.9: Kinetic profile of a stirred library of peptide 1 seeded with a) 10 mol % of 48,

b) 5 mol % of 48, c) 2 mol % of 48and d) 1 mol % of 48 on day 0. e) Comparison of growth

kinetics of 16 upon using diﬀerent percentages of 48 seed.

3.7 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

3.7. UPLC and LC-MS analyses 135

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 by dissolving 10µL of a 3.8 mM library in 10 µL DMF and then further diluting them by 180µL UPLC grade water.

During the analysis, all the peaks that have a peak area more than 1% 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.

3.7.1 UPLC Methods

Libraries were analysed using the following method:

Solvent A: ULC/MS grade water purchased from Biosolve (0.1% trifluoroacetic acid added)

Solvent B: ULC/MS grade acetonitrile purchased from Biosolve (0.1% trifluo-roacetic 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

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Figure 3.9: Kinetic profile of a stirred library of peptide 1 seeded with a) 10 mol % of 48,

b) 5 mol % of 48, c) 2 mol % of 48and d) 1 mol % of 48 on day 0. e) Comparison of growth

kinetics of 16upon using diﬀerent percentages of 48 seed.

3.7 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 by dissolving 10µL of a 3.8 mM library in 10 µL DMF and then further diluting them by 180µL UPLC grade water.

During the analysis, all the peaks that have a peak area more than 1% 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.

3.7.1 UPLC Methods

Libraries were analysed using the following method:

Solvent A: ULC/MS grade water purchased from Biosolve (0.1% trifluoroacetic acid added)

Solvent B: ULC/MS grade acetonitrile purchased from Biosolve (0.1% trifluo-roacetic 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

(19)

3.7.2 Elution times of macrocycles

Macrocycle Elution Time (min) 11 2.95 14 3.62 13 4.97 16 4.00 41 3.40 43 4.95 44 3.60 46 3.65 48 3.00 26 6.20 36 8.50 58 3.35

Table 3.3: Elution times of macrocycles using the solvent gradient in Table 3.2.

3.7.3 UPLC and LC-MS analysis of Peptide 1

Figure 3.10: UPLC traces (monitored at 254 nm) of monomer of the peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after purification.

(20)

Figure 3.11: Mass spectrum of monomer 11 from the LC-MS analysis of peptide 1

af-ter purification (corresponding to Figure 3.10). Calculated isotopic profile for [M+H]1+ (species, abundance): 714.38 (M, 100%), 715.39 (M+1, 58.48%); m/z calculated: 714.33 [M+H]1+; m/z observed: 714.38 [M+H]1+.

3.7.4 UPLC and LC-MS analysis of a DCL made from 1

Figure 3.12: UPLC traces (monitored at 254 nm) of the DCL made from 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 6 days.

(21)

Figure 3.13: Mass spectrum of cyclic trimer 13 from the LC-MS analysis of a stirred

li-brary made from peptide 1(corresponding to Figure 3.12). Calculated isotopic profile for [M+3H]3+_{(species, abundance): 712.89 (M, 100%), 713.23 (M+1, 97.6%), 713.56 (M+2,}

72.3%), 713.90 (M+3, 41.72%); m/z calculated: 1069.5 [M+2H]2+_{, 713.33 [M+3H]}3+_;

535.24 [M+4H]4+_{; m/z observed: 1068.86 [M+2H]}2+_{, 712.89 [M+3H]}3+_{; 534.91 [M+4H]}4+_.

Figure 3.14: Mass spectrum of cyclic tetramer 14 from the LC-MS analysis of a stirred

library made from peptide 1(corresponding to Figure 3.12). Calculated isotopic profile for [M+3H]3+_{(species, abundance): 950.43 (M, 100%), 950.78 (M+1, 86.02%), 951.12 (M+2,}

54.28%), 951.45 (M+3, 27.15%); m/z calculated: 1425.7 [M+2H]2+_{, 950.77 [M+3H]}3+_;

(22)

3.7. UPLC and LC-MS analyses 139 The small peaks could not be assigned to any of the macrocycles that can be produced by peptide 1 and are probably macrocycles of deletion product(s) or other impurities containing the 3,5-dithiobenzoic acid unit. In Figure 3.15 below a representative chromatogram is shown that contains two small peaks at around 4 minutes, where also the 16 replicator elutes. Figures 3.16 and 3.17 show the MS analysis of these

two peaks, which could unfortunately not be identified.

Figure 3.15: UPLC traces (monitored at 254 nm) of the DCL made from 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 6 days with an emphasis on the unidentified peaks.

Figure 3.16: Mass spectrum of the unidentified peak X1 from the LC-MS analysis of a stirred library made from peptide 1 (corresponding to Figure 3.15). m/z observed: 1456.08 [M+2H]2+_{, 971.04 [M+3H]}3+_{; 729.00 [M+4H]}4+_.

(23)

Figure 3.17: Mass spectrum of the unidentified peak X2 from the LC-MS analysis of a stirred library made from peptide 1 (corresponding to Figure 3.15). m/z observed: 1281.85 [M+2H]2+, 854.57 [M+3H]3+; 684.75 [M+4H]4+.

3.7.5 UPLC and LC-MS analysis of a DCL made from 1

equi-librated under an inert atmosphere.

Figure 3.18: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM) in borate buﬀer (50 mM, pH 8.12) after stirring at 1200 rpm for 120 days under an inert atmosphere.

3.7.6 UPLC and LC-MS analysis of a non-agitated DCL made

from 1

Figure 3.19: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after 18 days without agitation.

3.7.7 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 4

8

Figure 3.20: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) seeded with 10 mol % of 48 after stirring at 1200 rpm for 6 days.

(24)

Figure 3.17: Mass spectrum of the unidentified peak X2 from the LC-MS analysis of a stirred library made from peptide 1 (corresponding to Figure 3.15). m/z observed: 1281.85 [M+2H]2+, 854.57 [M+3H]3+; 684.75 [M+4H]4+.

3.7.5 UPLC and LC-MS analysis of a DCL made from 1

equi-librated under an inert atmosphere.

Figure 3.18: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM) in borate buﬀer (50 mM, pH 8.12) after stirring at 1200 rpm for 120 days under an inert atmosphere.

3.7.6 UPLC and LC-MS analysis of a non-agitated DCL made

from 1

Figure 3.19: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after 18 days without agitation.

3.7.7 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 4

8

Figure 3.20: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) seeded with 10 mol % of 48 after stirring at 1200 rpm for 6 days.

(25)

Figure 3.21: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 20 days. This octamer sample was used as seed.

Figure 3.22: Mass spectrum of cyclic octamer 16from the LC-MS analysis of a stirred

library made from peptide 1(corresponding to Figure 3.20). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1069.12 (M, 100%), 1069.36 (M+1, 93.97%), 1069.62 (M+2, 66.57%), 1069.87 (M+3, 53.72%), 1070.12 (M+4, 29.80%); m/z calculated: 1425.66 [M+3H]3+_{, 1069.50 [M+4H]}4+_{; 855.80 [M+5H]}5+_{; m/z observed: 1425.17 [M+3H]}3+_,

1069.12 [M+4H]4+_{; 855.48 [M+5H]}5+_.

Figure 3.23: Mass spectrum of cyclic hexamer 48from the LC-MS analysis of a stirred

library made from peptide 4 (corresponding to Figure 3.21). 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+_,

(26)

Figure 3.21: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 20 days. This octamer sample was used as seed.

library made from peptide 1(corresponding to Figure 3.20). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1069.12 (M, 100%), 1069.36 (M+1, 93.97%), 1069.62 (M+2, 66.57%), 1069.87 (M+3, 53.72%), 1070.12 (M+4, 29.80%); m/z calculated: 1425.66 [M+3H]3+_{, 1069.50 [M+4H]}4+_{; 855.80 [M+5H]}5+_{; m/z observed: 1425.17 [M+3H]}3+_,

1069.12 [M+4H]4+_{; 855.48 [M+5H]}5+_.

library made from peptide 4 (corresponding to Figure 3.21). 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+_,

(27)

3.7.8 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 5

8

Figure 3.24: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) and seeding with 10 mol % 58 after stirring at 1200 rpm for 44 days.

Figure 3.25: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 14 days. This octamer sample was used as a seed.

library made from peptide 5 (corresponding to Figure 3.25). 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+_,

(28)

3.7.8 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 5

8

Figure 3.24: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80% oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) and seeding with 10 mol % 58 after stirring at 1200 rpm for 44 days.

Figure 3.25: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 5 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 14 days. This octamer sample was used as a seed.

library made from peptide 5 (corresponding to Figure 3.25). 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+_,

(29)

3.7.9 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 2

6

Figure 3.27: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80 % oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) and seeding with 10 mol % 26 after stirring at 1200 rpm for 6 days.

Figure 3.28: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 2 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 20 days. This hexamer sample was used as seed.

Figure 3.29: Mass spectrum of cyclic trimer 1221from the LC-MS analysis of the library

made from peptide 1 seeded with 26(corresponding to Figure 3.27). Calculated isotopic

profile for [M+3H]3+ (species, abundance): 728.24 (M, 100%), 728.58 (M+1, 90.65%), 728.91 (M+2, 61.72%), 729.25 (M+3, 35.59%); m/z calculated: 1091.48 [M+2H]2+, 727.65 [M+3H]3+_{; 545.74 [M+4H]}4+_{; m/z observed: 1091.89 [M+2H]}2+_{, 728.24 [M+3H]}3+_{; 546.43}

[M+4H]4+_.

Figure 3.30: Mass spectrum of cyclic tetramer 1321from the LC-MS analysis of the library

vi made from peptide 1 seeded with 26(corresponding to Figure 3.27). Calculated isotopic

profile for [M+3H]3+ _{(species, abundance): 965.78 (M, 100%), 966.12 (M+1, 79.16%),}

966.45 (M+2, 49.42%), 966.78 (M+3, 28.32%); m/z calculated: 1447.63 [M+2H]2+_{, 965.09}

[M+3H]3+_{; 723.81 [M+4H]}4+_{; m/z observed: 1448.19 [M+2H]}2+_{, 965.78 [M+3H]}3+_{; 724.57}

(30)

3.7.9 UPLC and LC-MS analysis of a DCL made from 1 seeded

with 2

6

Figure 3.27: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80 % oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) and seeding with 10 mol % 26 after stirring at 1200 rpm for 6 days.

Figure 3.29: Mass spectrum of cyclic trimer 1221from the LC-MS analysis of the library

made from peptide 1 seeded with 26(corresponding to Figure 3.27). Calculated isotopic

profile for [M+3H]3+ (species, abundance): 728.24 (M, 100%), 728.58 (M+1, 90.65%), 728.91 (M+2, 61.72%), 729.25 (M+3, 35.59%); m/z calculated: 1091.48 [M+2H]2+, 727.65 [M+3H]3+_{; 545.74 [M+4H]}4+_{; m/z observed: 1091.89 [M+2H]}2+_{, 728.24 [M+3H]}3+_{; 546.43}

[M+4H]4+_.

Figure 3.30: Mass spectrum of cyclic tetramer 1321from the LC-MS analysis of the library

vi made from peptide 1 seeded with 26(corresponding to Figure 3.27). Calculated isotopic

profile for [M+3H]3+ _{(species, abundance): 965.78 (M, 100%), 966.12 (M+1, 79.16%),}

966.45 (M+2, 49.42%), 966.78 (M+3, 28.32%); m/z calculated: 1447.63 [M+2H]2+_{, 965.09}

[M+3H]3+_{; 723.81 [M+4H]}4+_{; m/z observed: 1448.19 [M+2H]}2+_{, 965.78 [M+3H]}3+_{; 724.57}

(31)

library made from peptide 2 (corresponding to Figure 3.28). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1137.76 (M, 100%), 1138.02 (M+1, 98.18%), 1138.27 (M+2, 80.84%), 1138.52 (M+3, 59.46%), 1138.75 (M+4, 39.19%); m/z calcu-lated: 1517.7 [M+3H]3+_{, 1138.53 [M+4H]}4+_{; 911.02 [M+5H]}5+_{; m/z observed: 1516.68}

[M+3H]3+_{, 1137.77 [M+4H]}4+_{; 910.41 [M+5H]}5+_.

3.7.10 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 3

6

Figure 3.32: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80 % oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) seeded with 10 mol % 36after stirring at 1200 rpm for 19 days.

Figure 3.33: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 3 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 42 days. This hexamer sample was used as a seed.

library made from peptide 3 (corresponding to Figure 3.33). Calculated isotopic pro-file 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 calcu-lated: 1529.80 [M+3H]3+_{, 1147.60 [M+4H]}4+_{; 918.28 [M+5H]}5+_{; m/z observed: 1529.66}

(32)

library made from peptide 2 (corresponding to Figure 3.28). Calculated isotopic pro-file for [M+4H]4+ (species, abundance): 1137.76 (M, 100%), 1138.02 (M+1, 98.18%), 1138.27 (M+2, 80.84%), 1138.52 (M+3, 59.46%), 1138.75 (M+4, 39.19%); m/z calcu-lated: 1517.7 [M+3H]3+_{, 1138.53 [M+4H]}4+_{; 911.02 [M+5H]}5+_{; m/z observed: 1516.68}

[M+3H]3+_{, 1137.77 [M+4H]}4+_{; 910.41 [M+5H]}5+_.

3.7.10 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 3

6

Figure 3.32: UPLC traces (monitored at 254 nm) of the product mixture obtained by 80 % oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) seeded with 10 mol % 36after stirring at 1200 rpm for 19 days.

Figure 3.33: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 3 (3.8 mM in 50 mM borate buﬀer, pH 8.12) after stirring at 1200 rpm for 42 days. This hexamer sample was used as a seed.

(33)

3.7.11 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 4

6

Figure 3.35: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 1 (3.8 mM in 50 mM borate buﬀer, pH 8.12) seeded with 10 mol % 46

after stirring at 1200 rpm for 19 days.

library made from peptide 4 (corresponding to Figure 3.36). 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}

[M+3H]3+_{, 1048.55 [M+4H]}4+_{; 839.02 [M+5H]}5+_.

3.7.12 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 1

6

(34)

3.7.11 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 4

6

after stirring at 1200 rpm for 19 days.

library made from peptide 4 (corresponding to Figure 3.36). 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}

[M+3H]3+_{, 1048.55 [M+4H]}4+_{; 839.02 [M+5H]}5+_.

3.7.12 UPLC and LC-MS analysis of a DCL made from 1,

seeded with 1

6

(35)

after stirring at 1200 rpm for 12 days. This hexamer sample was used as seed.

3.7.13 UPLC and LC-MS analysis of a non-agitated DCL made

from 4

Figure 3.40: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buﬀer, pH 8.12) left non-agitated for 30 days.

3.7.14 UPLC and LC-MS analysis of a DCL made from 4,

seeded with 1

6

left non-agitated for 13 days.

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+3H]3+ _{(species, abundance): 698.98 (M, 100%), 699.31 (M+1, 72.68%), 699.65 (M+2,}

40.71%), 699.98 (M+3, 17.83%),; m/z calculated: 1049.3 [M+2H]2+_{, 699.89 [M+3H]}3+_;

(36)

after stirring at 1200 rpm for 12 days. This hexamer sample was used as seed.

3.7.13 UPLC and LC-MS analysis of a non-agitated DCL made

from 4

Figure 3.40: UPLC traces (monitored at 254 nm) of the product mixture obtained by oxidation of peptide 4 (3.8 mM in 50 mM borate buﬀer, pH 8.12) left non-agitated for 30 days.

3.7.14 UPLC and LC-MS analysis of a DCL made from 4,

seeded with 1

6

left non-agitated for 13 days.

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+3H]3+_{(species, abundance): 698.98 (M, 100%), 699.31 (M+1, 72.68%), 699.65 (M+2,}

40.71%), 699.98 (M+3, 17.83%),; m/z calculated: 1049.3 [M+2H]2+_{, 699.89 [M+3H]}3+_;

(37)

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+3H]3+(species, abundance): 931.95 (M, 100%), 932.29 (M+1, 84.71%), 932.62 (M+2, 58.47%), 932.96 (M+3, 30.51%), m/z calculated: 1398.8 [M+2H]2+, 932.85 [M+3H]3+; 699.89 [M+4H]4+; m/z observed: 1397.40 [M+2H]2+, 931.95 [M+3H]3+; 698.93 [M+4H]4+

[M+3H]3+_{, 1048.55 [M+4H]}4+_{; 839.02 [M+5H]}5+_.

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+4H]4+(species, abundance): 1397.15 (M, 100%), 1397.40 (M+1, 94.58%), 1397.65 (M+2, 79.07%), 1397.90 (M+3, 61.90%), 1398.14 (M+4, 43.60%); m/z calculated: 1864.71 [M+3H]3+, 1398.78 [M+4H]4+; 1119.22 [M+5H]5+; m/z observed: 1862.51 [M+3H]3+, 1397.15 [M+4H]4+_{; 1117.92 [M+5H]}5+_.

(38)

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+3H]3+(species, abundance): 931.95 (M, 100%), 932.29 (M+1, 84.71%), 932.62 (M+2, 58.47%), 932.96 (M+3, 30.51%), m/z calculated: 1398.8 [M+2H]2+, 932.85 [M+3H]3+; 699.89 [M+4H]4+; m/z observed: 1397.40 [M+2H]2+, 931.95 [M+3H]3+; 698.93 [M+4H]4+

[M+3H]3+_{, 1048.55 [M+4H]}4+_{; 839.02 [M+5H]}5+_.

library made from peptide 4 (corresponding to Figure 3.41). Calculated isotopic profile for [M+4H]4+ (species, abundance): 1397.15 (M, 100%), 1397.40 (M+1, 94.58%), 1397.65 (M+2, 79.07%), 1397.90 (M+3, 61.90%), 1398.14 (M+4, 43.60%); m/z calculated: 1864.71 [M+3H]3+, 1398.78 [M+4H]4+; 1119.22 [M+5H]5+; m/z observed: 1862.51 [M+3H]3+, 1397.15 [M+4H]4+_{; 1117.92 [M+5H]}5+_.

(39)

3.8 UV-Vis spectra of diﬀerent sized macrocycles

of peptide 1

An aliquot of 10µL sample from a non-seeded DCL dominated by 13 and 14 or a

seeded DCL dominated by 16diluted with 600µL double distilled water in HELMA

10ˆ2 mm quartz cuvette. Spectra were obtained from 200 - 400 nm over 5 accumu-lations using a SPECORD S600 UV-Vis spectrometer.

Figure 3.46: UV-Vis spectra of diﬀerent sized macrocycles formed from peptide 1, showing that the extinction coeﬃcients at 254 nm are comparable.

(40)

3.9. Complete CD spectra of DCLs 157

3.9 Complete CD spectra of DCLs

Figure 3.47: CD spectra of libraries made from peptide 1 under diﬀerent conditions.

3.10 ThT Assay Results

Figure 3.48: Maximum thioflavin T fluorescence emission intensity (at 492 nm) for seeded and non-seeded DCLs.

(41)

3.11 Transmission Electron Microscopy Images

Figure 3.49: Transmission electron micrographs showing the 16 fibers produced by the

DCL made from peptide 1 seeded with 48.

3.12. References 159

3.12 References

[1] Mattia, E.; Otto, S. Nat. Nanotech-nol. 2015, 10, 111-119.

[2] Ruiz-Mirazo, K.; Briones, C.; de la Escosura, A. Chem. Rev. 2014, 114, 285-366.

[3] Ludlow, R. F.; Otto, S. Chem. Soc. Rev. 2008, 37, 101-108.

[4] Cougnon, F. B. L.; Sanders, J. K. M. Acc. Chem. Res. 2012, 45, 2211-2221.

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3.11 Transmission Electron Microscopy Images

Figure 3.49: Transmission electron micrographs showing the 16 fibers produced by the

DCL made from peptide 1 seeded with 48.

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