University of Groningen Structure-Function Relationships in Dynamic Combinatorial Libraries Altay, Meniz

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Structure-Function Relationships in Dynamic Combinatorial Libraries

Altay, Meniz

DOI:

10.33612/diss.90038152

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2019

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[15] Colomb-Delsuc, M.; Sadownik, J. W.; Otto, S. Nat. Commun. 2015, 6, 7427-7433.

[16] Carnall, J. M. A.; Waudby, C. A.; Belenguer, A. M.; Stuart, M. C.; Peyralans, J. J. P.; Otto, S. Science 2010, 327, 1502-1506.

[17] Colomb-Delsuc, M. Mechanistic Studies on a Peptide-Based Self-Replicating Sys-tem, Thesis, University of Groningen, 2015.

[18] Taylor, G. I. Phil. Trans. R. Soc. Lond. A 1923, 223, 289-343. [19] Mitchell, A. R. Biopolymers 2008, 90, 175-184.

[20] Collins, J. M.; Porter, K. A.; Singh, S. K.; Vanier, G. S. Org. Lett. 2014, 16, 940-943.

Manuscript in preparation.

Chapter 3 Spacer Length: A Factor for Tuning

Self-Replication in Dynamic Combinatorial

Libraries

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3.1 Introduction

T

he search for synthesizing life from non-living precursors is one of the most chal-lenging tasks for scientists. Since the RNA world hypothesis1,2 _{was first put}

forward in 1960s, RNA was proven to be both capable of storing genetic information and catalyzing certain chemical reactions and protein assemblies.3–6_{Although RNA}

was believed to be the first and main component of the early stages of evolution, more recent hypotheses have claimed that life could have emerged from much sim-pler entities.7,8_{With that, structure-function relationships of simpler molecules (like}

peptides, amino acids and nucleic acids) have gained much more attention in research aimed at understanding prebiotic evolution.9–12

Figure 3.1: a) Chemical structures of the building blocks that form replicators. b) Relative UPLC peak area (%) showing the emergence of corresponding replicators 16and 28over time.

Figure adapted from the reference.13

Our group previously showed that replicator size could be diversified by tuning overall peptide hydrophobicity by changing a single amino acid in the sequence.13

The same study also revealed two important selection criteria for new replicators to emerge: first, a certain number of building blocks is needed in a macrocycle to drive self-assembly and second, in order to call a specific macrocycle a ‘self-replicator’,

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its emergence should be slower in the DCL without an autocatalytic pathway. In the light of these findings, we selected the two of those previously reported build-ing blocks to study their self-assembly and self-replication behavior after makbuild-ing subtle structural changes (Figure 3.1). We focused on how the spacer length be-tween the aromatic core and the β-sheet forming tail of the building blocks influences self-assembly and self-replication. After investigating the DCLs made from individ-ual building blocks, we tested the replication behaviour where a specific macrocycle shifted the equilibrium towards producing more of itself. In Section 3.2.4, we tested replication behaviour in multi-building block systems in DCLs made from equimolar mixtures of ‘mutated’ building blocks.

3.2 Results and Discussion

We modified the spacer length of the two building blocks (1 and 2) by introducing an additional methylene unit to the backbone. By doing so, the overall hydrophobicity of the building block was slightly increased without introducing additional H-bond possibilities which may change the β-sheet propensity of the building blocks (Figure 3.2).

Figure 3.2: Chemical structures of the building blocks with diﬀerent spacer lengths. Mod-ifications are made by substitution of glycine with two unnatural amino acids having extra methylene units: β-alanine in 1a and 2a, γ-aminobutyric acid in 1b and 2b.

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blocks, we tested various external factors (building block purity, buﬀer type, use of denaturant) which were found out to aﬀect the resulting self-assembly in the solution and further self-replication behaviour.

3.2.1 DCLs made from building blocks 1a and 1b

Building block 1a was synthesized on solid phase via conventional SPPS with_{ą90 %} purity. Following the distribution of species in the DCL (1.0 mM in 50 mM borate buﬀer, pH 8.2) on a UPLC system over eight days, we observed a very rapid oxidation of the monomer (Figure 3.3a). The cyclic pentamer (1a)5 exceeded 80% of the

library material when the oxidation was complete after two days. We confirmed the suspected self-assembly of (1a)5into stacks with electron microscopy. As Figure 3.3b

shows, (1a)5forms long single fibers with approximately 5 nm width (Figure 3.3b).

These findings suggest that a new replicator with a smaller macrocycle size (cyclic pentamer) emerges compared to the behaviour observed for the parent building block which gave rise to the cyclic hexamer 16.

When compared to the exponential growth profile of 16, two processes happening

simultaneously prevented us from observing a similar behaviour for (1a)5: First one

is the very fast oxidation of monomer (1a)1and second is the very rapid nucleation of

the cyclic pentamer followed by the elongation of (1a)5stacks. From an evolutionary

point of view, such rapid emergence of a replicator could be an advantage. However, as we did not observe a lag phase in the early phase of the emergence of (1a)5, which

is a hallmark for an autocatalytic pathway,12 _{experimental proof for the replicating}

Figure 3.3: a) Relative UPLC peak area (%) showing the distribution of species in a DCL made from building block 1a with_{ą90% purity (1.0 mM in 50 mM borate buﬀer, pH 8.2),} b) TEM micrograph showing (1a)5 fibers.

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behaviour of (1a)5 remains elusive under these conditions. In order to gain more

insight, we first probed whether the oxidation rate of the monomer has an eﬀect on (1a)5 nucleation. We set up a DCL made from 1.0 mM building block (1a) and

kept it under an inert atmosphere. As Figure 3.4 shows that, although the DCL was exposed to oxygen from the air only during preparation, (1a)5 nucleation was still

very rapid; also in a sample that was only partially oxidized.

Figure 3.4: UPLC chromatograms for a DCL made from building block 1a (1.0 mM in 50 mM borate buﬀer pH 8.2) and kept agitated under inert atmosphere after preparation a) on day 0, b) on day 2.

Another possible way to slow down the growth of (1a)5 is to introduce a

denat-urant into the system. In biology, guanidinium chloride (GdmCl) is widely used to induce protein unfolding. Even though the exact denaturing mechanism of GdmCl is not known, there are studies showing it acts by disrupting salt bridges.14,15

As-suming a similar eﬀect on our peptide-based systems, we set up DCLs made from 1.0 mM building block (1a) with diﬀerent concentrations of GdmCl and monitored the library distribution over five days. When compared to the control sample which is GdmCl free (Figure 3.5h), we observed that the emergence of cyclic pentamer (1a)5

was inhibited in all samples. Instead, cyclic tetramer (1a)4 emerged as the main

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Figure 3.5: UPLC chromatograms after five days (monitored at 254 nm) of the DCLs made from peptide 1a (1.0 mM in 50 mM borate buﬀer, pH 8.2) containing varying amounts of GdmCl: a) 4.0 M, b) 3.0 M, c) 2.5 M, d) 2.0 M, e) 1.0 M, f) 400 mM, g) 50 mM and h) control sample without GdmCl.

This observation suggests that even small changes in the environment could shift the equilibrium in favour of a smaller cyclic species, (1a)4 and (1a)3 in this case.

After this point, we continued our investigation using a new batch of building block 1a with higher purity (from 90% to_{ą98%). We first tested the system using} diﬀer-ent concdiﬀer-entrations of the building block (Figure 3.6). Surprisingly, using the same concentration (1.0 mM) of 1a as before, cyclic tetramer (1a)4 was dominating the

library, instead of cyclic pentamer (1a)5. Note that (1a)5 was still formed in most

experiments but only transiently. When we compared the growth rate of cyclic tetramer, 0.5 mM, 1.0 mM and 2.0 mM libraries showed no significant diﬀerence and the relative percent of (1a)4 reached 80% in the first three days. In 0.1 mM and 3.8

mM libraries, the equilibrium was reached more slowly than in the other samples, possibly due to the emergence of the less stable (1a)3 and (1a)5 at an early stage,

which then decomposed in favour of (1a)4.

In the literature, the eﬀect of buﬀer on the nature of self-assembling structures has been widely studied.16 _{Additionally, NaCl was shown to be able to change the}

dimensions of assemblies through its eﬀect on the ionic strength of the solution.17

In the light of these observations, we compared the new emergent properties in a different buffer environment. We set up a DCL made from 1.0 mM building block 1a in phosphate buffered saline (PBS) and kept it agitated. Following the oxidation of

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Figure 3.6: Relative UPLC peak area (%) of species in agitated DCLs made from building block 1a (in 50 mM borate buﬀer, pH 8.2) with a concentration of a) 3.8 mM, b) 2.0 mM, c) 1.0 mM, d) 0.5 mM and e) 0.1 mM.

the monomer (1a)1, this time cyclic hexamer (1a)6dominated the library in the first

few days but then slowly decomposed to give way to cyclic tetramer (1a)4 (Figure

3.7b).

The above results revealed three diﬀerent self-assembling species all emerging from the same building block, depending on the environment, with the tetramer (1a)4

being the thermodynamically most stable one. Thus, upon increasing the spacer length, a smaller number of building blocks suﬃces for macrocycle self-assembly. We tested the autocatalytic behaviour of the diﬀerently sized macrocycles. As we discussed earlier, even though cyclic pentamer (1a)5showed features typical for

self-replication, we were unable to proof autocatalysis by self-seeding experiments. In contrast, we were able to proof the self-replication for (1a)4 and (1a)6. To a DCL

made from building block 1a (1.0 mM in 50 mM borate buﬀer, pH 8.2), we added 0.2 mol eq. (1a)4at day zero which induced a faster increase in the concentration of

cyclic tetramer (1a)4compared to its emergence in an unseeded library (Figure 3.8),

which strongly supports its self-replication behaviour.

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Figure 3.7: Relative UPLC peak areas (%) for the DCLs made from building block 1a in different buffer and salt environments: a) in borate buffer (50 mM, pH 8.2), b) in PBS (50 mM, 150 mM NaCl, pH 8.2).

Figure 3.8: Relative UPLC peak area (%) for (1a)4 growth in DCLs with and without

seed. Seeding experiment was performed in a DCL made from building block 1a (1.0 mM in 50 mM borate buﬀer, pH 8.2), mixed with 0.2 mol eq. (1a)4as seed and agitated at 1200

rpm.

aliquot from the DCL after two days of preparation in PBS when (1a)6 was still the

main species. We used it as seed in a separate DCL made from building block (1a) in borate buﬀer (50 mM, pH 8.2). While, without any seed, (1a)4 emerged as the

main species (Figure 3.6), when seeded with (1a)6 composition changed in favour of

(1a)6. Such behaviour confirmed that (1a)6 is also a self-replicator (Figure 3.9).

These results show that the slightly increased hydrophobicity of (1a) compared to 1 resulted in self-assembling species with smaller macrocycle sizes (cyclic pentamer and tetramer). We were also able to show self-replication of (1a)4 and (1a)6, under

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Figure 3.9: Relative UPLC peak area (%) in the DCLs made from building block 1a; a) 1.0 mM in 50 mM PBS +150 mM NaCl, pH 8.2 without any seed and b) 1.0 mM in 50 mM borate buﬀer, pH 8.2 and mixed with 0.2 mol eq. solution containing„80% (1a)6.

diﬀerent environmental conditions.

We also confirmed fiber formation as a result of self-assembly. Transmission elec-tron microscopy images showed that each diﬀerently sized macrocycle formed fibers with slight diﬀerences in their morphology (Figure 3.10). While (1a)5 assembled

into relatively long single fibers (Figure 3.3b), (1a)4gave shorter laterally associated

fibers (Figure 3.10a). In contrast to (1a)4 and (1a)5, (1a)6 fibers formed large

ag-gregates which was possibly a result of the high concentration of salt present in the solution as the DCL was prepared in PBS (Figure 3.10b).

Figure 3.10: TEM micrographs of a) (1a)4fibers formed in the stirred DCL made fromě

98% pure building block 1a in 50 mM borate buﬀer, pH 8.2; b) (1a)6fibers formed in the

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We characterized the secondary structures of the (1a)4and (1a)6assemblies with

circular dichroism (CD) and thioflavin T (ThT) fluorescence assays. Based on our previously reported analyses,18 _{the observed negative helicity at 196 nm and a}

pos-itive helicity around 208 nm suggests antiparallel β-sheet structure in the resulting fibers (Figure 3.11a and Figure 3.11b). Thioflavin T assays confirmed amyloid fibril-like structure for (1a)4and (1a)6fibers with increased emission intensities at 490 nm

compared to blank samples (Figure 3.11c and Figure 3.11d).

Figure 3.11: CD and ThT spectra for the self-replicators that from DCLs prepared from building block 1a; a) CD spectrum of (1a)4, b) CD spectrum of (1a)6, c) ThT spectrum

of (1a)4 and d) ThT spectrum of (1a)6.

3.2.2 DCLs made from building block 1b

To further test the impact of alkyl spacer length on self-assembly, we introduced γ-aminobutyric acid (GABA) which is a slightly more hydrophobic amino acid with three methylene units. All the DCLs were set up with building block (1b) with_ě98% purity to avoid any side product formation. We first tested the eﬀect of oxidation and mode of agitation. A set of DCLs were prepared by dissolving 1.0 mM building block (1b) in aqueous borate buﬀer (50 mM, pH 8.2). While two of the libraries were oxidized by oxygen from the air, the other two were_{ą90% oxidized with respect to} monomer concentration with sodium perborate. In addition, we kept two of these libraries agitated at 1200 rpm and the other two were kept without agitation.

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We monitored species distribution using UPLC for eight days (Figure 3.12). The results show that the unagitated DCLs contained cyclic trimers (1b)3 and tetramers

(1b)4 as the two major species. In contrast, in agitated DCLs, cyclic tetramer seems

to be a kinetic product of which the relative area decreased over time. Upon agitation, cyclic trimer (1b)3 and hexamer (1b)6were the dominant products. However, even

though the cyclic hexamer constituted up to 40% of the overall species distribution, it’s presumably not the most stable product as its concentration started to decrease after five days (Figure 3.12a and Figure 3.12c).

We discovered that composition of the DCLs made from building block (1b) could be shifted favouring one of the two self-assembling species. When we set up a 1.0 mM DCL in borate buﬀer (50 mM, pH 8.2) and oxidized more than 90% with respect to monomer with an aqueous solution of sodium perborate, cyclic trimer (1b)3emerged

as the major product (Figure 3.13a). In contrast, addition of a structurally related cross-seed (16) shifted the composition towards more of the cyclic hexamer (1b)6

Figure 3.12: Relative UPLC peak area (%) for the DCLs made from building block 1b (1.0 mM in 50 mM borate buﬀer, pH 8.2): a) agitated and air oxidized, b) unagitated and air oxidized, c) agitated and pre-oxidized, d) unagitated and pre-oxidized. For the libraries in (c) and (d), DCLs were oxidized more than 90% with respect to monomer concentration.

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(Figure 3.13c). For each species, we confirmed fiber formation by TEM. Both (1b)3

and (1b)6 formed bundles of fibers with similar morphologies (Figure 3.13b and

Figure 3.13d).

Figure 3.13: UPLC chromatograms (monitored at 254 nm) showing the distribution of species after reaching a stable composition in DCLs made from building block 1b (1.0 mM in 50 mM borate buﬀer, pH 8.2): a) pre-oxidized _{ą 90% with respect to monomer with} perborate and b) cross-seeded with 0.1 mol eq. 16 immediately after library preparation.

TEM micrographs of the samples dominated by c) (1b)3 and d) (1b)6.

3.2.3 DCLs made from building blocks 2a and 2b

As a next step in our investigation, we switched to a slightly less hydrophobic building block which contains an alanine residue instead of phenyl alanine and is known to form the 8-ring replicator 28. We followed a similar approach and introduced β-alanine

and GABA into building block 2. However, we did not observe any indication for self-assembly or self-replication in the DCLs made from 1.0 mM 2a or 2b in borate buﬀer (50 mM, pH 8.2). Only cyclic trimers (2a)3, (2b)3and tetramers (2a)4, (2b)4

emerged when the DCLs reached equilibrium (Figure 3.14a and 14c). We tested if we could trigger self-assembly by using the structurally closely related replicator solution 28. To DCLs made from building blocks 2a or 2b (1.0 mM in 50 mM borate

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distribution of species over time. These seeding experiments led to the formation of series of diﬀerent species, of which we could only identify the cyclic trimers and tetramers of 2a and 2b, together with cyclic hexamer of 2a in the first case.

Figure 3.14: UPLC chromatograms (monitored at 254 nm) for the DCLs made from a) 1.0 mM 2a, b) 1.0 mM 2a cross-seeded with 0.2 mol eq. 28, c) 1.0 mM 2b and d) 1.0 mM

2b cross-seeded with 0.2 mol eq. 28. Each DCL was prepared in 50 mM borate buﬀer (pH

8.2) and constantly agitated at room temperature.

3.2.4 Multi-component DCLs

One of the main requirements to build up replicator eco-systems is the presence of multiple building blocks. With that in mind, we set up a series of DCLs made from equimolar amounts of building blocks 1, 1a and 1b as binary or tertiary mixtures. Figure 3.15 shows the distribution of species in these mixed systems. Combined with the UPLC-MS results, we observed a range of macrocyclic species from trimers to hexamers. While equimolar mixtures of 1+1a and 1+1b both showed cyclic hexamers emerging as the main species (Figure 3.15a and Figure 3.15b), cyclic trimers dominated the overall distribution when both ‘mutated’ building blocks 1a and 1b were present in the DCL (Figure 3.15c and Figure 3.15d). We could not detect the self-replicators that contain only one type of building block which we discussed above. Instead, we observed that the emergence of self-replicators was strongly inhibited in those DCLs.

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Figure 3.15: UPLC chromatograms (monitored at 254 nm) after eleven days for the DCLs (in 50 mM borate buﬀer, pH 8.2) made from equimolar mixtures of building blocks 1, 1a and 1b: a) [1]=[1a]=0.5 mM, b) [1]=[1b]=0.5 mM, c) [1a]=[1b]=0.5 mM, d) [1]=[1a]=[1b]=0.33 mM.

We also characterized the assemblies of the cyclic hexamer and trimer mixtures with circular dichroism (CD) and thioflavin T (ThT) assays. Up to 4-fold increase in emission signals of ThT suggests β-sheet structures. Based on the signals at around 196 nm and 210 nm in the CD spectra, we conclude that all of the DCLs contain a mixture of parallel and anti-parallel β-sheets. Only the mixture of all three building blocks showed more anti-parallel character compared to the other DCLs (Figure 3.16).

Self-assembly into fibers was confirmed by electron microscopy. Figure 3.17 re-vealed relatively ordered mixtures of single fibers and/or bundles of fibers for samples made from (1+1a) and (1+1b) (Figure 3.17a and Figure 3.17b). In contrast, as the ‘mutated’ building blocks dominated the composition, fibers became more diverse in length and morphology (Figures Figure 3.17c and Figure 3.17d).

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Figure 3.16: CD and ThT spectra for the mixed species formed in the DCLs made from equimolar mixtures of building blocks in 50 mM borate buﬀer, pH 8.2: a) [1]=[1a]=0.5 mM , b) [1]=[1b]=0.5 mM, c) [1a]=[1b]=0.5 mM and d) [1]=[1a]=[1b]=0.33 mM.

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Figure 3.17: TEM images for the mixed species formed in the DCLs made from equimolar mixtures of building blocks in 50 mM borate buﬀer, pH 8.2: a) [1]=[1a]=0.5 mM , b) [1]=[1b]=0.5 mM, c) [1a]=[1b]=0.5 mM and d) [1]=[1a]=[1b]=0.33 mM.

Using a similar approach, we analysed DCLs constructed from equimolar mixtures of 2, 2a and/or 2b. UPLC-MS analyses revealed that all the DCLs were composed of mixtures of cyclic trimers and tetramers resembling a kinetic trap in the DCLs made from individual building blocks (Figure 3.18). CD and ThT assays showed no signs of a specific secondary structure but only random coils in the solution (Figure 3.19).

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Figure 3.18: UPLC chromatograms for the DCLs made from equimolar mixtures of build-ing blocks 2, (2a) and (2b) in aqueous borate buﬀer, pH 8.2: a) [2]=[2a]=0.5 mM, b) [2]=[2b]=0.5 mM, c) [2a]=[2b]=0.5 mM, d) [2]=[2a]=[2b]=0.33 mM.

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Figure 3.19: CD and ThT spectra for the mixed species formed in the DCLs made from equimolar mixtures of building blocks in 50 mM borate buﬀer, pH 8.2: a) [2]=[2a]=0.5 mM , b) [2]=[2b]=0.5 mM, c) [2a]=[2b]=0.5 mM and d) [2]=[2a]=[2b]=0.33 mM.

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3.3 Conclusions

In this chapter, we show that self-assembly can be aﬀected dramatically by small structural variations in building block design and by environmental changes. We in-vestigated these eﬀects using two model peptides which are known to form 6-ring (16)

and 8-ring (28) replicators. In the first set of DCLs made from mutants of building

block 1, the size of the self-assembling species became smaller and varied, depending on the conditions. When the β-sheet forming peptide was slightly less hydropho-bic, increasing spacer length inhibited self-assembly and the corresponding DCLs contained mostly cyclic trimers and tetramers (23/24). Surprisingly, replicators 16,

(1a)6, (1a)4, 28were not observed in the DCLs made from building block mixtures.

These observations suggest that the overall building block hydrophobicity can be a useful tool to control the size of the self-replicator, as increased hydrophobicity in-duced self-replicators with smaller macrocycle sizes to grow in case of building block 1 and its mutants. Moreover, depending on the amino-acid sequence this feature can be used to inhibit replicator emergence, in case of building block 2. Therefore, such small structural variations open up opportunities for investigating the evolution of multi-building block replicators in dynamic molecular environments.

3.4 Acknowledgements

Yi˘git Altay is gratefully acknowledged for the discussion on secondary structure anal-yses of the self-assembling species by circular dichroism (CD) and thioflavin T (ThT) assays.

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3.5 Experimental Section

3.5.1 Materials

Doubly distilled water was used in all experiments. Chemicals that are used in buﬀer preparation and peptide synthesis were purchased from Sigma Aldrich and/or Merck Chemicals. Peptide synthesis grade and UPLC grade solvents are purchased from Biosolve. Peptides were purchased from Cambridge Peptides Ltd. (Birmingham, UK) with_{ě90% and/or ě98% purities.}

3.5.2 Methods

Peptide Synthesis

Preliminary experiments (most of the DCLs made from individual peptides) were set up using building blocks synthesized on a solid support using Wang Resin (0.57-1.00 mmol/g substitution) at 0.1 mmol scale by following conventional solid phase peptide synthesis methodology. In each coupling step the DIC/Oxyma protocol was followed.19 _{Crude peptides were purified on a Schimadzu HPLC system using a}

Phe-nomenex, Jupiter prep-column (10µm, C5, 300 ˚A, 250 x 21.2 mm). Library and Sample Preparation

Building blocks were dissolved to 0.1-3.8 mM in borate buﬀer (50 mM in B4O7

2-anions, pH 8.2) or phosphate buﬀered saline (PBS) (50 mM with 150 mM NaCl, pH 8.2). Libraries were equilibrated in HPLC vials (12ˆ 32 mm) with Teflon caps and were left stirring on an IKA RCT hot plate stirrer at 1200 rpm.

Circular Dichroism (CD)

Samples were diluted to a concentration of 0.2 mM with respect to building block. Spectra were recorded at room temperature by using a JASCO J715 spectropho-tometer and HELMA quartz cuvettes with a path length of 1 mm. All spectra were recorded at room temperature from 190 nm to 300 nm, with 2 nm step interval and 3 scans with a speed of 200 nm/min. Solvent spectra were subtracted from all spectra. Thioflavin T (ThT) Fluorescence Assay

A ThT stock solution (2.2 mM) was prepared in phosphate buﬀer (50 mM with 150 mM NaCl, pH 8.2). A working solution was prepared by diluting the stock solution to 22µm. The fluorescence intensity of a 450 µL ThT solution (blank) was measured by excitation at 440 nm (5 nm slit width) and emission between 480 - 700 nm (5 nm slit

width). An aliquot of 80µL of the 100 µm peptide solution was added to a HELMA 10 _{ˆ 2 mm quartz cuvette and the sample was incubated for 2 minutes before the} measurement. All fluorescence measurements were performed by averaging 3 repeats on a JASCO FP6200 spectrophotometer with 480 nm cut-oﬀ filter on the emission channel to avoid high order diﬀractions resulting from the excitation.

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3.5 Experimental Section

3.5.1 Materials

Doubly distilled water was used in all experiments. Chemicals that are used in buﬀer preparation and peptide synthesis were purchased from Sigma Aldrich and/or Merck Chemicals. Peptide synthesis grade and UPLC grade solvents are purchased from Biosolve. Peptides were purchased from Cambridge Peptides Ltd. (Birmingham, UK) with_{ě90% and/or ě98% purities.}

3.5.2 Methods

Peptide Synthesis

Preliminary experiments (most of the DCLs made from individual peptides) were set up using building blocks synthesized on a solid support using Wang Resin (0.57-1.00 mmol/g substitution) at 0.1 mmol scale by following conventional solid phase peptide synthesis methodology. In each coupling step the DIC/Oxyma protocol was followed.19 _{Crude peptides were purified on a Schimadzu HPLC system using a}

Phe-nomenex, Jupiter prep-column (10µm, C5, 300 ˚A, 250 x 21.2 mm). Library and Sample Preparation

Building blocks were dissolved to 0.1-3.8 mM in borate buﬀer (50 mM in B4O7

2-anions, pH 8.2) or phosphate buﬀered saline (PBS) (50 mM with 150 mM NaCl, pH 8.2). Libraries were equilibrated in HPLC vials (12ˆ 32 mm) with Teflon caps and were left stirring on an IKA RCT hot plate stirrer at 1200 rpm.

Circular Dichroism (CD)

Samples were diluted to a concentration of 0.2 mM with respect to building block. Spectra were recorded at room temperature by using a JASCO J715 spectropho-tometer and HELMA quartz cuvettes with a path length of 1 mm. All spectra were recorded at room temperature from 190 nm to 300 nm, with 2 nm step interval and 3 scans with a speed of 200 nm/min. Solvent spectra were subtracted from all spectra. Thioflavin T (ThT) Fluorescence Assay

A ThT stock solution (2.2 mM) was prepared in phosphate buﬀer (50 mM with 150 mM NaCl, pH 8.2). A working solution was prepared by diluting the stock solution to 22µm. The fluorescence intensity of a 450 µL ThT solution (blank) was measured by excitation at 440 nm (5 nm slit width) and emission between 480 - 700 nm (5 nm slit

width). An aliquot of 80µL of the 100 µm peptide solution was added to a HELMA 10_{ˆ 2 mm quartz cuvette and the sample was incubated for 2 minutes before the} measurement. All fluorescence measurements were performed by averaging 3 repeats on a JASCO FP6200 spectrophotometer with 480 nm cut-oﬀ filter on the emission channel to avoid high order diﬀractions resulting from the excitation.

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3.6 Appendix

3.6.1 Kinetic profiles

Figure 3.20: Relative UPLC peak area (%) showing overall product distribution in DCLs made from building block 1a (1.0 mM in 50 mM borate buﬀer, pH 8.2) with varying amounts of GdmCl: a) 4.0 M, b) 3.0 M, c) 2.5 M, d) 2.0 M, e) 1.0 M, f) 400 mM, g) 50 mM, h) control sample without GdmCl.

Figure 3.21: Relative UPLC peak area (%) showing species distribution in agitated DCLs made from building block 2a (in 50 mM borate buﬀer, pH 8.2) with diﬀerent concentrations: a) 3.8 mM, b) 2.0 mM, c) 1.0 mM, d) 0.5 mM and e) 0.1 mM.

Figure 3.22: Relative UPLC peak area (%) showing species distribution in agitated DCLs made from building block 2b (in 50 mM borate buﬀer, pH 8.2) with diﬀerent concentrations: a) 3.8 mM, b) 2.0 mM, c) 1.0 mM, d) 0.5 mM and e) 0.1 mM.

(24)

3.6 Appendix

3.6.1 Kinetic profiles

Figure 3.20: Relative UPLC peak area (%) showing overall product distribution in DCLs made from building block 1a (1.0 mM in 50 mM borate buﬀer, pH 8.2) with varying amounts of GdmCl: a) 4.0 M, b) 3.0 M, c) 2.5 M, d) 2.0 M, e) 1.0 M, f) 400 mM, g) 50 mM, h) control sample without GdmCl.

Figure 3.21: Relative UPLC peak area (%) showing species distribution in agitated DCLs made from building block 2a (in 50 mM borate buﬀer, pH 8.2) with diﬀerent concentrations: a) 3.8 mM, b) 2.0 mM, c) 1.0 mM, d) 0.5 mM and e) 0.1 mM.

Figure 3.22: Relative UPLC peak area (%) showing species distribution in agitated DCLs made from building block 2b (in 50 mM borate buﬀer, pH 8.2) with diﬀerent concentrations: a) 3.8 mM, b) 2.0 mM, c) 1.0 mM, d) 0.5 mM and e) 0.1 mM.

(25)

3.6.2 LC-MS analyses of 1a

Figure 3.23: Mass spectrum of monomer (1a)1from the UPLC-MS analysis of an agitated

library made from peptide 1a. Calculated isotopic profile for [M+H]+(species, abundance):

774.34 (M, 96.78%), 775.34 (M+1, 46.57%); m/z calculated: 774.37 [M+H]+; m/z observed:

774.34 [M+H]+.

Figure 3.24: Mass spectrum of cyclic trimer (1a)3 from the UPLC-MS analysis of an

agitated library made from peptide 1a. Calculated isotopic profile (species, abundance)

for [M+3H]3+_{: 772.95 (M, 100%), 773.28 (M+1, 98.41%), 773.62 (M+2, 71.10%), 773.95}

(M+3, 41.49%); m/z calculated: 1158.53 [M+2H]2+_{, 772.35 [M+3H]}3+_{, 579.26 [M+4H]}4+_;

m/z observed: 1158.94 [M+2H]2+_{, 772.95 [M+3H]}3+_{, 580.20 [M+4H]}4+_.

Figure 3.25: Mass spectrum of cyclic tetramer (1a)4 from the UPLC-MS analysis of an

for [M+3H]3+_{: 1029.82 (M, 100%), 1030.15 (M+1, 88.34 %), 1030.48 (M+2, 64.09 %),}

1030.82 (M+3, 40.82 %); m/z calculated: 1544.70 [M+2H]2+_{, 1029.80 [M+3H]}3+_{, 772.35}

[M+4H]4+_{; m/z observed: 1543.95 [M+2H]}2+_{, 1029.82 [M+3H]}3+_{, 772.74 [M+4H]}4+_.

Figure 3.26: Mass spectrum of cyclic pentamer (1a)5 from the UPLC-MS analysis of an

agitated library made from peptide 1a. Calculated isotopic profile (species abundance)

for [M+4H]4+: 966.03 (M, 100%), 966.28 (M+1, 96.90%), 966.53 (M+2, 81.14%), 966.78

(M+3, 58.81%), 967.03 (M+4, 34.19%); m/z calculated: 1930.88 [M+2H]2+, 1287.26

[M+3H]3+_{, 965.44 [M+4H]}4+_{, 772.35 [M+5H]}5+_{; m/z observed:} _{1931.03 [M+2H]}2+_,

(26)

3.6.2 LC-MS analyses of 1a

library made from peptide 1a. Calculated isotopic profile for [M+H]+(species, abundance):

774.34 (M, 96.78%), 775.34 (M+1, 46.57%); m/z calculated: 774.37 [M+H]+; m/z observed:

774.34 [M+H]+.

for [M+3H]3+_{: 772.95 (M, 100%), 773.28 (M+1, 98.41%), 773.62 (M+2, 71.10%), 773.95}

for [M+3H]3+_{: 1029.82 (M, 100%), 1030.15 (M+1, 88.34 %), 1030.48 (M+2, 64.09 %),}

agitated library made from peptide 1a. Calculated isotopic profile (species abundance)

for [M+4H]4+: 966.03 (M, 100%), 966.28 (M+1, 96.90%), 966.53 (M+2, 81.14%), 966.78

(M+3, 58.81%), 967.03 (M+4, 34.19%); m/z calculated: 1930.88 [M+2H]2+, 1287.26

[M+3H]3+_{, 965.44 [M+4H]}4+_{, 772.35 [M+5H]}5+_{; m/z observed:} _{1931.03 [M+2H]}2+_,

(27)

Figure 3.27: Mass spectrum of cyclic hexamer (1a)6from the UPLC-MS analysis of an

agitated library made from peptide 1a. Calculated isotopic profile (species abundance) for

[M+4H]4+_{: 1158.63 (M, 100%), 1158.88 (M+1, 80.31%), 1159.12 (M+2, 60.91%), 1159.38}

(M+3, 43.63%), 1159.62 (M+4, 25.16%); m/z calculated: 1544.70 [M+3H]3+_{, 1158.53}

[M+4H]4+_{, 926.82 [M+5H]}5+_{; m/z observed: 1543.98 [M+3H]}3+_{, 1158.63 [M+4H]}4+_,

927.21 [M+5H]5+_.

3.6.3 LC-MS analyses of 1b

Figure 3.28: Mass spectrum of monomer (1b)1from the UPLC-MS analysis of an agitated

library made from peptide 1b. Calculated isotopic profile for [M+H]+(species, abundance):

788.73 (M, 100%), 789.74 (M+1, 57.44%); m/z calculated: 788.38 [M+H]+; m/z observed:

788.73 [M+H]+.

Figure 3.29: Mass spectrum of cyclic trimer (1b)3 from the UPLC-MS analysis of an

agitated library made from peptide 1b. Calculated isotopic profile (species, abundance)

for [M+3H]3+_{: 786.61 (M, 100%), 786.94 (M+1, 95.25%), 787.28 (M+2, 67.91%), 787.61}

(28)

[M+4H]4+_{: 1158.63 (M, 100%), 1158.88 (M+1, 80.31%), 1159.12 (M+2, 60.91%), 1159.38}

927.21 [M+5H]5+_.

3.6.3 LC-MS analyses of 1b

library made from peptide 1b. Calculated isotopic profile for [M+H]+(species, abundance):

788.73 (M, 100%), 789.74 (M+1, 57.44%); m/z calculated: 788.38 [M+H]+; m/z observed:

788.73 [M+H]+.

for [M+3H]3+_{: 786.61 (M, 100%), 786.94 (M+1, 95.25%), 787.28 (M+2, 67.91%), 787.61}

(29)

Figure 3.30: Mass spectrum of cyclic tetramer (1b)4 from the UPLC-MS analysis of an

for [M+3H]3+_{: 1048.54 (M, 100%), 1048.87 (M+1, 97.61 %), 1049.20 (M+2, 62.42 %),}

Figure 3.31: Mass spectrum of cyclic hexamer (1b)6from the UPLC-MS analysis of an

agitated library made from peptide 1b. Calculated isotopic profile (species abundance) for

[M+4H]4+_{: 1180.43 (M, 100%), 1180.68 (M+1, 83.13%), 1180.93 (M+2, 68.18%), 1181.17}

944.13 [M+5H]5+_.

3.6.4 LC-MS analyses of 2a

library made from peptide 2a. Calculated isotopic profile for [M+H]+_{(species, abundance):}

698.34 (M, 100%), 699.34 (M+1, 33.39%); m/z calculated: 698.34 [M+H]+_{; m/z observed:}

698.34 [M+H]+_.

for [M+3H]3+_{: 696.99 (M, 100%), 697.33 (M+1, 73.29%), 697.66 (M+2, 41.76%), 697.99}

(30)

for [M+3H]3+_{: 1048.54 (M, 100%), 1048.87 (M+1, 97.61 %), 1049.20 (M+2, 62.42 %),}

Figure 3.31: Mass spectrum of cyclic hexamer (1b)6from the UPLC-MS analysis of an

agitated library made from peptide 1b. Calculated isotopic profile (species abundance) for

[M+4H]4+_{: 1180.43 (M, 100%), 1180.68 (M+1, 83.13%), 1180.93 (M+2, 68.18%), 1181.17}

944.13 [M+5H]5+_.

3.6.4 LC-MS analyses of 2a

library made from peptide 2a. Calculated isotopic profile for [M+H]+_{(species, abundance):}

698.34 (M, 100%), 699.34 (M+1, 33.39%); m/z calculated: 698.34 [M+H]+_{; m/z observed:}

698.34 [M+H]+_.

for [M+3H]3+_{: 696.99 (M, 100%), 697.33 (M+1, 73.29%), 697.66 (M+2, 41.76%), 697.99}

(31)

for [M+3H]3+_{: 928.76 (M, 100%), 929.10 (M+1, 81.10%), 929.44 (M+2, 48.98%), 929.77}

[M+3H]3+_{: 1160.51 (M, 100%), 1160.85 (M+1, 91.45%), 1161.19 (M+2, 63.44%), 1161.52}

agitated library made from peptide 2a and cross-seeded with 0.2 mol eq. 28.

Calcu-lated isotopic profile (species abundance) for [M+4H]4+_{: 1044.46 (M, 100%), 1044.72}

(M+1, 98.85%), 1044.96 (M+2, 64.56%), 1045.19 (M+3, 50.32%), 1045.45 (M+4, 31.38%);

m/z calculated: 1392.64 [M+3H]3+_{, 1044.49 [M+4H]}4+_{, 835.59 [M+5H]}5+_{; m/z observed:}

1392.10 [M+3H]3+_{, 1044.46 [M+4H]}4+_{, 835.87 [M+5H]}5+_.

3.6.5 LC-MS analyses of 2b

library made from peptide 2b. Calculated isotopic profile for [M+H]+_{(species, abundance):}

712.35 (M, 85.52%), 713.36 (M+1, 31.26%); m/z calculated: 712.35 [M+H]+_{; m/z observed:}

(32)

for [M+3H]3+_{: 928.76 (M, 100%), 929.10 (M+1, 81.10%), 929.44 (M+2, 48.98%), 929.77}

[M+3H]3+_{: 1160.51 (M, 100%), 1160.85 (M+1, 91.45%), 1161.19 (M+2, 63.44%), 1161.52}

agitated library made from peptide 2a and cross-seeded with 0.2 mol eq. 28.

Calcu-lated isotopic profile (species abundance) for [M+4H]4+_{: 1044.46 (M, 100%), 1044.72}

(M+1, 98.85%), 1044.96 (M+2, 64.56%), 1045.19 (M+3, 50.32%), 1045.45 (M+4, 31.38%);

m/z calculated: 1392.64 [M+3H]3+_{, 1044.49 [M+4H]}4+_{, 835.59 [M+5H]}5+_{; m/z observed:}

1392.10 [M+3H]3+_{, 1044.46 [M+4H]}4+_{, 835.87 [M+5H]}5+_.

3.6.5 LC-MS analyses of 2b

library made from peptide 2b. Calculated isotopic profile for [M+H]+_{(species, abundance):}

712.35 (M, 85.52%), 713.36 (M+1, 31.26%); m/z calculated: 712.35 [M+H]+_{; m/z observed:}

(33)

for [M+3H]3+_{: 710.67 (M, 100%), 711.01 (M+1, 95.68 %), 711.34 (M+2, 68.28 %), 711.67}

(M+3, 37.27 %); m/z calculated: 1065.50 [M+2H]2+_{, 710.33 [M+3H]}3+_{, 532.75 [M+4H]}4+_;

for [M+3H]3+_{: 947.44 (M, 100%), 947.78 (M+1, 79.90 %), 948.12 (M+2, 47.73 %), 948.44}

3.6.6 LC-MS analyses of mixed libraries

Figure 3.40: Mass spectrum of cyclic hexamer (1)5(1b)1from the UPLC-MS analysis of

an agitated library made from peptides 1 and 1b. Calculated isotopic profile (species

abundance) for [M+4H]4+: 1144.87 (M, 100%), 1145.12 (M+1, 80.53 %), 1145.37 (M+2,

57.52 %), 1145.61 (M+3, 39.74 %), 1145.88 (M+4, 18.63 %); m/z calculated: 1526.01

[M+3H]3+_{, 1144.50 [M+4H]}4+_{, 915.60 [M+5H]}5+_{; m/z observed: 1525.64 [M+3H]}3+_,

(34)

for [M+3H]3+_{: 710.67 (M, 100%), 711.01 (M+1, 95.68 %), 711.34 (M+2, 68.28 %), 711.67}

for [M+3H]3+_{: 947.44 (M, 100%), 947.78 (M+1, 79.90 %), 948.12 (M+2, 47.73 %), 948.44}

3.6.6 LC-MS analyses of mixed libraries

abundance) for [M+4H]4+: 1144.87 (M, 100%), 1145.12 (M+1, 80.53 %), 1145.37 (M+2,

57.52 %), 1145.61 (M+3, 39.74 %), 1145.88 (M+4, 18.63 %); m/z calculated: 1526.01

(35)

abundance) for [M+4H]4+_{: 1151.63 (M, 100 %), 1151.87 (M+1, 92.32 %), 1152.12 (M+2,}

79.22 %), 1152.37 (M+3, 52.51%), 1152.63 (M+4, 34.19 %); m/z calculated: 1535.35

1151.63 [M+4H]4+_{, 921.81 [M+5H]}5+_.

Figure 3.42: Mass spectrum of cyclic hexamer (1)3(1b)3 from the UPLC-MS analysis of

71.94 %), 1159.37 (M+3, 54.45 %), 1159.63 (M+4, 36.23 %); m/z calculated: 1544.69

1158.63 [M+4H]4+_{, 927.41 [M+5H]}5+_.

78.18 %), 1166.39 (M+3, 55.70 %), 1166.63 (M+4, 37.39 %); m/z calculated: 1554.04

(36)

79.22 %), 1152.37 (M+3, 52.51%), 1152.63 (M+4, 34.19 %); m/z calculated: 1535.35

1151.63 [M+4H]4+_{, 921.81 [M+5H]}5+_.

71.94 %), 1159.37 (M+3, 54.45 %), 1159.63 (M+4, 36.23 %); m/z calculated: 1544.69

1158.63 [M+4H]4+_{, 927.41 [M+5H]}5+_.

78.18 %), 1166.39 (M+3, 55.70 %), 1166.63 (M+4, 37.39 %); m/z calculated: 1554.04

(37)

abundance) for [M+4H]4+: 1172.63 (M, 100 %), 1172.89 (M+1, 92.23 %), 1173.14 (M+2,

83.93 %), 1173.38 (M+3, 52.76 %), 1173.64 (M+4, 40.91 %); m/z calculated: 1563.38

[M+3H]3+, 1172.53 [M+4H]4+, 938.03 [M+5H]5+; m/z observed: 1563.00 [M+3H]3+,

1172.63 [M+4H]4+, 938.42 [M+5H]5+.

Figure 3.45: Mass spectrum of cyclic trimer (1)2(1b)1from the UPLC-MS analysis of

an agitated library made from peptides 1 and 1b. Calculated isotopic profile (species,

56.67 %), 768.93 (M+3, 33.35 %); m/z calculated: 1151.51 [M+2H]2+_{, 767.67 [M+3H]}3+_,

575.75 [M+4H]4+_{; m/z observed: 1151.65 [M+2H]}2+_{, 767.93 [M+3H]}3+_{, 576.29 [M+4H]}4+_.

Figure 3.46: Mass spectrum of cyclic trimer (1)1(1b)2 from the UPLC-MS analysis of

Figure 3.47: Mass spectrum of the mixture of cyclic hexamers (1)n(1)6-nfrom the

(38)

abundance) for [M+4H]4+: 1172.63 (M, 100 %), 1172.89 (M+1, 92.23 %), 1173.14 (M+2,

83.93 %), 1173.38 (M+3, 52.76 %), 1173.64 (M+4, 40.91 %); m/z calculated: 1563.38

[M+3H]3+, 1172.53 [M+4H]4+, 938.03 [M+5H]5+; m/z observed: 1563.00 [M+3H]3+,

1172.63 [M+4H]4+, 938.42 [M+5H]5+.

Figure 3.45: Mass spectrum of cyclic trimer (1)2(1b)1from the UPLC-MS analysis of

Figure 3.46: Mass spectrum of cyclic trimer (1)1(1b)2 from the UPLC-MS analysis of

Figure 3.47: Mass spectrum of the mixture of cyclic hexamers (1)n(1)6-nfrom the

(39)

(1)5(1a)1: Calculated isotopic profile (species, abundance) for [M+4H]4+: 1140.87

(M, 16.10 %), 1141.12 (M+1, 15.68 %), 1141.37 (M+2, 13.37 %), 1141.62 (M+3, 9.76 %), 1141.88 (M+4, 6.21 %); m/z calculated: 1521.34 [M+3H]3+_{, 1141.00 [M+4H]}4+_,

915.60 [M+5H]5+_{; m/z observed: 1520.64 [M+3H] 3+, 1140.87 [M+4H]}4+_{, 913.00}

[M+5H]5+_.

915.60 [M+5H]5+_{; m/z observed: 1525.30 [M+3H]}3+_{, 1144.37 [M+4H]}4+_{, 916.00}

[M+5H]5+_.

(M, 100 %), 1148.38 (M+1, 80.42 %), 1148.62 (M+2, 59.03 %), 1148.88 (M+3, 38.41 %), 1149.12 (M+4, 24.45 %); m/z calculated: 1530.68 [M+3H]3+_{, 1148.01 [M+4H]}4+_,

[M+5H]5+_.

(1a)6: Calculated isotopic profile (species, abundance) for [M+4H]4+: 1158.37

(M, 8.15 %), 1158.63 (M+1, 6.33 %), 1158.87 (M+2, 6.97 %), 1159.12 (M+3, 5.19 %), 1159.38 (M+4, 4.23 %); m/z calculated: 1158.53 [M+4H]4+_{, 926.82 [M+5H]}5+ _;

m/z observed: 1158.37 [M+4H]4+_{, 926.81 [M+5H]}5+_.

Figure 3.48: Mass spectrum of cyclic trimer (1)3 from the UPLC-MS analysis of an

agi-tated library made from peptides 1 and 1a. Calculated isotopic profile (species, abundance)

for [M+3H]3+_{: 758.58 (M, 100%), 758.91 (M+1, 86.00 %), 759.25 (M+2, 62.49 %), 759.58}

Figure 3.49: Mass spectrum of cyclic trimer (1)2(1a)1from the UPLC-MS analysis of

an agitated library made from peptides 1 and 1a. Calculated isotopic profile (species,

(40)

915.60 [M+5H]5+_{; m/z observed: 1520.64 [M+3H] 3+, 1140.87 [M+4H]}4+_{, 913.00}

[M+5H]5+_.

(M, 100 %), 1148.38 (M+1, 80.42 %), 1148.62 (M+2, 59.03 %), 1148.88 (M+3, 38.41 %), 1149.12 (M+4, 24.45 %); m/z calculated: 1530.68 [M+3H]3+_{, 1148.01 [M+4H]}4+_,

[M+5H]5+_.

(1a)6: Calculated isotopic profile (species, abundance) for [M+4H]4+: 1158.37

(M, 8.15 %), 1158.63 (M+1, 6.33 %), 1158.87 (M+2, 6.97 %), 1159.12 (M+3, 5.19 %), 1159.38 (M+4, 4.23 %); m/z calculated: 1158.53 [M+4H]4+_{, 926.82 [M+5H]}5+ _;

m/z observed: 1158.37 [M+4H]4+_{, 926.81 [M+5H]}5+_.

for [M+3H]3+_{: 758.58 (M, 100%), 758.91 (M+1, 86.00 %), 759.25 (M+2, 62.49 %), 759.58}

(41)

Figure 3.50: Mass spectrum of cyclic trimer (1)1(1a)2 from the UPLC-MS analysis of

Figure 3.51: Mass spectrum of cyclic trimer (1a)2(1b)1from the UPLC-MS analysis of

an agitated library made from peptides 1a and 1b. Calculated isotopic profile (species,

Figure 3.52: Mass spectrum of cyclic trimer (1a)1(1b)2 from the UPLC-MS analysis of

Figure 3.53: Mass spectrum of cyclic trimer (1)1(1a)1(1b)1from the UPLC-MS analysis

of an agitated library made from peptides 1, 1a and 1b. Calculated isotopic profile (species,

abundance) for [M+3H]3+_{: 773.00 (M, 100%), 773.34 (M+1, 97.11 %), 773.67 (M+2, 73.41}

%), 774.01 (M+3, 43.59 %); m/z calculated: 1158.52 [M+2H]2+_{, 772.35 [M+3H]}3+_{, 579.26}

(42)

Figure 3.51: Mass spectrum of cyclic trimer (1a)2(1b)1from the UPLC-MS analysis of

Figure 3.52: Mass spectrum of cyclic trimer (1a)1(1b)2 from the UPLC-MS analysis of

Figure 3.53: Mass spectrum of cyclic trimer (1)1(1a)1(1b)1from the UPLC-MS analysis

of an agitated library made from peptides 1, 1a and 1b. Calculated isotopic profile (species,

abundance) for [M+3H]3+_{: 773.00 (M, 100%), 773.34 (M+1, 97.11 %), 773.67 (M+2, 73.41}

%), 774.01 (M+3, 43.59 %); m/z calculated: 1158.52 [M+2H]2+_{, 772.35 [M+3H]}3+_{, 579.26}

(43)

Figure 3.54: Mass spectrum of cyclic tetramer (2)4 from the UPLC-MS analysis of an

agitated library made from peptide 2. Calculated isotopic profile (species, abundance) for

[M+3H]3+_{: 909.54 (M, 100%), 909.88 (M+1, 91.62 %), 910.21 (M+2, 82.08 %), 910.54}

Figure 3.55: Mass spectrum of cyclic tetramer (2)3(2a)1from the UPLC-MS analysis of

Figure 3.56: Mass spectrum of cyclic tetramer (2)2(2a)2 from the UPLC-MS analysis of

(44)

Figure 3.54: Mass spectrum of cyclic tetramer (2)4 from the UPLC-MS analysis of an

agitated library made from peptide 2. Calculated isotopic profile (species, abundance) for

[M+3H]3+_{: 909.54 (M, 100%), 909.88 (M+1, 91.62 %), 910.21 (M+2, 82.08 %), 910.54}

Figure 3.55: Mass spectrum of cyclic tetramer (2)3(2a)1from the UPLC-MS analysis of

(45)

for [M+3H]3+_{: 682.60 (M, 100 %), 682.93 (M+1, 87.55 %), 683.27 (M+2, 54.13 %), 683.60}

Figure 3.61: Mass spectrum of cyclic tetramer (2)3(2b)1from the UPLC-MS analysis of

(46)

for [M+3H]3+_{: 682.60 (M, 100 %), 682.93 (M+1, 87.55 %), 683.27 (M+2, 54.13 %), 683.60}

Figure 3.61: Mass spectrum of cyclic tetramer (2)3(2b)1from the UPLC-MS analysis of