University of Groningen Folding and replication in complex dynamic molecular networks Liu, Bin

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Folding and replication in complex dynamic molecular networks

Liu, Bin

DOI:

10.33612/diss.99784510

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Publication date: 2019

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Liu, B. (2019). Folding and replication in complex dynamic molecular networks. University of Groningen. https://doi.org/10.33612/diss.99784510

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Chapter 3 Dynamic Combinatorial Foldamers

Folding of macromolecules and fully synthetic chemical systems typically relies on the defined arrangement of secondary structure modules, such as helices, to give rise to often complex tertiary structures. Protein folding has been the result of millions of years of evolution through complex pathways. Research into synthetic foldamers has only recently started to focus on tertiary structures. In chapter 2, we have shown the emergence of a complex folded tertiary structure from DCLs made from amino acid‐nucleobase building blocks. These results prompted us to investigate dynamic folding using building blocks based on short peptides. In this chapter, we report the discovery of foldamers that form autonomously in aqueous media based on simple dipeptides using a systems chemistry approach. Crystallographic and spectroscopic analysis revealed unprecedented folding patterns, yielding complex tertiary structures. Remarkably, in one instance the folded structure does not feature any distinct secondary structure elements.

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3.1 Introduction

Establishing the fundamental principles that govern the emergence of structural complexity in well‐organized supramolecular systems remains a challenge.1 We know from protein chemistry that the way in which the twenty natural amino acids are inserted dictates dynamicity and the arrangement of local secondary segments, giving rise to unique molecular shapes, where atoms are located with atomic precision. Catalysis, binding, replication and information transfer arises at the level of tertiary assembly. Following a hierarchal approach,2 chemists have been able to build and manipulate molecular shapes obtained through folding. However, despite computational progress,3‐5 experimental manifestations of the synthesis and assembly of large complex molecules that can mimic and go beyond the shapes of proteins remain rare. The majority of structures of synthetic foldamers6‐12 reported to date rely on secondary structure motifs similar to those found in biopolymers, such as helices and, less abundantly, sheets, despite the broad repertoire of foldamer backbones. In biology folding has mainly arisen from sequences that have the propensity to assemble into helical13‐17 or β‐sheet like structures.18,19 Alterations of the amino‐acid sequence often dramatically impact non‐covalent interactions, leading to different shapes. Synthetic foldamers with chemical structures beyond the world of peptides and nucleotides have been shown to adopt complex tertiary structures.20‐23 Helically folded oligomeric backbones of defined length and high conformational stability have been utilized to bind DNA sequences,24 selectively encapsulate saccharides25 and induce charge‐transfer interactions.26 The field of foldamers has made great strides in producing artificial secondary structures. However given that much additional complexity and functional potential arises at the level of tertiary structure, accessing new types of folds using alternative chemistries might lead to the discovery of new shapes in which tertiary interactions not only stabilize local segments but actually govern them. In chapter 2, we have described a self‐synthesizing macrocyclic foldamer based on a chimeric amino acid‐nucleobase building block. The foldamer was composed of 15 identical subunits and exhibited a complex secondary and tertiary structure, consisting of repeats of five triads. Folding driven assembly27‐31 of molecules using a systems chemistry approach has received little attention, despite the fact that assembly phenomena can give rise to the selective formation of topologically defined structures,32,33 self‐replicating molecules34 and receptors for given targets.35

In this chapter, we report the discovery through dynamic combinatorial chemistry of highly ordered structures with, for synthetic foldamers, unrivalled complexity, by varying the molecular structure of simple dipeptide building blocks. By applying simple design rules, we

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Dynamic Combinatorial Foldamers

achieved the autonomous formation of giant foldamers (a 9, 12, 13, 16 and a 23mer), exhibiting remarkable interaction patterns.

Scheme 3.1. The formation of foldamers from DCLs made from dipeptide building blocks.

3.2 Results and Discussion

3.2.1 Design and synthesis of building blocks

We have extensively studied self‐replication based on building blocks featuring β‐sheet forming peptides (typically containing five amino‐acid residues) where small alterations of the sequence did not disrupt β‐sheet formation and assembly.36 We hypothesized that shortening the amino‐acid sequence and diminishing the propensity for β‐sheet formation might give more plasticity, potentially allowing for the formation of foldamers. Due to the vast combinatorial space available even for short peptide sequences, we decided to make use of simple dipeptides, the shortest possible sequences that have been reported to be organized into distinct assemblies.37 The dipeptides were composed of (un)natural amino acids appended with an aromatic dithiol at the N‐terminus of the sequence to enable the formation of different macrocyclic disulfides that interconvert through thiol‐disulfide exchange. From previous studies, we know that π‐π stacking is important for the stability of folded complex structures, so phenylalanine was introduced in the building block, expecting that the presence of the phenyl ring can assist the formation of folded structures. Besides, the para‐position of phenylalanine is easy to functionalize with substituents with different properties, which allows manipulating the supramolecular interactions by minor structural modifications. Lysine was introduced at the C‐terminus of the sequence to increase water solubility and the flexibility of the building block.

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In order to validate the design principles described above, a series of building blocks (1‐8, Scheme 3.1) composed of substituted phenylalanine and lysine were synthesized by Solid Phase Synthesis (SPS). Hydrophobic (fluorine, nitro and methoxy) and hydrophilic groups (hydroxyl, amino, carboxyl and guanidinium) were introduced at the para‐position of the phenylalanine residue.

3.2.2 Emergence of complex peptide foldamers

A series of libraries were set up by dissolving building blocks 1‐8 individually at a 2.0 mM concentration in borate buffer (50 mM, pH = 8.2) in the presence of air. The libraries were stirred until no further change in distribution was observed using UPLC/MS. In the library made from the unsubstituted building block 1, a 4mer macrocycle was selectively formed, and no complex cyclic products were observed. Similarly, 4mers were also the dominant products in libraries made from building blocks (2‐4) carrying hydrophobic groups, such as ‐F, ‐NO2 and ‐OCH3. No ordered supramolecular structure was observed using Transmission

Electron Microscopy (TEM) for these libraries, with the exception of that made from building block 4, which exhibited fibrillar structures (Figure S3.1). Seeding experiments suggested that the formation of 44 is autocatalytic (Figure S3.2), similar to previously reported peptide‐

based systems.36 However, replication is not the focus of the current chapter. Apparently, hydrophobic substituents do not lead to the formation of foldamers.

Various large macrocycles were observed in the libraries made from building blocks containing substituents featuring hydrophilic hydrogen‐bond donors or acceptors. A family of large macrocycles up to 23mers was observed in the library generated from building block 5, featuring tyrosine instead of phenylalanine (Figure S3.23). These results suggest that the introduction of potential hydrogen‐bonding sites can shift the product distribution of the library towards large ring sizes. Indeed, the selective formation of large macrocycles was observed in libraries constructed from building blocks 6‐8 featuring hydrophilic groups (carboxylate, amine and guanidinium respectively).

UPLC/MS analysis of the library made from carboxylic acid substituted building block 6 revealed the selective formation of a 9mer macrocycle (Figure 3.1a) (80%) accompanied by 4mer and 3mer (20%). More interestingly, a 23‐membered ring was formed with almost complete conversion (95%) in the library made from building block 7 featuring an amine group (Figure 3.1b). Finally, altering the overall charge of the monomer by adding a guanidinium moiety in building block 8 gave rise to the co‐existence of two 16mers. These two compounds had the exact same mass, yet exhibited a remarkable difference in

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rete pola a co Figu libra stirri In o mon (Figu fold sma bloc 3.2.3 In o puri Note attri inve ray c ention time arity (Figure onsequence o re 3.1. UPLC ries made fro ing for 14 day order to de nitored the o ure S3.4‐3.6 amers 69, 72 all cyclic 3me ck had oxidiz 3 Characteri

order to obt fied though e that UPLC ibute to par estigated usin

crystallograp

in the UPLC 3.1c). Overa of the amphi C analyses (ab om 2.0 mM bu ys. etermine the oxidation of 6). The result 23 and 816: th er and 4mer ed to form 3 ization of the tain insight automatic analysis of i tial unfoldin ng tandem m phy. C analysis, in all, the forma iphilic nature bsorption at uilding blocks e kinetic pr the libraries ts indicate th he emergenc ; in contrast 3mer and 4m e foldamers into the str flash colum solated 816 s ng of 816‐1 d mass spectro ndicating th ation of large e of these bu 254 nm) sho (a) 6, (b) 7, ( rofiles for t s made from here are two ce of 69 and t, the forma mer in the in ructure of m mn chromato showed peak uring the an ometry (MS/M

Dynamic

at the two e rings from uilding block

owing the fin c) 8 in borate the formatio m building bl o distinct pa d 723 do not ation of 816 o itial period. macrocycles ography usin ks for 816‐1 ( nalysis. Thus MS), circular

Combinato

compounds building blo s. nal product d e buffer (50 m

on of these ocks 6, 7 an athways for t depend on t occurs only a 69, 723 and ng a reverse 90%) and 81 the obtaine r dichrosim (

orial Foldam

had a diffe ocks 5‐8 cou distribution o mM, pH = 8.2) e foldamers, nd 8 by UPLC the formatio the formatio afer the bui 816, these w

e phase colu

16‐2 (10%), w ed samples w CD), NMR an

mers

erent ld be f the after , we C‐MS on of on of lding were umn. which were nd X‐

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The inclu sing fold likel The that that sign mac The ado Figu 23/7 upon pH = results of th uding large o le macrocyc amers 69 an y single mac CD spectrum t the dimerc t the CD spe ificantly enh crocycles. Sim CD bands of pt different f re 3.2. MS/M 7 for 723) and n CID activatio = 8.2) at 298 K he MS/MS s oligomers su cle rather th d 816 (Figure crocycles. m of 69 exhib captobenzen ctra of all m hanced as a c milarly enhan f these comp folding motif MS spectra of t (c) m/z=1655 on. (d) CD spe K. show that fo uch as 721, 7

han a caten e 3.2a, c), d

bited two ch ne core expe monomers sh consequence nced CD spe pounds were fs compared the precursor 5 (n/z = 16/5 ectrum of 69 ( oldamer 723 718 and so fo nane. Simila demonstratin aracteristic p eriences a c how only we e of the sup ectra were al e red‐shifted d to 69 (Figure r ion (a) m/z= for 816) show (black), 723 (gr fragmented rth (Figure 3 r results we ng that also peaks at 222 hiral enviro ak signals, in ramolecular lso observed d (240 and 2 e 3.2d). =1132 (n/z = 9 wing the form reen) and 816 into consec 3.2b), indica ere also obt

these comp 2 and 256 nm nment (Figu ndicating tha organization d for macrocy 68 nm), sugg 9/4 for 69), (b ation of cons (blue) in bora cutive fragm ating that 723 tained for o pounds are m, which ind ure 3.2d).38 at the chiral n (folding) o ycles 723 and gesting that b) m/z=1560 ( secutive fragm ate buffer (50 ents, 3 is a other most icate Note ity is f the d 816. they (n/z = ments mM,

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Figu 723, 8 Figu fold loca re 3.3. 1H‐NM 816. Aromatic ure 3.3 show amers 69, 7 ted at 7.0‐7 MR (D2O, 298K protons are s ws the 1H‐NM 23 and 816 (D .5 ppm, the K) spectra of b shown in red, MR spectra o D2O, 298K). α protons a building blocks α‐protons in of building b The aromat ppear in the

Dynamic

s 6, 7, 8 and t blue and alky blocks 6, 7 a tic protons o e range of 3.

Combinato

the correspon l protons in bl nd 8 and th of all mono 5‐5 ppm, an

orial Foldam

nding foldame lack. he correspon mers are m nd the other

mers

ers 69, nding ainly alkyl

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prot rang large the indic stru indic obse Figu diffe the f bond struc the c tons show si ge of chemic e macrocycle macrocycles cates that ctures. Inter cating the p erved in the re 3.4. X‐ray erent sets of π first set by a p ds connecting cture. (c), (d) chemical struc gnals at high cal shifts. All es adopt we s appear in t they are st restingly, fou presence of a single crysta crystal struct π‐stacks obser pseudo C2 ax g the phenyl ri and (e) Side v cture, respect h field. The p the spectra ell‐defined co the range of trongly shie ur sets of pr a 4‐fold sym al X‐ray struc ure of 723. (a) rved between is (dotted line ings. The colo views of 723 sh tively. protons of th a show rema onformation f 5.5‐8.5 ppm elded, furthe roton signals mmetry in th cture of this c ) Chemical st phenyls (solid e) are indicate ors of the phe howing the st he macrocycl arkably sharp s. The signal m. The shift er supportin s were obse he macrocyc compound (s ructure. Arrow d arrows). A s ed by dotted nyl rings corre tacks of pheny les are sprea p signals, sug ls of the aro of these sign ng the exist rved in the le. The same see below). ws and numb second set of π arrows. (b) Th espond to tho yl rings labele ad across a w ggesting tha matic proto nals to high tence of fo spectrum of e symmetry bers indicate t π‐stacks relat he ring of disu ose in the che ed as 1, 2, and wider t the ns of field olded f 816, was three ted to ulfide mical d 3 in

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Dynamic Combinatorial Foldamers

The structure of 723 was investigated by single crystal X‐ray diffraction. The poor quality of

the crystal structure and the disorder of the amino‐acid residues limit an analysis of bond lengths and angles. Nevertheless, the core part of the structure featuring the phenyl rings connected by disulfide bonds clearly confirms the macrocyclic nature of 723.

Figure 3.4b shows that the 23 building blocks in 723 are connected by 23 disulfide bonds to

form a single giant macrocycle containing 115 atoms. There is a pseudo 2‐fold symmetry in the core structure composed of 23 phenyls, with the pseudo C2 axis dissecting one of the phenyl rings (marked in gray in Figure 3.4a). One of the important interactions in the core structure is the π‐π stacking of the aromatic rings. There are three sets of stacks at different locations: two sets involve only two phenyl rings (Figure 3.4c and d), from i and i+2 residues (marked with solid arrows 1 and 2 in Figure 3.4a); the other stack involves three phenyl rings (Figure 3.4e), from i, i+2 and i+4 residues (marked with solid arrows 3 in Figure 3.4a). These stacks are related to another set of stacks (dashed arrows in Figure 3.4a) through the pseudo C2 axis. Apart from π‐stacking interactions, a helical arrangement can also be observed in the structure. The five phenyl rings at the bottom in Figure 3.4b form a helically folded motif. Like the 15mer described in the previous chapter, not only secondary structure elements (π‐stacks and a helix) are present, but also tertiary structure can be identified in 723: the molecule adopts a highly

ordered complex structure by long range noncovalent interactions.

The structure of 816 was also investigated using single crystal X‐ray diffraction. However, as

for 723, the poor quality of the crystal structure prevents an in‐depth analysis of the details

of the structure. It is clear that the core part of the structure is connected by 16 disulfide bonds constructing a single giant macrocycle containing 80 atoms (Figure 3.5b). Unlike 723,

the core structure of 816 has a 4‐fold symmetry, so the 16 phenyl rings are distributed over

four different environments (the phenyl rings are labeled in four different colors in Figure 3.5a).

The structure of 816 is remarkably complex, it only features two π‐stacking interactions in its

core structure (in contrast, a large number of such stacks were found in the 15mer and 23mer structures described above). Surprisingly, both sets of pi‐stacks involve only two phenyl rings that are recruited from opposite sites of the extended macrocycle (residues i and i+7, marked with solid arrows in chemical structure Figure 3.5a). No distinct secondary structure was found in the core structure, yet the entire foldamer exhibits tertiary structure

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due dyna stru stru Figu phen disul pane Note sam (Figu 1_H‐N diffe 3.2.4 Tem unfo solu

to the colle amic combi ctures direct cture modul

re 3.5. X‐ray nyl rings. The lfide bonds c el a. (c) Side v e that the la mple of the li ure S3.3), su NMR spectru erent confor 4 Unfolding mperature d olding. Figur ution at diffe ective action inatorial ch tly from sim les like in mo crystal struct e same‐colore onnecting the iew of 816 hig ater eluted 1 brary (conta uggesting tha um of 816‐2 s mations (Fig and refoldin ependent e re 3.6 show rent temper n of many n emistry as ple building ost other me ure of 816. (a) d phenyls of e phenyl ring hlighting two 16mer 816‐2 c aining 80% o at the prese shows broad gure S3.7). ng of the fold experiments s the CD sp ratures. The non‐covalent a powerfu blocks witho ethods. ) Chemical st the core par gs. The colors stacks of two can be ampl of 816‐1 and 9 ence of 816‐2 d signals, con damers (CD and pectra of th CD signals o t interaction l tool for out requiring ructure. Arrow rt are identica

of the pheny o phenyl rings. lified to 90% 9% of 816‐2 i 2 is due to p nsistent with NMR) were e foldamers of all foldam s. These fin obtaining c g to first obta ws indicate tw al by symmet

yl rings corre . % by dissolvin in the UPLC artial unfold h the presen e conducted 69, 723 and ers gradually nding establi complex ter aining secon wo sets of sta try. (b) The ri espond to tho

ng a freeze‐d analysis) in ding of 816‐1. nce of a rang d to invest d 816 in aqu y diminish as shed rtiary ndary acked ng of ose in dried DMF . The ge of igate eous s the

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tem app 723 a heat to b furth the the was aque whe upo conf cool Figu Chan upon perature in roximately li and 816, a m ted to 70 °C, be heated to her indicate resulting str solutions, as also probed eous solutio en the soluti n lowering firmed that l cycle.

re 3.6. CD sp nges in ellipti n heating from ncreases. Th inear (Figure more defined , the CD sign o 80 °C to o that, althou ructure is sig s was found d using 1H‐N ons of foldam

on were he the tempe the molecul

ectra of folda city at a spec m 20‐90°C. he decrease e 3.6a). In co d unfolding t nal of 723 was observe signi gh the build gnificantly di in the case MR experim mers show ated up to a rature, indic e compositi

amers (a) 69,

cific waveleng

e in the C ontrast, in th temperature s dramatical ificant spect ing blocks of ifferent. Refo of the 15me ments. Variab

that the spe around 80 °

cating that on of the sa

(b) 723 and (c

gth (260 nm)

Dynamic

CD signal o he temperatu e was observ ly reduced ( tral changes f all foldame olding was o er (Figure 3. ble temperat ectra of fold C. The spect unfolding i amples did n c) 816 in water ) in the CD sp

Combinato

of 69 with ure depende ved. When t Figure 3.6b) (Figure 3.6c ers are simila observed upo 6d). Unfoldi ture 1H‐NMR damers 723 a tra revert to s reversible not change f r at different pectra of diffe

orial Foldam

temperatur ent CD spect the solution , while 816 n c). These re ar, the stabili on cooling d ing and refo R experiment and 816 cha

o the initial s e. UPLC ana following a h temperature ferent macroc

mers

re is ra of was eeds esults ity of down lding ts on nged state alysis heat‐ es. (d) cycles

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3.2.5 We Con yield The 12m tem com Figu aque NaCl

3.3

We of u build desi 5 Effect of th investigate sidering the d of 69 by sh presence of mer (Figure plate effects mposition, wh re 3.7. UPLC eous borate b l, (c) MgCl2, (d

Conclusion

have establi unprecedent ding blocks, gn rules th he solvent e d whether overall nega ielding the e f 1.0 M MgC 3.7c) or a 1 s exerted by hereas guani traces (absor buffer (50 mM d) CaCl2, and (

ns

ished dynam ted folding

as a result at can be a

nvironment the solven ative charge electrostatic Cl2 and CaCl2 13mer (Figu y these salts dinium chlor rption at 254 M, pH 8.2) (a) e) guanidinium mic combinat assemblies of their the applied to a on the form nt environm of 69, salts w repulsion be 2 did not inc ure 3.7d) w s. Notably, c ride favored nm) of the D ) without add m chloride. torial chemis that form ermodynam access these mation of fold ment influen were added w etween the n crease the yi as observed hloride anio the formati DCLs made fro ded salt and in stry as a pow autonomou ic stability. W e highly ord

damers nced foldam with a view t negative char ield of the 9 d, respective ons did not a on of the 4m om 2.0 mM b n the presenc werful tool f usly from s We have fo dered topolo mer formati to enhancing rges (Figure 9mer. Howev ely, indicativ affect the lib mer (Figure 3 building block ce of 1.0 M o for the disco simple dipep ormulated si ogical struct on.33 g the 3.7). ver a ve of brary 3.7e). k 6 in of: (b) overy ptide mple tures

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Dynamic Combinatorial Foldamers

involving balancing the hydrophilic and hydrophobic parts of the monomers. Crystal structures revealed unique folding patterns, in one instance skipping hierarchic folding, adopting tertiary structure without significant secondary structure modules. Even though exact prediction of specific ring sizes remains challenging, our approach offers an attractive pathway for discovering fascinating new foldamers of substantial complexity. Further structure elucidation of these dynamic combinatorial foldamers might pave the way for elaborating them into catalysts.39 Furthermore, design and use of external templates may uncover yet more foldamers.

3.4. Acknowledgements

The research was performed in collaboration with Dr. Charalampos G. Pappas, who is most gratefully acknowledged for the synthesis of building blocks, preparation and UPLC analysis of the libraries, and characterization the structures. All the experiments involving MS analysis and Flash Chromatography were performed by Bin Liu. Prof. Ivan Huc and Dr. Pradeep Mandal are acknowledged for crystallization experiments and refinement of the crystal structures. The MS/MS experiments have been performed through collaboration with the research group of Prof. Kevin Pagel with the help of PhD. students, Waldemar Hoffmann and Christian Manz.

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3.5. Experimental section

3.5.1 General methods

All chemicals, unless otherwise stated, were purchased from Sigma‐Aldrich and used as received. Amino‐acid resins were purchased from Novabiochem and Fmoc modified phenylalanines from Chem Impex International and Iris Biotech. Acetonitrile (ULC‐MS grade), water (ULC‐MS grade) and trifluoroacetic acid (HPLC grade) were purchased from Biosolve BV. Flash column chromatography was performed on a Reveleris® X2 Flash Chromatography System (Grace Davison Discovery Sciences, Deerfield IL) on normal or reversed phase silica cartridges. NMR spectra were recorded on a 600 MHz spectrometer.

Buffer preparation

Borate buffer (12.5 mM in Na2B4O7, pH = 8.2) was prepared by dissolving boric acid

anhydride (87.0 mg, B2O3) in 50 mL doubly distilled water. The pH was adjusted to 8.2 using concentrated NaOH. Library preparation Peptide monomers were dissolved in borate buffer (12.5 and 50 mM, pH 8.0) to prepare a library. All libraries were set up in an HPLC vial (12×32 mm) with a Teflon‐coated screw cap. All HPLC vials were equipped with a cylindrical stirrer bar (2×5 mm, Teflon coated, purchased from VWR) and stirred at 1200 rpm using an IKA RCT basic hot plate stirrer. All experiments were performed at ambient conditions. UPLC and UPLC‐MS analysis

UPLC analyses were performed on a Waters Acquity H‐class system equipped with a PDA detector, at a detection wavelength of 254 nm. Samples were injected on a Phenomenex Aeris Peptide 1.7 μm (150 × 2.1 mm) column, using ULC‐MS grade water (eluent A) and ULC‐ MS grade acetonitrile (eluent B), containing 0.1 V/V % TFA as modifier. A flow rate of 0.3 mL/min and a column temperature of 35 °C were used.

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 in positive electrospray ionization mode with the following ionization parameters: capillary voltage: 3 kV; sampling cone voltage: 20 V; extraction cone voltage: 4 V; source gas temperature: 120 °C;

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Dynamic Combinatorial Foldamers

desolvation gas temperature: 450 °C; cone gas flow (nitrogen): 1 L/h; desolvation gas flow (nitrogen): 800 L/h.

Circular Dichroism spectroscopy

Spectra were recorded on a Jasco J‐810 spectrometer with a Peltier temperature controller. Sample concentrations (in building block) were 8×10–5 M. Heat‐cool cycles were applied from 20 to 90°C in steps of 1 degree at a rate of 0.1 degree/min and maintained for 10 min at every temperature before measuring. Spectra were obtained as averages of 3 measurements from 190 to 400 nm with a scanning speed of 150 nm/min and a bandwidth of 1 nm. MS/MS analysis MS/MS analysis was performed on a Synapt G2‐S HDMS (Waters Corporation, Manchester, UK), described in detail elsewhere (S. D. Pringle, K. Giles, J. L. Wildgoose, J. P. Williams, S. E. Slade, K. Thalassinos, R. H. Bateman, M. T. Bowers, J. H. Scrivens, Int. J. Mass Spectrom. 2007, 261, 1‐12.). Before MS/MS measurement the oligomeric stock solution isolated by UPLC (see library preparation and UPLC‐MS analysis) were typically diluted to 1‐5 mM with respect to building block with water/methanol (70%:30%) + 0.5% formic acid. A nano‐electrospray ionization source was used to ionize 35uL of sample from platinum‐palladium‐coated borosilicate capillaries prepared in‐house. Typical settings in positive ion mode were: 0.65 kV capillary voltage, 60 V sampling cone voltage, 5 V source offset voltage, 50°C source temperature, 500°C desolvation gas temperature, 50 L/h cone gas flow, 800 L/h desolvation gas flow, 10‐50 V trap CE, 0 V transfer CE. For MS/MS experiments either the trap collision energy or the transfer collision energy was increased to 10‐50 V.

Negative‐staining Transmission Electron Microscopy

Samples were diluted 40‐fold using UPLC grade water. A small drop (5 µL) of sample was then deposited on a 400 mesh copper grid covered with a thin carbon film (supplied by Agar Scientific). After 30 seconds, the droplet was blotted on filter paper. The sample was then stained with a solution of 2% uranyl acetate (4 µL) deposited on the grid, subsequently washed and blotted on filter paper after 30 seconds. The staining procedure was repeated a second time, this time without the washing and blotting step. 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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UPLC methods:

Method for the analysis of DCLs made from building blocks 1‐4: t / min % B 0 10 1 20 11 80 13 95 13.5 95 14 10 17 10

Method for the analysis of DCLs made from building blocks 5 and 6: t / min % B 0 10 1 15 11 50 13 95 13.5 95 14 10 17 10 Method for the analysis of DCLs made from building blocks 7 and 8: t / min % B 0 10 1 15 9 24 12.5 60 13 95 13.5 95 14 10 17 10 3.5.2 Synthesis and characterization of building blocks Synthesis of monomers has been performed using conventional Solid Phase Peptide Synthesis (SPPS) using the Fmoc/tBu strategy on Wang resin. Amino acids were introduced protected as Fmoc‐ Lys(Boc)‐OH, Fmoc‐Tyr(Boc)‐OH, Fmoc‐Phe‐OH, Fmoc‐Phe‐(4‐NH‐Boc)‐OH or Fmoc‐Phe‐(4‐COOtBu)‐ OH. Fmoc deprotection steps were carried out with 20 % piperidine in DMF (v/v) for 15 min. Coupling reactions of Fmoc amino acids were performed in DMF using N‐diisopropylcarbodiimide (DIC) and ethyl cyano(hydroxyimino)acetate (oxyma). Deprotection from resin and removal of the protecting groups from the side chains of the amino acids was performed using a cocktail of 95% TFA, 2,5% 1,2‐ ethanedithiol (EDT), 1,25% water and 1, 25% triisopropylsilane (TIS) for 3 hours. Crude peptides were purified using flash column chromatography and obtained at purity level> 97%. Impurities were mainly dimers and other small oligomers.

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Scheme S

Append

Figure S3 stirring a O O N H NH Boc HO O N H O NH2 R S3.1. Synthesi

dix

3.1. TEM imag t 1200 rpm. T 20% P 3 Fmoc N H H N O SH SH is route for pe ges for librari The building b Piperidine in DMF 3X for15 min 95% TFA 2,5% EDT 1,25% TIS 1,25% H2O Boc eptide‐based m ies made from block concent O O NH NH Boc O O N H O _H N NH R O monomers. m building blo tration was 2. O Fmoc N H 3 hours DIC, Oxy H2 R STrit STrit HO

Dyna

ocks: (a) 1, (b .0 mM in bor OH O s yma 3 hours DIC, oxyma B B O STrit STrit O

amic Combi

b) 2, (c) 3 and ate buffer (12 O O N H O _H N 20% Pip 3X fo NH Boc R O O N H O N NH Boc R

inatorial Fo

d (d) 4 after 1 2.5 mM, pH = H N Fmoc peridine in DMF or15 min NH2

oldamers

inatorial Fo

LC‐MS analysi H]4+. LC‐MS analysi m/z: 2182.17

oldamers

s of a DCL s of a DCL [M+3H]3+,

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Figure S3 made fro 1133.09 [ Figure S3 made fro 2013.83 [ 3.34. Mass spe om 6 (2.0 mM) [M+4H]4+. 3.35. Mass spe om 6 (2.0 mM) [M+3H]3+, 151 ectrum of 69 ( ). Calculated m ectrum of 612 ). Calculated m 10.73 [M+4H]4 (retention tim m/z: 1510.36 (retention tim m/z: 2013.48 4+_{, 1208.76 [M} me 4.38 min in [M+3H]3+, 113 me 4.67 min i [M+3H]3+, 151 M+5H]5+. n Figure S3.31 33.02 [M+4H] n Figure S3.31 10.36 [M+4H] 1e) from the L 4+ ; observed m 1e) from the L 4+ , 1208.49 [M LC‐MS analysi m/z: 1510.44 LC‐MS analysi M+5H]5+; obse s of a DCL [M+3H]3+, s of a DCL erved m/z:

(36)

Figure S3 made fro 671.88 [M Figure S3 made fro 3.36. Mass spe om 6 (2.0 mM M+3H]3+. 3.37. Mass spe om 6 (2.0 mM 2+ ectrum of 64 ( ). Calculated ectrum of 63 ( M). Calculated (retention tim m/z: 1007.24 (retention tim m/z: 1510.36 me 5.56 min in 4 [M+2H]2+, 67 me 6.88 min in 6 [M+1H]+, 75

Dyna

amic Combi

1e) from the L 3+ ; observed m 1e) from the L 2+_; observed

inatorial Fo

oldamers

s of a DCL [M+2H]2+, s of a DCL 5 [M+1H]+,

(37)

Figure S3 after diss Figure S3 made fro [M+7H]7+ [M+7H]7+ 3.38. UPLC tra solving, (b) aft 3.39. Mass spe om 7 (2.0 mM + , 1364.16 [M + , 1364.37 [M+ ces of a DCL m ter stirring for ectrum of 723 M). Calculate +8H]8+; obser +8H]8+. made from 7 ( r 20 days. (retention tim d m/z: 2727. rved m/z: 272 (2.0 mM) in b me 8.62 min i .31 [M+4H]4+, 27.57 [M+4H]4 orate buffer ( n Figure S3.38 , 2182.05 [M 4+ , 2182.11 [M (12.5 mM, pH 8b) from the L +5H]5+, 1818 M+5H]5+, 1818 = 8.0): (a) im LC‐MS analysi 8.54 [M+6H]6+ 8.40 [M+6H]6+ mediately s of a DCL +_{, 1558.90} + , 1558.87

(38)

Figure S3 made fro 475.20 [M Figure S3 after diss 3.40. Mass spe om 7 (2.0 mM M+3H]3+. 3.41. UPLC tra solving, (b) aft ectrum of 73 ( M). Calculated ces of a DCL m ter stirring for retention tim d m/z: 712.22 made from 8 ( r 14 days. me 11.34 min i 2 [M+2H]2+, 4 (2.0 mM) in b

Dyna

n Figure S3.38 475.15 [M+3H orate buffer (

amic Combi

8b) from the L ]3+; observed (12.5 mM, pH

inatorial Fo

LC‐MS analysi d m/z: 712.11 = 8.0): (a) im

oldamers

is of a DCL 1[M+2H]2+, mediately

(39)

Figure S3 DCL made m/z: 2065 Figure S3 DCL made m/z: 2065 3.42. Mass sp e from 8 (2.0 5.52 [M+4H]4 3.43. Mass sp e from 8 (2.0 5.49 [M+4H]4 ectrum of 816 mM). Calcula +_{, 1652.78 [M} ectrum of 816 mM). Calcula +_{, 1652.77 [M} 6‐1 (retention ated m/z: 2065 +5H]5+, 1377. 6‐2 (retention ated m/z: 2065 +5H]5+, 1377. time 3.74 m 5.65 [M+4H]4 .57 [M+6H]6+. time 8.29 m 5.65 [M+4H]4 .57 [M+6H]6+. in in Figure S 4+ , 1652.72 [M in in Figure S 4+_{, 1652.72 [M} 3.41b) from t M+5H]5+, 1377. 3.41b) from t M+5H]5+, 1377. the LC‐MS an .44 [M+6H]6+; the LC‐MS an .44 [M+6H]6+; alysis of a observed alysis of a observed

(40)

Figure S3 made fro 689.54 [M Figure S3 made fro 517.25 [M 3.44. Mass spe om 8 (2.0 mM M+3H]3+. 3.45. Mass spe om 8 (2.0 mM M+3H]3+. ectrum of 84 ( ). Calculated ectrum of 83 ( M). Calculated retention tim m/z: 1033.33 retention tim m/z: 775.25 me 10.44 min i [M+2H]2+, 68 me 11.67 min i [M+2H]2+, 51

Dyna

amic Combi

1b) from the L 3+ ; observed m 1b) from the L ]3+; observed

inatorial Fo

oldamers

is of a DCL [M+2H]2+, is of a DCL [M+2H]2+,

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3.6 References

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(24) Ziach, K.; Chollet, C.; Parissi, V.; Prabhakaran, P.; Marchivie, M.; Corvaglia, V.; Bose, P. P.; Laxmi‐Reddy, K.; Godde, F.; Schmitter, J. M.; Chaignepain, S.; Pourquier, P.; Huc, I. Nat. Chem. 2018, 10, 511. (25) Saha, S.; Kauffmann, B.; Ferrand, Y.; Huc, I. Angew. Chem. Int. Ed. 2018, 57, 13542. (26) Wolffs, M.; Delsuc, N.; Veldman, D.; Van Anh, N.; Williams, R. M.; Meskers, S. C. J.; Janssen, R. A. J.; Huc, I.; Schenning, A. P. H. J. J. Am. Chem. Soc. 2009, 131, 4819. (27) Case, M. A.; McLendon, G. L. J. Am. Chem. Soc. 2000, 122, 8089. (28) Oh, K.; Jeong, K. S.; Moore, J. S. Nature 2001, 414, 889. (29) Krishnan‐Ghosh, Y.; Balasubramanian, S. Angew. Chem. Int. Ed. 2003, 42, 2171. (30) Lafuente, M.; Atcher, J.; Sola, J.; Alfonso, I. Chem. Eur. J. 2015, 21, 17002.

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Dynamic Combinatorial Foldamers

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