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 4 Dynamic Peptide Nucleic Acid Self‐
Replicators
The conditions that led to the formation of the first organisms and the ways that life originates from a lifeless chemical soup are poorly understood. The hypothesis of ‘‘RNA‐ peptide coevolution’’ suggests a close relationship between amino acids and nucleotides that may extend to the origin of life. We now report two strategies for the fabrication of amino acid and nucleobase‐based self‐replicating nanostructures by using dynamic combinatorial chemistry. The first one relies on mixing nucleobase‐ and peptide‐based building blocks, where the assimilation of these two gives rise to highly ordered structures and the second one depends on the synthesis of peptide nucleic acids (PNAs). Unlike the other nucleic acid self‐replicating systems that rely on base pairing and pre‐synthesis of (short) oligonucleotide sequences, self‐replication in the dynamic combinatorial libraries is driven by the self‐ assembly of ordered supramolecular nanostructures. Cross‐ and auto‐catalysis were observed, highlighting that the interaction between these two essential building blocks of life can trigger emergent behaviors.
4.1. Introduction
One of the grand challenges in contemporary science is the transition from chemistry (inanimate matter) to biology (living matter) during the origin of life.1 It still remains unclear how simple chemicals could have transformed into functional biological machinery, although, there are various hypotheses addressing this transition. The RNA world hypothesis suggests RNA plays an important role because of its ability to store, transmit, and replicate genetic information.2,3 More importantly, the evolution of RNA and its closely related derivatives can be mediated by RNA.4‐7 However, the prebiotic pathway to synthesize RNA still remains quite challenging, and it may be that RNA is a product of complex evolutionary processes.8
The RNA‐peptide coevolution hypothesis emphasizes the synergy between oligonucleotides and peptides, as nucleic acids and proteins work together to perform a stunning array of functions such as catalysis and information transfer.9‐13 The co‐existence of peptides and RNA on the early earth is plausible, highlighting the role of small peptides in the evolution of early genetic systems. The enormous chemical space offered by combining the 20 amino acids provides the possibility for the formation of a broad range of primary sequences and subsequent potential functions, including catalysis14 or selective binding.15 However, the way in which nucleic acids and peptides are connected to facilitate the emergence of life on the prebiotic earth remains unclear.
Regardless the hypotheses, self‐replication plays central role in all evolutionary scenarios.16‐
18 The development of self‐replicating systems containing nucleic acids and peptides may
provide a way to improve our understanding of the origin of life. The most straightforward approach to develop nucleobase‐ and peptide‐based self‐replicating systems is to introduce nucleic acids and peptides in a single system simultaneously. Yet, the lack of suitable self‐ replicating systems has hampered the implementation of this strategy. Another way to incorporate nucleic acids and peptides into a self‐replicating system is using Peptide Nucleic Acids (PNAs), which result from the substitution of the charged sugar phosphate backbone of DNA by a neutral amino ethyl glycine linker.19‐21 After the development of the first non‐ enzymatic self‐replicating system, von Kiedrowski‘s group developed a PNA‐based self‐ replicating system in which a hexameric sequence acts as a template for the synthesis of a complementary hexa‐PNA strand from trimeric building blocks.22,23 Nielsen and coworkers also used PNAs to trigger self‐replication by template directed cross‐catalytic ligation.24 Moreover, Ghadiri’s lab demonstrated dynamic sequence exchange of PNA thioesters via self‐pairing with complementary strands or cross‐pairing with external short DNA/RNA
oligo repl sequ We (five that oxyg one We dive from build poss Here chim oligo mac lead inco cons nucl build omers.25 Wh icating syste uences. have previou e amino‐acid t are equippe gen to yield particular r have furthe ersification29 m dynamic co ding‐block l sibility to inc ein, we repo meric buildin onucleotides crocycles. We ding to the a orporating b sisting of tw leobases, res ding block 3 hile these re ems, most o usly discover d residues‐co ed with two a dynamic co ring occurs t rmore demo in these sys ombinatoria ibrary, but corporate dif ort the fabric ng blocks, w s to serve e have inves utocatalytic both nucleob wo building spectively), 3. We also s eports have of them rely
red self‐repl omposed of thiol group ombinatoria hrough stac onstrated se stems. The g l libraries is t also from m fferent buildi cation of self without the as template tigated a var formation o bases and p g blocks (1 but also from show that t incorporate y on base p
icating mole alternating s which can l mixture of cking of the equence sele great advant that self‐rep multi‐buildin ing blocks in f‐replicating need of ba es, as a res riety of nucle of ordered su peptides ca and 2 are m the librar he newly fo ed peptides pairing and ecules based hydrophobi form disulfi disulfide ma appended p ection,27 env age of the e plicators can g‐blocks libr to a self‐rep systems by u ase‐pair inte
sult of dyn eic and amin upramolecula n not only functionaliz ies that con ormed self‐re and nucleic pre‐synthesi on β‐sheet f c and charg de bonds in acrocycles.26 peptide chain ironmental a emergence o not only em raries,29 whi licator. using amino eractions or amic exchan no acid seque ar structures emerge fro zed by shor tain only PN eplicators, t
c acids into is of short P
forming pep ged amino a n the presenc Self‐assemb ns into β‐sh adaptation28 of self‐replica merge from s ich provides acid‐nucleo pre‐synthes nge of disu ences (Table s. Self‐replica om the libr
rt peptides NA functiona the structure self‐ PNAs tides acids) ce of bly of eets. 8 and ators ingle s the obase sized ulfide e 4.1), ators aries and alized es of
whic othe Sche nucle bloc eme
4.2.
4.2. In ch a co iden com stru and func indic ch are strong er replicators eme 4.1. Sc eobase‐peptid ks 1 and 2, w ergence of PNA. Results an
1. Emergenc hapter 2, we omplex and u ntical nucleo mbinatorial ch cture is a co hydrogen ctionalized b cated that, u gly depende s. hematic repr de replicators which are funcA replicators f
nd discussio
ce of nucleob e have show unprecedent obase‐peptid hemistry app ombination o bonding. Si building block upon additio nt on the nu resentation o s: (a) The eme ctionalized by from the libraon
base‐peptide n the format ed secondar de‐functiona proach. The d of intramolec imilar obser ks (2b, 2c an n of the pref ucleobase, arof the prop ergence of rep
y a short pep ary made from e self‐replica tion of a self ry and tertiar alized buildi driving force cular noncov rvations we nd 2d). Howe formed gian re able to cro osed mecha plicators from ptide and a nu m building bloc ators f‐synthesizin ry structure t ing blocks e for the form valent intera ere made w ever, seeding t macrocycle oss‐catalyze nisms for th m the library m ucleobase, res ck 3 (3a3 in th ng macrocycl that constru (2a) by us mation of the ctions includ with the oth g experimen e, growth is n the formatio he emergenc made from bu spectively. (b is case). lic foldamer cts itself fro sing a dyn e complex fo ding
π-π
stac her nucleob nts for the 15 not faster, w on of ce of ilding ) The with m 15 amic olded cking base‐ 5mer whichsuggests that the foldamer cannot make copies of itself. In order to develop a self‐replicating system capable of incorporating nucleobases and peptides, we speculated that this can be achieved by introducing nucleobase‐peptide‐functionalized building blocks into a self‐ replicating system featuring a similar chimeric structure. As mentioned earlier, we have previously reported the emergence of self‐replicator (16) from DCLs made from dithiol functionalized peptide building block (1), which is driven by β‐sheet forming self‐assembly.26 Building blocks 1 and 2a have the same aromatic dithiol core which allows them to exchange with each other and form different disulfide macrocycles. We reasoned that the presence of self‐replicating peptides may lead to the formation of new replicating species involving nucleobase‐functionalized building blocks.
To test our hypothesis, we performed experiments by dissolving equimolar amounts of building blocks 1 and 2a in a total concentration of 2.0 mM in 12.5 mM borate buffer (pH = 8.2) in a capped vial and stirred the solution in the presence of air. The library reached a stable composition after three weeks. Notably, a rather complex product distribution was observed, composed of mixed species (mainly cyclic 15mers), where a broad peak was noticed and separation was challenging (Figure 4.1f). The system tends to form complex folded structures (mixed 15mers) due to the presence of nucleobase‐peptide building block
2a, facilitated by the fact that building blocks 2a and 1 have the same core structure
(dimercaptobenzamide). Note that the formation of a folded structure is mainly driven by
π-π
stacking of the core phenyl and the hydrogen bonding network involving the amides attached to the core phenyl. Other cyclic oligomers, such as mixed 3mers and 4mers, were also observed in the library. No aggregates were observed in the library made from equimolar amounts of building blocks 1 and 2a using TEM, indicating that even peptides capable of inducing β‐sheets are not able to shift the product distribution towards self‐ replicating assemblies under these conditions.Figu pH = (b) 1 In o thes libra 30/7 wee Inte bloc 4.1h obse afte 30 m dicta re 4.1. UPLC = 8.2) at a tota 10, (c) 20, (d) 3
order to furt se complex m aries were se 70, 20/80, 1 eks, until n restingly, a s ck 2a (122a1) h, i and j), w erved with a r the librarie mol %. Thes ate the non‐ analysis of lib al concentrati 30, (e) 40, (f) 5 her explore mixtures, we et up at diff 10/90) at a t no change
specific libra ) was forme while all othe
a ratio of 70 es reached e
e results ind ‐covalent int braries made f ion of 2.0 mM 50, (g) 60, (h) the potenti decided to v ferent ratios total concen in distribut ary member d in the libr er libraries fo 0/30 (Figure equilibrium w dicate that t eractions tha from building M at different 70, (i) 80, (j) 9
al for formi vary the pro s (1/2a = 90/ ntration of 2 tion was o consisting o raries with th ormed mixed e 4.1h). Note when the con the ratio of at drive the g blocks 1 and ratios stirred 90 and (k) 100 ng folded an portion of th /10, 80/20, 2.0 mM. The bserved usi of two buildin he ratios of d 15mers. Th e that no m ncentration building blo formation of d 2a in borate at 1200 rpm 0 mol % of bu nd self‐replic he building b 70/30, 60/4 e libraries w ing UPLC/M ng blocks 1 a 90/10, 80/2 he highest y ixed 15mers of building b ocks in dynam
f different st e buffer (12.5 m for 25 days: ilding block 2 cating speci blocks. A seri 40, 50/50, 40 were stirred f MS (Figure and one bui 20, 70/30 (Fi yield of 122a s were obse block 2a is b mic libraries tructures. Ind mM, (a) 0, a. es in es of 0/60, for 3 4.1). lding igure 1 was erved elow s can deed,
in the libraries where a significant amplification of 122a1 was observed, a transition was noticed from a clear to an opaque solution, suggesting the formation of a supramolecular structure.
We then focused on libraries made from 1 and 2a in a ratio of 2/1. In the first 10 days, a variety of different macrocycles was formed. However, subsequently the concentration of
122a1 increased until it became the main product of the library, while the concentration of the other rings decreased dramatically (Figure S4.1). The kinetic profile for the formation of
122a1 exhibits a sigmoidal shape, which is indicative of the presence of auto‐catalytic species. To further probe this, a seeding experiment was performed by adding 10 mol % of preformed 122a1 to a library which had been stirred for three days. UPLC analysis revealed that, upon seeding, the growth of the trimer is significantly faster (Figure 4.2e). The formation of 122a1 was highly depended on mechanical agitation. Libraries prepared without stirring or shaking, showed no amplification, even after 30 days (Figure S4.1), in agreement with previously reported observations on peptide based systems.27 These results suggest that the emergence of self‐replicator 122a1 has a similar fiber growth‐breakage mechanism. Interestingly, the new replicator has a specific ratio of building blocks, which is unprecedented in mixed libraries based on the same aromatic dithiol core.29‐31
Subsequently, the other nucleobase building blocks containing thymine (2b), guanine (2c) and cytosine (2d) were also tested and the corresponding libraries were set up (Figure S4.23,
S4.36 and S4.46). The results showed that in mixed systems trimers 122b1, 122c1 and 122d1 are amplified in ratios of 90/10 and 80/20, with the highest amplification observed at a ratio of 80/20 (Figure 4.2b, c and d). The ratio of the building blocks required to selectively amplify the self‐replicators produced from different nucleobases is slightly different. The most favorable ratio for the emergence of self‐replicator 122a1 is 30 mol%, while for the other nucleobases this ratio is 20 mol%. These results indicate that the nature of the nucleobase plays a crucial role in the replicating systems, where different nucleobases have different aromatic rings and hydrogen bonding sites which can influence the interactions with the peptide building block. It is quite challenging to establish the exact organization of the nucleobase and the peptide segments in the final structure in these dynamic systems.
Figu the r at a 1200 122b imag 122c Seed re 4.2. Histog ratio of the bu total concent 0 rpm for (a) b1, (g) 122c1 a ges of librarie 1 and (l) 122d1 ding experim grams of the f uilding blocks tration of 2.0 122a1, (b) 122 nd (h) 122d1 s made from 1, respectively ments for the formation of t (1/2 = 90/10 mM in borat 2b1, (c) 122c1 a upon adding building bloc y. e other mixe the mixed rep 0, 80/20, 70/3 e buffer (12.5 and (d) 122d1 10 mol % of cks 1 and 2 th ed trimers (1 plicators (122a 0, 60/40, 50/5 5 mM, pH = 8 . Seeding ind f the preform hat were dom 122b1, 122c1 a1, 122b1, 122c1 50, 40/60, 30/ .2) under con uced formatio med macrocyc inated by (i) 1 and 122d1) a 1 and 122d1) v /70, 20/80, 1 ntinuous stirri on of (e) 122a
cles at day 3.
122a1, (j) 122b also confirm versus 0/90) ing at a1, (f) TEM b1, (k) that
their formation is autocatalytic (Figure 4.2f‐h). The emergence of these trimers coincides with the formation of ordered fibrillar assemblies, as evident from Transmission Electron Microscopy (TEM) data (Figure 4.2i‐l). Circular Dichroism (CD) experiments were also used to study the supramolecular chiral ordering of the assemblies (Figure S4.2). The results suggest a β‐sheet type arrangement, as previously observed for peptide based systems.32
4.2.2. Cross‐catalysis between nucleobase containing self‐replicators
In biology, replication fidelity is critical for cell survival, evolution, and disease prevention. High fidelity helps maintaining multiple generations of genetic information and helps avoiding mutations that can trigger and promote disease. In contrast, low fidelity facilitates the evolution of species, produces diversity, and promotes the development of the immune system. In order to develop a self‐replicating chemical system in the direction of de‐novo life, it is necessary that such system is capable of maintaining and developing information. The process of replication of genetic information in all currently known living systems relies on base‐pairing between nucleobases. Having developed self‐replicating systems which incorporate both nucleobases and peptides, we decided to investigate whether these self‐ replicators are able to cross‐catalyze the formation of other replicators through base‐pairing. A series of cross‐seeding experiments were performed on the libraries made from building blocks 1 and 2 in a 2:1 ratio. In every case, a 10 mol% seed was added (e.g. the libraries of 1 (1.0 mM) and 2a (0.5 mM) were seeded by 10 mol% of other nucleobase nanostructure
122b1, 122c1 and 122d1 after the libraries had been stirred for 3 days). As shown in Figure 4.3, in case of building blocks 2a (Figure 4.3a), 2b (Figure 4.3b) and 2d (Figure 4.3d), the yields for the mixed replicators (122a1, 122b1 and 122d1) are similar.
Notably, for the library containing building block 1 and guanine building block 2c (Figure
4.3c), the formation of 122c1 can be amplified by all other replicating trimers, even though the yield of the library seeded by 122d1 is remarkably lower than upon seeding by 122a1 or
122b1. Regardless the relative formation and the catalytic activity of the peptide nucleobase systems, overall the formation of self‐replicating structures can be cross‐catalyzed by other nucleobase assemblies, highlighting that new species may be derived from pre‐existing replicators, involving nucleobase and peptide structural motifs. Despite the fact that we are not able to exclude the existence of base‐pairing in these systems, the results suggest that base‐paring does not occur, or does not have an obvious impact on cross‐replication. However, these results also indicate that the new self‐replicating system is able to achieve self‐replicator diversity through mutation.
Figu mol% In o seed the from for t that only repl nucl 0.12 show seed re 4.3. Cross % of other pre rder to test t ding experim preformed m building bl the emergen t auto‐ and c y catalyze t icators (Figu leobase buil 25 mM) wer wn in Figure ded library a s‐seeding indu eformed self‐r the replicatio ments for libr replicators 1 locks 1, 2a a nce of mixed cross‐catalys the formatio ure 4.4a). Fu ding blocks e also perfo
e 4.4b, the c re higher tha uced growth o replicators at on fidelity of raries which 122a1 and 12 and 2b ([1] = d trimer repl sis is preserv on of them urthermore, 1, 2a, 2b, 2 ormed by ad concentratio an the library of (a) 122a1, (b day 3. f this new se contain diffe 22b1 were se = 1.0 mM, [2 icators), res ved in comp mselves, but seeding exp 2c and 2d ([1 dding one of ns of replica y without se b) 122b1, (c) 1 elf‐replicating erent nucleo eeded into t a] = [2b] = 0 pectively (Fi lex systems also accele periments of 1] = 1.0 mM f preformed ators 122a1, eed after 30 d 122c1 and (d) 1 g system, we obase buildin two identica 0.25 mM, the gure S4.3). T in which rep erate the g f a library co M, [2a] = [2b replicators 122b1, 122c1 days stirring. 122d1 by addin e also perfor ng blocks. Ini al libraries m e preferred The results s plicators can growth of o ontaining all b] = [2c] = [2 (Figure S4.4 and 122d1 in . ng 10 rmed tially, made ratio show n not other four 2d] = 4). As n the
Figu ([1] = = [2c amo Mor som evol than Thes resu asse pref The furth re 4.4. Summ = 1.0 mM, [2a c] = [2d] = 0.1 ount of seed. reover, the a mewhat highe lution of the n cross‐replic se results in ulting supra emblies inco ference for s balance of her developm mary of seedin a] = [2b] = 0.2 125 mM), com addition of t er efficiency e cross‐seedi cation at the ndicate that molecular s orporating d elf‐replicatio high and low ment of new ng experimen 5 mM), (b) bu mpared with n he preforme y than the fo
ng experime e early stages t self‐replica structures ca different nu on relative to w replication w self‐replicat nts on libraries uilding blocks non‐seeded li ed replicator ormation of ents also sho s of the grow ation is pref
an also ind cleobase se o cross‐replic n fidelity in t ting systems s made from 1, 2a, 2b, 2c a braries. The s r promoted t the other n ows that the wth in cross‐ ferred in th duce the fo egments, th cation sugge this system s to maintain (a) building b and 2d ([1] = 1 striped areas the formatio nucleobase r self‐replicati seeded libra ese systems rmation of ough cross‐ ests some de shows the p n and develo blocks 1, 2a an 1.0 mM, [2a] correspond t on of itself w replicators. T ion rate is hi aries (Figure s. However, higher ord ‐replication. egree of here possibility fo p informatio nd 2b = [2b] o the with a Time igher 4.5). , the dered The edity. r the on.
Figu bloc of th 4.2.3 In o nucl follo Simi How from nucl high repl re 4.5. Seedi ks 1, 2a, 2b, 2 he preformed 3. Importanc
order to inv leobase was owing a prot
ilar experime wever, no se m 90/10 to 1 leobase trim hlighting the ication proce ng induced g 2c and 2d ([1] self‐replicato ce of the nu vestigate th s replaced by tocol analog ents were pe lective form 0/90 (Figure mer replicato role of the esses. growth of nu = 1.0 mM, [2 ors (a) 122a1, (b cleobase he significan y a simple p gous to that erformed as ation of trim
e S4.57). Add
ors were als nucleobases
cleobase self
2a] = [2b] = [2
b) 122b1, (c) 1
nce of the henyl ring. T used for pr s discussed a mer was obse ditionally, cro so performe s in the mixe f‐replicators i 2c] = [2d] = 0.1 122c1 and (d) 1 nucleobase Thus, buildin reparing the above for the erved at diff oss‐seeding d and no cr ed systems i n libraries ma 125 mM) in by 22d1 at day 3. motif in o ng block 2e w nucleobase e nucleobase ferent ratios experiments ross‐catalysi n driving the ade from bu by adding 10 m . our system, was synthes e building blo
e‐based syst s of 1:2e, ran s with prefor s was obser e self‐ and c ilding mol % the sized, ocks. ems. nging rmed rved, ross‐
4.3. Emergence of PNAs based replicators
4.3.1 Emergence of amino acid functionalized PNA replicators
We investigated whether we can fabricate self‐replicating peptide nucleic acids (PNAs), where replicators may assembly to give a stack of nucleobases without the need of β‐sheet forming peptides. In this case, we slightly changed our design moving to PNAs. We maintained the aromatic dithiol at the N‐terminus of the sequence, appended to an aminoethyl glycine linker and a nucleobase, terminated by an amino acid (Table 4.1). All the building blocks were synthesized by using Fmoc/tBu solid phase peptide synthesis (SPPS). All libraries were set up using 12.5 mM sodium borate buffer (pH = 8.2) under continuous stirring (1200 rpm). Rapid oxidation (80% conversion of thiols to disulfides) of the solutions containing 3.8 mM building block was performed by using sodium perborate solution (80 mM), followed by slower further oxidation mediated by oxygen present in the air.31 A cyclic tetramer emerged in high yield (85%) as evident from UPLC/MS analysis after 30 minutes of reaction in case of the adenine building block (3a). Over time, the tetramer macrocycle became the sole product (Figure 4.6a), with the composition remaining unchanged up to 6 days. Upon replacing adenine with thymine (3b), a completely different composition emerged, consisting of cyclic trimer, tetramer, hexamer and other oligomers. Notably, after 2 days of stirring, only the pentamer macrocycle was observed at the expense of the others (Figure 4.6b). In contrast, a cyclic trimer was observed in the case of cytosine (3d) (Figure
4.6c).
In order to investigate whether the dynamic PNA macrocycles are capable of self‐replication, seeding experiments were performed by adding 10 mol% of the preformed macrocycle to a fresh library that was not treated with sodium perborate. In case of 3a, upon addition of the seed, time dependent UPLC analysis revealed that the formation of the tetramer is significantly faster compared to its growth in the non‐seeded library (Figure 4.6d). For thymine and cytosine building blocks (3b and 3d), seeding experiments were performed in 80% oxidized libraries. UPLC analysis established the autocatalytic nature of the formed macrocycles, as they grew rapidly and accounted for 80% of the library material within 1 day, diminishing the lag phase observed for the first days of the reaction in the absence of seed (Figure 4.6e and f).
Figu distr = 8.2 by ad and TEM mac foun afte by t 350 re 4.6. Eme ribution of the 2) after 80 % o dding 10 mol 3d that were M analysis rev crocycles of nd for all the r the format the aromatic nm) in the a ergence of ly e libraries ma oxidation by s % preformed dominated by vealed the fo various leng e monomers tion of the n c dithiol (pea assembly 33 ( ysine function de from 3.8 m sodium perbo macrocycles. y (g) 3a4, (h) 3 ormation of gth and diam , while a sign nanostructur aks observed Figure S4.6). nalized PNA mM (a) 3a, (b) rate. Seeding . TEM images 3b5 and (i) 3c3 fibrillar asse meter (Figure nificant enha res, highlight d at 250 and . replicators. T ) 3b and (c) 3c g induced grow of libraries m , respectively emblies for t e 4.6g, h an ancement of ting the chir d 275 nm) a Time evolutio c in borate bu wth of (d) 3a4 made from bui . the lysine fun nd i). Silent C f the CD sign ral environm nd the nucle
on of the pro uffer (12.5 mM 4, (e) 3b5 and ( lding blocks 3 nctionalized CD spectra w nal was obse ment experie eic acids (30 oduct M, pH (f) 3c3 3a, 3b PNA were erved nced 00 to
Figu distr = 8.2 by ad and We For resp wer in Fi wer 4.7a asse re 4.7. Emer ribution of the 2) after 80 % o dding 10 mol 3h that were also investig the histidine pectively, wh e performed igure 4.7d, e e observed a, b and c). embly (Figure
rgence of hist e libraries ma oxidation by s % preformed dominated by
gated the eff e PNAs, we hile for 3f, als d for the new
e and f, conf
in the librar CD spectra e S4.7). tidine functio de from 3.8 m sodium perbo d macrocycles y (g) 3e4, (h) 3 fect of the am observed th so a trimer e wly formed m firm the aut ries made fr of the repli
onalized PNA mM (a) 3e, (b) orate. Seeding . TEM images 3f3 and (i) 3h3, mino acid on he formation emerged (Fig macrocycles ocatalytic na rom the hist
icators also replicators. T ) 3f and (c) 3h g induced grow s of libraries m , respectively. n library com n of tetrame gure 4.7a, b (3e4, 3f4 and ature of the tidine conta showed cle Time evolutio h in borate bu wth of (d) 3e4 made from bui position and er and trime and c). Seed d 3h3) and th ir formation ining buildin ar signals ar
on of the pro uffer (12.5 mM
4, (e) 3f3 and (
ilding blocks 3
d self‐replica er for 3e an ding experim he results sh . Nanostruct ng blocks (Fi rising from oduct M, pH f) 3h3 3e, 3f ation. d 3h ments hown tures igure their
4.3.2 In ca mad mac days was com mM mac stru trim Figu from perb brom imag after In ca the tape indic 2 Cation‐intr ase of guanin de from 3c a crocycles up s. TEM analy obtained fo mplex second potassium croscopical t ctural analy mer (Figure 4. re 4.8. Cation m (a) 3c and (d borate at day mide (KBr). CD ges of DCLs pr r 10 days of th ase of library tetramer m e‐like structu cating that t roduced gua ne (G) conta a completely to 14mers ysis of the s or the mixtur dary structur bromide (KB ransition fro sis showed a .8b) and bun n‐induced gua d) 3g (3.8 mM 1 (upper lin D spectra of D repared from he reaction co y made from macrocycle g ures (Figure 4 the amino ac anine based ining PNAs, a y different co (Figure 4.8a ample revea re (Figure 4. res upon add Br), only the om a clear s a characteris ndled of fiber anine based s M in 12.5 mM b e), day 10 (m DCLs made fro (c) 3c upon t orresponding t m 3g, a family rew over ti 4.8e and f). A cid residues supramolec a rather unu omposition
a). The com
aled ill‐defin
.8b and S4.8
dition of cat e trimer mac
solution to a stic CD signa rs, respectiv supramolecula borate buffer middle line) a om (b) 3c and the addition o to the tetram y of large olig me (Figure Addition of play a key ro ular assemb sual behavio emerged, co position rem ed aggregat 8). Guanines ions.34 Nota crocycle was a viscous su
al with a pe ely (Figure 4
ar assembly. U r pH 8.12) afte and after the (e) 3g before of KBr, corresp er. gomers was 4.8d), whic KBr did not a ole in the fo bly or was obser onsisting of mained unch
es and a CD are known t bly, upon th s observed, spension. Sp ak at 268 nm 4.8c). UPLC analysis er oxidation (8 addition of 5 e and after ad ponding to the also found in h self‐assem alter the libr rmation of t rved. For libr a family of hanged up t D silent spect to assemble he addition o accompanie pectroscopic m in the cas
s of DCLs prep 80%) using so 50 mM potas dition of KBr. e trimer and nitially, howe mbled into c rary composi these topolo aries large o 30 trum e into of 50 ed by c and se of pared odium ssium TEM (f) 3g ever, chiral ition, ogical
structures.
4.4. Conclusion
In summary, we have shown how self‐replicators containing both amino‐acid and nucleobase building blocks can spontaneously emerge from dynamic combinatorial libraries. The presence of nucleobases in the building blocks is essential for the emergence of self‐ replicators. Unlike in living systems or previous artificial nucleic acid replicating systems, the process of self‐replication of this new system is driven by stacking rather than base‐pairing. These results provide a new way for auto‐catalytically generating linear arrays of nucleobases. Self‐replicators with different nucleobases can be selectively amplified from multi‐component libraries by self‐ and cross‐replication, where auto‐catalysis is more efficient than cross‐catalysis. No cross‐catalysis was observed when nucleobase in the building blocks were replaced by a phenyl ring. Thus, self‐replicating molecules can not only achieve relatively accurate copying of themselves, but can also catalyze the formation of new replicators through cross‐replication (mutation leading to diversity). Diversity and mutation are two of the most important ingredients to achieve Darwinian evolution. Therefore, our results constitute a step towards Darwinian evolution in fully synthetic chemical systems.
4.5. Acknowledgements
The research was performed in collaboration with Dr. Charalampos G. Pappas, who is most gratefully acknowledged for the experiments with the PNA replicators. Christoph Jurissek is acknowledged for the synthesis of building block 2d. Jim Ottelé and Meniz Altay are acknowledged for the measurement of Transmission Electron Microscopy. Dr. Gaël Schaeffer
4.6. Experimental section
4.6.1 General methodsAll chemicals, unless otherwise stated, were purchased from Sigma‐Aldrich and used as received. Fmoc‐based PNAs were purchased from Link Technologies UK. Acetonitrile (ULC‐ MS grade), water (ULC‐MS grade) and trifluoroacetic acid (HPLC grade) were purchased from Biosolve BV. Anhydrous solvents used in synthesis were freshly collected from a dry solvent purification system prior to use. Flash column chromatography was performed on a Reveleris® X2 Flash Chromatography System (Grace Davison Discovery Sciences, Deerfield IL) on normal or reverse phase silica cartridges. NMR spectra were recorded on 400, 500 and 600 MHz, cryo‐NMR spectrometers, locked on deuterated solvents and referenced to the solvent peak. HRMS spectra were recorded on a LTQ Orbitrap XL instrument in ESI mode.
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. Then the pH was adjusted to 8.2 using concentrated NaOH. Library preparation and sampling Building blocks were dissolved in borate buffer (12.5 mM, pH 8.2). Where necessary, the pH of the solution was adjusted by the addition of 1.0 M NaOH solution such that the final pH was 8.2. 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 were 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 or I‐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, purchased from Phenomenex, 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; desolvation gas temperature: 450 °C; cone gas flow (nitrogen): 1 L/h; desolvation gas flow (nitrogen): 800 L/h. Circular Dichroism Spectra were recorded at room temperature using a JASCO J715 spectrophotometer 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. Samples were diluted to a concentration of 0.15 mM with respect to building block concentration.
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. UPLC methods: Method for the analysis of DCLs made from building blocks 1 and 2a: t / min % B 0 10 1 20 3 31 12 37 12.5 90 13.5 90 14 10 17 10
Method for the analysis of DCLs made from building blocks 3a, 3b, 3c, 3d, 3e, 3g and 3h: t / min % B 0 10 1 10 1.3 15 11 30 11.5 95 12 95 12.5 10 17 10 Method for the analysis of DCLs made from building blocks 1 and 2b: t / min % B 0 10 1 20 3 31 4 32 12 39 12.5 90 13.5 90 14 10 17 10 Method for the analysis of DCLs made from building blocks 1 and 2c: t / min % B 0 10 1 20 3 31 4 32 12 36 12.5 90 13.5 90 14 10 17 10 Method for the analysis of DCLs made from building blocks 1 and 2d: t / min % B 0 10 1 25 3 32 12 35 12.5 90 13.5 90 14 10 17 10 Method for the analysis of DCLs made from building blocks 1 and 2e: t / min % B 0 10 1 30 9 65 10 70 12 90 13 90 13.5 10 17 10 Method for the analysis of DCLs made from building block 3f: t / min % B 0 10 1 10 1.3 20 6 28 11 32 11.5 95 12 95 12.5 10 17 10
4.6.2 Synthesis and characterization of building blocks
The synthesis of building blocks 2a and 2c are described in chapter 2.
Building blocks 3a‐3i were synthesized by conventional peptide synthesis using pre‐loaded Wang resins then purified by Flash column chromatography.
To a solution of thymine (1.26 g, 10 mmol) and pyridine (5.0 mL, 50 mmol) in 50 mL CH3CN at 0 °C was slowly added benzoyl chloride (2.3 mL, 20
mmol) and the mixture was stirred at room temperature for 2 days under N2. Methanol (2.0 mL) was added to the mixture and stirring was
continued at room temperature for 1 h. Then the solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford product S2‐1 as a colorless solid. Yield = 65%, 1.5 g. 1H NMR: (400 MHz, DMSO‐d6, 298K) δ H = 1.81 (s, 3H, ‐CH3), 7.51 (q, J = 1.1 Hz, 1H), 7.61‐7.55 (m, 2H), 7.77‐7.72 (m, 1H), 7.95‐7.90 (m, 2H), 11.35 (s, 1H, thymine NH). 13C NMR (101 MHz, DMSO‐d6, 298K) δC = 14.8, 111.0, 132.5, 133.3, 134.5, 138.4, 141.9, 153.1, 166.7, 173.2. HRESI‐MS calc. for C12H11N2O3+ 231.0674, found 231.0681.
To a solution of S2‐1 (1.1 g, 5 mmol), K2CO3 (0.7 g, 5 mmol) and
tetrabutylammonium iodide (TBAI, 0.18 g, 0.5 mmol) in dry dimethylformamide (DMF, 30 mL) was added tert‐butyl (2‐ bromoethyl)carbamate (1.17 g, 5 mmol). The resulting solution was stirred at room temperature for 48 h. Then the solvent was evaporated under
vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford product S2‐2 as a colorless solid. Yield = 65%, 1.5 g. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.41 (s, 9H, ‐Boc), 1.88 (s, 3H, ‐CH3), 3.35 (d, J = 6.1 Hz, 2H, ‐CH2), 3.76 (t, J = 5.7 Hz, 2H, ‐CH2), 5.11‐4.99 (m, 1H), 7.08 (q, J = 1.2 Hz, 1H), 7.47 (dd, J1 = 8.3, J2 =7.4 Hz, 2H), 7.62 (t, J = 7.5 Hz, 1H), 8.03 (d, J = 7.7 Hz, 2H). 13C NMR (101 MHz, CDCl3, 298K) δC = 14.9, 31.0, 41.4, 51.9, 82.4, 112.6, 131.8, 133.3, 134.3, 137.7, 143.7, 152.6, 158.8, 166.0, 172.0. HRESI‐MS calc. for C7H24N3O5+ 374.1710, found 374.1707. S2‐2 (1.8 g, 0.5 mmol) was dissolved in 30 mL 75% TFA/DCM under a nitrogen
atmosphere and the mixture was stirred overnight. After that, the solvents were evaporated under reduced pressure and the crude product was washed three times with hexane (20 mL). Colorless solid powder S2‐3 was collected by filtration.
Yield = 85%, 1.35 g.
1H NMR: (400 MHz, CD
3OD, 298K) δH = 1.87 (s, 3H, ‐CH3), 3.28 (t, J = 5.7 Hz, 2H, ‐CH2), 4.04 (t, J = 5.7 Hz, 2H, ‐CH2), 7.42 (d, J = 1.6 Hz, 1H). 13C NMR (101 MHz, CD3OD, 298K) δC = 13.5,
41.4, 48.4, 113.3, 143.9, 154.9, 168.0. HRESI‐MS calc. for C7H12N3O2+ 170.0924, found
170.0927.
L‐Fmoc‐aspartic acid alpha‐t‐butylester (411 mg, 1.00 mmol), 2‐(1H‐
benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU, 379 mg, 1.00 mmol), S2‐3 (292 mg, 1.00 mmol) and triethylamine (0.30 mL, 2.15 mmol) were dissolved in 10 mL dry acetonitrile and the mixture was stirred at room temperature overnight under a nitrogen atmosphere. The solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford S2‐4 as a white powder. Yield = 46%, 0.27 g. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.44 (s, 9H, ‐(CH3)3), 1.77 (s, 3H, ‐CH3), 2.67 (dd, J1 = 17.0 Hz, J2 = 5.2 Hz, 1H), 3.08‐3.00 (m, 1H), 3.52 (d, J = 10.5 Hz, 1H), 3.74‐3.60 (m, 2H), 4.05 (d, J = 13.5 Hz, 1H), 4.16 (t, J = 6.6 Hz, 1H), 4.44 (d, J = 6.7 Hz, 2H), 4.57 (dt, J1 = 10.7 Hz, J2 =
5.2 Hz, 1H), 6.22 (d, J = 9.3 Hz, 1H), 7.14 (s, 1H), 7.27‐7.18 (m, 2H), 7.39‐7.27 (m, 2H), 7.53 (dd, J1 = 7.6 Hz, J2 = 4.9 Hz, 2H), 7.67 (t, J = 6.5 Hz, 2H), 7.99 (s, 1H), 10.63 (s, 1H, thymine NH). 13C NMR (101 MHz, CDCl 3, 298K) δC = 11.3, 14.4, 30.7, 40.1, 41.7, 48.9, 49.8, 50.9, 54.2, 69.5, 84.3, 112.6, 122.6, 127.6, 129.55, 129.61, 130.3, 143.9, 145.5, 146.3, 146.4, 154.6, 158.9, 173.8, 174.3. HRESI‐MS calc. for C30H35N4O7+ 563.2500, found 563.2505. S2‐4 (562 mg, 1.00 mmol) was dissolved in 20 mL 20% piperdine in DMF (20:80/piperidine:DMF) and stirred for 0.5 h at room temperature. Then the solvents were evaporated under vacuum and the crude powder was washed with hexane (30 mL x 3) to afford derivative S2‐5 as a slightly yellow powder. Yield = 51%, 0.17 g. 1H NMR: (400 MHz, CD 3OD, 298K) δH = 1.43 (s, 9H, ‐(CH3)3), 1.87 (d, J = 1.2 Hz, 3H, ‐CH3), 2.43 (dd, J1 = 16.5 Hz, J2 = 7.9 Hz, 1H, ‐CH2), 2.66‐2.60 (m, 1H, ‐CH2), 3.51‐3.47 (m, 2H, ‐CH2), 3.55 (dd, J1 = 7.9 Hz, J2 = 4.9 Hz, 1H), 4.09 (t, J = 5.6 Hz, 2H), 7.21 (d, J = 1.3 Hz, 1H, thymine CH). 13C NMR (101 MHz, CD 3OD, 298K) δC = 14.2, 29.57, 29.59, 39.9, 42.2, 42.3, 54.3, 83.5, 111.2, 138.8, 177.4. HRESI‐MS calc. for C15H25N4O5+ 341.1819, found 341.1817. S0 (670 mg, 1.00 mmol), 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐
tetramethyluronium hexafluorophosphate (HBTU, 379 mg, 1.00 mmol), S2‐5 (341 mg, 1.00 mmol) and triethylamine (0.30 mL, 2.15 mmol) were dissolved in 10 mL dry DMF and the mixture was stirred at room temperature overnight under a nitrogen atmosphere. The solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐ 10% methanol in DCM), followed by removal of solvent to afford S2‐6 as a white powder. Yield = 59%, 0.58 g. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.42 (s, 9H, ‐(CH3)3), 1.80 (s, 3H, ‐CH3), 2.60 (dd, J1 = 16.6 Hz, J2 = 6.3 Hz, 1H, ‐CH2), 2.75 (dd, J1 = 16.6 Hz, J2 = 5.3 Hz, 1H, ‐CH2), 3.50 (tt, J1 = 8.1 Hz, J2 = 5.2 Hz, 2H), 4.10 (ddt, J1 = 17.1 Hz, J2 = 13.5 Hz, J3 = 6.7 Hz, 2H), 4.76 (dt, J1 = 8.5 Hz, J2 = 5.7 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 6.79 (dd, J1 = 5.5 Hz, J2 = 1.5 Hz, 1H), 6.98 (d, J = 1.6 Hz, 2H), 7.24‐7.06 (m, 20H), 7.26‐7.32 (m, 12H), 9.56 (s, 1H, thymine NH). 13C NMR (101 MHz, CDCl3, 298K) δC = 15.6, 16.8, 30.7, 52.4, 74.2, 84.2, 129.5, 130.5, 132.49, 132.51, 135.6, 135.8, N H O O O NH N O STrit TritS S2-6 NH O O
137.5, 145.5, 146.6, 146.7, 155.5, 168.4, 173.1, 173.4. HRESI‐MS calc. for C60H57N4O6S2+
993.3714, found 993.3716.
S2‐6 (100 mg, 0.10 mmol) was dissolved in 10 mL 50% TFA in DCM
and stirred for 12 h at room temperature under N2. Et3SiH (0.50 mL,
3.1 mmol) was added to the reaction mixture which was stirred for another hour. Solvents were evaporated under vacuum and the crude mixture was washed with hexane (10 mL x 2). The product was purified by reverse phase flash column chromatograph (RP C18, 0‐90% acetonitrile in water with 0.1% TFA), and the desired product 2b was obtained after lyophilization as a white powder Yield = 41%, 20 mg. 1H NMR: (400 MHz, CD 3OD, 298K) δH = 2.75 (dd, J1 = 16.7 Hz, J2 = 8.4 Hz, 1H, ‐CH2), 2.88 (dd, J1 = 16.8 Hz, J2 = 5.2 Hz, 1H, ‐CH2), 3.48‐3.52 (m, 2H, ‐CH2), 4.02‐4.16 (m, 2H, ‐CH2), 4.81‐4.83 (m, 1H, ‐CH), 7.17 (d, J = 1.2 Hz, 1H, thymine), 7.37 (t, J = 1.7 Hz, 1H, ArH), 7.52 (d, J = 1.7 Hz, 2H, ArH). No 13C NMR could be obtained due to the poor solubility of 2b. HRESI‐MS calc. for C14H21N4O6S2+ 453.0897, found 453.0899.
To a solution of cytosine (1.0 g, 9 mmol) and 4‐dimethylaminopyridine (DMAP, 0.1 g, 0.9 mmol) in dry tetrahydrofuran (THF, 30 mL) was added di‐
tert‐butyl dicarbonate (7.85 g, 36 mmol). The resulting solution was stirred at
room temperature for 12 h under N2. Then the solvent was evaporated
HCl (1 N, 1 x 20 mL) and brine (2 x 20 mL), dried with Na2SO4, followed by removal of solvent to afford product S4‐1 as a colorless solid. Yield = 91%, 3.37 g. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.54 (s, 18H, ‐Boc), 1.59 (s, 9H, ‐Boc), 7.06 (d, J = 7.9 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H). 13C NMR (101 MHz, CDCl3, 298K) δC = 30.3, 30.4, 88.0, 89.4, 99.6, 146.1, 151.8, 152.9, 153.9, 165.0. HRESI‐MS calc. for C19H30N3O7+ 412.2078, found 412.2074. To a solution of S4‐1 (3.0 g, 7.3 mmol) in MeOH (120 mL) was added
saturated NaHCO3 aq. (35 mL). The resulting turbid solution was stirred at 50
for 1 h. Then MeOH was evaporated under vacuum and water (50 mL) was added into the crude mixture, the aqueous layer was extracted with DCM (3 x 80 mL). The organic layer was dried with Na2SO4 and the solvent was
evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford
product S4‐2 as a colorless solid. Yield = 75%, 1.7 g. 1H NMR: (400 MHz, DMSO‐d6, 298K) δ H = 1.46 (s, 18H, ‐Boc), 6.68 (d, J = 7.0 Hz, 1H), 7.87 (d, J = 7.0 Hz, 1H). 13C NMR (101 MHz, DMSO‐d6, 298K) δC = 30.4, 87.4, 98.2, 150.7, 152.4, 158.2, 165.7. HRESI‐MS calc. for C14H22N3O5+ 312.1554, found 312.1555.
To a solution of S4‐2 (1.6 g, 5 mmol), K2CO3 (0.7 g, 5 mmol) and TBAI
(0.18 g, 0.5 mmol) in dry DMF (40 mL) was added N‐(2‐ bromoethyl)phthalimide (1.3 g, 5 mmol). The resulting solution was stirred at room temperature for 48 h under N2. Then the solvent was
evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by
removal of solvent to afford product S4‐3 as a colorless solid. Yield = 53%, 1.28 g. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.52 (s, 18H, ‐Boc), 4.07 (ddd, J1 = 6.2 Hz, J2 = 5.0, J3 = 1.2 Hz, 2H, ‐CH2), 4.14 (ddd, J1 = 6.3 Hz, J2 = 4.9, J3 = 1.1 Hz, 2H, ‐CH2), 6.89 (d, J = 7.4 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.70 (dd, J1 = 5.5 Hz, J2 = 3.1 Hz, 2H), 7.81 (dd, J1 = 5.5 Hz, J2 = 3.1 Hz, 2H). 13C NMR (101 MHz, CDCl3, 298K) δC = 27.8, 49.2, 84.9, 96.6, 123.7, 131.8, 134.3, 147.5, 149.6, 155.0, 168.0. HRESI‐MS calc. for C24H29N4O7+ 485.2031, found 485.2031. N N N O Boc Boc N O O S4-3
To a solution of S4‐3 (0.5 g, 1 mmol) in methanol (25 mL) was added hydrazine monohydrate (100 mg, 2 mmol). The reaction mixture was stirred at 80 for 1 h and the resulting solid was separated. Then the solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford
product S4‐4 as a colorless oil. Yield = 67%, 0.24 g. 1H NMR: (400 MHz, CD 3OD, 298K) δH = 1.52 (s, 9H, ‐Boc), 2.99 (d, J = 6.5 Hz, 2H, ‐CH2), 3.93 (t, J = 6.1 Hz, 2H, ‐CH2), 7.23 (dd, J1 = 7.3, J2 = 1.4 Hz, 1H, cytosine), 7.91 (dd, J1 = 7.3, J2 = 1.0 Hz, 1H, cytosine). 13C NMR (101 MHz, CD3OD, 298K) δC = 29.6, 42.3, 55.1, 84.3, 98.0, 110.0, 152.1, 154.8, 160.1, 166.4. HRESI‐MS calc. for C11H19N4O3+ 255.1452, found 255.1456.
To a solution of L‐Fmoc‐aspartic acid alpha‐t‐butylester (411 mg, 1 mmol), HBTU (379 mg, 1 mmol) and triethylamine (0.30 mL, 2 mmol) in 20 mL dry acetonitrile was added S4‐4 (250 mg, 1 mmol). The mixture was stirred at room temperature overnight under N2. Then
the solvent was evaporated under vacuum and the crude mixture was dissolved in ethyl acetate (50 mL), washed with brine (2 x 30 mL), dried with Na2SO4,
and the solvent was evaporated under vacuum. The crude mixture was dissolved in 20 mL 20% piperdine in DMF (20:80/piperidine:DMF) and stirred for 0.5 h at room temperature. The solvents were evaporated under vacuum and the crude powder was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford S4‐5 as oil. Yield = 35%, 0.15 g. 1H NMR: (400 MHz, CD 3OD, 298K) δH = 1.45 (s, 9H), 1.52 (s, 9H), 2.57‐2.50 (m, 1H), 2.66‐2.59 (m, 1H), 3.59‐3.51 (m, 3H), 4.05‐3.93 (m, 2H), 7.21 (dd, J1 = 7.3 Hz, J2 = 2.2 Hz, 1H, cytosine CH), 7.87 (dd, J1 = 7.3 Hz, J2 = 1.2 Hz, 1H, cytosine CH). 13C NMR (101 MHz, CD3OD, 298K) δC = 29.58, 29.60, 29.62, 40.3, 52.4, 54.3, 83.5, 84.2, 97.9, 152.3, 166.4, 173.6, 178.1. HRESI‐MS calc. for C19H32N5O6+ 426.2347, found 426.2343. The procedure for the synthesis of S4‐6 was the same as that described for the synthesis of S2‐6.
Yield = 43%, 460 mg. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.44 (s, 9H), 1.46 (s, 9H), 2.70 (dd, J1 = 15.1 Hz, J2 = 8.2 Hz, 1H), 2.78 (dd, J1 = 15.1 Hz, J2 = 5.1 Hz, 1H), 3.50‐3.42 (m, 1H), 3.60 (dd, J1 = 13.8 Hz, J2 = 7.1 Hz, 1H), 3.77‐3.79 (m, 2H), 4.98 (td, J1 = 8.3 Hz, J2 = 5.0 Hz, 1H), 6.92 (d, J = 1.6 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 7.02 (d, J = 1.6 Hz, 2H), 7.20‐7.08 (m, 20H), 7.21‐7.24 (m, 11H), 8.42 (s, 1H), 8.71 (s, 1H). 13C NMR (101 MHz, CDCl3, 298K) δC = 16.8, 25.3, 27.9, 29.6, 30.71, 30.74, 30.83, 31.7, 34.2, 37.3, 40.0, 41.2, 52.9, 53.6, 74.0, 84.4, 84.9, 110.0, 129.4, 130.4, 132.5, 132.6, 135.7, 135.8, 137.2, 145.0, 146.7, 152.1, 154.2, 158.2, 165.9, 168.3, 173.7, 174.2. HRESI‐MS calc. for C64H64N5O7S2+ 1078.4242, found 1078.4249.
The procedure for the synthesis of 2d was the same as that described for the synthesis of 2b. Yield = 33%, 13 mg. 1H NMR: (400 MHz, CD 3OD, 298K) δH = 2.79 (dd, J1 = 16.9 Hz, J2 = 7.9 Hz, 1H, ‐CH2), 2.90 (dd, J1 = 16.9 Hz, J2 = 5.8 Hz, 1H, ‐CH2), 3.51‐3.54 (m, 2H, ‐CH2), 3.87‐ 3.91 (m, 1H, ‐CH2), 3.93‐4.01 (m, 1H, ‐CH2), 4.74 (dd, J1 = 7.8, J2 = 5.8 Hz, 1H, ‐CH), 5.95 (d, J = 7.6 Hz, 1H, cytosine), 7.39 (q, J = 1.8 Hz, 1H, ArH), 7.48‐7.52 (m, 2H, ArH), 7.79 (dd, J1 = 7.6 Hz, J2 = 0.9 Hz, 1H, cytosine). 13
C NMR (101 MHz, CD
3OD, 298K) δ
C= 37.6, 39.9, 52.2,
53.5, 95.2, 126.5, 133.3, 136.6, 137.7, 150.6, 153.0, 163.0, 170.2, 175.1, 175.2.
HRESI‐ MS calc. for C17H20N5O5S2+ 438.0900, found 438.0903. The procedure for the synthesis of S5‐1 was the same as that described for the synthesis of S2‐4.Yield = 49%, 252 mg. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.44 (s, 9H, ‐C(CH3)3), 2.61 (dd, J1 = 16.8 Hz, J2 = 9.8 Hz, 1H, ‐CH2), 2.83 (dd, J1 = 16.8 Hz, J2 = 4.0 Hz, 1H, ‐CH2), 3.45‐3.52 (m, 2H, ‐CH2), 4.19 (t, J = 6.9 Hz, 1H, ‐CH), 4.34‐ 4.40 (m, 2H, ‐CH2), 4.47‐4.49 (m, 1H, ‐CH), 6.00 (s, 1H), 6.59 (s, 1H), 7.14‐7.22 (m, 3H, ArH), 7.23‐7.34 (m, 4H, ArH), 7.40 (t, J = 7.5, 2H, ArH), 7.57 (d, J = 7.5 Hz, 2H, ArH), 7.76 (d, J = 7.5Hz, 2H). 13C NMR (101 MHz, CDCl3, 298K) δC = 30.7, 38.3, 40.2, 41.3, 43.6, 49.8, 53.9, 69.8, 84.5, 122.7, 127.7, 129.2, 130.4, 131.2, 131.37, 131.40, 131.6, 141.3, 143.95, 143.96, 169.3, 173.1. HRESI‐MS calc. for C31H35N2O5+ 515.2540, found 515.2541. S5‐1 (515 mg, 1 mmol) was dissolved in 20 mL 20% piperdine in DMF (20:80/piperidine:DMF) and stirred for 0.5 h at room temperature. Then the solvents were evaporated under vacuum and the crude powder was washed 2 times with hexane (10 mL). Then the crude product was dissolved in a mixture of S0 (670 mg, 1.00 mmol), 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU, 379 mg, 1.00 mmol) and triethylamine (0.30 mL, 2.15 mmol) in 10 mL dry DMF and the mixture was stirred at room temperature overnight under a nitrogen atmosphere. The solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography (SiO2, 0‐10% methanol in DCM), followed by removal of solvent to afford S5‐2 as a white powder. Yield = 33%, 312 mg. 1H NMR: (400 MHz, CDCl 3, 298K) δH = 1.44 (s, 9H, ‐C(CH3)3), 2.53 (dd, J1 = 16.8 Hz, J2 = 7.2 Hz, 1H, ‐CH2), 2.71‐2.79 (m, 3H, ‐CH2), 3.49 (q, J = 6.8 Hz, 2H, ‐CH2), 2.53 (td, J1 = 7.4 Hz, J2 = 4.5 Hz, 1H, ‐CH), 6.50 (t, J = 5.9 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 1.6 Hz, 1H), 7.06 (d, J = 1.6 Hz, 1H), 7.13‐7.22 (m, 22H, ArH), 7.28‐7322 (m, 11H, ArH). 13C NMR (101 MHz, CDCl3, 298K) δC = 30.7, 38.3, 43.5, 52.3, 62.2, 74.0, 84.4, 129.2, 129.5, 130.4, 131.26, 131.34, 132.5, 135.0, 135.4, 137.7, 146.7, 172.2, 172.7, 173.7. HRESI‐MS calc. for C61H57N2O4S2+ 945.3754, found 945.3761. The procedure for the synthesis of 2e was the same as that described for the synthesis of 2b. Yield = 46%, 20 mg.
1H NMR: (400 MHz, CDCl 3, 298K) δH = 2.75 (dd, J1 = 16.8 Hz, J2 = 7.2 Hz, 1H, ‐CH2), 2.78 (t, J = 7.2 Hz, 2H, ‐CH2), 2.86 (dd, J1 = 16.8 Hz, J2 = 4.8 Hz, 1H, ‐CH2), ), 3.43 (t, J = 7.2 Hz, 2H, ‐CH2), 4.74‐4.76 (m, 1H, ‐CH), 7.12‐7.24 (m, 5H, ‐ph), 7.38 (t, J = 2.0 Hz, 1H, ‐ArH), 7.45 (t, J = 2.0 Hz, 2H, ‐ArH). 13C NMR (101 MHz, CD3OD, 298K) δC = 37.6, 37.9, 43.4, 53.3, 126.6, 128.6, 130.7, 131.1, 133.3, 136.5, 137.8, 141.6, 170.0, 174.1, 175.3. HRESI‐MS calc. for C19H21N2O4S2+ 405.0937, found 405.0935. 4.6.3 Appendix 0 4 8 12 16 20 0 20 40 60 80 100 Pe rcentage of Peak Area of 1 2 2a1 (%) Time (days)
no agitation no agitation (seed 15 mol %) stirred stirred (seed 15 mol %) shaken shaken (seed 15 mol %)
Figure S4.1. Kinetic profile of the emergence of mixed trimers 122a1 in libraries made from building blocks 1 (1.0 mM) and 2a (0.5 mM) under different conditions. 200 250 300 350 400 -10 -5 0 5 10 Ellip tic ity (m d e g ) Wavelength (nm) 122a1 122b1 122c1 122d1 Figure S4.2. CD spectra of libraries made from building blocks 1 and 2 that were dominated by 122a1, 122b1, 122c1 and 122d1, respectively. N H OH O O NH O SH HS 2e
Figu and Figu mM, (c) 1 re S4.3. Seed 2b ([1] = 1.0 m re S4.4. UPLC , [2a] = [2b] = 22b1, (d) 122c1 ding induced g mM, [2a] = [2b C analysis of t = [2c] = [2d] = 1 and (e) 122d growth of 122 b] = 0.25 mM the libraries m 0.125 mM) b 1 after 30 day 2a1 and 122b1 ) by adding 10 made from bu by adding 10 m ys stirring. in libraries m 0 mol % prefo uilding blocks mol % preform made from bu ormed (a) 122b 1, 2a, 2b, 2c med (a) witho uilding blocks b1 and (b) 122a c and 2b ([1] out seed, (b) 1 1, 2a a1. = 1.0 122a1,
Figu mM) 30 d Figu 3a4, re S4.5. UPLC ) by adding 10 ays stirring. re S4.6. CD sp 3b5 and 3d3, r C analysis of th 0 mol % prefo pectra of libra respectively, c he libraries ma ormed (a) with aries made fro compared wit ade from buil hout seed, (b om building b th monomer. ding blocks 1 ) 122a1, (c) 122 blocks 3a, 3b a and 2e ([1] = 2b1, (d) 122c1 and 3d that w 1.0 mM, [2e] and (e) 122d1 were dominate = 0.5 after ed by
Figu 3e4, Figu after re S4.7. CD sp 3f3 and 3h3, r re S4.8. TEM r 30 days stirr pectra of libra respectively, c images of lib ring. aries made fro compared with rary made fro om building b h monomer. om building b blocks 3e, 3f a block 3c in bor and 3h that w rate buffer (1 were dominate 12.5 mM, pH = ed by = 8.2)
4.6.4 Figu a DC 1378 Figu analy [M+5 4 UPLC and re S4.9. Mass CL made from 8.24 [M+5H]5+ re S4.10. Ma ysis of a DCL m 5H]5+; observe UPLC‐MS an s spectrum of 1 (0.4 mM) a + ; observed m ass spectrum made from 1 ed m/z: 1797. nalysis 2a15 (retentio and 2a (1.6 m /z: 2296.18 [M of 112a14 (re (0.4 mM) and 30 [M+4H]4+, on time 3.53 m M). Calculate M+3H]3+, 1722 etention time d 2a (1.6 mM) 1438.07 [M+ min in Figure 4 d m/z: 2296.4 2.99 [M+4H]4+ e 4.13 min in ). Calculated m 5H]5+. 4.1c) from the 40 [M+3H]3+, + , 1378.56 [M+ n Figure 4.1c m/z: 1797.11 e LC‐MS analy 1722.55 [M+4 +5H]5+. ) from the L [M+4H]4+, 143 ysis of 4H]4+, C‐MS 37.89
Figu analy [M+5 Figu analy [M+5 re S4.11. Ma ysis of a DCL m 5H]5+; observe re S4.12. Ma ysis of a DCL m 5H]5+; observe ass spectrum made from 1 ed m/z: 1872. ass spectrum made from 1 ed m/z: 1946. of 122a13 (re (0.4 mM) and 36 [M+4H]4+, of 132a12 (re (0.4 mM) and 64 [M+4H]4+, etention time d 2a (1.6 mM) 1497.91 [M+ etention time d 2a (1.6 mM) 1557.73 [M+ e 4.20 min in ). Calculated m 5H]5+. e 4.28 min in ). Calculated m 5H]5+. n Figure 4.1c m/z: 1871.68 n Figure 4.1c m/z: 1946.23 ) from the L [M+4H]4+, 149 ) from the L [M+4H]4+, 155 C‐MS 97.54 C‐MS 57.19
Figu of a obse Figu from time 1871 [M+5 re S4.13. Mas DCL made fro erved m/z: 919 re S4.14. Mas m the LC‐MS a e of these ma 1.68 [M+4H]4+ 5H]5+, 1248.43 ss spectrum o om 1 (0.4 mM 9.14 [M+2H]2 ss spectrum o analysis of a D acrocycles, the +, 1497.54 [M 3 [M+6H]6+. C of 2a4 (retenti M) and 2a (1.6 +, 613.75 [M+ of 122a13, 132a DCL made fro ey are analyz M+5H]5+, 1248 alculated m/z ion time 4.58 mM). Calcula +3H]3+. a12 and 142a11 om 1 (0.4 mM zed in a single 8.12 [M+6H]6+ z for 132a12: 1 8 min in Figure ated m/z: 919 (retention tim M) and 2a (1.6
e mass spectr
+; observed m 946.24 [M+4H e 4.1c) from t .16 [M+2H]2+, me 4.81‐4.85 6 mM). Due to rum. Calculate m/z: 1872.08 [ H]4+, 1557.19 the LC‐MS an , 613.11 [M+3 min in Figure o similar rete ed m/z for 12 [M+4H]4+, 149 [M+5H]5+, 129 alysis 3H]3+; 4.1c) ntion 22a13: 97.71 97.83
