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Selective amide bond formation in redox-active coacervate protocells

9 Membraneless compartments, like complex coacervates droplets, are promising protocell models 10 because of their ability to sequester a wide range of guest molecules and their catalytic properties. 11 However, it remains unclear how the building blocks of life, including peptides, could be 12 synthesized from primitive precursor molecules inside such protocells. Here, we develop a new 13 protocell model formed by phase separation of prebiotically relevant small redox-active 14 ferricyanide (Fe(CN)6)/ferrocyanide (Fe(CN)6) molecules and a cationic peptide. The assembly 15 of these coacervate protocells can be regulated by redox chemistry and they act as oxidizing hubs 16 for sequestered metabolites, such as NAD(P)H, and fiber precursors. Interestingly, we show that 17 the oxidizing potential of ferricyanide inside coacervates can be harnessed to drive the selective 18 formation of amide bonds between prebiotically relevant amino thioacids and amino acids or 19 peptides. We demonstrate that aminoacylation is enhanced in Fe(CN)6/peptide coacervate 20 dispersions compared to the surrounding dilute phase, and selective for amino acids that interact 21 less strongly with the coacervates. We finally use this amide bond formation to create self22


29
Amide bond formation is an essential chemical reaction in all forms of life that is catalyzed by 30 highly evolved biomolecular machinery. However, before ribosomes and specialized enzymes 31 became capable of protein synthesis, 1,2 alternative, simple prebiotic routes to create peptide bonds 32 in a spatiotemporally controlled way likely existed. Protocellular compartments provide a 33 promising platform to localize chemical reactions relevant to life. [3][4][5] What the nature of such 34 protocellular compartments capable of peptide synthesis could be, remains unknown. As plausible 35 precursors to peptides, α-aminothioacids (AA-SH) 6 and acetylated aminothioacids (Ac-AA-SH) 7 36 have been shown to be formed in aqueous conditions at near-neutral pH. They are considered 37 interesting alternatives to biological thioesters for prebiotic peptide ligation, and can be ligated into 38 peptide with the aid of an oxidizing catalyst, such as ferricyanide. 7-10 However, high concentrations 39 of reactants and catalysts are typically required for these oxidative peptide ligations, which may 40 not have been easy to reach. Recent work has demonstrated that some micro-compartments and 41 protocell systems could compartmentalize (bio)chemical reactions, and act as catalytic 42 microreactors or reaction localization centers with potential for prebiotic peptide ligation. 11-16 43 In particular, membraneless compartments based on complex coacervates have been 44 considered as versatile protocell models in the origins of life research. 5,16-19 Coacervate droplets 45 temperature, 26,27 light, 28 enzymatic reactions, 23,29-31 or fuel-driven chemical reaction cycles. 18 54 To date, research on coacervates as reaction localization centers has mostly focused on 55 enzymatic reactions, or reactions involving complex RNAzymes (ribozymes). However, the 56 fundamental biochemical reactions that lead to the formation of peptides, which themselves are 57 often the building blocks of coacervates, have not been studied. For coacervates to be a plausible 58 protocell model, they must be able to support a prebiotically relevant ligation reaction of peptides, 59 and ultimately, link this reaction to the construction of new coacervates. Here, we show that 60 ferricyanide-containing coacervates can potentially fulfill this role. These coacervates can be used 61 to oxidize not only a variety of common metabolites, but also amino thioacids, which can 62 subsequently be aminoacylated yielding a product with a new amide bond. 63 Coacervates with ferrocyanide, the reduced form of ferricyanide, as multivalent anion have 64 already been reported by Bungenberg-de Jong and Kruyt in 1929. 32 They used gelatin at low pH 65 as a long polycation and observed small punctated droplets upon mixing with potassium 66 ferrocyanide. However, the redox potential of the iron centers inside these coacervates remains 67 unclear. More recently, it was shown that coacervates can be formed from much smaller cationic 68 oligopeptides, such as oligolysine and oligoarginine with as little as five amino acids, complexed 69 salt-free solution. The difference between (Lys)10 and (Arg)10 can be explained by the higher pKa 117 of the basic residue in arginine compared to lysine, which may generate more stable ionic 118 interactions at a given chain length. 36 Increasing the length of polylysine to (Lys)30 resulted in the 119 formation of coacervates with trivalent ferricyanide as well ( Supplementary Fig. 4c The obtained coacervates with a diameter of about 2 to 3 µm were observed in a PLL-g-PEG 131 functionalized microchamber (Figure 1b-c). These microdroplets exhibited liquid-like properties: 132 they wetted the bottom glass surface and sedimenting coacervates coalesced with coacervates 133 already present at the bottom of the microchambers to form bigger droplets. The droplets 134 sequestered negatively charged client solutes, including pyranine, NAD(P)H, and ribonucleic acids 135 Supplementary Fig. 4). Determination of the partitioning of ferricyanide and 136 ferrocyanide by Uv-vis spectroscopy (see Methods) showed that both these multivalent scaffolding 137 anions were highly concentrated in the coacervate droplets: the internal concentration in coacervate 138 droplets was 100 times higher than the surrounding dilute aqueous phase. For example, for samples 139 prepared from 2 mM ferricyanide and pLys (5mM lysine monomers) solutions, we found an 140 internal ferricyanide concentration of ~30 mM, compared to ~0.3 mM in the surrounding aqueous 141 phase. (Supplementary Fig. 5). Surprisingly, the concentration ratio for ferri-and ferrocyanide was 142 the same, despite their different valency. We attribute this to the formation of a tighter complex 143 between ferricyanide and polycations, which results in a more hydrophobic coacervate, 27 an effect 144 that has previously been observed with ferricyanide in polyelectrolyte brushes. 37 A similar 100x 145 concentration was previously found for a negatively charged bis-carboxylic acid ligand, which can 146 exist in a tetravalent (4-) ring configuration, in coacervates with cationic polymers. 38 These initial 147 observations suggest that the spontaneous assembly of peptides and ferricyanide or ferrocyanide 148 could be developed as potential prebiotic membraneless compartments. 149 Typically, coacervates formed by charge-charge interaction are sensitive to the ionic strength 150 and they exhibit a critical salt concentration (CSC) above which phase separation does not take 151 place. Since ferricyanide and ferrocyanide have a different net charge, their CSC is likely different, 152 even though the previously discussed hydration differences may decrease that effect. A difference 153 in CSC would allow for selective compartmentalization controlled by redox chemistry. To 154 completely disappear. Figure 1g  illustrates the remarkable reversibility of this process: droplets could be generated and dissolved 200 up to eight times, and we were able to carry out identical transitions when starting from either 201 Fe(CN)6 4or Fe(CN)6 3-. After eight cycles, the system loses its ability to condense into droplets, 202 which is mainly caused by accumulation of the waste products from GSH or NADH and S2O8 2-. 203 The level of redox control over droplet generation shown in Figure 2 has not been achieved before 204 and holds great promise for the development of dynamic protocell models. 205 For use of these ferricyanide coacervates as potential oxidizing hubs in which oxidation 206 reactions could be localized, it is important to know where the redox reactions utilized in Fig. 2  207 take place. We can take advantage of the fluorescence of common redox-active metabolites such 208 as NADPH, in combination with the observed pH dependence, to monitor the conversion ferri-and 209 ferrocyanide. We incubated ferricyanide/(Arg)10 coacervates (8 mM ferricyanide/24 mM Arg) with 210 2 mM NADPH at neutral pH and observed clear NADPH fluorescence inside the coacervates, 211 indicating that NADPH is sequestered by the coacervates (Supplementary Fig. 7). At this pH, the 212 reduction of ferricyanide by NADPH is suppressed (Fig. 2c), which allows for equilibration and 213 focusing of the microscope. We then decreased the outer pH by addition of a fixed amount of acid 214 intensity first disappeared from the center of the coacervate droplets ( Supplementary Fig. 7), 217 indicating that the oxidation of NADPH to non-fluorescence NADP + by ferricyanide took place 218 predominantly inside the droplets instead of in solution, 18 in which case an exchange of 219 NADPH/NADP + and reduction of fluorescence at the droplet interface would be expected. Taken 220 together, these data show our ferricyanide-based coacervates are redox-active compartments that 221 can locally oxidize sequestered metabolites. 222 223

Fiber self-assembly inside ferricyanide-based coacervates 224
We further investigated if the redox activity of ferricyanide-based coacervates could lead to 225 spatially controlled higher order assembly. Self-assembly of filaments inside or at the periphery of 226 (proto)cellular compartments is key to many transport processes, locomotion and division. 39,40 227 Spatially controlled assembly of analogous model filaments in cell-like compartments is therefore 228 an interesting goal in protocell and synthetic cell research. [40][41][42] We selected an amino acid 229 derivative benzoyl cysteine as precursor for filaments (Fig. 3). N,N′-dibenzoyl-L-cystine (DBC) is 230 a well-known redox-active supramolecular gelator, 43,44 which has been used to make filaments in 231 aqueous solution at low pH. To create the precursor form, we reduced the water soluble DBC 2at 232 pH 7 with 1 equivalent of dithiothreitol (DTT) to give non-active monomer N-benzoyl-L-cysteine 233 (BC -), which is highly soluble at both high and low pH and does not stack to form fibers. 234 We flushed a solution of BC -(with dye Nile red) in a microchamber containing 235 ferricyanide/(Arg)10 coacervates (final concentration: 10 mM BC -, 4 mM ferricyanide, 12 mM 236 (Arg)10 (monomer basis)) at pH 7. Upon addition of the coacervates, we observed a gradual increase 237 in the total fluorescent intensity inside coacervates, as newly formed non-active monomer 2-238 sequester Nile red ( Supplementary Fig. 4h). Upon addition of BC to an aqueous dispersion of 242 ferricyanide/(Arg)10 coacervates at pH 3 (below the pKa of the carboxylate groups of DBC, pKa ∼ 243 3.6), 44 we observed oxidized, bright fluorescent DBC filaments assembled into shells around the 244 coacervate droplets, and several bundles of filaments present inside the droplets (Fig. 3b). The 245 bundled filaments inside the coacervates are clearly visible in transmission, and are strongly stained 246 by Nile red. These bundles can be seen to pierce the interface of the coacervate droplets. In control 247 with ferricyanide but without polycations, we observed a fiber dispersion without clear bundling 248 or formation of shells (Fig. 3c). giving an aster-like shape ( Supplementary Fig. 8c). In short, these findings show that ferricyanide 261 coacervates can drive the formation of self-assembled filaments via oxidation of the filament 262 precursors. 263 264

Amide bond formation through amino thioacid oxidation in coacervate protocells 265
Having established the potential of ferricyanide-based coacervates as oxidizing hubs for redox-266 active guest molecules, we sought to use the oxidizing potential to synthesize peptides by 267 catalyzing the formation of amide bonds. Ferricyanide has been described as a prebiotically 268 abundant oxidizing agent, 33 and has been used to activate amino thioacids by oxidation to facilitate 269 the formation of an amide bond upon reaction with nucleophilic aminonitriles and amino acids. 6-270 9,34 However, the oxidative aminoacylation of thioacids is usually performed with high reactant and 271 ferricyanide concentrations (sometimes close to 100 mM), which could have been difficult to reach 272 everywhere on Early Earth. Therefore, the highly concentrated ferricyanide coacervate droplets 273 could be interesting model compartments for prebiotic amide bond formation (Fig. 4a). As a proof 274 of principle, we first prepared ferricyanide coacervate droplets by direct mixing of aqueous 275 solutions of ferricyanide and trimethylated poly-L-lysine (pLys(Me)3) at charge stoichiometry. The 276 pLys(Me)3 was chosen to avoid the ε-coupling of Lys-NH2. To study the amide bond formation in 277 coacervate droplets, we incubated N-acetyl-glycine thioacid (Ac-Gly-SH) (8 mM) with Gly (3 eq.) 278 of coacervates, while in dilute phase at most 5% of ligation product was observed. In controls with 282 peptides but without ferricyanide, we found no ligation product ( Supplementary Fig. 9), and also 283 in the presence of ferrocyanide/pLys coacervate droplets, no ligation product was observed 284 ( Supplementary Fig. 10). The significantly higher yield in the presence of coacervates can be 285 attributed to the high local ferricyanide concentration inside coacervate droplets and implies that 286 the ligation reaction takes place predominantly inside the coacervates. Ferricyanide-based 287 coacervate droplets can thus act as microreactors that enhance the rates of oxidative aminoacylation. 288 We found that the ligation reaction was most enhanced in droplets formed from shorter peptides, 289 (Lys(Me)3)20 and (Lys(Me)3)30, compared to longer pLys(Me)3 (Fig. 4c). We attribute this to the 290 lower multivalency of the polycation, which results in weaker complexation with the ferricyanide, 291 making it more available for the reaction with the amino thioacids. 292 From an origins of life perspective, it is interesting to explore the selectivity of aminoacylation 293 when more than one amino acid can react with an available amino thioacid inside coacervate 294 droplets. Instead of only glycine (Gly), stoichiometric (1:1) competition reactions between glycine 295 and glutamic acid (Glu), Alanine (Ala), and Phenylalanine (Phe) were investigated. All competition 296 reactions demonstrated a significant selectivity for one of the ligation products at pH 9 297 (Supplementary Table 1). For example, when we added both glycine (12 mM) and glutamic acid 298 (12 mM) to ferricyanide-based droplets (8 mM ferricyanide) containing Ac-Gly-SH (8 mM), we 299 observed a strong selectivity for peptide ligation with glycine. Glutamic acid alone yields the Ac-300 Gly-Glu-OH dipeptide in similar yields as glycine (Fig. 4d, Supplementary Fig. 11a), but when 301 308 309 glycine and glutamic acid were incubated together in a 1:1 ratio with Ac-Gly-SH in 310 (Lys(Me)3)20/ferricyanide coacervates, more than 90% of the ligation products was Ac-Gly-Gly-311 OH (Fig. 4d, Supplementary Fig. 11b). Glycine thus outcompeted glutamic acid very effectively 312 in the ligation process. We reasoned that the activity of glycine inside coacervates is higher than 313 glutamic acid. Although glycine and glutamic acid have similar nucleophilicities, 45 glutamic acid 314 is bound more strongly to the cationic lysine residues inside the coacervate and therefore has a 315 lower activity. As a result, glycine reacts faster than glutamic acid inside the coacervate 316 environment and the outcome we observe is an example of kinetic pathway selection, caused by 317 the local protocell environment. Likewise, we observed selective incorporation of glycine in 318 mixtures with alanine (83% Gly/17% Ala) and phenylalanine (78% Gly/22% Phe) (Supplementary  319   Table 1), because the latter have a slightly stronger interaction with the peptide backbone in the 320 coacervates. Alanine is incorporated with slight preference over glutamic acid (60% Ala/40% Glu), 321 but surprisingly, glutamic acid is incorporated more very slightly effectively than phenylalanine 322 (63% Glu/37% Phe). 323 The products of the above reactions with simple amino (thio)acids are small molecules that 324 interact only weakly with the coacervates. Therefore, they can quickly escape the protocell and the 325 evolutionary advantage for the protocells is limited. To establish a feedback between the formation 326 of amide bonds inside protocells and protocell fitness, we sought to retain the reaction product 327 inside the coacervates. We tested if the scaffold peptides used to form the coacervates could 328 themselves react as nucleophiles with oxidized aminothioacids. We added Ac-Phe-SH or Phe-SH 329 (4 mM) to ferricyanide (4 mM)/ pLys (12 mM) droplets, leading to ε-NH2 ligation of the protocell 330 building blocks (pLys) (Fig. 5a, Supplementary Fig. 12). Anchoring of the aromatic phenylalanine 331 to the pLys scaffold makes the coacervate interior more hydrophobic, and leads to additional 332 cation-π interactions between the coacervate components, both of which result in an expected 333 increase in salt resistance. 27 Indeed, we found that ferricyanide/pLys-g-Phe coacervates remain 334 stable far beyond the CSC of the original ferricyanide/pLys coacervates. Fig. 5b shows the turbidity 335 of the coacervates for increasing the salt concentrations. In the control sample, to which we added 336 GSH (4 mM) instead of an amino thioacid to reduce the ferricyanide, the turbidity decreases and 337 reaches background levels around 180 mM NaCl, where all droplets had disappeared completely 338 (Fig. 5c). In contrast, when Ac-Phe-SH/Phe-SH was added and reacted with the ε-NH2 of pLys, 339 the turbidity transition shifted to a significantly higher salt concentration and the absolute intensity 340 of the plateau at high salt concentration was higher (Fig. 5b). Even at high salt concentrations (220 341 mM), above the original CSC, microscope images show the presence of gel-like droplets, which 342 do not fuse (Fig. 5c)       Samples for turbidity measurements were prepared directly into 96-well plates, by adding, respectively, Milli-Q water, Tris buffer (pH 7.4, 50 mM), pLys, and ferricyanide or ferrocyanide to a total volume of 100 µL. Mixing was done by gentle pipetting (3×) before each measurement.
Samples for the microscopy experiments were prepared in microcentrifuge tubes. After addition of the substrate, a 20 µL aliquot was immediately taken for imaging on a glass slide.

Turbidity measurements
Turbidity measurements were performed in triplicate using a Berthold Tristar (2) LB 942 microplate reader. The temperature was kept constant at 25 ± 1°C. The absorbance was measured at 520 nm, where none of the mixture components absorbed significantly. The absorbance of a well filled with the same volume of water was used as a blank. Samples were shaken for 5 s before every readout. The critical point was determined by extrapolating the first-order derivative at the inflection point to zero turbidity. Note that this critical salt concentration does not take into account ions from other sources than the added NaCl, and the actual critical ionic strength may be slightly higher.

Ferricyanide and ferrocyanide partitioning
In a typical procedure, the coacervates dispersion was centrifuged in an Eppendorf tube at 3000x g until the dilute phase was transparent under microscope, the coacervate phase had sedimented to the bottom of the cell. The concentration of the ferricyanide/ferrocyanide in the dilute phase was quantitatively analyzed with Uv-vis at wavelength of 320 nm and 420 nm ( Supplementary Fig. 5).
We measured the volume of the top (dilute) solution and calculated the ferricyanide/ferrocyanide concentrations of the top solution from the standard curve, the volume and the moles of ferricyanide /ferrocyanide in coacervate phase can be calculated from the total feed, then the ferricyanide and ferrocyanide concentrations inside the coacervate phase can be obtained.

Confocal fluorescence microscopy
Optical and fluorescence microscopy images were recorded on an Olympus UIS2 microscope, equipped with a motorized stage (Prior, Optiscan II). Fluorescent images were recorded with an EMCCD camera (Andor, iXon), using illumination from a mercury lamp, an excitation filter of 482/18 nm (Semrock BrightLine) and an emission filter of 525/45 nm (Semrock BrightLine).
Samples were loaded into the wells of PLL-g-PEG-functionalized Ibidi μ-slides and closed with a lid (microscopy chambers).

Microscopy chambers preparation
The Ibidi μ-slides used for imaging were functionalized with PLL-g-PEG to minimize wetting and spreading of the coacervate droplets. Each slide was first activated by oxygen plasma treatment (Diener electronic, Femto). The PLL-g-PEG (0.01 mg/mL in 10 mM HEPES buffer, pH 7.4) solution was added into each well and incubated at room temperature for 1 h. After that, the glass slides were cleaned by rinsing (3 times with 10 mM HEPS, pH 7.4, 3 times with MQ). The slides were dried by using compressed air.

NMR measurements
Nuclear Magnetic Resonance (NMR) spectra were measured on a Bruker-AVANCE III 400 MHz spectrometer.

Mass spectrometry
Mass spectra were obtained from a Thermo Scientific TM LCQ Fleet TM ion trap mass spectrometer with Gemini-NX C18 110A 150 x 2.0 mm column and JEOL Accurate Time of Flight (ToF) instruments, both using linear ion trap electrospray ionisation (ESI).

Synthesis of trimethylated oligolysine and poly-L-lysine
Trimethylated oligolysines (Lys(Me)3)n and poly-L-lysine (pLys(Me)3) were prepared from (Lys)n and pLys, according to literature methods. 3 Briefly, 2 mL DMS was added to 50 mg (Lys)n, in 20 mL H2O and 6 mL ethanol. The pH was adjusted to 9.5 and maintained by the addition of 1 M NaOH. The reaction was considered complete when the pH remained nearly constant. The pLys(Me)3 was further purified by dialysis against 2 times 1 L of a 2 M NaCl aqueous solution and subsequently, 3 times 1 L of water in a dialysis membrane (MWCO, 3500). After that, the residue was freeze-dried to yield a white powder.