C-Terminal-Modified Oligourea Foldamers as a Result of Terminal Methyl Ester Reactions under Alkaline Conditions

Hybrids of short oligourea foldamers with residues of α, β and γ-amino acids esters at the C-terminus were obtained and subjected to a reaction with LiOH. There are two possible transformations under such conditions, one of which is ester hydrolysis and the formation of a carboxylic group and the other is the cyclization reaction after abstraction of a proton from urea by a base. We have investigated this reaction with difference C-terminal residue structures, as well as under different work-up conditions, especially for oligourea hybrids with α-amino acid esters. For these compounds, an oligourea–hydantoin combination is the product of cyclization. The stability of the hydantoin ring under alkaline conditions has been alsotested. Furthermore, this work reports data related to the structure of C-terminal-modified oligourea foldamers in solution and, for one compound, in the solid state. Helical folding is preserved both for cyclized and linear modifications, with oligourea–acid hybrids appearing to be more conformationally stable, as they are stabilized by an additional intramolecular hydrogen bond in comparison to cyclic derivatives.


Introduction
In recent years, oligourea foldamers with the general formula [NHCH(R)CH 2 NHCO] n have gained much attention as structural and functional mimetics of peptides [1][2][3][4][5][6][7][8]. It has been shown that oligoureas can mimic the helical secondary structure of peptides. Oligourea foldamers fold into 2.5 helices, stabilized by a network of 3-centered hydrogen bonds, forming 12-and 14-membered pseudorings [2,4]. The secondary structure is independent of the sequence, and even short oligomers tend to form stable spatial structures in solution and in the solid state [2]. It has been shown that water-soluble oligourea foldamers with carefully designed sequences are also able to form higher-ordered structures such as bundles or channels [9][10][11][12]. Moreover, it is possible to encapsulate guest molecules inside the bundle [13,14]. It has also been demonstrated that oligoureas can act as electron transport mediators, similar to peptides and proteins [15][16][17]. The stability of the helical conformation of these oligomers has enabled studies which have elucidated the mechanism of electron transfer to be length dependent, changing from tunneling to hopping with the length of the molecule and thereby the thickness of the monolayer [15,16].
Over the years the (oligo)urea backbone has undergone various modifications and the impact of these changes on the structural properties of the new analogs has been investigated. This has led to the conclusion that the 2.5-helical structures of oligoureas are robust and it is possible to substitute the urea residue with γ-amino acid [6,18], carbamate [18,19], thiourea [20], guanidinium [21] and also N-methylated [22] or urea residues with noncanonical substitution patterns [23] without the loss of the structural integrity of the new molecule. It has also been shown that urea fragments as short as three residues can induce a helical conformation when fused to a peptide too short to fold into a stable secondary 2 of 17 structure [24,25]. Such α-peptide/urea chimeras adopt a continuous and well-defined helical structure spanning the entire sequence stabilized by a H bond network, regardless of the position (e.g., N-terminal or C-terminal) of the urea segment on the peptide fragment [24].
The aim of the work reported here was to obtain oligourea foldamers containing carboxyl groups at the C-terminus under alkaline conditions. Similar connections of urea fragments with α-amino acid residues were reported by Liskamp and co-workers [26]. They developed a solid phase synthetic methodology allowing them to obtain the desired compounds [26]. We performed our syntheses in solution and decided to expand the types of C-terminal residues to include βand γ-amino acids. It turned out that in addition to the expected product 1, compound 2 (Figure 1), as the result of the cyclization of the last residue, was also formed. The type of the resultant product (1 or 2) depended on the structure of the C-terminal residue, as well as on the work-up conditions. In this paper we also report the influence of C-terminal modifications on the secondary structure of the obtained compounds. new molecule. It has also been shown that urea fragments as short as three resid induce a helical conformation when fused to a peptide too short to fold into a sta ondary structure [24,25]. Such α-peptide/urea chimeras adopt a continuous and fined helical structure spanning the entire sequence stabilized by a H bond netw gardless of the position (e.g., N-terminal or C-terminal) of the urea segment on the fragment [24].
The aim of the work reported here was to obtain oligourea foldamers contain boxyl groups at the C-terminus under alkaline conditions. Similar connections fragments with α-amino acid residues were reported by Liskamp and co-work They developed a solid phase synthetic methodology allowing them to obtain the compounds [26]. We performed our syntheses in solution and decided to expand t of C-terminal residues to include β-and γ-amino acids. It turned out that in add the expected product 1, compound 2 (Figure 1), as the result of the cyclization of residue, was also formed. The type of the resultant product (1 or 2) depended on th ture of the C-terminal residue, as well as on the work-up conditions. In this paper report the influence of C-terminal modifications on the secondary structure of tained compounds.

Synthesis of Oligourea Methyl Esters and Their Transformations under Alkaline Co and Stability Studies of Oligourea-Hydantoin Derivatives
Short chain oligoureas were obtained following well-established methods tion, utilizing Boc-protected activated succinimidyl carbamates [4,27]. At the C-te the methyl esters of varied amino acids in the form of succinimidyl carbamates w pled (see the Supporting Information, Figure S1) to form a urea bond. To facil coupling of different amino acids residues, oligourea synthesis was initiated by B tected 1-isobutyl-2-azidoethylamine and then carried out by sequential linkage of ual succinimidyl carbamates from the so-called C-terminus to the N-teminus to ob desired tetramer, as it has previously been reported [15]. To incorporate the Cresidues, the azide function in compound 3 (Scheme 1) was reduced to an amin with hydrogen in the presence of Pd/C, and then succinimidyl carbamate deriv methyl esters of α-, β-and γ-amino acids (BB1-BB4) were used in the coupling r Compounds of type 5 were reacted at RT with excess (10 eq.) LiOH in a 4:1 mi MeOH and H2O (Scheme 1). The reaction progress was monitored by RP-HPLC Supporting Information, Figures S49-S60). Under such conditions, two types of p were possible: a linear acid as a result of the hydrolysis reaction and/or a cyclic as a result of proton abstraction of the urea group and intramolecular 5-or 6-me ring formation (Figure 1, see also the Supporting Information, Figures S2 and S3 case, the result of the reaction strongly depended on the structure of the last Cresidue of the foldamers as well as on the work-up conditions. For derivative goureas containing α-amino acids, the disappearance of the substrate was observ 2h, and for the Gly residue at the C-terminus (5b), both products 1 and 2 cont

Synthesis of Oligourea Methyl Esters and Their Transformations under Alkaline Conditions and Stability Studies of Oligourea-Hydantoin Derivatives
Short chain oligoureas were obtained following well-established methods in solution, utilizing Boc-protected activated succinimidyl carbamates [4,27]. At the C-terminus, the methyl esters of varied amino acids in the form of succinimidyl carbamates were coupled (see the Supporting Information, Figure S1) to form a urea bond. To facilitate the coupling of different amino acids residues, oligourea synthesis was initiated by Boc-protected 1-isobutyl-2-azidoethylamine and then carried out by sequential linkage of individual succinimidyl carbamates from the so-called C-terminus to the N-teminus to obtain the desired tetramer, as it has previously been reported [15]. To incorporate the C-terminal residues, the azide function in compound 3 (Scheme 1) was reduced to an amine group with hydrogen in the presence of Pd/C, and then succinimidyl carbamate derivatives of methyl esters of α-, βand γ-amino acids (BB1-BB4) were used in the coupling reaction. Compounds of type 5 were reacted at RT with excess (10 eq.) LiOH in a 4:1 mixture of MeOH and H 2 O (Scheme 1). The reaction progress was monitored by RP-HPLC (see the Supporting Information, Figures S49-S60). Under such conditions, two types of products were possible: a linear acid as a result of the hydrolysis reaction and/or a cyclic product as a result of proton abstraction of the urea group and intramolecular 5-or 6-membered ring formation (Figure 1, see also the Supporting Information, Figures S2 and S3). In our case, the result of the reaction strongly depended on the structure of the last C-terminal residue of the foldamers as well as on the work-up conditions. For derivatives of oligoureas containing α-amino acids, the disappearance of the substrate was observed after 2h, and for the Gly residue at the C-terminus (5b), both products 1 and 2 containing a hydantoin ring [28][29][30] were formed in the ratio 3:7. For Aib, Ala, Val and Leu (5a,c,d,e) at the C-terminus, products of type 2 were formed in more than 95% yield in the crude reaction mixture. In all cases we observed the epimerization of the stereogenic center of the amino acid residue and 3 of 17 so a mixture of diastereomeric products was formed. According to the NMR spectra, the ratio of diastereomers was defined as approximately 1:1 (see the Supporting Information, Figure S47). For Ala derivative 5c, in the crude product after the work-up, the amount of acid 1c increased to 25% (see the explanation below). For foldamers with Aib, Val and Leu at the C-terminus, acid 1 was not observed after the work-up. hydantoin ring [28][29][30] were formed in the ratio 3:7. For Aib, Ala, Val and Leu (5a,c,d,e) at the C-terminus, products of type 2 were formed in more than 95% yield in the crude reaction mixture. In all cases we observed the epimerization of the stereogenic center of the amino acid residue and so a mixture of diastereomeric products was formed. According to the NMR spectra, the ratio of diastereomers was defined as approximately 1:1 (see the Supporting Information, Figure S47). For Ala derivative 5c, in the crude product after the work-up, the amount of acid 1c increased to 25% (see the explanation below). For foldamers with Aib, Val and Leu at the C-terminus, acid 1 was not observed after the work-up.
Scheme 1. Synthesis of methyl esters of oligourea foldamers and subsequent reaction under alkaline conditions.
It is known from the literature that ureido esters form hydantoin analogs under alkaline conditions, but most of the examples refer to solid phase synthetic procedures [26,31,32]. Liskamp and co-workers [26] observed that resin-bound oligourea was cleaved off the Tentagel resin as substituted hydantoin under basic conditions in the presence of KCN in MeOH. Subsequently, various hydroxides were tested by them on the model compound and it was discovered that the highest amount of hydantoin was formed with NaOH (45%), whereas TBAOH favored the acid form (>98%) of the peptidomimetic.
Next, we decided to investigate the outcome of the reaction of β-and γ-amino acid residues at the C-terminus. The linear compound 1f was formed for unbranched βhGly, whereas for β 3 hAla, a mixture (around 1:1, see the Supporting Information, Figure S56) of two products (1g and 2g) was obtained. For γ-amino acid derivatives, only compounds 1h-j were formed as products of the simple hydrolysis of methyl esters and no cyclic products were observed.
We speculate that the outcome (cyclisation versus hydrolysis) of the reaction of oligourea methyl esters of type 5 with LiOH and the dependence on the structure of the last amino acid residue may be caused, among other reasons, by steric factors. The reaction occurs with oligomers long enough to fold into 2.5 helices, as confirmed by 2D NMR Scheme 1. Synthesis of methyl esters of oligourea foldamers and subsequent reaction under alkaline conditions. It is known from the literature that ureido esters form hydantoin analogs under alkaline conditions, but most of the examples refer to solid phase synthetic procedures [26,31,32]. Liskamp and co-workers [26] observed that resin-bound oligourea was cleaved off the Tentagel resin as substituted hydantoin under basic conditions in the presence of KCN in MeOH. Subsequently, various hydroxides were tested by them on the model compound and it was discovered that the highest amount of hydantoin was formed with NaOH (45%), whereas TBAOH favored the acid form (>98%) of the peptidomimetic.
Next, we decided to investigate the outcome of the reaction of βand γ-amino acid residues at the C-terminus. The linear compound 1f was formed for unbranched βhGly, whereas for β 3 hAla, a mixture (around 1:1, see the Supporting Information, Figure S56) of two products (1g and 2g) was obtained. For γ-amino acid derivatives, only compounds 1h-j were formed as products of the simple hydrolysis of methyl esters and no cyclic products were observed.
We speculate that the outcome (cyclisation versus hydrolysis) of the reaction of oligourea methyl esters of type 5 with LiOH and the dependence on the structure of the last amino acid residue may be caused, among other reasons, by steric factors. The reaction occurs with oligomers long enough to fold into 2.5 helices, as confirmed by 2D NMR ROESY spectra [2]. All urea groups adopt a trans,trans conformation (see the Supporting Information, Figure S48), which is necessary for helical folding and formation of the intramolecular hydrogen bonding network. For terminal amino acid residues with short distances between the last urea group at the C-terminus and the methyl ester moiety, as present in α-amino acids, the attack of the OH − anion on the carbonyl carbon may be hampered because of the helix. On the other hand, cyclization is favored by the proximity of the reacting groups. For Gly derivatives, the unbranched chain seems to be flexible enough to give both products. The elongation of the terminal amino acid residues by additional methylene groups, as in βand further γ-derivatives, causes more lability of the carbon chain and extends the distance of the ester group, which facilitates alkaline hydrolysis and the formation of products of type 1.
The reaction of oligourea 5b was then studied in more detail. We have discovered that the ratio of cyclic to linear products before and after isolation strongly depended on the work-up method used. In the crude reaction mixture after 2 h, the ratio of 1b and 2b determined by HPLC was 3:7. The crude was then highly diluted with water and DCM and after extraction, the compound 2b was obtained from the organic phase, whereas 1b was isolated from the aqueous phase after acidification. On the other hand, when the crude reaction mixture was evaporated to dryness and then re-dissolved in a mixture of DCM and water, acidified with 1M HCl and extracted, the only isolated product was compound 1b (Figure 2a). It was evident that during evaporation, the hydantoin ring in 2b underwent hydrolysis and oligourea 1b with a glycine residue at the C-terminus was formed [29,33,34]. Interestingly, the foldamer with a cyclic residue at the C-terminus is slightly more polar than the foldamer with a carboxylic moiety (Figure 2a). ROESY spectra [2]. All urea groups adopt a trans,trans conformation (see the Supporting Information, Figure S48), which is necessary for helical folding and formation of the intramolecular hydrogen bonding network. For terminal amino acid residues with short distances between the last urea group at the C-terminus and the methyl ester moiety, as present in α-amino acids, the attack of the OHanion on the carbonyl carbon may be hampered because of the helix. On the other hand, cyclization is favored by the proximity of the reacting groups. For Gly derivatives, the unbranched chain seems to be flexible enough to give both products. The elongation of the terminal amino acid residues by additional methylene groups, as in β-and further γ-derivatives, causes more lability of the carbon chain and extends the distance of the ester group, which facilitates alkaline hydrolysis and the formation of products of type 1.
The reaction of oligourea 5b was then studied in more detail. We have discovered that the ratio of cyclic to linear products before and after isolation strongly depended on the work-up method used. In the crude reaction mixture after 2 h, the ratio of 1b and 2b determined by HPLC was 3:7. The crude was then highly diluted with water and DCM and after extraction, the compound 2b was obtained from the organic phase, whereas 1b was isolated from the aqueous phase after acidification. On the other hand, when the crude reaction mixture was evaporated to dryness and then re-dissolved in a mixture of DCM and water, acidified with 1M HCl and extracted, the only isolated product was compound 1b (Figure 2a). It was evident that during evaporation, the hydantoin ring in 2b underwent hydrolysis and oligourea 1b with a glycine residue at the C-terminus was formed [29,33,34]. Interestingly, the foldamer with a cyclic residue at the C-terminus is slightly more polar than the foldamer with a carboxylic moiety ( Figure 2a). To investigate the influence of the temperature on the conversion of the hydantoin residue into a carboxylic acid moiety, we took compound 2b and performed the reaction at 45 °C ( Figure 2b). The concentration of compound 2b was twice as high, but the excess of LiOH relative to the compound was the same. The reaction was stirred in a 1:1 MeOH:H2O mixture, i.e., with a higher amount of water. This was to mimic the conditions during the evaporation and reduction of organic solvent in favor of water. By HPLC, we To investigate the influence of the temperature on the conversion of the hydantoin residue into a carboxylic acid moiety, we took compound 2b and performed the reaction at 45 • C (Figure 2b). The concentration of compound 2b was twice as high, but the excess of LiOH relative to the compound was the same. The reaction was stirred in a 1:1 MeOH:H 2 O mixture, i.e., with a higher amount of water. This was to mimic the conditions during the evaporation and reduction of organic solvent in favor of water. By HPLC, we observed a gradual disappearance of hydantion derivative 2b and its conversion into oligourea acid 1b (Figure 2b). After 4 h of stirring at 45 • C, the conversion was complete. The reaction using the same concentration and mixture of solvents was also performed at RT (23 • C). After 2 h of stirring, compound 2b still dominated (64% by HPLC) and even after 24 h it was still observed in the reaction mixture (5%, see the Supporting Information, Figure S95a).
To test the influence of the substituent at position 5 of the hydantoin ring and its size, we performed the same reaction on diastereomers of 2e (see the Supporting Information, Figure S95b,c). After 2 h at 45 • C, we observed the formation of only 9% of the acidic form, whereas at RT, the hydantoin ring was stable and did not undergo hydrolysis. These observations confirmed that the isolated products after the reaction with LiOH strongly depended on the work-up conditions for oligoureas with unsubstituted residues (such as Gly) or those with small side chains (such as Ala).

Structural Studies of Hydantoin and Carboxylic Acid Derivatives of Oligoureas
The structural consequences of the presence of a hydantoin ring or carboxylic group at the C-terminus of short oligourea chains were investigated in solution by NMR and ECD. Additionally, for compound 2a, we obtained a monocrystal suitable for X-ray analysis.
NMR spectra of all compounds were recorded at a concentration of ca. 3 mM in 10% DMSO-d 6 in CD 3 CN. Proton resonances were assigned by using a combination of COSY, TOCSY, ROESY and heteronuclear HSQC 2D experiments (see the Supporting Information, Tables S1 and S2). To compare the propensity of folding into a 2.5 helix for C-terminal-modified oligoureas, compounds 1f and 2a were chosen.
As shown in Figure 3b, the chemical shifts of urea protons are visible between 5.2 and 6.3 ppm for 1f, whereas for 2a, the highest chemical shift is less than 6.0 ppm. Only slight changes in chemical shifts (0.03-0.09 ppm) were observed for urea protons at the N-terminus not involved in intramolecular hydrogen bonds. The highest downfield shifts (0.16-0.44 ppm) for oligourea acids in comparison to oligourea hydantoins were observed for the middle urea group N'H(Ala2u) and NH(Ala3u) (marked in green in Figure 3b, (u) means urea residue). This is because the urea moiety in oligourea acid is involved in helix stabilization both as a donor and acceptor of intramolecular hydrogen bonds (Figure 3a). Based on ROESY spectra, it was confirmed that all urea groups of the oligourea hydantoin and oligourea acid are in trans,trans conformations (see the Supporting Information, Figure S48), known to be necessary for helical folding of the backbone. Additionally, strong ROE connectivity between CH 3 (tBu) and both N'H(Ala2u) and NH(Ala3u) was observed, further supporting the formation of a 2.5 helix [22]. observed a gradual disappearance of hydantion derivative 2b and its conversion into oligourea acid 1b (Figure 2b). After 4 h of stirring at 45 °C, the conversion was complete. The reaction using the same concentration and mixture of solvents was also performed at RT (23 °C). After 2 h of stirring, compound 2b still dominated (64% by HPLC) and even after 24 h it was still observed in the reaction mixture (5%, see the Supporting Information, Figure S95a). To test the influence of the substituent at position 5 of the hydantoin ring and its size, we performed the same reaction on diastereomers of 2e (see the Supporting Information, Figure S95b,c). After 2 h at 45 °C, we observed the formation of only 9% of the acidic form, whereas at RT, the hydantoin ring was stable and did not undergo hydrolysis. These observations confirmed that the isolated products after the reaction with LiOH strongly depended on the work-up conditions for oligoureas with unsubstituted residues (such as Gly) or those with small side chains (such as Ala).

Structural Studies of Hydantoin and Carboxylic Acid Derivatives of Oligoureas
The structural consequences of the presence of a hydantoin ring or carboxylic group at the C-terminus of short oligourea chains were investigated in solution by NMR and ECD. Additionally, for compound 2a, we obtained a monocrystal suitable for X-ray analysis.
NMR spectra of all compounds were recorded at a concentration of ca. 3 mM in 10% DMSO-d6 in CD3CN. Proton resonances were assigned by using a combination of COSY, TOCSY, ROESY and heteronuclear HSQC 2D experiments (see the Supporting Information, Tables S1 and S2). To compare the propensity of folding into a 2.5 helix for Cterminal-modified oligoureas, compounds 1f and 2a were chosen.
As shown in Figure 3b, the chemical shifts of urea protons are visible between 5.2 and 6.3 ppm for 1f, whereas for 2a, the highest chemical shift is less than 6.0 ppm. Only slight changes in chemical shifts (0.03-0.09 ppm) were observed for urea protons at the Nterminus not involved in intramolecular hydrogen bonds. The highest downfield shifts (0.16-0.44 ppm) for oligourea acids in comparison to oligourea hydantoins were observed for the middle urea group N'H(Ala2u) and NH(Ala3u) (marked in green in Figure 3b, (u) means urea residue). This is because the urea moiety in oligourea acid is involved in helix stabilization both as a donor and acceptor of intramolecular hydrogen bonds (Figure 3a). Based on ROESY spectra, it was confirmed that all urea groups of the oligourea hydantoin and oligourea acid are in trans,trans conformations (see the Supporting Information, Figure S48), known to be necessary for helical folding of the backbone. Additionally, strong ROE connectivity between CH3(tBu) and both N'H(Ala2u) and NH(Ala3u) was observed, further supporting the formation of a 2.5 helix [22]. It is well known that the differences in the chemical shifts (∆δ) of CH 2 protons of the main chain are indicative of 2.5 helix formation [1-3]. We compared ∆δ values for C-terminal-modified oligoureas and discovered that for oligourea hydantoin 2a, the diastereotopicity values were noticeably lower than for oligourea acid 1f (Table 1). This is expected to be due to the difference in the number of possible intramolecular hydrogen bonds (two for hydantoin derivatives and three for acid derivatives) and it is in agreement with the literature reports on similar length compounds [2]. To further investigate the folding propensity of oligoureas 2a and 1f in solution, ECD spectra were measured (Figure 4, see also Supporting Information, Figure S94) at a concentration of 0.2 mM in 2,2,2-trifluoroethanol (TFE). Both types of oligourea derivatives exhibited the typical CD signature of oligoureas [2], with positive bands at λ = 200 and 201 nm for hydantoin and acid derivatives, respectively. Interestingly, the intensity of the band was significantly lower for 2a than for 1f, strongly suggesting that the secondary structure of oligourea hydantoins is less stabilized and therefore less stable in solution than the oligourea acids. It is well known that the differences in the chemical shifts (Δδ) of CH2 protons of the main chain are indicative of 2.5 helix formation [1-3]. We compared Δδ values for C-terminal-modified oligoureas and discovered that for oligourea hydantoin 2a, the diastereotopicity values were noticeably lower than for oligourea acid 1f (Table 1). This is expected to be due to the difference in the number of possible intramolecular hydrogen bonds (two for hydantoin derivatives and three for acid derivatives) and it is in agreement with the literature reports on similar length compounds [2]. To further investigate the folding propensity of oligoureas 2a and 1f in solution, ECD spectra were measured (Figure 4, see also Supporting Information, Figure S94) at a concentration of 0.2 mM in 2,2,2-trifluoroethanol (TFE). Both types of oligourea derivatives exhibited the typical CD signature of oligoureas [2], with positive bands at λ = 200 and 201 nm for hydantoin and acid derivatives, respectively. Interestingly, the intensity of the band was significantly lower for 2a than for 1f, strongly suggesting that the secondary structure of oligourea hydantoins is less stabilized and therefore less stable in solution than the oligourea acids. For compound 2a, we were able to obtain a crystal structure, providing definite confirmation of the secondary structure of compound 2a in the solid state. The crystal structure was solved in the P21 space group. There are four independent molecules (A-D) and 2.5 water molecules present in the asymmetric unit ( Figure 5, see the Supporting Information, Table S3 and Figures S96-S99). For compound 2a, we were able to obtain a crystal structure, providing definite confirmation of the secondary structure of compound 2a in the solid state. The crystal structure was solved in the P2 1 space group. There are four independent molecules (A-D) and 2.5 water molecules present in the asymmetric unit ( Figure 5, see the Supporting Information, Table S3 and Figures S96-S99).
The oligourea fragment is seen to be stabilized by two intramolecular hydrogen bonds,  Table S4), typical values for canonical oligourea 2.5 helices [19]. Molecules A and B, as well as C and D, are hydrogen bonded by head to tail intermolecular interactions (Figure 5b). These intermolecular hydrogen bonds are formed between O (Ala3u) and NH (tBu)/NH (Leu1u) and also O (Leu4u) and N'H (Leu1u). The intermolecular O-N distances for molecules A and B are around 2.9 Å, whereas for molecules C and D, this distance is slightly longer at around 3 Å. Additionally, the hydantoin ring is involved in an intermolecular hydrogen bonding network between A and B as well as C and D molecules, and CO (4) (for numeration of the hydantoin ring, see Scheme 1) is bonded to NH (Ala2u). Furthermore, NH (1) of the hydantoin ring in A and B is linked to O (Leu4u) of the C and D molecules. The same hydrogen in C and D is bonded to the oxygen of water molecules. All O-N distances described above are around 3 Å. Thanks to the network of intermolecular interactions, the crystal lattice of molecules A and D as well as B and C form superhelices (Figure 5c).  Table S4), typical values for canonical oligourea 2.5 helices [19]. Molecules A and B, as well as C and D, are hydrogen bonded by head to tail intermolecular interactions (Figure 5b). These intermolecular hydrogen bonds are formed between O (Ala3u) and NH (tBu)/NH (Leu1u) and also O (Leu4u) and N'H (Leu1u). The intermolecular O-N distances for molecules A and B are around 2.9 Å, whereas for molecules C and D, this distance is slightly longer at around 3 Å. Additionally, the hydantoin ring is involved in an intermolecular hydrogen bonding network between A and B as well as C and D molecules, and CO (4) (for numeration of the hydantoin ring, see Scheme 1) is bonded to NH (Ala2u). Furthermore, NH (1) of the hydantoin ring in A and B is linked to O (Leu4u) of the C and D molecules. The same hydrogen in C and D is bonded to the oxygen of water molecules. All O-N distances described above are around 3 Å. Thanks to the network of intermolecular interactions, the crystal lattice of molecules A and D as well as B and C form superhelices (Figure 5c).
The results obtained from the crystal structure, together with the solution studies, reveal that the hydantoin ring is somewhat labile and may adopt a number of different orientations relative to the folded part of the molecule.

General Consideration
Solvents and reagents are commercially available. 1 H and 13 C NMR spectra were recorded on a Bruker Avance 300 or 500 MHz for 1 H and 75 MHz for 13 C. Two-dimensional NMR spectra (COSY, TOCSY, HSQC and ROESY) were recorded on a Bruker Avance III HD 500 MHz. The chemical shifts were reported in ppm (δ) and coupling constants (J) were given in Hz. To indicate multiplicity, the abbreviations singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), doublet of doublets (dd) and multiplet (m) were used. Splitting patterns which were not easy to interpret were labelled as multiplets (m). The The results obtained from the crystal structure, together with the solution studies, reveal that the hydantoin ring is somewhat labile and may adopt a number of different orientations relative to the folded part of the molecule.

General Consideration
Solvents and reagents are commercially available. 1 H and 13 C NMR spectra were recorded on a Bruker Avance 300 or 500 MHz for 1 H and 75 MHz for 13 C. Two-dimensional NMR spectra (COSY, TOCSY, HSQC and ROESY) were recorded on a Bruker Avance III HD 500 MHz. The chemical shifts were reported in ppm (δ) and coupling constants (J) were given in Hz. To indicate multiplicity, the abbreviations singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), doublet of doublets (dd) and multiplet (m) were used. Splitting patterns which were not easy to interpret were labelled as multiplets (m). The HRMS data were obtained on a QExactive mass spectrometer with electrospray ionization. Column chromatography was carried out on silica gel (70-230 mesh). Analytical RP-HPLC analyses were performed using a Jupiter 4u Proteo 90Å column (4.6 × 250 mm) at a flow rate of 1 mL × min −1 . The mobile phase was composed of 0.1% (v/v) TFA/H 2 O (phase A) and 0.1% (v/v) TFA−CH 3 CN (phase B). The detection was performed at 200 nm and the method used was 10% of B for 5 min and a 10-97% gradient of B in 30 min. Activated (S)-succinimidyl-{2-{[(tert-butoxy)carbonyl]amino}-2-X-ethyl}carbamate monomers for the introduction of urea bonds were prepared from corresponding N-Boc-protected ethylene diamine derivatives using recently reported procedures [27,35]. Tetramer 3 was synthesized as previously described [15]. Known compounds synthesized by literature methods were confirmed by comparing reported characterization data.

General Procedure for the Synthesis of Building Blocks BB1-BB4
All methyl ester carbamate building blocks (BB1, BB2b, BB2c, BB2d, BB2e, BB3f, BB3g and BB4h) were obtained from appropriate amino acids using the same two step procedure.
Step I: Thionylchloride (2 eq.) was slowly added to a cooled (−78 • C) suspension of amino acid (1 eq.) in CH 3 OH (20 mL/g) under an inert atmosphere. Then, the reaction mixture was warmed up to ambient temperature and stirred at this temperature overnight. Following this, the solvent was removed under reduced pressure and the solid residue was filtered and washed with diethyl ether. The product was taken to the next step without further purification.
Step II: The product (1 eq.,) from the previous step was dissolved in dry DCM (20 mL/g) at 0 • C, and DIPEA (3 eq.) was added. After stirring for 10 min, the solution was added dropwise into a flask containing solid N,N -disuccinimidyl carbonate (DSC, 1.2 eq.). The reaction was stirred for 3 h under Ar at RT. Following this, the reaction mixture was washed with 1M KHSO 4 (3×) and brine (1×). In some cases, the products remained in the aqueous layer, so this layer was extracted with DCM until all the desired compound was in the organic layer. Combined organic layers were dried over Na 2 SO 4 . After evaporation under reduced pressure, the crude product was obtained as a colorless/yellowish oil. It was purified by dissolving it in small amount of DCM, followed by precipitation with diethyl ether and/or petroleum ether. The pure product was obtained as a white solid (except BB4h, which was a yellowish oil used without further purification). Copies of 1 H and 13 C NMR spectra, as well as HRMS spectra of building blocks are given in the Supporting Information, Figures S4-S23  Both methyl ester carbamate building blocks, BB4i and BB4j, were obtained starting from Boc-protected γ-amino acids. Boc deprotection procedure: protected γ-AA was treated with TFA (6 mL/g) and stirred at 0 • C for 1 h under Ar. When the deprotection reaction was complete, TFA was removed and the residue was co-evaporated with cyclohexane (3×) and diethyl ether (1×) and then reacted with thionyl chloride.

Synthesis of Amine 4
To a solution of compound 3 (100 mg, 0.17 mmol) in CH 3 OH (15 mL), palladium on charcoal (10% w/w, 10 mg) in 2 mL of CH 3 OH was added. The reaction mixture was stirred under a 1 bar H 2 atmosphere (balloon) for 5h. Then, it was filtered through Celite, concentrated under vacuo and co-evaporated with diethyl ether (2×). Product 4 was used without further purification. RP-HPLC: t R = 24.48 min.

Synthesis of Oligourea Esters 5
Amine 4 (1 eq.) was dissolved in CH 3 CN (40 mL/g) and DMF (10 mL/g) under Ar, and DIPEA (3 eq.) was added at basic pH. The mixture was left for 15 min, followed by a dropwise addition of appropriate building block BB1-4 (1.2 eq.) in CH 3 CN solution (7 mL/g). The reaction was left overnight at ambient temperature. When the reaction was complete, solvents were removed under vacuum and co-evaporated with toluene (3×) to remove DMF. The residue was redissolved in EtOAc, washed with 1M KHSO 4 (4×), saturated NaHCO 3 (1×), brine (1×) and water (1×) and then evaporated. The crude product was purified by silica gel chromatography (CH 2 Cl 2 :CH 3 OH). The pure product was obtained as a white solid. Copies of 1 H NMR spectra, as well as HRMS spectra of oligourea esters are given in the Supporting Information, Figures S24-S33

Hydrolysis of Oligourea Ester 5
The corresponding ester 5 (1 eq.) was dissolved in MeOH (7 mM). LiOH × H 2 O (10 eq) was dissolved in water (the ratio of MeOH to H 2 O was 4:1) and added to the ester solution. The reaction mixture was stirred for 2 h at RT. The progress of the reaction was monitored by HPLC (for 5g, the reaction mixture was stirred overnight). The work-up conditions depended on the obtained product/products. To isolate oligourea acid 1, the solvents were completely and slowly removed under vacuum (water bath at 45 • C). The residue was redissolved in CH 2 Cl 2 and distilled water. The water layer was extracted with CH 2 Cl 2 (2×) with the addition of brine because of the formation of an emulsion. The organic layers were removed. HCl (1M) was added to water layer until an acidic pH and it was extracted with CH 2 Cl 2 until all the product was completely extracted into the organic layer. The solvent was evaporated, and the crude product was dissolved in CH 3 CN, quickly passed through a syringe filter (PA 0.2µm) and lyophilized from CH 3 CN with addition of water. The product of type 1 was obtained as a white solid. To isolate oligourea hydantoin/dihydrouracil 2, the reaction mixture was diluted with CH 2 Cl 2 and distilled water. The layers were separated, and the water layer was extracted with CH 2 Cl 2 with the addition of brine (emulsion formation) until all the cyclic product was completely extracted into the organic layer. The solvent was evaporated, and the crude product was dissolved in CH 3 CN, quickly passed through a syringe filter (PA 0.2µm) and lyophilized from CH 3 CN with the addition of water. The product of type 2 was obtained as a white solid. Copies of 1 H NMR spectra, as well as HRMS spectra of oligourea acids and oligourea hydantoins/dihydrouracils are given in the Supporting Information, Figures S34-S46   2b or 2e. The samples were incubated (with mixing) at 45 • C or RT (set as 23 • C) in the thermomixer (Eppendorf). At specific time intervals (30 min, 1 h, 2 h, 4 h or 24 h), aliquots were collected and the samples for RP-HPLC analyses were prepared as follows: 25 µL of the sample was added to 150µL of MeOH. It was checked by HPLC that the reaction at such a dilution was slow enough not to proceed before injection.

Circular Dichroism
Circular dichroism (CD) experiments were performed on a Jasco J-1500 spectrometer. Data are expressed in terms of the total molar ellipticity (deg·cm 2 ·dmol −1 ). CD spectra of oligomers (0.2 mM) were acquired in 2,2,2-trifluoroethanol between 190 and 250 nm using a rectangular quartz cell with a path length of 1 mm.

Crystallographic Data
See the supporting information for details (Table S3). CCDC-2237296 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif (accessed on 20 January 2023).

Conclusions
Herein, we have investigated the reaction of oligoureas with methyl esters at the Cterminus under basic conditions. Under such conditions, two possible transformations can occur: the hydrolysis of methyl ester with the formation of an oligourea acid or cyclization of the final residue and the formation of 5-or 6-membered rings at the C-terminus of the oligomer. We discovered that the result of the reaction is strongly dependent on the structure of the terminal residue. Oligoureas modified with αand β-amino acid esters form two types of products, whereas γ-amino acid esters undergo only a hydrolysis reaction. Moreover, for Gly and Ala derivatives, the yield of isolated products depends on the work-up conditions, influenced strongly by the chemical stability of the hydantoin ring.
Conformational studies of short acid and hydantoin oligoureas in solution as well as in the solid state revealed that both types of compound fold into 2.5 helices. The stability of the secondary structure of the acid derivatives was observed to be high, as confirmed by NMR and ECD experiments. The hydantoin ring seems to be noticeably labile and is not involved in the intramolecular hydrogen bonding network. Even in the crystal structure, it occupies two positions, either folded "inside" the helix or flipped outside of the helix.
The C-terminal-modified oligoureas described in this work expand the family of synthetic oligomers able to fold into stable structures. Moreover, these compounds may find application in molecular recognition and in the formation of supramolecular polymers or more complicated architectures [36,37], as well as in the field of biochemistry, as many hydantoin derivatives show biological activity [28,29]. The hydantoin foldamer hybrids reported here seem to be promising candidates for further studies, as they contain a helical portion which, when properly designed, may be able to mimic biologically active peptides, with the modifiable hydantoin moiety providing a further avenue to enable the design of molecules with biological activities.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article and its Supplementary Information.