Improved Stability and Tunable Functionalization of Parallel β‐Sheets via Multicomponent N‐Alkylation of the Turn Moiety

Abstract In contrast to the myriad of methods available to produce α‐helices and antiparallel β‐sheets in synthetic peptides, just a few are known for the construction of stable, non‐cyclic parallel β‐sheets. Herein, we report an efficient on‐resin approach for the assembly of parallel β‐sheet peptides in which the N‐alkylated turn moiety enhances the stability and gives access to a variety of functionalizations without modifying the parallel strands. The key synthetic step of this strategy is the multicomponent construction of an N‐alkylated turn using the Ugi reaction on varied isocyano‐resins. This four‐component process assembles the orthogonally protected turn fragment and incorporates handles serving for labeling/conjugation purposes or for reducing peptide aggregation. NMR and circular dichroism analyses confirm the better‐structured and more stable parallel β‐sheets in the N‐alkylated peptides compared to the non‐functionalized variants.

Many advances in modern peptide/protein science rest on the ability of synthetic chemists to ligate peptides,f unctionalize their side chains in ac hemoselective manner and construct peptidomimetics capable of reproducing regular protein secondary structures in water. Whereas av ariety of methods are available for producing water-stable helical peptides [1] and antiparallel b-sheets, [2] the synthesis of parallel b-sheets that are stable in water is an otable endeavor. [3] Understanding the stability and biological behavior of this subclass of secondary structure is crucial for modern science, but model parallel b-sheets are more difficult to produce by synthetic means than the antiparallel variants,asthey cannot be fully composed of a-amino acids. [2,3] Problems associated with the inability to fold and the tendencyt oa ggregate also make their study in water very challenging. [3][4][5][6] In this regard, many efforts have been devoted to producing non-and pseudo-peptidic turns capable of properly aligning two parallel strands and promoting b-sheet nucleation. [3][4][5][6] However,j ust af ew non-cyclic peptides are known to autonomously fold into ap arallel b-sheet in aqueous solution. [3,5b, 7] Besides the early contributions from Nowick [4] and Kelly, [5] Gellmans [3,7] group has provided the major advances by developing an effective pseudo-peptidic turn. [7a,b] This group has also assessed the effect of strand length [7c] and number [7d] on the stability of non-cyclic parallel b-sheets in water.
As shown in Scheme 1, Gellmansa pproach [7] comprises the utilization of acentral turn moiety composed of d-Pro and 1,2-diamino-1,1-dimethylethane( DADME). Different variants of parallel b-sheets have been produced using this turn, [8] including examples that rely on backbone cyclization [8a] and interstrand disulfide cross-linking [8b] to impose further macrocyclic constraints and improve stability.Considering the turninducing capacity of amide N-alkylation in short peptides, [9] we hypothesized that the combination of d-Pro with an Nalkylated DADME moiety could serve as an effective template for assembling very stable parallel b-sheets without the need of cyclization constraints.The rationale for this is the presence of an additional tertiary amide with ab ulky Nalkylation at the turn fragment, which should bend the Scheme 1. Comparison of state-of-the-art with the novel multicomponent reaction (MCR) approach towards stable and functionalized parallel b-sheets.
peptide chain more easily and enforce the interstrand interactions to ag reater degree.
Herein, we introduce amulticomponent reaction (MCR) approach for the synthesis of water-stable and functionalized parallel b-sheets.O ur strategy stands over key structural evidence previously validated by Gellmansg roup, [7] but it provides as eries of innovations in terms of synthetic efficiency,v ersatility,a nd b-sheet stability,t hat make it very relevant for the peptide/protein community.A sd epicted in Scheme 1, we devised an approach relying on the Ugimulticomponent reaction [10] to assemble the d-Pro-DADME turn moiety and simultaneously link this N-alkylated fragment to the resin. In contrast to previous stepwise protocols, comprising the initial solution-phase coupling of the d-Pro-DADME fragment to as hort peptide followed by anchoring to the resin by aside chain, [7] our strategy is very convergent and fully conducted on solid phase.I nt his sense,e ach Ugicomponent has ad ifferent purpose,i .e., while Fmoc-d-Pro and Alloc-DADME serve as carboxylic acid and amine components,r espectively,t he isocyanidee nables the attachment to the resin and introduces the functional linker.T he choice of the carbonyl component is also relevant since the use of acleavable aldehyde allows access to non-alkylated bsheets.
Previously,M CRs have been used to produce conformationally constrained macrocyclic peptides [11] featuring stable protein secondary structures,such as a-helices [12] and reverse turns. [13] However,t his chemistry has never been employed for the construction of non-cyclic b-sheets.I nc ontrast to the clear conformational bias imposed by am acrocyclization, here we aim at addressing the effect of the Ugi-derived Nalkylation of amodel turn moiety on the stability of non-cyclic parallel b-sheets.E vidence for the effectiveness of d-Pro-l-Pro as at emplate of antiparallel b-sheets suggests that two consecutive tertiary amides at the central turn fragment should confer ap reorganization for folding antiparallel bhairpin conformations. [2,3] We wondered whether the turn connecting two parallel strands might follow the same tendency and benefit from an additional N-alkylation of the artificial DADME residue.P revious reports of our group on the turn-forming propensity of Ugi-derived N-alkylated peptide fragments shed light on this possibility. [14] By focusing on an MCR approach, we also seek to provide aflexible protocol for producing parallel b-sheets with varied types of functionalization at the turn moiety.A sb-sheet peptides are endowed with low solubility and tendency to aggregate,weenvisioned that an additional functionalization at ap osition not affecting the parallel strands would be beneficial not only for the solubility but also for late-stage derivatization purposes (for example,l abeling). As depicted in Scheme 2, ak ey advantage of this approach is that the complete sequence can be carried out on resin, which needs to be initially functionalized with an isocyanide group. [15] Four different commercially available amino resins,i .e., Rink-MBHA, Gly-Wang, ethylenediamine-trityl, and 2-aminoethyl-polystyrene,w ere initially modified by af ormylation/ Scheme 2. A) Solid-phase synthesis of parallel b-sheets via multicomponent assembly and resin anchoring of the N-alkylated turn-inducing moiety.B )Circular dichroism spectra of the peptides evaluating their parallel b-sheet content. AcB:acetate buffer,DIC:d iisopropylcarbodiimide, SPPS:solid-phasepeptide synthesis.

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Communications 260 www.angewandte.org dehydration protocol, thus setting dissimilar cleavable sites before assembling the two parallel strands.I nfrared analysis unequivocally proved the formation of the isocyanide group, according to the band around 2100 cm À1 .For the Ugi reaction to be carried out on resin, preformation of the imine is achieved in solution by stirring the Alloc mono-protected diamine and the aldehyde component in DCM/MeOH 1:1f or 1h.T he mixture is added together with Fmoc-d-Pro-OH (both in four-fold excess) to the isocyano-resin and the reaction is shaken for maximum 1day at room temperature. Thes uccess of the Ugi reactions was assessed by RP-HPLC/ ESI-MS analysis after acidic mini-cleavage and by quantitative cleavage of the Fmoc group and determination of the absorption at 301 nm (see the Supporting Information). This latter analysis also enabled the calculation of the loading of the different peptide-functionalized resins 1-4,w hich is crucial for the subsequent solid-phase peptide synthesis (SPPS). All four types of amino-alkyl-modified resin proved suitable to be converted into isonitriles and to perform the subsequent Ugi reactions,t hus providing optimum flexibility with respect to solid support and cleavage sensitivity.
Three degrees of orthogonality were employed during the assembly of the two peptide strands using the Fmoc/tBu methodology.Initial cleavage of the Fmoc group enabled the growth of the upper strand, keeping the Alloc-protected residue unaffected. Removal of this latter protecting group and assembly of the lower strand completed the synthesis of peptides 5-8 in good overall yield and purity of the crude products (Scheme 1). Thea mino acid sequence chosen for peptides 5-8 follows some previously established criteria. [7,8,16] These are,a )having an intrinsic propensity for bsheet formation, b) choosing positions to enable favorable (hydrophobic or electrostatic) interactions between paired residues in each strand, and c) adding some basic amino acids to generate positive charges and therefore reduce aggregation.
As shown in Scheme 2, parallel b-sheets bearing acarboxamide (5a,b), ac arboxylic acid (6a,b), and an amine (7a,b) were produced, either with or withou the geminal dimethyl groups in the diamine residue.T hus,weaimed at finding out whether the turn-inducing capacity of the N-alkylated peptidomimetic fragment is sufficient to fold the peptides into parallel b-sheets without the positive influence of the Thorpe-Ingold effect provided by the geminal methyl substituents. Thev ersatility of the MCR method also enabled the facile synthesis of the non-N-alkylated b-sheet, peptide 8,using the acid-labile 2,4-dimethoxyphenyl substituent [17] (arising from the aldehyde component), which allows the cleavage at that specific position.
Scheme 2B (upper panel) shows ac omparison of the circular dichroism (CD) spectra in acetate buffer (AcB) of the N-alkylated peptides 5a, 6a,and 7a with the non-N-alkylated one (8), all based on the DADME residue.T he increment in the intensity of the maximum at 198 nm and the minimum at 215 nm, in peptides 5a, 6a,a nd 7a suggests that the Nalkylation of the turn-inducing moiety leads to ah igher bsheet population. It is plausible to accept ap ositive effect of the additional amide N-alkylation on the parallel b-sheetfolding propensity;s imilar to its role in antiparallel b-sheets (b-hairpins) when a l-Pro is added in the turn following the key d-Pro residue. [2] It is noteworthy that there are no significant differences between the N-alkylated b-sheet peptides 5a, 6a,a nd 7a, bearing either neutral or charged moieties as part of the turn N-modification. This indicates that the N-alkylation, and not the nature of the functionality, plays ap ositive effect on the folding.O nt he other hand, comparison of the CD spectra (Scheme 2B,l ower panel) of the N-alkylated peptides 5b, 6b,a nd 7b without the additional geminal methyl groups to reference peptide 8 reveals loss of b-sheet content. Indeed, the conformational preorganization imposed by the geminal methyl groups of the d-Pro-DADME turn is crucial for b-sheet folding in aqueous solution, regardless of the presence of an N-alkylation.
To our knowledge,t here are no methods available for labeling or ligating a b-sheet peptide without affecting the strand termini or side chains,w hich are known to be crucial for the b-sheet stability.I nt his regard, the capability of the MCR approach to easily functionalize ap arallel b-sheet without modifying the strands shows great prospect in the pursuit of biological applications.T oe xemplify this possibility,t he commercially available cysteamine-trityl resin was converted into the isocyano-resin and subjected to the Ugi reaction to assemble the resin-linked d-Pro-DADME fragment 9.A ss hown in Scheme 3, subsequent assembly of the two peptide strands followed by mild acidic cleavage from the resin led to the thiol-functionalized parallel b-sheet 10 in good yield and moderate purity.P urified peptide 10 was readily conjugated to the maleimide-functionalized dansyl tag 11 in presence of tris(2-carboxyethyl)phosphine (TCEP) to furnish the fluorescently labeled parallel b-sheet 12 in excellent yield. TheC Ds pectra of both peptides 10 and 12 show the characteristic b-sheet pattern described above, confirming that the type of functionality attached to the turn N-alkylation does not significantly affect the b-sheet content in water.
Further to the CD evidence for ahigher b-sheet content in Ugi-peptides,wesought confirmation by NMR spectroscopy. Initially,t he NMR analysis was employed to assess the tendency of the peptides to aggregate and whether the Nalkylation functionality has an influence on it. This was accomplished following am ethodology used by Gellman to evaluate this phenomenon in model parallel b-sheets. [7,8] Thus, the NMR spectra of all peptides bearing the d-Pro-DADME turn fragment were acquired at 2.5 mm concentration in 9:1 H 2 O/D 2 Oc ontaining 100 mm of sodium acetate buffer (pH 3.8). Peptide 7a,w hich bears an additional positive charge,s hows the sharpest and most defined signal lines in the 1 HNMR spectrum, with no change in the chemical shift and remaining stable in solution (i.e., without precipitation) even after aging for 6months.T he superposition of three mono-dimensional 1 HNMR spectra of peptide 7a at concentrations 0.5 mm,1mm, and 2.5 mm,showed no changes in the in the chemical shifts of the amide hydrogens,i ndicating absence of self-association (see the Supporting Information). In contrast, the 1 HNMR spectrum of peptide 6a,w ith ac arboxylic acid at the N-alkylation moiety,s hows broader signal lines and signs of precipitation after aging. Interestingly,p eptides 5a and 8 exhibit signal lines broader than 7a, but no sign of aggregation after aging in solution for 6months.
Chemical shift deviation (CSD) of the amino acid aprotons of aw ell-structured peptide with respect to the random coil state,i .e., Dd CaH = d CaH (observed)Àd CaH (random coil) is ap owerful tool in secondary structure determination. It is known that having aset of three or more residues with d aH shifted downfield by ! 0.1 ppm can be taken as evidence of b-sheet conformation. [18] Accordingly,w e focused on assessing the influence of the N-alkylation in the b-sheet content by comparing the CSD of peptides 7a and 8, using the random coil data from the original report by Richards group. [18] As shown in Figure 1A,t he Dd CaH data confirm that both peptides occur as parallel b-sheets in aqueous buffer, with most of the residues having a Dd CaH ! 0.1 ppm. Importantly,t he higher Ddc aH values for the Nalkylated peptide 7a corroborate the evidence provided by the CD spectra, that this peptide has ab etter structured bsheet conformation than the non-N-alkylated one.
To determine the NMR structure of peptide 7ain aqueous solution, the full resonance assignment was accomplished to look for the presence of NOEs between nonadjacent residues. Such interstrand NOEs are decisive to corroborate the bsheet conformation and to construct the NOE-restrained dynamics of this peptide. Figure 1Bshows the NMR structure of 7a,b uilt using 11 NOEs between adjacent residues,s ix interstrand NOEs and 19 dihedral angle restrictions.Only one rotamer of the N-alkylated (tertiary) amide is observed. The superposition of the ten lowest-energy conformations displays the good alignment of the two parallel strands,confirming the well-structured nature of this b-sheet (RMSD among backbone atoms 0.64 AE 0.02 ). As seen in other non-cyclic parallel b-sheets, [7] them ajor deviations were observed at the N-termini, but the interstrand NOE between the penultimate Va la nd Leu residues confirms the well-developed bsheet conformation alongside the two strands.B esides the steric bias provided by the turn N-alkylation that helps to bend the peptide backbone,t his modification removes the possibility of g-turn formation, which would not favor folding into ap arallel b-sheet due to its tight nature.
To evaluate the stability of the N-alkylated b-sheet structures,w ed ecided to compare the CD spectra of peptides 7a and 8 in aqueous solution at different temperatures. Figure 2depicts the CD spectra at 15, 35, 55, and 75 8 8C, clearly showing that the N-alkylated peptide 7amaintains the parallel b-sheet conformation even at higher temperatures, while non-N-alkylated 8 shows asteep decrease of the b-sheet population already at 55 8 8C. This corroborates the greater capacity of the N-alkylated turn to keep the desired fold intact, not only at physiological temperature but also under thermic stress.
In conclusion, we have developed an efficient multicomponent approach for the on-resin assembly of N-alkylated parallel b-sheet peptides with improved stability and varied functionalization. Thekey step in this methodology is the Ugi four-component reaction that allows the construction of the N-alkylated turn fragment in one step by simultaneously incorporating d-Pro and DADME residues onto the iso-  cyano-modified resins.T he procedure shows great versatility, as it enables not only the variation of the functionalization of the N-alkylation moiety at will, but also the access to non-Nalkylated b-sheets in am ore convergent manner than previously described. CD analysis confirmed the positive effect of the turn N-alkylation on b-sheet stability,e ven at high temperatures.F urthermore,t he NMR structure of as elected N-alkylated peptide confirmed the formation of aw ell-structured parallel b-sheet in water. Overall, we demonstrated that the functionality attached to the turn Nalkylation can be tuned to reduce aggregation (adding positive charges) or used for fluorescent labeling without modification of the strand backbone and side chains and without affecting the b-sheet stability.B yp roviding an efficient, all-on resin approach toward stable b-sheet-bearing tunable functionalizations,w eo pen av ariety of possibilities for the-eventually bioorthogonal-construction of miniproteins based either on multiple parallel b-sheets or on ac ombination of parallel b-sheets with other types of secondary structures,s uch as a-helices and antiparallel bhairpins.