δ-Peptides from RuAAC-Derived 1,5-Disubstituted Triazole Units

FULL PAPER

DOI: 10.1002/ejoc.201400018

δ-Peptides from RuAAC-Derived 1,5-Disubstituted Triazole Units

Johan R. Johansson,[a,b] Elin Hermansson,[a,b] Bengt Nordén,[a] Nina Kann,[b] andTamás Beke-Somfai*[a]

Keywords: Foldamers / Conformation analysis / Peptidomimetics / Nitrogen heterocycles / Cycloaddition / Click chemistry

Non-natural peptides with structures and functions similar tonatural peptides have emerged lately in biomedical as wellas nanotechnological contexts. They are interesting for phar-maceutical applications since they can adopt structures withnew targeting potentials and because they are generally notprone to degradation by proteases. We report here a new setof peptidomimetics derived from δ-peptides, consisting of nunits of a 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl).The monomer was prepared using ruthenium-catalyzedazide–alkyne cycloaddition (RuAAC) chemistry using[RuCl2Cp*]x as the catalyst, allowing for simpler purificationand resulting in excellent yields. This achiral monomer wasused to prepare peptide oligomers that are water soluble in-

Introduction

Non-natural biocompounds, capable of mimicking natu-ral biomolecules and displaying similar properties, havebeen experiencing an exponential increase in scientific at-tention because of their potential applications in diverseareas ranging from pharmacology to nanotechnology. Themost successful advances in the future are anticipated inareas where the synthetic compounds are capable of retain-ing the beneficial properties of natural molecules, yet dis-play significant enhancements in some areas where the lat-ter have limitations. For instance, foldamers, non-naturalfolded peptidomimetic oligomers, have become a separatefield;[1] enzyme resistance,[2] diverse stereochemistry,[1,3] andantiviral, antifungal and antibacterial activities are all ad-vantageous qualities of these compounds.[2b,4] Althoughthese properties have an obvious influence on the pharma-ceutical potential of foldamers, recent advances have alsodemonstrated their high affinity for self-assembly[5] leadingto possible applications in nanotechnology.[6]

[a] Department of Chemical and Biological Engineering, PhysicalChemistry, Chalmers University of Technology,41296 Göteborg, SwedenE-mail: [email protected]://www.chalmers.se

[b] Department of Chemical and Biological Engineering, OrganicChemistry, Chalmers University of Technology,41296 Göteborg, SwedenSupporting information for this article is available on theWWW under http://dx.doi.org/10.1002/ejoc.201400018.

Eur. J. Org. Chem. 2014, 2703–2713 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2703

dependent of peptide chain length. Conformational analysisand structural investigations of the oligomers were per-formed by 2D NOESY NMR experiments, and by quantumchemical calculations using the ωB97X-D functional. Thesedata indicate that several conformations may co-exist withslight energetic differences. Together with their increasedhydrophilicity, this feature of homo-5Tzl may prove essentialfor mimicking natural peptides composed of α-amino acids,where the various secondary structures are achieved by sidechain effects and not by the rigidity of the peptide backbone.The improved synthetic method allows for facile variation ofthe 5Tzl amino acid side chains, further increasing the versa-tility of these compounds.

The structural and constitutional composition of mostfoldamers can be traced back to natural peptide backbones,where α-amino acids and their homologues, β-, γ-, etc.amino acids, are used. As elongated hydrocarbon chains re-sult in increased backbone flexibility, to facilitate foldinginto various, mainly helical, secondary structures, both ali-phatic and aromatic cyclic insertions have been applied.[1a,7]

These structural changes have resulted in diverse examplesof new foldamers and foldamer-like compounds. However,the elongated hydrocarbon chains and the aliphatic cyclicinsertions have raised solubility problems.[1a,8] Accordingly,despite showing some very interesting potential in thefield,[1c,8a] foldamer derivatives built from longer homo-logues, such as δ-peptides are still rare. Further explorationof δ-amino acid substituents is expected to have significantimportance, as the backbone constitution of δ-amino acidderivatives easily fits into sequences of natural α-peptides.[9]

Another common problem with non-natural amino acidderivatives is that synthesis of various building units withdifferent side chains can be difficult, thus, limiting wide-spread studies of structural and functional properties. Inthe last decade, click-chemistry,[10] in particular the forma-tion of 1,4-disubstituted 1,2,3-triazoles from an alkyne andan azide,[11] has had a substantial impact on many areas ofchemistry.[12] The benign conditions for triazole formationcoupled with high efficiency has resulted in widespread ap-plications also in the fields of chemical biology[13] and“click peptidomimetics”.[7c,14] Additional benefits of theseheterocyclic structures are their low toxicity,[15] high sta-

T. Beke-Somfai et al.FULL PAPERbility, as well as their potential capacity for hydrogen bond-ing, π-stacking, and polar interactions.[16] Accordingly,using mainly 1,4-substituted triazoles, several peptidic sub-stitutions have been made, substituting either a single resi-due in a natural peptide sequence or using mixed naturalamino acids with multiple triazole insertions, the aim beingto enhance certain structural and functional changes.[17]

These successful attempts also suggest that triazole-basedoligomers could be an interesting set of compounds, thatmay expand the set of longer backbone chain homologuesand could create a new set of foldamers. However, to thebest of our knowledge, there are no reported foldamersbuilt entirely from click chemistry-derived peptidomimet-ics.[18] This is most likely due to the fact that, although tri-azoles may have advantages both in terms of ease of synthe-sis and in increased solubility of certain peptide chains, theoligomers of 1,4-disubstituted triazoles result in extendedconformers with little conformational flexibility.[7c] On theother hand, the much less studied 1,5-substituted triazoles,prepared by ruthenium-catalyzed reaction of an organic az-ide with an alkyne,[19] could provide an alternative with aflexible enough backbone to adopt several conformations.Byusse et al. have reported the synthesis of a bicyclic pepti-domimetic compound containing a 1,5-disubstituted 1,2,3-triazole,[20] whereas Appella and co-workers have employeda similar unit to induce a turn in a peptoid oligomer.[21]

However, applications of the ruthenium-catalyzed cycload-dition reaction in this context are still rare and further workis needed to develop suitable building blocks and syntheticmethods within this area.

Here we present a new set of peptides built from δ-aminoacid derivatives using 1,5-disubstituted 1,2,3-triazole aminoacids. The required monomer was synthesized using the ruth-enium-catalyzed azide-alkyne cycloaddition (RuAAC) reac-tion. The use of a new catalyst for this reaction enabled acqui-sition of the monomer in fewer steps than are required for arelated building block[21] and in an excellent yield of 96%.

Using this monomer, di- tri- and tetramers were synthe-sized using classical solution phase peptide synthesis meth-ods employing Boc and ester protecting groups. The re-sulting peptides have the same number of atoms in the pept-ide backbone as δ-peptides, but the 1,5-substituted ring re-stricts conformations in a fashion similar to the cis-peptidebond in natural α-amino acid peptides.

To assess potential future uses of this new set of clickoligomers, rigorous structural characterization will be cru-cial. As a first step, we have here investigated structuralproperties of these achiral compounds both by 2D NMRspectroscopy, and by computational methods. Quantummechanical calculations represent an important tool for in-vestigating structural properties of novel foldamers. Indeed,ab initio methods can directly address these exotic com-pounds and can support various experimental structure de-termining methods during analysis of possible conform-ers.[1c,9,22] Here, we employed the ωB97X-D density func-tional which includes dispersion correction[23] and usedboth polar and apolar solvents to test relative stabilities ofthe obtained secondary structures.

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The resulting compounds are characterized by severaladvantageous properties including: a) modular synthesis ofthe monomers, allowing variation of the side chains; b) in-creased solubility of the oligomers both in polar solventsand in water, where the solubility does not decrease, butrather increases slightly with increasing peptide length; andc) high flexibility of the achiral backbone enabling this newset of foldamers to adopt several conformations with com-peting relative energies.

Results

Synthesis

5Tzl monomer 1 was synthesized by a RuAAC reactionstarting from methyl 2-azidoacetate and N-Boc-propar-gylamine (Scheme 1). The reaction works well with com-monly used [RuClCp*(PPh3)2] as the catalyst. However,using flash chromatography for purification, we were un-able to separate the catalyst from triazole product 1. Basedon our previous work with the RuAAC reaction,[24] wefound that a RuIII catalyst,[19a,25] [RuCl2Cp*]x, could be ap-plied with good results in this reaction. By employing 4mol-% of [RuCl2Cp*]x with THF as the solvent, triazole 1was obtained in 96% yield after heating to 100 °C for20 min in a microwave reactor; these conditions allowed theseparation problems encountered earlier to be circum-vented. This set of conditions provides a great improvementrelative to previously reported routes to similar com-pounds.[20,21] The regiochemistry of 1,5-disubstituted tri-azole 1 was confirmed by 2D NOESY (see Supporting In-formation). From triazole monomer 1, dimer 4, trimer 7and tetramer 8 were synthesized using standard deprotec-tion and coupling methods (Scheme 2). The Boc-group of1 was quantitatively removed by treatment with TFA inCH2Cl2. The ester hydrolysis to form acid 3 was then opti-mized; the best results were achieved with 1 m LiOH (aq)(1.5 equiv.) in MeOH at ambient temperature. Amide cou-pling of building blocks 2 and 3 using T3P[26] as the cou-pling agent gave dimer 4 in moderate yield. Although de-protection of 4 to amine 5 proceeded without any problemusing either TFA in CH2Cl2 or HCl in dioxane, selectivehydrolysis of the methyl ester of 4 to 6 was found to bemore problematic since the amide bond was also hydrolyzedupon heating. Performing the reaction at room temperature,however, afforded carboxylic acid 6 in 93% yield. Trimer 7could then be formed by coupling of dimer 5 with monomer3 using T3P as the coupling reagent, albeit in low yield(unoptimized). Compound 7 has a solubility in water of up

Scheme 1. One step synthesis of triazole monomer 1 by applicationof the RuAAC reaction, using [RuCl2Cp*]x as the catalyst.


Scheme 2. Synthetic route to oligomers based on 1,5-disubstitued triazole monomers (5Tzl). Reagents and conditions: a) TFA/CH2Cl2(1:2), 22 °C 1.5 h (quant.); b) 1 m NaOH (3 equiv.), THF, microwave heating 100 °C, 15 min (78%); c) T3P (1.05 equiv.), Et3N (5 equiv.),22 °C 65 h (32%); d) TFA/CH2Cl2 (1:2), 22 °C 1.5 h (quant.) or 4 m HCl in dioxane (3 equiv.), MeOH 22 °C 2 h (quant.); e) 1 m LiOH(1.5 equiv.) MeOH, 22 °C 1 h (93%); f) T3P (1.1 equiv.), DIPEA (9 equiv.), 22 °C 5 d (9%); g) T3P (2 equiv.), DIPEA (10 equiv.), 22 °C64 h (5%).

to 2 mg/mL, but could also be dissolved in MeOH, DMSOand DMF. Coupling of dimers 5 and 6 produced tetramericstructure 8, also water soluble, using the same reaction con-ditions as noted earlier.

Theoretical Investigations

We have investigated the structural and energetic proper-ties of the 5Tzl-containing oligomers by quantum chemicalcalculations. To explore the potential secondary structuresand to support NMR studies, four different models wereemployed including: a heptamer model, which is longenough to form hydrogen bonds and thus stabilize the widerhelices, two tetramer models, and one trimer model. Onetetramer was built with terminal protecting groups used

Table 1. Average backbone dihedral angles for the investigated sec-ondary strucures in the heptamer models obtained at the ωB97X-D/6-31G(d) (M1) level of theory in DMSO. Nomenclature of thedihedral angles is the same as used for δ-amino acids.[8a]

Conf.[a] φ[b] θ ζ ρ ψ

Ext –77.6 168.0 0.5 67.0 179.4(2.9) (5.4) (0.8) (2.8) (9.0)

H8 145.5 –49.8 7.6 92.7 124.1(11.0) (3.3) (0.4) (1.7) (6.3)

H10 62.5 –142.6 –0.7 87.8 –17.8(18.7) (1.3) (0.7) (2.6) (4.5)

H14[c] 136.6/ –72.4/ –4.2/ 84.6/ 51.2/–152.5 –100.3 –5.0 67.4 126.8

(25.9/2.0) (6.8/1.1) (4.4/0.6) (9.2/2.9) (8.8/5.7)H16 89.6 –87.2 –7.5 118.8 –11.7

(8.8) (9.2) (6.2) (16.1) (13.6)H20[c] 145.1/ –63.2/ –0.7/ 96.6/ –3.8/

–131.3 –156.5 –3.4 84.7 36.6(31.6/32.8) (14.1/37.5) (2.5/9.3) (16.5/25.7) (26.2/17.4)

[a] Investigated conformers. [b] Torsional angles are displayed indegrees. Values in parenthesis show the standard deviation values.[c] For H14 and H16 two conformers were identified as buildingunits. For both of these, the averages and standard deviations werecalculated separately.

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commonly in modelling studies,[1c,3,22,27] whereas the othertetramer as well as the trimer models were built using Bocand methyl ester protecting groups to model 7 and 8 whichwere also used in the NMR studies [Tetra(Boc-Est),Tri(Boc-Est)]. Based on the peptide backbone scaffolds de-scribed for acyclic δ-peptides,[8a] most of the identified heli-cal secondary structures of a single peptide strand, (e.g. H8,

Table 2. Relative energies obtained for the trimer and tetramermodels using different theoretical methods (M2–M4) in DMSO.All values are in kcalmol–1.

Conformer Trimer (Boc-Est) Tetra (Boc-Est) TetramerM2[a] M3 M2 M3 M2 M3 M4

Ext 15.3 15.7 20.2 20.5 21.7 22.1 22.0H8 9.7 10.1 11.5 12.1 11.9 12.5 12.2H10 9.1 9.6 10.1 10.6 9.4 9.9 9.9H14 0.0 0.0 0.0 0.0 0.0 0.0 0.0H16 1.9 2.0 1.3 1.3 –1.2 –1.1 –1.1

T1 5.0 5.0T2 6.9 7.0T3 7.1 7.3

[a] M2: ωB97X-D/6-31+G(d,p)//ωB97X-D/6-31G(d), M3: ωB97X-D/6-311++G(d,p)//ωB97X-D/6-31G(d), M4: ωB97X-D/6-311++G(d,p). All calculations considered effects of DMSO usingthe IEFPCM model. For more details on the calculations see Exp.Section.

Table 3. Relative energies of the investigated secondary structuresobtained for the heptamer models using different theoretical meth-ods and solvent models. All values are in kcalmol–1.

Conformer M2[a] (DMSO) M2 (Aq) M2 (decanol)

Ext 39.9 39.2 48.1H8 24.0 24.1 23.2H10 18.3 18.1 21.2H14 0.0 0.0 0.0H16 –5.0 –5.0 –4.8H20 – –4.5 –5.0

[a] M2: ωB97X-D/6-31+G(d,p)//ωB97X-D/6-31G(d). For more de-tails on the calculations see Exp. Section.

T. Beke-Somfai et al.FULL PAPERH10, H14, H16 and H20), an extended conformation with-out H-bonds (Ext), three different turn structures (T1–3)for the trimer model as well as their potential to form multi-stranded assemblies, were addressed (see Tables 1, 2, 3, and4, Figures 1, 2, and 3). For the helical structures, thenumber in the nomenclature indicates the number of atomsforming a pseudo-ring in each hydrogen bond.

Table 4. Average backbone dihedral angles of the investigated hep-tamer double-stranded structures obtained at the ωB97X-D/6-31G(d) (M1) level of theory in DMSO. Nomenclature of the dihe-dral angles is the same as used for δ-amino acids.[8a]

Conformer[a] φ[a] θ ζ ρ ψ

2-Helix –137.2 76.6 0.5 103.2.0 157.1(30.7) (17.1) (3.5) (31.9) (25.7)

Sheet –80.5 171.2 0.2 68.3 –173.6(4.7) (10.1) (2.0) (5.9) (13.4)

[a] Investigated two-stranded conformers. [b] Torsional angles aredisplayed in degrees. Values in parenthesis show the standard devia-tion.

Figure 1. Investigated secondary structures of the heptamer modeland the lowest energy turn conformer, T1, for model Tri(Boc-Est).Formed H-bonds are marked by dashed lines.


Figure 2. Relative energies of selected secondary structures dividedby the number of residues as obtained at the ωB97X-D/6-31+G(d,p)//ωB97X-D/6-31G(d) (M2) level of theory in DMSO.

Figure 3. Investigated multi-stranded assemblies of the heptamermodel. A: Side and top view of a double-stranded helix (2-Helix)with a scaffold similar to that of gramicidin. B: Two side views ofa parallel sheet secondary structure motif (Sheet).

Single-Stranded Conformers

The cyclic insertion renders the middle dihedral angle ζclose to zero for all secondary structures making the tor-sional angles of the investigated conformers substantiallydifferent from those observed for acyclic δ-peptides.[8a] Nev-ertheless, the remaining four freely rotating dihedral anglesprovide sufficient flexibility for the compounds. As a result,the investigated secondary structures could all be locatedfor the heptamer model, and, with the exception of H20,for the tetramer and trimer models as well.

Considering the structural properties of the investigatedmodels, the Ext., H8, H10 and H16 structures are built outof homoconformer residues, where each has similar tor-sional angles with low standard deviation. However, for


H14 and H20, the residues have two different types of con-formers that follow each other in an alternating mannerthroughout the peptide backbone (Table 1). In the case ofthe H20 structure, this results in the absence of selected H-bonds, most likely due to prohibitively strong steric repul-sions between the triazole rings. This is also represented bymuch larger standard deviation values relative to the otherstructures (Table 1). The turn structures investigated in thetrimer model all form a 10-membered pseudo-ring in themiddle residue. For T1 and T2, this positions the first andlast residues in such a way as to form a second H-bondwhere the triazole groups are either on the opposite side(T1) (Figure 1), or on the same side (T2). In T3 the C-ter-minal residue is more bent and does not form an H-bondwith the N-terminal residue.

Considering energetic properties, the H16 and H14 heli-ces are low energy conformers for all shorter oligomers.There is only a small change in energetic preference betweenH14 and H16, as the latter has one less H-bond in the C-terminal in the Tetra(Boc-Est) model because of the esterprotecting group. In the case of the Trimer(Boc-Est) model,the T1 turn has its relative energy within 5.0 kcal/mol ofthe lowest energy H14 structure. Thus, the Trimer(Boc-Est)model may also be considered a low energy conformer(Table 2). For the heptamer model, the relative energy dis-tribution remains similar to the shorter ones, although theenergy differences are larger due to the fact that more resi-dues are present in the peptide chain (Table 3). This peptideis long enough to stabilize H20, resulting in the H14, H16and H20 helices being within 5 kcal/mol of each other inrelative energy. To address the potential solubility of thedifferent conformers, besides the polar DMSO used in theexperimental investigations, the energies of the conformerswere also obtained in the very polar aqueous matrix (Aq)and in the hydrophobic decanol, which can be used to ap-proximate a lipid bilayer environment.[28] We found that thethree different solvents do not have a dramatic effect on therelative energy distribution of the conformers, and onlysome destabilization of H8 and H10 was observed whenchanging from the use of water to decanol as solvent.

The energetic preferences of the different models can becompared on a “per residue” basis if the obtained energiesare divided by the number of residues present in the dif-ferent peptide models (Figure 2). The per residue relativeenergy differences show very close agreement between themodels indicating that the energetic differences betweenmost of the secondary structures change linearly with thenumber of residues in the peptide chain. Using such a com-parison, the investigated conformers could be catagorizedinto three energetic regions. The extended conformer hasno hydrogen bonds, and thus on a per residue basis it is 5–6 kcal/mol less stable than reference H14. The helices aresplit into two categories: the H8 and H10 are destabilizedby ca. 2.5–3.5 kcal/mol per residue, whereas the group de-scribing H14, H16, and H20 helices have low relative ener-gies (Figure 2). The only deviation from the linear energychange per residue may be observed for H16, where someextra stabilization seems to occur as the length of the oligo-


mers increases. Overall, the very different end protectinggroups for the two tetramer models do not affect the rela-tive energy distributions significantly. It should also benoted that the use of M2–M4 levels of theory for the twotetramer models show only slight differences in terms ofboth structure and energy, thus indicating that the M2 levelis sufficiently accurate to describe these systems (Table 2).

Multi-Stranded Assemblies

Two double-stranded structures were optimized to inves-tigate whether steric, conformational, as well as energeticproperties of these compounds may allow formation ofhigher order assemblies. One structure was an intertwineddouble helix similar to that of the antimicrobial channel-forming gramicidin[29] (2-Helix). The other was a parallelsheet (Sheet), an analogue of the parallel β-sheet observedfor natural peptides and proteins (Figure 3 and Table 4).These two scaffolds were optimized using both the tetramerand the heptamer models. In the 2-Helix, the conformationof the individual strands was found to differ from thoseobtained for the single peptide conformers. However, forthe sheet structure, the dihedral angles are in close agree-ment with those obtained for the Ext single-stranded con-former.

The obtained relative energies of the multi-stranded sys-tems were –7.7 kcal/mol for the 2-Helix and –2.2 kcal/molfor Sheet model at the M2 level of theory. These valuesfurther support that such asemblies may have comparablestability to the investigated low-energy helices formed froma single strand. The quantum mechanical calculations high-light that, upon adequate selection of side chains and sur-rounding environment, higher order self-assembled struc-tures could be manifested from 5Tzl oligomers. Notably,these preliminary QM studies do not consider conforma-tional entropy contributions, which may be significant whenobtaining accurate free energies, a point beyond our focusin the present study.

Structural Investigation with NMR Spectroscopy

2D NOESY NMR methods were employed to study theconformational behaviour of trimer 7 and tetramer 8 insolution. The 1H-1H NOESY experiments were performedat 13 mm and 23 mm concentrations, for 7 and 8, respec-tively, in [D6]DMSO at 25 °C using a mixing time of600 ms. The NOESY spectrum of trimer 7 exhibits severalshort range NOEs but also a few mid-long range cross-peaks (Figure 4). Strong inter-residue NOE cross-peakswere found between methylene H6 and the terminal meth-oxy protons H1 (H1–H6) as well as between H6 and thetriazole proton H3 (H3–H6). Cross-peaks were also seen be-tween the central triazole proton H7 and the methylenessurrounding the third triazole unit, (i.e. H10 and H12). TheBoc-group (H14) showed inter-residue NOE-crosspeaks tothe central triazole proton (H7) and also to either methyleneH4 or the central methylene H8 (for more details see Sup-porting Information and Figure 4). Due to signal overlapbetween the carbonyl methylene protons H6 and H10, as

T. Beke-Somfai et al.FULL PAPER

Figure 4. Important sections of the 1H-1H NOESY spectra (400 MHz, 600 ms mixing time) of 13 mm trimer 7 in [D6]DMSO at 25 °C,showing inter-residual NOE-crosspeaks, including 1H NMR spectra. Identified peaks are highlighted on the structure with red arrows,whereas for crosspeaks where the chemical shifts overlap, the alternative pairs of atoms possibly giving rise to the NOE peaks areconnected with dashed blue lines. See text for more details.

well as the amine methylene protons H4 and H8, all NOEcross-peaks could not be fully assigned relying solely onNMR spectroscopic data. In addition to [D6]DMSO,NOESY was also performed in pure CD3OD as well as in15% CD3OD in D2O. The experiment in CD3OD sufferedfrom greater signal overlap than observed when using [D6]-DMSO as the solvent; no additional information could beobtained. The use of 15% CD3OD in D2O gave essentiallythe same results as the DMSO studies, but without visual-ization of the amide proton signals and with slightly lowerresolution.

Although it is not expected that short achiral oligomerswould adopt a well-defined conformation, it is apparentfrom NMR spectra that several conformers might coexist.Using the secondary structures obtained from the theoreti-cal calculations, the most likely conformers may be iden-tified. To help in this assignment, we analyzed the smallestdistances between corresponding pairs of groups of hydro-gens on the secondary structures calculated using theTri(Boc-Est) model.

Considering overlaps arising from methylene peaks H6

and H10, out of the possible structures, the H14 helix is the


only one that may enable the crosspeak between H10 andH1, with the closest distance of 4.5 Å between these. How-ever, this structure should also generate a strong crosspeakbetween the methoxy H1 and the central triazole proton H7;in H14 these atoms have an intervening distance of 3.0 Åalthough this peak is missing from the spectra. Therefore,the crosspeak in question is more likely to correspond toan NOE between protons H1 and H6, which could arisemainly from helix forming conformers, such as H8, H10,H16 or the turn T3, which in the Tri(Boc-Est) model haveclosest H1–H6 distances of 3.2 Å, 5.0 Å, 3.8 Å, and 5.0 Å,respectively. For the other pair of ambiguous overlaps, (i.e.between the Boc-group (H14) and methylenes H4 or H8), thepresence of an interaction between H14 and central triazoleproton H7 and the analysis of the calculated structures ex-clude all conformers with a H4–H14 NOE, except for H16,which has H14 within 3 Å of both H4 and H7. The co-pres-ence of the H7–H14 and H8–H14 NOEs would be consistentwith H10, for which these are within 3.1 Å, and, based onthe heptamer models, also with H20. Interestingly, a weakNOE cross-peak was also observed between the two endsof the oligomer, (i.e. between the methyl ester protons H1


and the Boc-group hydrogens H14) (Figure 4). This suggeststhe presence of a turn-like conformer as for T1–3; thesehydrogens are within ca. 3.5 Å of each other. The H1–H6,H7–H14 or H8–H14 crosspeaks, however, cannot originatefrom such turn-like conformers.

These observations lead us to conclude that 7 has neithera random conformation without any folded structure, nora single, well-defined conformer. Most likely we are seeinghere the co-existence of at least two conformers that appearto enable formation of H10, H16 as well as turn-like struc-tures.

The NOESY spectra of tetramer 8 has even more exten-sive overlaps of the carbonyl methylene protons (H6, H10

and H14), the amine methylene protons (H4, H8 and H12),as well as the amide (H5, H9 and H13) and triazole protons(H7 and H11), of the different units. Overall, NOESYdata for 8 shows a very similar pattern to that of trimer7, with no additional qualitative information. These datasuggest that 8 most likely assumes secondary structuressimilar to those of 7 (see Figure S1 in the Supporting Infor-mation).

Discussion

Oligomers built from achiral building block 5Tzl (1) pro-vide an excellent framework to address the backbone char-acteristics of these peptidomimetics. By combining NMRspectroscopic data with the computed secondary structures,the NMR spectra of the tri- and tetra-oligomers with Bocand methyl ester protecting groups indicate that structuresup to tetramers built with 1 do not force the structure intoone preferred conformation. Rather, these oligomers popu-late several helical and turn-like conformers simultaneously.The co-presence of several stable conformers is supportednot only by observed NOEs, but also by the relative energydistributions obtained using computational methods. Con-sidering the calculated relative energies, 7 and 8 most prob-ably form H16, T1 and similar turn-like conformers, or evenconformers building up to H20. Although formation ofH10 may be fully justified on the basis of observed NMRpeaks, its relative energy is greater than 9 kcal/mol for theTri(Boc-Est) model, indicating that this conformer is lesslikely to form. For short achiral oligomers, such conforma-tional flexibility is an advantageous property since it indi-cates that the backbone structure is flexible enough to pre-vent predominant folds that might limit structural diversitypotentially generated later by sequential modifications.These qualities make 5Tzl oligomers somewhat similar tonatural peptides, where the peptide backbone is neither tooflexible, nor too rigid thus allowing several secondary struc-ture elements to be identified for shorter peptides with sim-ilar energetic properties.[30]

Considering energy values, the longer peptide chainseems to stabilize wider helices (Table 3). In contrast, theappearance of alternating conformers, and the high stan-dard deviation in torsional angles observed for these struc-tures may also indicate that this structure could be de-


stroyed by solvent dynamics. It should also be noted that,although the experimental investigations did not indicatesigns of dimerization or aggregation, the preliminary calcu-lations propose that, upon choosing suitable sequences andconditions, the backbone may allow formation of higherorder assemblies with stabilities comparable to those of sin-gle stranded structures. The structural information ob-tained here provides important first insight into what fold-ing capabilities these 5Tzl oligomers may have.

Introducing substituents onto the methylene carbons ofelongated sequences would be highly interesting. Suchmodifications would afford the opportunity to study sub-stituent effects on the conformational behaviour of 5Tzl-based oligomers. In addition, both positions become chiralupon substitution, allowing the additional benefit of beingable to study different combinations of enantiomers if themonomers are prepared in optically pure form. Fortunately,our improved synthetic route is well adapted to incorporatesuch changes. With regards to the organic azide precursor,a number of chiral 2-substituted 2-azidopropanoic acid de-rivatives are commercially available with different side-chain functionalities such as alkyl, heteroalkyl, aryl and es-ter moieties, allowing evaluation of structural properties.Alternatively, commercially available methyl 2-bromoacet-ate derivatives with pendant side chains could also be em-ployed directly as precursors using our sequential one-potRuAAC reaction.[24] In terms of the alkyne component, chi-ral α-substituted propargylic amines can be prepared in onestep from a wide variety of commercially available proparg-ylic alcohols using asymmetric copper-catalyzed methodsreported in the last few years.[31] Ready access to such chiralazide and alkyne building blocks is envisioned to grant ra-pid access to structurally diverse foldamers for both struc-tural and functional peptidomimetic studies.

Conclusions

We have synthesized a new set of peptidomimetic com-pounds constructed from 1,5-disubstituted 1,2,3-triazole(5Tzl) units. These monomer δ-amino acid derivatives wereprepared by application of the RuAAC reaction, using[RuCl2Cp*]x which facilitated purification, affording the de-sired compounds in up to 96 % yield. The building blockswere subsequently assembled to form dimeric, trimeric andtetrameric structures. These pseudopeptides show good sol-ubility in polar solvents, irrespective of the number of resi-dues. Structural characterizations were performed using 2-dimensional NMR spectroscopy and quantum mechanicalcalculations. Both approaches indicate that these short achi-ral oligomers simultaneously adopt several conformers withcomparable stabilities. Overall, considering the above quali-ties of these 5Tzl oligomers and that the click reaction pro-vides easy access to several amino acids derivatives, we an-ticipate that these new sets of click oligomers or“clickomers” will become a valuable addition to existingpeptidic foldamers and provide a highly suitable molecularscaffold to investigate structure-function relationships using

T. Beke-Somfai et al.FULL PAPERmore advanced sequences. Accordingly, we currently aim toexpand our knowledge using a new set of 5Tzl oligomers,testing structure and potential bioactivity of chiral oligo-mers with structurally diverse side chains.

Experimental SectionComputational Details: Initial geometries of the investigated tetra-mer and heptamer models were obtained at the RHF/3-21G levelof theory. Considering that the larger models could experience sig-nificant structural changes due to dispersive forces of the aromatictriazole moieties, the obtained geometries were submitted to fur-ther optimizations at the ωB97X-D/6-31G(d) level of theory (M1).The recently developed ωB97X-D functional[23] was chosen becauseit incorporates both empirical dispersion correction, with a dampedfunction, as well as long-range corrections for electron-electron in-teractions, and also showed good results in benchmark tests onpeptide models.[32] All the obtained structures were finally opti-mized at the M1 level in dimethyl sulfoxide (DMSO), which wasconsidered with the Polarizable Continuum Model using the Inte-gral Equation Formalism (IEFPCM).[33] The only exception wasthe H20 helix of the heptamer model, which had convergence prob-lems in DMSO, and thus, for this model the gas phase structurewas used. Energy values for the tetramer models were further re-fined at the ωB97X-D/6-311++G(d,p)//ωB97X-D/6-31G(d) (M3)level of theory. The largest double-stranded heptamer models con-sisted of 242 atoms. Therefore energies for these were obtained atthe ωB97X-D/6-31+G(d,p)//ωB97X-D/6-31G(d) level of theory(M2). For the heptamer model, solvent effects of three solventswith different polarity, water (Aq), DMSO, and decanol, were con-sidered. As optimization was performed at the M1 level using the6-31G(d) basis set, the presence of basis set superposition error(BSSE) may have led to inappropriate geometry and thus to lessaccurate relative energies. To validate the accuracy of the singlepoint energies obtained at M2 and M3 level, the single-strandedtetramer structures were also fully optimized at the ωB97X-D/6-311++G(d,p) level of theory (M4) in DMSO. This did not resultin significant variations in geometry and the M2–M3 energies ap-pear to describe the relative energy distribution of the different con-formers with very close correlations to those of M4 (Table 2). Allcalculations were performed using the Gaussian 09 software pack-age.[34]

Experimental: General information: Chemicals were obtained fromcommercial suppliers and used without purification unless other-wise noted. All microwave reactions were carried out with a BiotageSeries 60 Initiator (actual vial temperature was monitored using anIR sensor), using a fixed hold time. Abbreviations for NMR are: s,singlet; d, doublet; t, triplet; q, quartet; br. s, broad singlet; app,apparent. Chemical shifts (δ) are given in ppm relative to the sol-vent residual peak (CDCl3: 7.26 ppm for 1H NMR and 77.23 ppmfor 13C NMR, [D6]DMSO: 2.50 ppm for 1H NMR and 39.51 ppmfor 13C NMR) or an internal standard (TMS: 0.00 ppm for 1HNMR). 2D NOESY experiments were carried out in 10–25 mm

[D6]DMSO solution at 25 °C, using a NOESY mixing time of 600ms and were acquired with 1024 points in the ƒ2 domain and 256points in the ƒ1 domain. The data were processed using Mes-tReNova software and baseline correction was applied to both di-mensions using Bernstein polynomial fit (3 orders). A 90° sine bellwindow function was applied in ƒ2 and the data was zero-filled inƒ2 domain to give a final matrix of 1024 by 1024 real points.

Methyl 2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetate (1): In a microwave vial were placed N-Boc-propar-


gylamine (627 mg, 4.0 mmol), methyl 2-azidoacetate (475 mg,4.1 mmol) and THF (15 mL). [RuCl2Cp*]x (48 mg, 0.16 mmol) wasadded and the vial was sealed and flushed with N2 for 1–2 min.The reaction mixture was heated to 100 °C for 20 min in a micro-wave reactor. The reaction mixture was filtered through a syringefilter and concentrated. The crude product was purified by flashchromatography on silica gel (using a gradient from 0 to15% MeOH in CH2Cl2) to obtain methyl 2-(5-{[(tert-butoxycarb-onyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetate (1) as a whitesolid, yield 1.03 g (96%). 1H NMR (400 MHz, CDCl3, 25 °C): δ =7.47 (s, 1 H), 5.63 (t, J = 6.0 Hz, 1 H), 5.21 (s, 2 H), 4.27 (d, J =6.2 Hz, 2 H), 3.70 (s, 3 H), 1.35 (s, 9 H) ppm. 1H NMR (400 MHz,[D6]DMSO, 25 °C): δ = 7.55 (s, 1 H), 7.42 (br. t, J = 5.5 Hz, 1 H),5.39 (s, 2 H), 4.21 (d, J = 6.0 Hz, 2 H), 3.70 (s, 3 H), 1.37 (s, 9H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C): δ = 167.92,156.02, 137.11, 133.00, 78.88, 53.00, 48.94, 32.99, 28.55 ppm.HRMS (ESI): m/z calcd. for C11H18N4O4: 271.1401 [M + H]+,found 271.1408 [M + H]+.

Methyl 2-[5-(Aminomethyl)-1H-1,2,3-triazol-1-yl]acetate (2):Methyl 2-(5-{[(tert-butoxycarbonyl)amino]methyl}-1H-1,2,3-tri-azol-1-yl)acetate (1) (3.02 g, 11.2 mmol) was dissolved in CH2Cl2(20 mL) and TFA (10 mL, 130 mmol) was added. The reactionmixture was stirred at ambient temperature for 1.5 h and concen-trated under reduced pressure to obtain the crude product as theTFA salt. The crude product was used as such without further puri-fication. 1H NMR (400 MHz, [D6]DMSO, 25 °C): δ = 8.31 (br. s,3 H), 7.83 (s, 1 H), 5.49 (s, 2 H), 4.22 (br. d, J = 4.9 Hz, 2 H), 3.71(s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C): δ = 167.97,134.53, 132.62, 53.18, 49.33, 31.44 ppm. HRMS (ESI): m/z calcd.for C6H10N4O2: 171.0877 [M + H]+, found 171.0875 [M +H]+.

2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)-acetic Acid (3): In a microwave vial were placed methyl 2-(5-{[(tert-butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetate (1)(445 mg, 1.65 mmol) and THF (5 mL). 1 m NaOH (5.0 mL,5.0 mmol) was added and the reaction mixture was heated to100 °C for 15 min in a microwave reactor. CH2Cl2 was added and1 m HCl (5 mL) was added dropwise. The mixture was extractedwith CH2Cl2 three times and the combined organic phase was fil-tered through a phase separator and concentrated to obtain the 2-(5-{[(tert-butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)-acetic acid (3) as a white solid, yield 330 mg (78%). 1H NMR(400 MHz, [D6]DMSO, 25 °C): δ = 13.41 (br. s, 1 H), 7.50 (s, 1 H),7.41 (br. t, J = 5.8 Hz, 1 H), 5.24 (s, 2 H), 4.18 (d, J = 6.0 Hz, 2H), 1.35 (s, 9 H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C): δ= 168.80, 156.03, 136.97, 132.82, 78.88, 49.18, 33.12, 28.56 ppm.HRMS (ESI): m/z calcd. for C10H16N4O4: 257.1244 [M + H]+,found 257.1242 [M + H]+.

Methyl 2-(5-{[2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetate (4): 2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)-acetic acid (3) (2.25 g, 8.78 mmol) was dissolved in EtOAc (40 mL)and Et3N (3 mL, 21.5 mmol). T3P (5.50 mL, 50 wt.-% in EtOAc,9.24 mmol) was added dropwise and the mixture was stirred at22 °C for 30 min. Methyl 2-[5-(aminomethyl)-1H-1,2,3-triazol-1-yl]acetate (2) was suspended in EtOAc (30 mL) and Et3N (3.5 mL,25.1 mmol) was added. The reaction mixture was stirred at ambienttemperature for 65 h. Water (20 mL) was added and the mixturewas stirred for another 30 min. NH4Cl (aq) was added and themixture was extracted with EtOAc three times. The combined or-ganic phase was dried with anhydrous Na2SO4, filtered and concen-trated. EtOAc was added and the product was precipitated, filtered


and washed with EtOAc to obtain methyl 2-(5-{[2-(5-{[(tert-but-oxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]-methyl}-1H-1,2,3-triazol-1-yl)acetate (4) as a white solid (1.03 g).The filtrate was concentrated and purified by flash chromatographyon silica gel (50 g Si-column using a stepwise gradient from 0–10 % MeOH/CH2Cl2 over 8 column volumes followed by 10–30 % MeOH/CH2Cl2 over 6 column volumes) to obtain another122 mg of the product after precipitation. Yield 1.15 g (32%). 1HNMR (400 MHz, [D6]DMSO, 25 °C): δ = 8.88 (t, J = 5.7 Hz, 1 H),7.64 (s, 1 H), 7.50 (s, 1 H), 7.37 (br. t, 1 H), 5.39 (s, 2 H), 5.13 (s,2 H), 4.40 (d, J = 5.7 Hz, 2 H), 4.18 (d, J = 5.8 Hz, 2 H), 3.68 (s,3 H), 1.35 (s, 9 H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C):δ = 167.94, 166.29, 156.01, 137.39, 136.16, 133.37, 132.69, 78.87,53.05, 49.98, 49.04, 33.31, 31.79, 28.55 ppm. HRMS (ESI): m/zcalcd. for C16H24N8O5: 409.1942 [M + H]+, found 409.1944 [M +H]+.

Methyl 2-[5-({2-[5-(Aminomethyl)-1H-1,2,3-triazol-1-yl]acet-amido}methyl)-1H-1,2,3-triazol-1-yl]acetate (5): Methyl 2-(5-{[2-(5-{[(tert-butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acet-amido]methyl}-1H-1,2,3-triazol-1-yl)acetate (4) (322 mg,0.79 mmol) was suspended in CH2Cl2 (5 mL) and TFA (3.0 mL,39 mmol) was added dropwise. The reaction mixture was stirred atambient temperature for 3 h and concentrated to obtain the crudemethyl 2-[5-({2-[5-(aminomethyl)-1H-1,2,3-triazol-1-yl ]-acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetate (5) as a TFA-salt.The crude product was used as such without further purification.1H NMR (400 MHz, [D6]DMSO, 25 °C): δ = 9.12 (t, J = 5.8 Hz,1 H), 8.23 (br. s, 3 H), 7.80 (s, 1 H), 7.65 (s, 1 H), 5.40 (s, 2 H),5.26 (s, 2 H), 4.43 (d, J = 5.7 Hz, 2 H), 4.21 (q, J = 5.5 Hz, 2 H),3.67 (s, 3 H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C): δ =167.95, 166.59, 158.48, 136.01, 134.46, 133.40, 132.79, 53.09, 50.34,49.05, 31.91, 31.75 ppm. HRMS (ESI): m/z calcd. for C11H16N8O3:309.1418 [M + H]+, found 309.1429 [M + H]+.

2-(5-{[2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetic Acid (6):Methyl 2-(5-{[2-(5-{[(tert-butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetate(4) (148 mg, 0.36 mmol) was dissolved in MeOH (6 mL) and 1 m

LiOH (0.55 mL, 0.55 mmol) was added. The reaction mixture wasstirred at ambient temperature for 1 h and 15 min. Water andEtOAc was added followed by 1 m HCl (0.55 mL, 0.55 mmol) andthe mixture was extracted with EtOAc four times. The combinedorganic phase was dried with anhydrous Na2SO4, filtered and con-centrated to obtain 2-(5-{[2-(5-{[(tert-butoxycarbonyl)amino]-methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetic acid (6) as a white solid, yield 133 mg (93%). 1H NMR(400 MHz, [D6]DMSO, 25 °C): δ = 13.45 (br. s, 1 H), 8.90 (t, J =5.7 Hz, 1 H), 7.62 (s, 1 H), 7.50 (s, 1 H), 7.36 (br. t, J = 5.6 Hz, 1H), 5.24 (s, 2 H), 5.14 (s, 2 H), 4.39 (d, J = 5.7 Hz, 2 H), 4.18 (d,J = 5.9 Hz, 2 H), 1.36 (s, 9 H) ppm. 13C NMR (101 MHz, [D6]-DMSO, 25 °C): δ = 168.82, 166.28, 156.01, 137.41, 136.07, 133.15,132.68, 78.88, 50.00, 49.32, 33.33, 32.02, 28.56 ppm. HRMS (ESI):m/z calcd. for C15H22N8O5: 395.1786 [M + H]+, found 395.1786[M + H]+.

Methyl 2-(5-{[2-(5-{[2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)-acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetate (7): 2-(5-{[(tert-Butoxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetic acid(3) (202 mg, 0.79 mmol) was suspended in EtOAc (3 mL) andDIPEA (0.3 mL, 1.72 mmol). T3P (0.52 mL, 50 wt.-% in EtOAc,0.87 mmol) was added dropwise and the reaction mixture wasstirred at ambient temperature for 35 min. A suspension of 2-[5-


({2-[5-(aminomethyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetate (5) (0.79 mmol) in EtOAc (5 mL), DMF(2 mL) and DIPEA (1 mL, 5.7 mmol) was added and the reactionmixture was stirred at ambient temperature for 5 days. Water(5 mL) was added and the mixture was stirred for another 40 min.NH4Cl (aq) was added and the mixture was extracted with EtOActhree times. 1 m HCl was added to the water phase and the mixturewas extracted with EtOAc three times. The combined organic phasewas dried with anhydrous Na2SO4, filtered and concentrated.EtOAc was added and the product was precipitated, filtered andwashed with EtOAc to obtain methyl 2-(5-{[2-(5-{[2-(5-{[(tert-but-oxycarbonyl)amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]-methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetate (7) as a white solid, yield 40.1 mg (9 %). 1H NMR(400 MHz, [D6]DMSO, 25 °C): δ = 8.92 (t, J = 5.7 Hz, 1 H), 8.89(t, J = 5.6 Hz, 1 H), 7.64 (s, 1 H), 7.61 (s, 1 H), 7.50 (s, 1 H), 7.37(s, 1 H), 5.39 (s, 2 H), 5.14 (s, 4 H), 4.40 (d, J = 5.7 Hz, 2 H), 4.39(d, J = 5.6 Hz, 2 H), 4.18 (d, J = 5.8 Hz, 2 H), 3.67 (s, 3 H), 1.35(s, 9 H) ppm. 13C NMR (101 MHz, [D6]DMSO, 25 °C): δ = 167.93,166.26 (two signals overlapping), 156.01, 137.39, 136.42, 136.12,133.40, 133.02, 132.69, 78.88, 53.06, 50.10, 50.02, 49.04, 33.32,3 2 . 1 9 , 3 1 . 76 , 2 8 . 56 pp m. HR MS (E SI ) : m / z c a l cd . forC21H30N12O6: 547.2484 [M + H]+, found 547.2501 [M + H]+.

Methyl 2-{5-[(2-{5-[(2-{5-[(2-{5-[tert-Butoxycarbonyl]amino}-methyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl-1H-1,2,3-triazol-1-yl)cetate (8): 2-(5-{[2-(5-{[(tert-Butoxycarbonyl)-amino]methyl}-1H-1,2,3-triazol-1-yl)acetamido]methyl}-1H-1,2,3-triazol-1-yl)acetic acid (6) (130 mg, 0.33 mmol) was dissolved inEtOAc (15 mL), DMF (3 mL) and DIPEA (0.23 mL, 1.32 mmol).T3P (0.39 mL, 50 wt.-% in EtOAc, 0.66 mmol) was added dropwiseand the reaction mixture was stirred at ambient temperature for1 h. A suspension of methyl 2-[5-({2-[5-(aminomethyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetate (5)(0.48 mmol) in EtOAc (5 mL), DMF (5 mL), and DIPEA(0.33 mL, 1.89 mmol), was added and the reaction mixture wasstirred at ambient temperature for 64 h. Water (10 mL) was addedand the mixture was stirred for another 1.5 h. 1 m HCl (3.0 mL)was added and the mixture (pH ≈ 4) was extracted with EtOAcfive times. The combined organic phase was dried with anhydrousNa2SO4, filtered and concentrated to recover 39 mg (30%) of dimeracid (6). The water phase was concentrated and purified by prepar-ative HPLC (Kromasil C8 column, 10 μm particles, 250�50 mm,using a gradient from 5–45 % acetonitrile in water/acetonitrile/for-mic acid 95:5:0.2 buffer) to obtain methyl 2-{5-[(2-{5-[(2-{5-[(2-{5-[tert-butoxycarbonyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acet-amido}methyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl)-1H-1,2,3-triazol-1-yl]acetamido}methyl-1H-1,2,3-triazol-1-yl)acetate (8) as awhite solid, yield (11 mg, 5%). [13 mg (6%) of the correspondingacid was also isolated together with 33 mg (25%) of dimer acid(6)]. 1H NMR (400 MHz, [D6]DMSO, 25 °C): δ = 8.96–8.86 (m, J

= 5.8 Hz, 3 H), 7.65 (s, 1 H), 7.62 (s, 1 H), 7.61 (s, 1 H), 7.51 (s, 1H), 7.39 (br. t, 1 H), 5.39 (s, 2 H), 5.17 (s, 2 H), 5.16 (s, 2 H), 5.15(s, 2 H), 4.41 (d, J = 5.7 Hz, 6 H), 4.19 (d, J = 5.8 Hz, 2 H), 3.67(s, 3 H), 1.36 (s, 9 H) ppm. 13C NMR (101 MHz, [D6]DMSO,25 °C): δ = 167.93, 166.29, 166.27, 166.26, 156.03, 137.42, 136.46,136.40, 136.15, 133.41, 133.03, 132.72, 78.90, 53.06, 50.14, 50.09,50.04, 49.04, 33.33, 32.24, 32.20, 31.78, 28.56 ppm. HRMS (ESI):m/z calcd. for C26H36N16O7: 685.3026 [M + H]+, found 685.3049[M + H]+.

Supporting Information (see footnote on the first page of this arti-cle): (see footnote on the first page of this article): 1H NMR and

T. Beke-Somfai et al.FULL PAPER13C NMR spectra of compounds 1–8. NOESY of compounds 1, 4,6–8.

Acknowledgments

This work was funded by the King Abdullah University of Scienceand Technology (KAUST Kuk No1) and by the European Re-search Council (ERC). The authors would like to thank Dr.Thomas Antonsson for useful discussions, Peter Bonn for assist-ance with HPLC purification of 8 and Annika Langborg Wein-mann for running HRMS analyses.

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Received: January 3, 2014Published Online: February 14, 2014

δ-Peptides from RuAAC-Derived 1,5-Disubstituted Triazole Units

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