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A Minimalistic Hydrolase Based on Co-Assembled Cyclic DipeptidesAlexander Kleinsmann, Boris Nachtsheim
Submitted date: 30/09/2019 • Posted date: 01/10/2019Licence: CC BY-NC-ND 4.0Citation information: Kleinsmann, Alexander; Nachtsheim, Boris (2019): A Minimalistic Hydrolase Based onCo-Assembled Cyclic Dipeptides. ChemRxiv. Preprint.
This paper describes minimalistic cyclic dipeptides acting as esterase-mimicks in a self-assembled hydrogelstate. It demonstrates that cyclic dipeptides could have acted as enzyme-precursors on a primordial earth andhence be important for abiogenesis.
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A Minimalistic Hydrolase based
on Co-Assembled Cyclic
Dipeptides
Alexander J. Kleinsmann[b] and Boris J.
Nachtsheim*[a]
Abstract: The self-assembly of small peptides into larger
aggregates is an important process for the fundamental
understanding of abiogenesis. In this article we demonstrate that
blends of cyclic dipeptides (2,5-diketopiperazines – DKPs) bearing
either histidine or cysteine in combination with a lipophilic amino acid
form highly stable aggregates in aqueous solution with esterase-like
activity. We demonstrate that the catalytic activity is based on an
intermolecular cooperative behavior between histidine and cysteine.
A high control of the molecular arrangement of the peptide
assemblies was gained by C-H-π interactions between Phe and Leu
or Val sidechains, resulting in a significant increase in catalytic
activity. These interactions were strongly supported by Hartree-Fock
calculations and finally confirmed via 1H-NMR HRMAS NOE
spectroscopy.
The transition of simple small molecular building blocks, inparticular fatty-, amino- and nucleic acids, into self-replicatingsystems with an autonomous metabolism is the critical step forthe emergence of the first living cells with a minimalisticgenotype and phenotype.[1] The initial manifestation of smallpeptides as enzyme precursors that could have providedimportant catalytic properties for autonomous self-replicatingsystems is still underexplored.[2],[3] Here, self-assemblyprocesses that form higher ordered aggregates fromspontaneously formed small oligopeptides throughintermolecular H-bonding interactions is believed to be an
important initial step.[4a–c,2,4d] In this regard, cyclic dipeptides (2,5-diketopiperazines – DKPs) are observed frequently as undesiredside-products during peptide formation and under prebioticconditions,[5] in particular as degradation products of smalloligopeptides.[6] In addition, they have been found on theYamato-791198 and Murchison carbonaceous chondrites.[7] Werecently demonstrated that a variety of Phe-containing DKPsform highly stable aggregates in aqueous solutions. [8] Their self-aggregation is the result of strong H-bonding interactionsbetween the cyclic amides and additional π-π or C-H-π-interactions between the Phe sidechains. For proving therelevance of DKPs in the context of abiogenesis, their catalyticproperties must be elucidated. So far their catalytic activity hasonly been demonstrated by Lipton and co-workers in solution forthe asymmetric Strecker reaction.[9] Presuming their hightendency to aggregate in water into a defined moleculararrangement, we proposed that simple blends of two DKPscomposed of proteinogenic α-amino acids with lipophilic sidechains and differing “functional” side chains should renderenzyme-like catalytic activity in the co-assembled state throughintermolecular cooperative effects.
Figure 1. (a) A DKP-based mimic of a catalytic dyade (b) Investigated DKPstructures
To verify this working hypothesis, we generated a minimalistichydrolase mimic (Figure 1a). In the catalytically active side ofhydrolases, imidazoles of His-residues are in close proximity toSer, Cys or Asp side-chains as the structural basis for catalyticdyads or triades. Artificial enzymes, in particular esterasesbased on the self-assembly of short oligopeptides have beendescribed frequently.[10] Commonly, lipophilic tripeptides,amphiphilic oligopeptides or amyloid-forming peptides arenecessary to generate self-assembled nanostructures withesterase-like activity.[11] Catalytically active aggregates can alsobe formed based on artificial dendrimers, by fixation of a peptide
[a] Prof. Dr. Boris J. NachtsheimInstitut für Organische und Analytische ChemieUniversität BremenLeobener Straße 7, 28359 Bremen, [email protected]
[b] Dr. Alexander J. KleinsmannInstitut für Organische ChemieUniversität TübingenAuf der Morgenstelle 18, 72076 Tübingen, Germany
onto nanoparticles,[12] or the generation of other amino acid-derived hybrids.[13],[14] The relevance of these approaches inabiogenesis is questionable due to the artificial nature of theunderlying molecular building blocks. To the best of ourknowledge simple dipeptides without non-natural syntheticmodifications are not known as minimalistic esterase mimics.Based on our recent findings towards the outstanding self-aggregation properties of DKPs, we combined His-DKP 1 andCys-DKPs (2, 3 and 4) as shown in Figure 1b. These threedifferent blends [1+2], [1+3] and [1+4] should give co-assembled nanostructures with a putative esterase activity. [15]
We first investigated the principle co-aggregation properties ofall three blends. Co-assembly was verified through hydrogelformation and subsequent investigation of the freeze-driedhydrogels via SEM (Figure 2). All three blends formed stablehydrogels through a simple heating/cooling cycle in pure waterat concentrations between 80 and 106 mM. SEM and TEMimages showed the appearance of nanofibers with varyingaverage diameters ([1+2]: 12.3 nm, [1+3]: 32.6 nm and [1+4]:21.4 nm) (for detailed analysis of representative SEM-imagessee ESI).
Figure 2. SEM- and TEM-images of co-assembled DKP-blends A: [1+2]; B:[1+3]; C: [1+4].
To investigate esterase-like activity of the co-aggregates, thehydrolysis of sodium 4-acetoxy-3-nitrobenzenesulfonate (ANBS,Figure 1a), a water- soluble derivative of the common modelcompound 2,4-dinitrophenyl acetate (DNPA), was chosen as themodel reaction. A solution of ANBS was added on top of thepreformed hydrogel and reaction kinetics were monitored byUV/Vis. Initial Job’s plot analysis revealed a maximum initial rateconstant v0 at = 0.4 for blend [1+2] and = 0.5 for blends[1+3] and [1+4] (Figure 3 - A). We then investigated the pH-dependency of the ester hydrolysis (Figure 3 - B). While with[1+2] v0 reaches a maximum at pH = 7.50, co-assemblies of[1+3] and [1+4] reached explicit maxima at slightly lower pH-values (7.25 and 7.38).
Figure 3. A: Job’s plot analysis of DKP-blends [1+2], [1+3] and [1+4]. B: pH-dependency of the initial rate constants.
In sharp contrast, v0 of pure self-assembled 1 has a maximum at6.50 which corresponds well to the pKa-value of His. For self-assembled DKP 2 v0 increases until pH 7.5 and reaches aplateau.The broad maximum of Job’s plot analysis for blend [1+2]together with the slight shift from the theoretical optimal ratio ofboth DKPs from 1:1 as observed for [1+3] and [1+4] is indicativefor a random distribution of 1 and 2 within the fibrous network(Figure 4 – A). The sharp maxima at = 0.5 for [1+3] and [1+4]on the other hand indicate a highly defined co-assembly of bothDKPs (Figure 4 - B).
Figure 4. A: Random distribution of DKPs 1 and 2 within the co-assembly. B:Alternating distribution of DKPs 1 and 3 or 4 within the co-assembly.
This defined alternating co-assembly should also result in highercatalytic performance of blends [1+3] and [1+4], as alreadyindicated by the significantly higher v0-values. Next, we wantedto compare v0 of the DKPs between the co-assembled hydrogelstate and a solution by disturbing the co-assembly processthrough DMF addition. In general, v0 should be reduced for thehydrogels since substrate availability is initially strongly limitedby diffusion processes. In addition, the accessibility of thecatalytically active His and Cys residues should be stronglylimited in the self-assembled hydrogel state throughintermolecular interactions of individual strands to form thethree-dimensional network. As an initial control experiment, we
tested the catalytic activity of pure His-DKP 1 in solution (Figure5 – A, dotted lines). Even though 1 accelerated ANBS hydrolysis,it cannot be defined as catalyst. The solution exhibits a fast initialreaction turnover in the first 10 minutes and finally approachesasymptotically a substrate conversion that matches the totalDKP concentration. Hence, only one turnover is observed. Withthe same absolute molarity, the corresponding hydrogel of 1shows an inferior substrate conversion, also with a stronglydecelerating slope finally converging to an overall conversionclose to the DKP concentration. This diminished reactivitystrongly indicates the lower accessibility of the His-residues inthe aggregated state. The observed saturation in both thesolution and the gel state of 1 indicates a quick N-acetylation ofthe His-residue followed by a very slow deacetylation, excludinga truly catalytic behaviour. Next we investigated thecorresponding blends (Figure 5, A-C). In all cases the self-assembled blended DKPs were compared with thecorresponding DKPs kept in solution as a control. As alreadyobserved for pure 1, all blended solutions, even thoughaccelerating ester hydrolysis, provided only one turnover. Realcatalytic behaviour is only observed for blended hydrogels. For[1+2] total conversion of the solution again converges to theinitial DKP-concentration while in the co-assembled stateproduct concentration exceeds DKP-concentration after 35 min(Figure 5 – B). A similar catalytic behaviour was observed for[1+3] and [1+4], although, as already indicated in Figure 3 - A,ANBS hydrolysis was throughout faster, exceeding the initialDKP concentration after 20-25 min. In sharp contrast to pure 1and [1+2], initial hydrolysis rates using the co-assembled blends[1+3] and [1+4] were comparable to the solution phaseexperiments (Figure 5 - C and D). Overall, the co-assembledblend [1+4] shows the best results in direct comparison with thecorresponding solution phase and in direct comparison to theother blends.
Figure 5. Product formation in ANBS hydrolysis, c (ANBS) = 60 mM; solidlines: reaction was performed in the self-assembled hydrogel (gel); dashedlines: reaction was performed in solution (sol) (HEPES:DMF = 1:1, V = 1.25ml); dotted lines: total DKP concentration referenced to the total volume; A: 1,pH = 6.50, c (1-hydrogel) = 92 mM; B: [1+2] (1.5:1), pH = 7.50, c = 92 mM; C:[1+3] (1:1), pH = 7.25, c = 106 mM; D: [1+4] (1:1), pH = 7.38, c = 80 mM.Product conversion was detected via UV/Vis at = 406 nm. In all experimentsbackground hydrolysis of ANBS was measured in the corresponding buffers atthe same pH with identical substrate concentration and subtracted from themeasured values.
For a more precise comparison of their catalytic efficiency, v0
was investigated in dependence of the substrate concentrationat the optimal pH and ratio for each blend. The Michaelis-Menten enzyme kinetics model was used to calculate the rateconstants for all co-assembled hydrogels. In all blends, catalystturnover became the rate-limiting step at very high substrateconcentrations, a typical behaviour for enzyme-catalysedreactions (see ESI – Table S2). Michaelis Menten constants(KM), rate constants (Kcat) as well as the catalytic efficiencies (Kcat
/ KM) are given in Table 1. The highest substrate-affinity and thehighest catalytic efficiency was once again observed for blend[1+4] (KM = 6.81). Kcat values between [1+3] and [1+4] differ onlyinsignificantly but KM is twofold higher for [1+3]. This is indicativefor a significantly weaker substrate affinity and might be theresult of sterically more favourable or multiple C-H-π-interactionsbetween 1 and 4 which subsequently leads to a closer proximityof the imidazole and thiol functionalities at the opposite site ofthe DKP.
Table 1: Summary of Michaelis-Menten kinetics.
Hydrogel KM
(10-3 M)K
cat
(10-3 s-1)Kcat/KM
(10-1 M-1 s-1)
[1+2]a 8.51 0.73 0.86
[1+3]b 12.18 1.60 1.31
[1+4]c 6.81 1.46 2.14
a 1.5:1 ratio of 1 and 2, pH = 7.50; b: 1:1 ratio of 1 and 3, pH = 7.25; c: 1:1ratio of 1 and 4, pH = 7.38.
Figure 6. Calculated structures of DKP-dimers. A: [1+2]; B: [1+3]; C: [1+4].Structures were calculated using the semi-empirical HF-3c functional in thegas phase.
To verify this hypothesis, gas phase calculations based on thelow cost Hartree-Fock/minimal basis set composite method HF-3C which shows excellent performance for noncovalentinteractions[16] have been accomplished for dimers of [1+2],[1+3] and [1+4] (Figure 6). Each energy minimized structureconfirms two central intermolecular H-bonds between the twocyclic amides with typical O-H-distances ranging from 1.74 to1.92 Å. H-Bonds between the lipophilic amino acids aresignificantly longer (1.89-1.92 Å) than the H-bonds between theHis and Cys amino acids (1.74 – 1.76 Å). As predicted, alllipophilic side chains show significant C-H-π-interactions. For[1+2] two C-H-π-interactions of the ortho- and meta protons ofthe Phe side chain in 2 and the π-system of the Phe side chainin 1 give a disordered T-shape geometry between the twobenzene rings with C-H-π-distances of 2.80 and 3.17 Å. In thecalculated structure of [1+3] two significant C-H-π-interactionbetween two C-H protons of the terminal CH3-group of the Valside chain in 3 and the benzene ring in 1 exist. The calculatedC-H-π-distances to the centroid of the benzene ring is 3.04 Åand 3.05 Å to the centroid of the C3-C4-π-bond. For [1+4], twoC-H-π-interaction are calculated with C-H-π-distance of 2.70and 2.77 Å between C-Hprotons of both terminal CH3 groupsand two distinct C-C-π-bonds of Phe. All distances are in goodagreement with typical average distances of C-H-π interactionsas observed in solid state protein structures.[17] Obviously, theadditional methylene group in the side chain of 4 allows asignificantly stronger C-H-π-interaction as implicated by shorter
C-H-centroid distances. In all blends, combination of the twocentral amide hydrogen bonds and the additional C-H-π-interaction arranges the functional imidazole and thiolfunctionalities into close proximity. Calculated S-H-N-distancesvary from 2.18 Å in [1+2] and [1+3], and 2.07 Å for [1+4]. It hasto be mentioned, that the horizontal dimension of thesecalculated single-strands (approx. 1 nm) is one dimension belowthe observed fibre thickness as observed via SEM and TEM.Thus, further inter-strand interactions must be operational whichstrongly limits the true accessibility of the catalytically activesides in the self-assembled state. Under this premise it is evenmore surprising that blends [1+3] and [1+4] show similar initialhydrolysis rates in comparison to the corresponding DKPs keptin solution. To finally verify that the calculated alternating co-assembly in [1+3] and [1+4] is based on C-H-π-interactions, 1HHRMAS NOESY experiments of the co-assembled hydrogelswere performed in D2O (Figure 7). Clearly, the strongest NOEcorrelation was observed between C-H of 3 or C-H of 4 and C-Haryl of 1.
Figure 7 A: Detail magnifications of 1H HRMAS NOE spectra in D2O of A:Hydrogel [1+3] (1:1) and B: Hydrogel [1+4] (1:1); diamonds: aromatic protonsof the Phe sidechain of 1, triangles: C-H protons of the Val sidechain of 3,circles: C-H protons of the Leu sidechain of 4.
In summary we described the most minimalistic peptide self-assembly with an enzyme-like activity. It is based on abiogenesisrelevant cyclic dipeptides solely build from the proteinogenicamino acids Phe, His, Val, Leu and Cys. A high catalytic turnoveris exclusively observed in the self-aggregated state forheterologous mixtures (blends) of His- and a Cys-containingcyclic dipeptides. Hartree-Fock calculations as well as HRMASNOE experiments strongly indicate that C-H-π-interactions aswell as intermolecular amide hydrogen bonds are responsible forthe heterologous self-aggregation which finally leads to a closeproximity of His and Cys side chain to give a catalytic dyade.These findings offer a new perceptive toward a potential role ofthe so far undervalued role of cyclic dipeptides in chemicalevolution and further implies the importance of self-assembledpeptide aggregates in the pre-Darwian evolution.
Experimental Section
Experimental Detail can be found in the Supporting Information.
Keywords: Self-assembly; Esterase; Molecular Evolution;
Hydrogel; Abiogenesis
[1] T. A. E. Jakschitz, B. M. Rode, Chem. Soc. Rev. 2012, 41, 5484.
[2] G. Danger, R. Plasson, R. Pascal, Chem. Soc. Rev. 2012, 41, 5416.
[3] a) A. Brack, Pure Appl. Chem. 1993, 65, 1143; b) A. Brack, Chem.
Biodiversity 2007, 4, 665.
[4] a) B. M. Rode, Peptides 1999, 20, 773; b) I. Ghosh, J. Chmielewski,
Curr. Opin. Chem. Biol. 2004, 8, 640; c) O. Carny, E. Gazit, FASEB J.
2005, 19, 1051; d) J. Greenwald, M. P. Friedmann, R. Riek, Angew.
Chem. Int. Ed. 2016, 55, 11609.
[5] a) A. Brack, K. W. Ehler, L. E. Orgel, J. Mol. Evol. 1976, 8, 307; b) A. L.
Weber, L. E. Orgel, J. Mol. Evol. 1978, 11, 189; c) A. L. Weber, L. E.
Orgel, J. Mol. Evol. 1979, 13, 185; d) A. L. Weber, L. E. Orgel, J. Mol.
Evol. 1979, 13, 193; e) S. Steinberg, J. L. Bada, Science 1981, 213,
544; f) M. Nagayama, O. Takaoka, K. Inomata, Y. Yamagata, Orig. Life
Evol. Biosph. 1990, 20, 249; g) J. Ying, R. Lin, P. Xu, Y. Wu, Y. Liu, Y.
Zhao, Sci. Rep. 2018, 8, 936; h) Y. Furukawa, T. Otake, T. Ishiguro, H.
Nakazawa, T. Kakegawa, Orig. Life Evol. Biosph. 2012, 42, 519.
[6] a) K. Kawamura, T. Nishi, T. Sakiyama, J. Am. Chem. Soc. 2005, 127,
522; b) K. Kawamura, H. Takeya, T. Kushibe, Adv. Space Res. 2009,
44, 267.
[7] A. Shimoyama, R. Ogasawara, Orig. Life Evol. Biosph. 2002, 32, 165.
[8] A. J. Kleinsmann, B. J. Nachtsheim, Chem. Commun. 2013, 49, 7818.
[9] a) M. S. Iyer, K. M. Gigstad, N. D. Namdev, M. Lipton, J. Am. Chem.
Soc. 1996, 118, 4910; b) M. S. Iyer, K. M. Gigstad, N. D. Namdev, M.
Lipton, Amino Acids 1996, 11, 259.
[10] a) M. Raynal, P. Ballester, A. Vidal-Ferran, P. W. N. M. van Leeuwen,
Chem. Soc. Rev. 2014, 43, 1734; b) E. Kuah, S. Toh, J. Yee, Q. Ma, Z.
Gao, Chem. Eur. J. 2016, 22, 8404; c) N. Singh, M. Kumar, J. F.
Miravet, R. V. Ulijn, B. Escuder, Chem. Eur. J. 2017, 23, 981; d) M. D.
Nothling, Z. Xiao, A. Bhaskaran, M. T. Blyth, C. W. Bennett, M. L.
Coote, L. A. Connal, ACS Catal. 2018, 9, 168; e) O. Zozulia, M. A.
Dolan, I. V. Korendovych, Chem. Soc. Rev. 2018, 47, 3621.
[11] a) K. S. Broo, H. Nilsson, J. Nilsson, A. Flodberg, L. Baltzer, J. Am.
Chem. Soc. 1998, 120, 4063; b) B. Baumeister, N. Sakai, S. Matile,
Org. Lett. 2001, 3, 4229; c) A. J. Nicoll, R. K. Allemann, Org. Biomol.
Chem. 2004, 2, 2175; d) M. O. Guler, S. I. Stupp, J. Am. Chem. Soc.
2007, 129, 12082; e) Z. Huang, S. Guan, Y. Wang, G. Shi, L. Cao, Y.
Gao, Z. Dong, J. Xu, Q. Luo, J. Liu, J. Mater. Chem. B 2013, 1, 2297; f)
C. Zhang, X. Xue, Q. Luo, Y. Li, K. Yang, X. Zhuang, Y. Jiang, J. Zhang,
J. Liu, G. Zou et al., ACS nano 2014, 8, 11715; g) M. Matsumoto, S. J.
Lee, M. L. Waters, M. R. Gagné, J. Am. Chem. Soc. 2014, 136, 15817;
h) M. Bélières, N. Chouini-Lalanne, C. Déjugnat, RSC Adv. 2015, 5,
35830; i) C. G. Pappas, I. R. Sasselli, R. V. Ulijn, Angew. Chem. Int. Ed.
2015, 54, 8119; j) M. P. Friedmann, V. Torbeev, V. Zelenay, A. Sobol, J.
Greenwald, R. Riek, PloS one 2015, 10, e0143948; k) A. J. Burton, A.
R. Thomson, W. M. Dawson, R. L. Brady, D. N. Woolfson, Nat. Chem.
2016, 8, 837; l) G. Gulseren, M. A. Khalily, A. B. Tekinay, M. O. Guler, J.
Mater. Chem. B 2016, 4, 4605; m) Y. Maeda, J. Fang, Y. Ikezoe, D. H.
Pike, V. Nanda, H. Matsui, PloS one 2016, 11, e0153700; n) M. Wang,
Y. Lv, X. Liu, W. Qi, R. Su, Z. He, ACS Appl. Mater. Interfaces 2016, 8,
14133; o) Y.-M. Wong, H. Masunaga, J.-A. Chuah, K. Sudesh, K.
Numata, Biomacromolecules 2016, 17, 3375; p) A. M. Garcia, M.
Kurbasic, S. Kralj, M. Melchionna, S. Marchesan, Chem. Commun.
2017, 53, 8110; q) R. Song, X. Wu, B. Xue, Y. Yang, W. Huang, G.
Zeng, J. Wang, W. Li, Y. Cao, W. Wang et al., J. Am. Chem. Soc. 2018.
[12] D. Zaramella, P. Scrimin, L. J. Prins, J. Am. Chem. Soc. 2012, 134,
8396.
[13] T. Darbre, J.-L. Reymond, Acc. Chem. Res. 2006, 39, 925.
[14] N. Singh, M. P. Conte, R. V. Ulijn, J. F. Miravet, B. Escuder, Chem.
Commun. 2015, 51, 13213.
[15] a) B. Escuder, F. Rodríguez-Llansola, J. F. Miravet, New J. Chem.
2010, 34, 1044; b) X. Du, J. Zhou, J. Shi, B. Xu, Chem. Rev. 2015, 115,
13165.
[16] a) R. Sure, S. Grimme, J. Comput. Chem. 2013, 34, 1672; b) J. G.
Brandenburg, M. Hochheim, T. Bredow, S. Grimme, J. Phys. Chem.
Lett. 2014, 5, 4275.
[17] a) M. Brandl, M. S. Weiss, A. Jabs, J. Sühnel, R. Hilgenfeld, J. Mol.
Biol. 2001, 307, 357; b) M. Nishio, J. Mol. Struct. 2012, 1018, 2.
download fileview on ChemRxivmanuscript_DKPs_ChemRxiv.docx (1.24 MiB)
A Minimalistic Hydrolase based
on Co-Assembled Cyclic
Dipeptides
Alexander J. Kleinsmann[b] and Boris J.
Nachtsheim*[a]
Abstract: The self-assembly of small peptides into larger aggregates
is an important process for the fundamental understanding of
abiogenesis. In this article we demonstrate that blends of cyclic
dipeptides (2,5-diketopiperazines – DKPs) bearing either histidine or
cysteine in combination with a lipophilic amino acid form highly stable
aggregates in aqueous solution with esterase-like activity. We
demonstrate that the catalytic activity is based on an intermolecular
cooperative behavior between histidine and cysteine. A high control
of the molecular arrangement of the peptide assemblies was gained
by C-H-π interactions between Phe and Leu or Val sidechains,
resulting in a significant increase in catalytic activity. These
interactions were strongly supported by Hartree-Fock calculations
and finally confirmed via 1H-NMR HRMAS NOE spectroscopy.
The transition of simple small molecular building blocks, in
particular fatty-, amino- and nucleic acids, into self-replicating
systems with an autonomous metabolism is the critical step for
the emergence of the first living cells with a minimalistic genotype
and phenotype.[1] The initial manifestation of small peptides as
enzyme precursors that could have provided important catalytic
properties for autonomous self-replicating systems is still
underexplored.[2],[3] Here, self-assembly processes that form
higher ordered aggregates from spontaneously formed small
oligopeptides through intermolecular H-bonding interactions is
believed to be an important initial step.[4a–c,2,4d] In this regard,
cyclic dipeptides (2,5-diketopiperazines – DKPs) are observed
frequently as undesired side-products during peptide formation
and under prebiotic conditions,[5] in particular as degradation
products of small oligopeptides.[6] In addition, they have been
found on the Yamato-791198 and Murchison carbonaceous
chondrites.[7] We recently demonstrated that a variety of Phe-
containing DKPs form highly stable aggregates in aqueous
solutions.[8] Their self-aggregation is the result of strong H-
bonding interactions between the cyclic amides and additional π-
π− or C-H-π-interactions between the Phe sidechains. For
proving the relevance of DKPs in the context of abiogenesis, their
catalytic properties must be elucidated. So far their catalytic
activity has only been demonstrated by Lipton and co-workers in
solution for the asymmetric Strecker reaction. [9] Presuming their
high tendency to aggregate in water into a defined molecular
arrangement, we proposed that simple blends of two DKPs
composed of proteinogenic α-amino acids with lipophilic side
chains and differing “functional” side chains should render
enzyme-like catalytic activity in the co-assembled state through
intermolecular cooperative effects.
Figure 1. (a) A DKP-based mimic of a catalytic dyade (b) Investigated DKP
structures
To verify this working hypothesis, we generated a minimalistic
hydrolase mimic (Figure 1a). In the catalytically active side of
hydrolases, imidazoles of His-residues are in close proximity to
Ser, Cys or Asp side-chains as the structural basis for catalytic
dyads or triades. Artificial enzymes, in particular esterases based
on the self-assembly of short oligopeptides have been described
frequently.[10] Commonly, lipophilic tripeptides, amphiphilic
oligopeptides or amyloid-forming peptides are necessary to
generate self-assembled nanostructures with esterase-like
activity.[11] Catalytically active aggregates can also be formed
based on artificial dendrimers, by fixation of a peptide onto
nanoparticles,[12] or the generation of other amino acid-derived
hybrids.[13],[14] The relevance of these approaches in abiogenesis
is questionable due to the artificial nature of the underlying
[a] Prof. Dr. Boris J. Nachtsheim
Institut für Organische und Analytische Chemie
Universität Bremen
Leobener Straße 7, 28359 Bremen, Germany
[b] Dr. Alexander J. Kleinsmann
Institut für Organische Chemie
Universität Tübingen
Auf der Morgenstelle 18, 72076 Tübingen, Germany
molecular building blocks. To the best of our knowledge simple
dipeptides without non-natural synthetic modifications are not
known as minimalistic esterase mimics. Based on our recent
findings towards the outstanding self-aggregation properties of
DKPs, we combined His-DKP 1 and Cys-DKPs (2, 3 and 4) as
shown in Figure 1b. These three different blends [1+2], [1+3] and
[1+4] should give co-assembled nanostructures with a putative
esterase activity.[15] We first investigated the principle co-
aggregation properties of all three blends. Co-assembly was
verified through hydrogel formation and subsequent investigation
of the freeze-dried hydrogels via SEM (Figure 2). All three blends
formed stable hydrogels through a simple heating/cooling cycle in
pure water at concentrations between 80 and 106 mM. SEM and
TEM images showed the appearance of nanofibers with varying
average diameters ([1+2]: 12.3 nm, [1+3]: 32.6 nm and [1+4]:
21.4 nm) (for detailed analysis of representative SEM-images see
ESI).
Figure 2. SEM- and TEM-images of co-assembled DKP-blends A: [1+2]; B:
[1+3]; C: [1+4].
To investigate esterase-like activity of the co-aggregates, the
hydrolysis of sodium 4-acetoxy-3-nitrobenzenesulfonate (ANBS,
Figure 1a), a water- soluble derivative of the common model
compound 2,4-dinitrophenyl acetate (DNPA), was chosen as the
model reaction. A solution of ANBS was added on top of the
preformed hydrogel and reaction kinetics were monitored by
UV/Vis. Initial Job’s plot analysis revealed a maximum initial rate
constant v0 at = 0.4 for blend [1+2] and = 0.5 for blends [1+3]
and [1+4] (Figure 3 - A). We then investigated the pH-
dependency of the ester hydrolysis (Figure 3 - B). While with [1+2]
v0 reaches a maximum at pH = 7.50, co-assemblies of [1+3] and
[1+4] reached explicit maxima at slightly lower pH-values (7.25
and 7.38).
Figure 3. A: Job’s plot analysis of DKP-blends [1+2], [1+3] and [1+4]. B: pH-
dependency of the initial rate constants.
In sharp contrast, v0 of pure self-assembled 1 has a maximum at
6.50 which corresponds well to the pKa-value of His. For self-
assembled DKP 2 v0 increases until pH 7.5 and reaches a plateau.
The broad maximum of Job’s plot analysis for blend [1+2]
together with the slight shift from the theoretical optimal ratio of
both DKPs from 1:1 as observed for [1+3] and [1+4] is indicative
for a random distribution of 1 and 2 within the fibrous network
(Figure 4 – A). The sharp maxima at = 0.5 for [1+3] and [1+4]
on the other hand indicate a highly defined co-assembly of both
DKPs (Figure 4 - B).
Figure 4. A: Random distribution of DKPs 1 and 2 within the co-assembly. B:
Alternating distribution of DKPs 1 and 3 or 4 within the co-assembly.
This defined alternating co-assembly should also result in higher
catalytic performance of blends [1+3] and [1+4], as already
indicated by the significantly higher v0-values. Next, we wanted to
compare v0 of the DKPs between the co-assembled hydrogel
state and a solution by disturbing the co-assembly process
through DMF addition. In general, v0 should be reduced for the
hydrogels since substrate availability is initially strongly limited by
diffusion processes. In addition, the accessibility of the
catalytically active His and Cys residues should be strongly limited
in the self-assembled hydrogel state through intermolecular
interactions of individual strands to form the three-dimensional
network. As an initial control experiment, we tested the catalytic
activity of pure His-DKP 1 in solution (Figure 5 – A, dotted lines).
Even though 1 accelerated ANBS hydrolysis, it cannot be defined
as catalyst. The solution exhibits a fast initial reaction turnover in
the first 10 minutes and finally approaches asymptotically a
substrate conversion that matches the total DKP concentration.
Hence, only one turnover is observed. With the same absolute
molarity, the corresponding hydrogel of 1 shows an inferior
substrate conversion, also with a strongly decelerating slope
finally converging to an overall conversion close to the DKP
concentration. This diminished reactivity strongly indicates the
lower accessibility of the His-residues in the aggregated state.
The observed saturation in both the solution and the gel state of
1 indicates a quick N-acetylation of the His-residue followed by a
very slow deacetylation, excluding a truly catalytic behaviour.
Next we investigated the corresponding blends (Figure 5, A-C). In
all cases the self-assembled blended DKPs were compared with
the corresponding DKPs kept in solution as a control. As already
observed for pure 1, all blended solutions, even though
accelerating ester hydrolysis, provided only one turnover. Real
catalytic behaviour is only observed for blended hydrogels. For
[1+2] total conversion of the solution again converges to the initial
DKP-concentration while in the co-assembled state product
concentration exceeds DKP-concentration after 35 min (Figure 5
– B). A similar catalytic behaviour was observed for [1+3] and
[1+4], although, as already indicated in Figure 3 - A, ANBS
hydrolysis was throughout faster, exceeding the initial DKP
concentration after 20-25 min. In sharp contrast to pure 1 and
[1+2], initial hydrolysis rates using the co-assembled blends [1+3]
and [1+4] were comparable to the solution phase experiments
(Figure 5 - C and D). Overall, the co-assembled blend [1+4]
shows the best results in direct comparison with the
corresponding solution phase and in direct comparison to the
other blends.
Figure 5. Product formation in ANBS hydrolysis, c (ANBS) = 60 mM; solid lines:
reaction was performed in the self-assembled hydrogel (gel); dashed lines:
reaction was performed in solution (sol) (HEPES:DMF = 1:1, V = 1.25 ml);
dotted lines: total DKP concentration referenced to the total volume; A: 1, pH =
6.50, c (1-hydrogel) = 92 mM; B: [1+2] (1.5:1), pH = 7.50, c = 92 mM; C: [1+3]
(1:1), pH = 7.25, c = 106 mM; D: [1+4] (1:1), pH = 7.38, c = 80 mM. Product
conversion was detected via UV/Vis at = 406 nm. In all experiments
background hydrolysis of ANBS was measured in the corresponding buffers at
the same pH with identical substrate concentration and subtracted from the
measured values.
For a more precise comparison of their catalytic efficiency, v0 was
investigated in dependence of the substrate concentration at the
optimal pH and ratio for each blend. The Michaelis-Menten
enzyme kinetics model was used to calculate the rate constants
for all co-assembled hydrogels. In all blends, catalyst turnover
became the rate-limiting step at very high substrate
concentrations, a typical behaviour for enzyme-catalysed
reactions (see ESI – Table S2). Michaelis Menten constants (KM),
rate constants (Kcat) as well as the catalytic efficiencies (Kcat / KM)
are given in Table 1. The highest substrate-affinity and the highest
catalytic efficiency was once again observed for blend [1+4] (KM
= 6.81). Kcat values between [1+3] and [1+4] differ only
insignificantly but KM is twofold higher for [1+3]. This is indicative
for a significantly weaker substrate affinity and might be the result
of sterically more favourable or multiple C-H-π-interactions
between 1 and 4 which subsequently leads to a closer proximity
of the imidazole and thiol functionalities at the opposite site of the
DKP.
Table 1: Summary of Michaelis-Menten kinetics.
Hydrogel KM
(10-3 M)
K cat
(10-3 s-1)
Kcat/KM
(10-1 M-1 s-1)
[1+2]a 8.51 0.73 0.86
[1+3]b 12.18 1.60 1.31
[1+4]c 6.81 1.46 2.14
a 1.5:1 ratio of 1 and 2, pH = 7.50; b: 1:1 ratio of 1 and 3, pH = 7.25; c: 1:1 ratio
of 1 and 4, pH = 7.38.
Figure 6. Calculated structures of DKP-dimers. A: [1+2]; B: [1+3]; C: [1+4].
Structures were calculated using the semi-empirical HF-3c functional in the gas
phase.
To verify this hypothesis, gas phase calculations based on the low
cost Hartree-Fock/minimal basis set composite method HF-3C
which shows excellent performance for noncovalent
interactions[16] have been accomplished for dimers of [1+2], [1+3]
and [1+4] (Figure 6). Each energy minimized structure confirms
two central intermolecular H-bonds between the two cyclic
amides with typical O-H-distances ranging from 1.74 to 1.92 Å. H-
Bonds between the lipophilic amino acids are significantly longer
(1.89-1.92 Å) than the H-bonds between the His and Cys amino
acids (1.74 – 1.76 Å). As predicted, all lipophilic side chains show
significant C-H-π-interactions. For [1+2] two C-H-π-interactions
of the ortho- and meta protons of the Phe side chain in 2 and the
π-system of the Phe side chain in 1 give a disordered T-shape
geometry between the two benzene rings with C-H-π-distances
of 2.80 and 3.17 Å. In the calculated structure of [1+3] two
significant C-H-π-interaction between two C-H protons of the
terminal CH3-group of the Val side chain in 3 and the benzene
ring in 1 exist. The calculated C-H-π-distances to the centroid of
the benzene ring is 3.04 Å and 3.05 Å to the centroid of the C3-
C4-π-bond. For [1+4], two C-H-π-interaction are calculated with
C-H-π-distance of 2.70 and 2.77 Å between C-H protons of both
terminal CH3 groups and two distinct C-C-π-bonds of Phe. All
distances are in good agreement with typical average distances
of C-H-π interactions as observed in solid state protein
structures.[17] Obviously, the additional methylene group in the
side chain of 4 allows a significantly stronger C-H-π-interaction as
implicated by shorter C-H-centroid distances. In all blends,
combination of the two central amide hydrogen bonds and the
additional C-H-π-interaction arranges the functional imidazole
and thiol functionalities into close proximity. Calculated S-H-N-
distances vary from 2.18 Å in [1+2] and [1+3], and 2.07 Å for
[1+4]. It has to be mentioned, that the horizontal dimension of
these calculated single-strands (approx. 1 nm) is one dimension
below the observed fibre thickness as observed via SEM and
TEM. Thus, further inter-strand interactions must be operational
which strongly limits the true accessibility of the catalytically active
sides in the self-assembled state. Under this premise it is even
more surprising that blends [1+3] and [1+4] show similar initial
hydrolysis rates in comparison to the corresponding DKPs kept in
solution. To finally verify that the calculated alternating co-
assembly in [1+3] and [1+4] is based on C-H-π-interactions, 1H
HRMAS NOESY experiments of the co-assembled hydrogels
were performed in D2O (Figure 7). Clearly, the strongest NOE
correlation was observed between C-H of 3 or C-H of 4 and C-
Haryl of 1.
Figure 7 A: Detail magnifications of 1H HRMAS NOE spectra in D2O of A:
Hydrogel [1+3] (1:1) and B: Hydrogel [1+4] (1:1); diamonds: aromatic protons
of the Phe sidechain of 1, triangles: C-H protons of the Val sidechain of 3,
circles: C-H protons of the Leu sidechain of 4.
In summary we described the most minimalistic peptide self-
assembly with an enzyme-like activity. It is based on abiogenesis
relevant cyclic dipeptides solely build from the proteinogenic
amino acids Phe, His, Val, Leu and Cys. A high catalytic turnover
is exclusively observed in the self-aggregated state for
heterologous mixtures (blends) of His- and a Cys-containing
cyclic dipeptides. Hartree-Fock calculations as well as HRMAS
NOE experiments strongly indicate that C-H-π-interactions as
well as intermolecular amide hydrogen bonds are responsible for
the heterologous self-aggregation which finally leads to a close
proximity of His and Cys side chain to give a catalytic dyade.
These findings offer a new perceptive toward a potential role of
the so far undervalued role of cyclic dipeptides in chemical
evolution and further implies the importance of self-assembled
peptide aggregates in the pre-Darwian evolution.
Experimental Section
Experimental Detail can be found in the Supporting Information.
Keywords: Self-assembly; Esterase; Molecular Evolution;
Hydrogel; Abiogenesis
[1] T. A. E. Jakschitz, B. M. Rode, Chem. Soc. Rev. 2012, 41, 5484.
[2] G. Danger, R. Plasson, R. Pascal, Chem. Soc. Rev. 2012, 41, 5416.
[3] a) A. Brack, Pure Appl. Chem. 1993, 65, 1143; b) A. Brack, Chem.
Biodiversity 2007, 4, 665.
[4] a) B. M. Rode, Peptides 1999, 20, 773; b) I. Ghosh, J. Chmielewski, Curr.
Opin. Chem. Biol. 2004, 8, 640; c) O. Carny, E. Gazit, FASEB J. 2005,
19, 1051; d) J. Greenwald, M. P. Friedmann, R. Riek, Angew. Chem. Int.
Ed. 2016, 55, 11609.
[5] a) A. Brack, K. W. Ehler, L. E. Orgel, J. Mol. Evol. 1976, 8, 307; b) A. L.
Weber, L. E. Orgel, J. Mol. Evol. 1978, 11, 189; c) A. L. Weber, L. E.
Orgel, J. Mol. Evol. 1979, 13, 185; d) A. L. Weber, L. E. Orgel, J. Mol.
Evol. 1979, 13, 193; e) S. Steinberg, J. L. Bada, Science 1981, 213, 544;
f) M. Nagayama, O. Takaoka, K. Inomata, Y. Yamagata, Orig. Life Evol.
Biosph. 1990, 20, 249; g) J. Ying, R. Lin, P. Xu, Y. Wu, Y. Liu, Y. Zhao,
Sci. Rep. 2018, 8, 936; h) Y. Furukawa, T. Otake, T. Ishiguro, H.
Nakazawa, T. Kakegawa, Orig. Life Evol. Biosph. 2012, 42, 519.
[6] a) K. Kawamura, T. Nishi, T. Sakiyama, J. Am. Chem. Soc. 2005, 127,
522; b) K. Kawamura, H. Takeya, T. Kushibe, Adv. Space Res. 2009, 44,
267.
[7] A. Shimoyama, R. Ogasawara, Orig. Life Evol. Biosph. 2002, 32, 165.
[8] A. J. Kleinsmann, B. J. Nachtsheim, Chem. Commun. 2013, 49, 7818.
[9] a) M. S. Iyer, K. M. Gigstad, N. D. Namdev, M. Lipton, J. Am. Chem. Soc.
1996, 118, 4910; b) M. S. Iyer, K. M. Gigstad, N. D. Namdev, M. Lipton,
Amino Acids 1996, 11, 259.
[10] a) M. Raynal, P. Ballester, A. Vidal-Ferran, P. W. N. M. van Leeuwen,
Chem. Soc. Rev. 2014, 43, 1734; b) E. Kuah, S. Toh, J. Yee, Q. Ma, Z.
Gao, Chem. Eur. J. 2016, 22, 8404; c) N. Singh, M. Kumar, J. F. Miravet,
R. V. Ulijn, B. Escuder, Chem. Eur. J. 2017, 23, 981; d) M. D. Nothling,
Z. Xiao, A. Bhaskaran, M. T. Blyth, C. W. Bennett, M. L. Coote, L. A.
Connal, ACS Catal. 2018, 9, 168; e) O. Zozulia, M. A. Dolan, I. V.
Korendovych, Chem. Soc. Rev. 2018, 47, 3621.
[11] a) K. S. Broo, H. Nilsson, J. Nilsson, A. Flodberg, L. Baltzer, J. Am. Chem.
Soc. 1998, 120, 4063; b) B. Baumeister, N. Sakai, S. Matile, Org. Lett.
2001, 3, 4229; c) A. J. Nicoll, R. K. Allemann, Org. Biomol. Chem. 2004,
2, 2175; d) M. O. Guler, S. I. Stupp, J. Am. Chem. Soc. 2007, 129, 12082;
e) Z. Huang, S. Guan, Y. Wang, G. Shi, L. Cao, Y. Gao, Z. Dong, J. Xu,
Q. Luo, J. Liu, J. Mater. Chem. B 2013, 1, 2297; f) C. Zhang, X. Xue, Q.
Luo, Y. Li, K. Yang, X. Zhuang, Y. Jiang, J. Zhang, J. Liu, G. Zou et al.,
ACS nano 2014, 8, 11715; g) M. Matsumoto, S. J. Lee, M. L. Waters, M.
R. Gagné, J. Am. Chem. Soc. 2014, 136, 15817; h) M. Bélières, N.
Chouini-Lalanne, C. Déjugnat, RSC Adv. 2015, 5, 35830; i) C. G. Pappas,
I. R. Sasselli, R. V. Ulijn, Angew. Chem. Int. Ed. 2015, 54, 8119; j) M. P.
Friedmann, V. Torbeev, V. Zelenay, A. Sobol, J. Greenwald, R. Riek,
PloS one 2015, 10, e0143948; k) A. J. Burton, A. R. Thomson, W. M.
Dawson, R. L. Brady, D. N. Woolfson, Nat. Chem. 2016, 8, 837; l) G.
Gulseren, M. A. Khalily, A. B. Tekinay, M. O. Guler, J. Mater. Chem. B
2016, 4, 4605; m) Y. Maeda, J. Fang, Y. Ikezoe, D. H. Pike, V. Nanda,
H. Matsui, PloS one 2016, 11, e0153700; n) M. Wang, Y. Lv, X. Liu, W.
Qi, R. Su, Z. He, ACS Appl. Mater. Interfaces 2016, 8, 14133; o) Y.-M.
Wong, H. Masunaga, J.-A. Chuah, K. Sudesh, K. Numata,
Biomacromolecules 2016, 17, 3375; p) A. M. Garcia, M. Kurbasic, S.
Kralj, M. Melchionna, S. Marchesan, Chem. Commun. 2017, 53, 8110;
q) R. Song, X. Wu, B. Xue, Y. Yang, W. Huang, G. Zeng, J. Wang, W. Li,
Y. Cao, W. Wang et al., J. Am. Chem. Soc. 2018.
[12] D. Zaramella, P. Scrimin, L. J. Prins, J. Am. Chem. Soc. 2012, 134, 8396.
[13] T. Darbre, J.-L. Reymond, Acc. Chem. Res. 2006, 39, 925.
[14] N. Singh, M. P. Conte, R. V. Ulijn, J. F. Miravet, B. Escuder, Chem.
Commun. 2015, 51, 13213.
[15] a) B. Escuder, F. Rodríguez-Llansola, J. F. Miravet, New J. Chem. 2010,
34, 1044; b) X. Du, J. Zhou, J. Shi, B. Xu, Chem. Rev. 2015, 115, 13165.
[16] a) R. Sure, S. Grimme, J. Comput. Chem. 2013, 34, 1672; b) J. G.
Brandenburg, M. Hochheim, T. Bredow, S. Grimme, J. Phys. Chem. Lett.
2014, 5, 4275.
[17] a) M. Brandl, M. S. Weiss, A. Jabs, J. Sühnel, R. Hilgenfeld, J. Mol. Biol.
2001, 307, 357; b) M. Nishio, J. Mol. Struct. 2012, 1018, 2.
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Supporting Information
A Minimalistic Hydrolase based on Co-Assembled
Cyclic Dipeptides
Alexander J. Kleinsmann[b] and Boris J. Nachtsheim*[a]
a Institut für Organische und Analytische Chemie, Universität Bremen, Leobener Straße 7, 28359 Bremen, Germanyb Institut für Organische Chemie, Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
S1
Table of Contents
1.1Materials.........................................................................................................................3
1.2UV/Vis experiments........................................................................................................4
1.3Synthesis........................................................................................................................6
1.3.1General procedures for the Synthesis of DKPs 1-4:.................................................6
1.3.2Cyclo[l-His-l-Phe] (DKP 1)........................................................................................6
1.3.3Cyclo[l-Cys(PMB)-l-Phe]..........................................................................................6
1.3.4Cyclo[l-Cys-l-Phe] (DKP 2).......................................................................................6
1.3.5Cyclo[l-Cys(PMB)-l-Val]............................................................................................7
1.3.6Cyclo[l-Cys-l-Val] (DKP 3)........................................................................................7
1.3.7Cyclo[l-Cys(PMB)-l-Leu]...........................................................................................7
1.3.8Cyclo[l-Cys-l-Leu] (DKP 4).......................................................................................8
1.3.9Sodium 4-hydroxy-3-nitrobenzenesulfonate.............................................................8
1.3.10Sodium 4-acetoxy-3-nitrobenzenesulfonate (ANBS)..............................................8
2Nanofiber Diameter Determination with SEM Images.........................................................10
3Enzyme Kinetics Model.......................................................................................................12
4Computational Studies........................................................................................................14
4.1General Details.............................................................................................................14
4.2Coordinates..................................................................................................................14
4.2.1[1+2].......................................................................................................................14
4.2.2[1+3].......................................................................................................................17
4.2.3[1+4].......................................................................................................................18
5NMR Spectra.......................................................................................................................21
5.11H and 13C NMR spectra of ANBS and DKPs 1-4..........................................................22
5.21H HR-MAS NOE spectra of co-assembled hydrogels..................................................28
5.31H NOE spectra of blended DKP solutions....................................................................31
6 References.........................................................................................................................34
S2
1.1 Materials
Unless otherwise stated, all chemicals were used as received from a commercial supplier.The water used for the preparation for the hydrogels and buffers was of Millipore Milli-Qgrade. Buffer solutions were stored under exclusion from light and used up within five days.MES buffer (0.25 M) was used for pH 6.25 and 6.50, HEPES buffer (0.25 M) was used frompH 6.75 to 8.50.
For scanning electron microscopy (SEM), the DKP-samples were dissolved in water of Milli-Q grade in 4 ml screw-cap vials and the warm solution was applied to pre-cooled aluminumsheets. The samples were allowed to mature at 4°C for 20 minutes, lyophilized and coatedwith a thin layer of platinum by using a Balzers SCD 050 sputter coater. A Hitachi SU8030scanning electron microscope was used to record the images of the xerogels with anaccelerating voltage of 1 kV.
Transmission electron microscopy (TEM) images were recorded with a Hitachi SU8030scanning electron microscope in STEM mode at an accelerating voltage of 30 kV. Thehydrogel samples were prepared in a 4 ml screw-cap vial, lyophilized and distributed onto aTEM grid (200 mesh copper grid) that was coated with carbon film.1H and 13C NMR spectra were recorded on a Bruker Advance 400 MHz instrument in DMSO-d6 or D2O. The 1H chemical shifts are reported as (parts per million) relative to the quintetsignal of DMSO at 2.50 ppm or to the singlet signal of 3-(trimethylsilyl)propionic-2,2,3,3-d4
acid sodium salt in D2O at 0.00 ppm. The 13C chemical shifts are reported as (parts permillion) relative to the DMSO septet at 39.43 ppm or to the singlet signal of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt in D2O at 0.00 ppm. The followingabbreviations have been used to describe splitting patterns: br=broad, s=singlet, d=doublet,t=triplet, q=quartet, qi=quintet, m=multiplet. Coupling constants J are given in Hz. 1H HR/MAS NMR NOE spectra of the hydrogels in D2O were recorded on a Bruker ARX 400MHz instrument with a 4 mm triple resonance HR/MAS probehead. The samples weremeasured at room temperature at spinning frequencies of 2.5 or 4.0 kHz.
IR spectra were recorded with a Jasco FT/IR-4100 spectrometer. UV/Vis spectra wererecorded with a Perkin-Elmer Lambda 2 UV/Vis spectrometer. Mass spectra were recordedon a Finnigan MAT95 spectrometer. High-resolution mass spectra were recorded by usingESI method with a Bruker Daltonics Apex II FT-ICR mass analyzer. Optical rotations weremeasured with sodium light on a Jasco P‐1020 polarimeter. Elemental analysis was carriedout on an Elementar Vario MICRO Cube analyzer. Melting points were determined with aBüchi B‐540 melting point analyzer.
S3
1.2 UV/Vis experiments
Kinetic experiments
For experiments comprising DKPs with cysteine, the buffer was degassed before DKPaddition by sparging argon through. DKPs were dissolved in the corresponding buffer (0.25M) by heating in a 4 ml screw cap vial, the solution was allowed to reach room temperatureand the pH of the mixture was checked and adjusted if necessary. The mixture was heatedagain, dipped in a water bath at 50°C for a few seconds to avoid burst of the vial andimmediately cooled in an ice bath for 20 minutes. The hydrogel was subsequently allowed toreach room temperature for additional 20 minutes. The corresponding buffer (950 µl) wascarefully added on top of the hydrogel followed by substrate solution (50 µl in DMF) andgently mixed by agitation. For higher substrate concentrations (more than 60 mM in thebuffer-DMF solution), applied for the substrate concentration dependent initial ratemeasurements, the substrate was directly dissolved in buffer/DMF (950:50 µl) mixtures andplaced on top of hydrogel. The reaction mixture was gently agitated every 10-15 secondsduring the measurements and samples were diluted with the corresponding buffer. Theproduct formation was measured at 406 nm and extinction coefficients for the calculation ofthe product concentration were determined experimentally (Table S1). All experiments wererepeated at least three times and the average value was used for further calculations. In allexperiments background hydrolysis of ANBS was measured in the corresponding buffers atthe same pH with identical substrate concentration and subtracted from the measuredvalues.
Table S 1: Experimentally determined pH dependent extinction coefficients of ANBS in 0.25 M HEPES buffer(measured at = 406 nm).
pH valueMolar Extinction Coefficient
[m2/mol]6.25 335.556.50 358.116.75 378.107.00 400.467.25 410.007.38 413.72
S4
7.50 416.787.75 420.068.00 424.138.25 426.798.50 427.49
S5
1.3 Synthesis
1.3.1 General procedures for the Synthesis of DKPs 1-4:
Boc‐His(Boc)‐OH was synthesized following the procedure by Castro and co‐workers.[1]
DKP 1 and the S-4-methoxybenzyl (PMB) protected precursors of DKP 2-4 were preparedaccording to our previously reported procedure.[2]
The PMB-protected DKPs (2.50 mmol) were dissolved in 25 ml of a TFA/H2O/phenol (90:5:5)mixture and refluxed for one hour. The reaction mixture concentrated to a fourth part andcooled to 4°C and an excess of diethylether was added. The precipitate collected by filtration,washed three times with diethylether and dried under reduced pressure. The crude productwas dissolved in a dithiothreitol (DTT) solution (100 ml, 10 mM in THF/H2O (8:2)) and 1.5 mlof saturated sodium bicarbonate solution were added. The solution was stirred for 30minutes and subsequently THF was removed under reduced pressure. The resultingsuspension was cooled in an ice bath and the precipitate was collected by filtration andwashed with water.
1.3.2 Cyclo[L-His-L-Phe] (DKP 1)
DKP 1 was synthesized according to the general procedure usingBoc‐Phe‐OH (23.70 g, 89.33 mmol, 2.0 eq.) and Boc‐His(Boc)‐OH(47.62 g, 134.00 mmol, 3.0 eq.). The crude product solution in THF-water (8:1) was concentrated under reduced pressure until all volatilecomponents were removed. The aqueous suspension was cooled in
an ice bath under strong stirring and an excess of diethylether was added. The solid wascollected by filtration and washed with water and diethylether. The crude product wasrecrystallized from water, subsequently recrystallized from methanol and dried under reducedpressure. DKP 1 was received as a white solid (6.48 g, 22.79 mmol, 51%).1H NMR (400 MHz, D2O) δ 8.52 (s, 1H), 7.46-7.33 (m, 3H), 7.19 (d, J=6.9 Hz, 2H), 6.95 (s,1H), 4.54-4.44 (m, 1H), 4.26-4.12 (m, 1H), 3.17 (dd, J=14.0, 3.2 Hz, 1H), 2.98 (dd, J=14.0,4.4 Hz, 1H), 2.53 (dd, J=15.3, 4.4 Hz, 1H), 1.89 (dd, J=15.3, 7.6 Hz, 1H). 13C NMR (101MHz, D2O) δ 172.0, 170.7, 137.7, 136.7, 133.5 (2x), 131.9 (2x), 130.7, 130.1, 120.7, 58.6,56.2, 41.2, 31.2. MS (FAB) calculated for C15H17N4O2 [M+H]+: m/z 285.1, found: 285.2.[2]
1.3.3 Cyclo[L-Cys(PMB)-L-Phe]
Cyclo[L-Cys(PMB)-L-Phe] was synthesized according to the generalprocedure using Boc‐Phe‐OH (17.46 g, 65.83 mmol, 2.0 eq.) andBoc‐Cys(PMB)‐OH (33.72 g, 98.75 mmol, 3.0 eq.). The crude productsolution in THF-water (8:1) was concentrated under reduced pressureat 50°C, until the THF was removed completely, and stirred strongly
at 4 °C. The product was collected by filtration, washed with water and dried under reducedpressure. Cyclo[L-Cys(PMB)-L-Phe] was received as a white solid (5.82 g, 15.72 mmol,48%).1H NMR (400 MHz, DMSO-d6) δ 8.28-8.17 (m, 1H), 8.07-7.95 (m, 1H), 7.28-7.14 (m, 7H),6.85 (d, J=8.6 Hz, 2H), 4.23-4.14 (m, 1H), 3.87-3.80 (m, 1H), 3.72 (s, 3H), 3.51 (s, 2H), 3.14(dd, J=13.5, 4.7 Hz, 1H), 2.94 (dd, J=13.5, 4.9 Hz, 1H), 2.31 (dd, J=13.7, 3.9 Hz, 1H), 1.50(dd, J=13.7, 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.2, 165.9, 158.1, 136.3, 130.2(2x), 130.1, 130.0 (2x), 128.1 (2x), 126.7, 113.7, 55.4, 55.0, 53.6, 39.5, 35.2, 34.7. MS (FAB)calculated for C20H23N2O3S [M+H]+: m/z 371.1, found 371.2.[2]
1.3.4 Cyclo[L-Cys-L-Phe] (DKP 2)
S6
Cyclo[L-Cys(PMB)-L-Phe] (1.00 g, 2.70 mmol) was deprotectedaccording to the general procedure. DKP 2 was received as a white solid(0.58 g, 2.30 mmol, 85%).1H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.04 (s, 1H), 7.30-7.14 (m,5H), 4.24-4.17 (m, 1H), 3.93-3.86 (m, 1H), 3.15 (dd, J=13.6, 4.3 Hz, 1H),
2.92 (dd, J=13.6, 4.9 Hz, 1H), 2.36-2.29 (m, 1H), 2.02-1.92 (m, 1H), 1.74 (t, J=8.5 Hz, 1H).13C NMR (101 MHz, DMSO-d6) δ 166.5, 165.5, 136.3, 130.3 (2x), 128.1 (2x), 126.6, 55.9,55.2, 38.2, 27.4. MS (FAB) calculated for C12H14N2O2SNa [M+H]+: m/z 251.1, found: 251.1.[2]
1.3.5 Cyclo[L-Cys(PMB)-L-Val]
Cyclo[L-Cys(PMB)-L-Val] was synthesized according to the generalprocedure using Boc‐Cys(PMB)‐OH (16.55 g, 48.48 mmol, 2.0 eq.) andBoc‐Val‐OH (15.80 g, 72.72 mmol, 3.0 eq.). The crude product solution inTHF-water (8:1) was concentrated under reduced pressure at 50°C, untilthe THF was removed completely and stirred strongly at 4 °C. The product
was collected by filtration, washed with water and dried under reduced pressure. Cyclo[L-Cys(PMB)-L-Val] was received as a white solid (3.36 g, 10.43 mmol, 43%).1H NMR (400 MHz, DMSO-d6) δ 8.18-8.11 (m, 1H), 8.11-8.06 (m, 1H), 7.27-7.20 (m, 2H),6.90-6.81 (m, 2H), 4.19-4.13 (m, 1H), 3.76-3.65 (m, 6H), 2.84 (dd, J=13.8, 4.9 Hz, 1H), 2.75(dd, J=13.8, 4.1 Hz, 1H), 2.26-2.15 (m, 1H), 0.98 (d, J=7.1 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 166.8, 166.5, 158.2, 130.3, 130.0 (2x), 113.8 (2x), 59.4,55.0, 54.2, 35.5, 34.4, 31.2, 18.6, 17.3. HRMS (ESI) calculated for C16H22N2O3SNa [M+Na]+:m/z 345.12433, found: 345.12463. FT‐IR (cm‐1): 3188.7, 3090.4, 3055.7, 2969.8, 1655.6,1608.8, 1582.8, 1510.5, 1444.9, 1303.2, 1241.5, 1175.9, 1107.9, 1031.7, 831.7, 787.8,676.9. α 22
D = -20.8 (c=0.67, DMSO). Mp: 219 -222 °C (decomp.).
1.3.6 Cyclo[L-Cys-L-Val] (DKP 3)
Cyclo[L-Cys(PMB)-L-Val] (1.00 g, 3.10 mmol) was deprotected according tothe general procedure. DKP 3 was received as a white solid (0.43 g, 2.13mmol, 69%).1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 8.03 (s, 1H), 4.20-4.15 (m, 1H),3.78-3.74 (m, 1H), 2.96-2.86 (m, 1H), 2.80-2.72 (m, 1H), 2.28-2.19 (m, 1H),
2.18-2.11 (m, 1H), 0.97 (d, J=7.2 Hz, 3H), 0.87 (d, J=6.8 Hz, 3H). 13C NMR (101 MHz,DMSO-d6) δ 167.3, 166.4, 59.2, 55.3, 30.7, 26.7, 18.5, 17.2. HRMS (ESI) calculated forC8H14N2O2SNa [M+Na]+: m/z 225.06682, found: 225.06702. FT‐IR (cm‐1): 3182.9, 3049.9,2956.8, 2872.5, 2564.4, 1656.6, 1446.8, 1340.3, 1241.5, 1196.6, 1175.9, 1160.9, 1122.9,1106.9, 973.4, 837.0, 800.3, 758.9, 737.2, 651.8. α 22
D = -89.9 (c=1.0, DMSO). Mp: 251-253°C (decomp.).
1.3.7 Cyclo[L-Cys(PMB)-L-Leu]
Cyclo[L-Cys(PMB)-L-Leu] was synthesized according to the generalprocedure using Boc‐Leu‐OH*H2O (12.47 g, 50.00 mmol, 2 eq.) and Boc‐Cys(PMB)‐OH (25.61 g, 75.00 mmol, 3 eq.). The crude product solution inTHF-water (8:1) was concentrated under reduced pressure at 50°C untilthe product did start to precipitate. Subsequently, an excess of n-hexanewas added and the suspension was strongly stirred at 4°C until it became
homogeneous. The product was collected by filtration and dried under reduced pressure.Cyclo[L-Cys(PMB)-L-Leu] was received as a white solid (2.27 g, 6.76 mmol, 27%).
S7
1H NMR (400 MHz, DMSO-d6) δ 8.39-8.30 (m, 1H), 8.14-8.06 (m, 1H), 7.27-7.18 (m, 2H),6.91-6.82 (m, 2H), 4.17-4.09 (m, 1H), 3.83-3.76 (m, 1H), 3.75-3.68 (m, 5H), 2.86 (dd, J=13.9,4.6 Hz, 1H), 2.71 (dd, J=14.0, 4.1 Hz, 1H), 1.94-1.83 (m, 1H), 1.72-1.58 (m, 2H), 0.90-0.84(m, 6H). 13C NMR (101 MHz, DMSO-d6) δ 167.9, 166.1, 158.2, 130.1, 130.0 (2x), 113.8 (2x),55.0, 54.6, 52.6, 44.1, 35.5, 34.6, 23.4, 23.1, 21.8. HRMS (ESI) calculated forC17H24N2O3SNa [M+Na]+: m/z 359.13998, found: 359.14022. FT‐IR (cm‐1): 3183.4, 3046.0,2956.3, 2895.1, 1662.8, 1610.3, 1511.0, 1458.9, 1328.7, 1300.3, 1243.9, 1175.4, 1097.3,1033.2, 827.3, 762.7, 690.4. α 22
D = -26.9 (c=1.0, DMSO). Mp: 177-180°C (decomp.).[3]
1.3.8 Cyclo[L-Cys-L-Leu] (DKP 4)
Cyclo[L-Cys(PMB)-l-Leu] (0.90 g, 2.67 mmol) was deprotected according tothe general procedure. DKP 4 was received as a white solid (0.41 g, 1.89mmol, 71%).1H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.02 (s, 1H), 4.20-4.11 (m, 1H),3.86-3.76 (m, 1H), 2.95-2.86 (m, 1H), 2.78-2.69 (m, 1H), 2.24-2.16 (m, 1H),
1.94-1.81 (m, 1H), 1.72-1.63 (m, 1H), 1.61-1.52 (m, 1H), 0.91-0.82 (m, 6H). 13C NMR (101MHz, DMSO-d6) δ 168.4, 166.0, 55.5, 52.4, 43.0, 26.9, 23.4, 23.0, 21.9. HRMS (ESI)calculated for C9H16N2O2SNa [M+Na]+: m/z 239.08247, found: 239.08257. FT‐IR (cm‐1):3480.4, 3430.7, 3185.4, 3045.5, 2956.3, 2873.4, 1662.3, 1459.9, 1386.1, 1364.9, 1324.9,1117.6, 1094.9, 971.5, 846.6, 810.9, 681.7. α 22
D = -66.4 (c=1.0, DMSO). Mp: 221-224°C(decomp.).[3]
1.3.9 Sodium 4-hydroxy-3-nitrobenzenesulfonate
5.95 g of 2-Nitrophenol (42.77 mmol, 1.0 eq.) were dissolved in 50 ml drycarbon disulfide under argon atmosphere, the flask was sealed with septumand an injection needle was applied to provide hydrogen chloride removalduring the reaction. The solution was cooled in an ice bath for 10 minutes
and 2.84 ml of chlorosulfonic acid (42.77 mmol, 1.0 eq) were added dropwise. The reactionmixture was stirred for another 10 minutes at 4°C, allowed to reach room temperature andstirred for another 20 minutes. The resulting precipitate was collected by filtration andwashed three times with hexane. The received 4-hydroxy-3-nitrobenzenesulfonic acid wasevaporated to dryness, suspended in 10 ml of water and cooled in an ice bath. Thesuspension was treated carefully with saturated sodium bicarbonate solution under stirringuntil pH = 4-5 was reached. The crude product was collected by filtration and washed withacetone (x3) which was collected separately from the aqueous filtrate and disposed. Theaqueous filtrate was concentrated and the former process was repeated. The resultingsodium 4-hydroxy-3-nitrobenzenesulfonate was dried under vacuum, dissolved in boilingacetic acid and filtered while hot. The product was recrystallized from acetic acid/benzene,washed with benzene and dried under vacuum to yield 8.35 g (34.63 mmol, 80%) of a yellowsolid.1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.02 (d, J=2.1 Hz, 1H), 7.72 (dd, J=8.6, 2.1Hz, 1H), 7.09 (d, J=8.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 152.3, 139.8, 135.3,132.5, 122.2, 118.6. Anal. calcd. for C6H4NNaO6S: C 29.88, H 1.67, N 5.81, S 13.29 %;found: C 29.48, H 1.59, N 5.86, S 13.18. HRMS (ESI) calculated for [M]-: m/z 217.97648,found: 217.97636.[4]
1.3.10 Sodium 4-acetoxy-3-nitrobenzenesulfonate (ANBS)
3.00 g of sodium 4-hydroxy-3-nitrobenzenesulfonate (12.44 mmol) weresuspended in 75 ml of acetic anhydride and refluxed for 15 hours. The
S8
reaction mixture was cooled in an ice bath and the precipitate was collected by filtration. Thecrude product was first recrystallized from methanol and washed with ethanol, then fromacetic acid/benzene and washed with benzene to yield 2.97 g (10.49 mmol, 84%) of sodium4-acetoxy-3-nitrobenzenesulfonate (ANBS) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J=2.0 Hz, 1H), 7.98 (dd, J=8.4, 2.1 Hz, 1H), 7.44 (d,J=8.3 Hz, 1H), 2.34 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.47, 147.19, 143.08,140.61, 132.27, 125.26, 122.46, 20.55. Anal. calcd. for C8H6NNaO7S: C 33.93, H 2.14, N4.95, S 11.32; found: C 33.89, H 2.03, N 5.10, S 11.39. HRMS (ESI) calculated for [M]-: m/z259.98705, found: 259.98723.[5]
S9
2 Nanofiber Diameter Determination with SEM Images
Figure S 1: SEM image of xerogel 1; nanofiber diameter measurement represented.
Figure S 2: SEM image of xerogel 2; nanofiber diameter measurement represented.
Figure S 3: SEM image of xerogel [1+2]; nanofiber diameter measurement represented.
S10
Figure S 4: SEM image of xerogel [1+3]; nanofiber diameter measurement represented.
Figure S 5: SEM image of xerogel [1+4]; nanofiber diameter measurement represented.
S11
3 Enzyme Kinetics Model
Table S 2: Initial rates of ANBS hydrolysis depending on the substrate concentration catalyzed by co-assembledhydrogels in 0,25 M HEPES buffer. Hydrogel [1+2] (1 : 1,5 (n/n); pH = 7.50; c (hydrogel) = 92 mM); c (totalvolume) = 18.4 mM. Hydrogel [1+3] (1 : 1 (n/n); pH = 7.25; c (hydrogel) = 92 mM); c (total volume) = 18.4 mM.Hydrogel [1+4] (1 : 1 (n/n); pH = 7.38; c (hydrogel) = 80 mM); c (total volume) = 16.0 mM.
c(ANBS) [mM]Hydrogel [1+2]
v0 [mM/min]Hydrogel [1+3]
v0 [mM/min]Hydrogel [1+4]
v0 [mM/min]10 0,436 - 0,8320 0,559 1,10 1,0440 0,665 1,33 1,2060 0,712 1,48 1,2590 0,727 1,53 1,30
100 0,739 1,58 1,31120 - 1,60 -
Figure S 6: Initial rates of ANBS hydrolysis plotted against the substrate concentration (top) and thecorresponding Lineweaver-Burk plots (bottom). (A+D) Hydrogel [1+2] (1 : 1,5 (n/n); pH = 7.50; c (hydrogel) = 92mM); c (total volume) = 18.4 mM. (B+E) Hydrogel [1+3] (1 : 1 (n/n); pH = 7.25; c (hydrogel) = 92 mM); c (totalvolume) = 18.4 mM. (C+F) Hydrogel [1+4] (1 : 1 (n/n); pH = 7.38; c (hydrogel) = 80 mM); c (total volume) = 16.0mM.
S12
S13
4 Computational Studies
4.1 General Details
Structure calculations were performed by employing Orca 4.1 software. [6] The structures were m withHartree-Fock/minimal basis set composite HF-3C.[7] Harmonic vibrational frequency calculations wereperformed at the same level of theory to characterize the nature of the stationary points along thereaction coordinates. For all optimized structures, no imaginary frequencies were found. The density-fitting RI-J approach for the Coulomb integrals was applied for the geometry optimization andfrequencies calculations.[8]
4.2 Coordinates
4.2.1 [1+2]
C 1.64660503369833 0.71829933000705 -1.90593427631273
N 1.51085892125377 1.12244835856581 -0.51646986848090
C 0.49120335538741 0.65671641644035 0.29850071672167
C -0.76141256304809 0.08532458316751 -0.43849829500061
N -0.80426038401843 0.42266615110435 -1.84278883062057
C 0.26156562499085 0.81207849065733 -2.60898212354175
O 0.14506600487860 1.19681601682318 -3.75635481500535
O 0.52826847469930 0.71759068264233 1.50684583579751
C 2.24686600003256 -0.72674248574964 -2.01018698364000
C 2.22998722331780 -1.31462212691193 -3.41390569566369
C 3.38520267562308 -1.34044922642227 -4.18179804028309
S14
C 3.37792773783534 -1.91167888874418 -5.44785824146546
C 2.20828512800229 -2.45806994752320 -5.95636128191051
C 1.04723897661953 -2.43256199642000 -5.19245912368440
C 1.06053683727092 -1.86422875081248 -3.92846295495896
C -0.90084367074323 -1.45358017602799 -0.16946421449641
C -2.15025340913867 -1.98746354175916 -0.83009240704359
C -2.32107306535719 -2.82956289610631 -1.88261674016280
N -3.67974727908135 -2.96195132200242 -2.16439094859990
C -4.31049892969649 -2.21923348684530 -1.30261535880759
N -3.41994905392255 -1.59781548466811 -0.45201522524692
H 2.30666563370193 1.41471520736752 -2.42259604715242
H 2.32392078098049 1.48850030553561 -0.04274696864257
H -1.61218089536888 0.56368510113811 0.05093224475531
H -1.70280454156177 0.33434037917251 -2.32678265432067
H 1.68768745165817 -1.37097449298647 -1.34485920496635
H 3.26568704304435 -0.68515745623729 -1.64249078773981
H 4.29677595417095 -0.91991274192387 -3.78656452927043
H 4.28349486926205 -1.92831780616441 -6.03614554454839
H 2.19879150475456 -2.89968833977376 -6.94193679400725
H 0.13015606795288 -2.85278690229709 -5.58074394302694
H 0.15567587453081 -1.85133268149010 -3.33990205242821
H -0.04920309311397 -1.98946170471788 -0.56997971860710
H -0.92178771529744 -1.61098363789290 0.90731965992073
H -1.56520619796956 -3.33723892163097 -2.45766405124006
H -5.38238510919346 -2.07805474508916 -1.22711445569619
C -2.67875437973291 1.77582514653845 -6.46517560247304
N -2.40598680660589 1.17868691828695 -5.17044007620811
C -3.26056064146341 0.34706036425195 -4.50025764194374
C -4.52172734487733 -0.10163601505665 -5.29509073497136
N -5.03302230717474 1.02446937882951 -6.05292413424848
C -4.20487679324940 1.85658522537133 -6.79185904925004
O -4.62422693869664 2.62285144277786 -7.62900308922521
O -3.09260143726117 -0.01700759538637 -3.35135571193316
C -1.90475414213058 1.09280358676869 -7.64465894928756
H -2.34496209935111 2.81536636414360 -6.43785974182801
H -1.49964198066446 1.36154728471496 -4.73299948559716
H -6.01764663810652 1.05890546976589 -6.27178483747476
C -4.24497281422948 -1.33202499635258 -6.22937654142929
S -3.08318153417440 -2.58872163696337 -5.56254336830732
H -3.81192126324700 -0.97027108082032 -7.15762793563683
H -5.20242315441674 -1.78287845980319 -6.47958978616223
H -3.68784819668220 -2.77706531063072 -4.33442453198125S15
H -5.25640032714105 -0.39498945197652 -4.54549520913612
H -2.33744036566487 1.43817099133037 -8.57772831480508
H -2.01707827804209 0.01814493201129 -7.58134657538323
H -3.64540674106952 -0.96425357554104 0.30382580222999
C -0.43200309346510 1.46890849224963 -7.58071898193529
C 0.01852431427203 2.61845883884382 -8.21996142343962
C 0.46071114814154 0.70375681513235 -6.84703578095652
C 1.34888774748983 2.99579470371671 -8.13088266276857
C 1.79409808697255 1.08147235376961 -6.74963596201109
C 2.24108373953952 2.22655212446780 -7.39218532556238
H -0.67681817775375 3.21652078736218 -8.78871115948479
H 0.11894334044251 -0.18588447740971 -6.34045669340166
H 1.69086991896143 3.88789850237495 -8.63389327583496
H 2.47351646451629 0.47755937523624 -6.16727520694033
H 3.27689241370978 2.52089544857156 -7.31872570123643
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4.2.2 [1+3]
C 1.51808168679845 0.50609125942352 -1.39576731152621
N 1.27898821320987 0.50893154661653 0.03598748747933
C 0.19557256100104 -0.10049023445895 0.64581783549675
C -0.97191708971099 -0.48272904122959 -0.31322188831350
N -0.95096725106844 0.28240603202795 -1.53743299296353
C 0.18436897403405 0.69057988751241 -2.18184952632196
O 0.17533348219148 1.20087327818711 -3.28685578619174
O 0.13572219785788 -0.30615637476712 1.83702144929831
C 2.28751359728117 -0.77769244827668 -1.85345512562246
C 2.46357849733890 -0.87164628487239 -3.36280981075565
C 3.12191780839248 0.13258666061324 -4.06417796153411
C 3.30018945181920 0.03019810804905 -5.43466777349874
C 2.82485067220024 -1.08291721991230 -6.11907507509162
C 2.16653621713943 -2.08631935776667 -5.42435597828764
C 1.98514382471633 -1.97631475650606 -4.05160401046036
C -0.97850735798576 -2.03278190092548 -0.57124764800826
C -2.03142593142656 -2.37313001962193 -1.59797148892286
C -1.94435995220407 -2.50138891338098 -2.94896764312529
N -3.21307394666411 -2.71587375836415 -3.47991228473689
C -4.04344105011451 -2.71620625061655 -2.47753600252334
N -3.36984223259846 -2.51057458594599 -1.29223795973998
H 2.12812868164652 1.37247393543175 -1.65142260534031
H 2.04461420597247 0.76228202227112 0.64291286650292
H -1.89129854816945 -0.23945003054444 0.22123069335938
H -1.84430911271107 0.38789088770125 -2.03249995505374
H 1.76623846923743 -1.65423455211575 -1.49650343065263
H 3.25974101054383 -0.76197740434301 -1.36778328698693
H 3.49572588351675 0.99744386133513 -3.53897180875097
H 3.81029732984543 0.81708978090183 -5.97086910475793
H 2.96284530809469 -1.16234429367950 -7.18720200802656
H 1.78931354964160 -2.95193446026367 -5.94896440501196
H 1.47347872939257 -2.75921161938749 -3.51424132596844
H -0.01418019428908 -2.35032258806873 -0.94738319272127
H -1.15599898660833 -2.54465070565767 0.37122469977012
H -1.06397780138249 -2.44400510478601 -3.56711475163641
H -5.11763440950286 -2.85498547345242 -2.52528579465346
C -2.20966306362052 1.66073507701354 -6.14092698111233
N -2.29171053775885 1.36218548323930 -4.72294794497013
C -3.41005876295533 1.00467747475856 -4.03589600250354
C -4.78871150279694 1.25857881560107 -4.71094832653188
S17
N -4.68985733653564 1.94097076440993 -5.97966268530409
C -3.54540819310990 2.21925744159463 -6.70646139458826
O -3.59212237125628 2.85088479885196 -7.73927283818731
O -3.38748963257435 0.56350250777811 -2.89614654289976
C -1.75716129195863 0.43163579164860 -7.00472426844734
H -1.46378832205219 2.44508515960845 -6.27395991878365
H -1.39825011064139 1.27677520652569 -4.23155095880085
H -5.54924674681106 2.27067877154249 -6.39226965393414
C -5.61406015455317 -0.06745652934129 -4.78017612992917
S -5.00467965499002 -1.28806368042201 -6.00954315036964
H -6.63759155704826 0.18835919368132 -5.04470640037539
H -5.62837798165340 -0.50285930471746 -3.78372180945850
H -4.13893671721996 -1.95221500927912 -5.15562849576705
H -5.31730473931509 1.90379399344188 -4.00053021083875
H -2.61549571232459 -0.21144689884182 -7.15556881370443
H -3.77444580112171 -2.45337030892007 -0.36598119726549
C -0.66084018988513 -0.37315608624677 -6.26833952900768
H -1.05121612449416 -0.82198503436724 -5.36395073974449
H -0.29577827457560 -1.16377479973191 -6.91205495358927
H 0.17522184638222 0.26380882102616 -6.00276994693171
C -1.24183542374906 0.92306255212584 -8.37794586066276
H -0.34561462208194 1.51742130314967 -8.24676997712270
H -1.00207149497481 0.07494689564952 -9.00808065027702
H -1.98976401375987 1.53181571909356 -8.87482771361471
4.2.3 [1+4]
C 1.47850710030332 0.50201924909328 -1.53622521535497
N 1.23924653047458 0.65579066667923 -0.11240904452897
C 0.15967770544121 0.10711218153402 0.55955360965050
C -1.02108332259017 -0.34230496337237 -0.35205414025225
N -0.99144440301670 0.30087227867214 -1.64316204087760
C 0.14511921571087 0.60794422240282 -2.33894821788326
O 0.13336853299350 0.97499088220466 -3.49954096534541
O 0.11166693932376 0.00616980838943 1.76471945858018
C 2.23704754053863 -0.82876518937435 -1.85497911983508
C 2.54564022295821 -0.99478241688103 -3.33524783497589
C 3.45362600631817 -0.14825228068369 -3.96094622685852
C 3.76093341969903 -0.31650791910291 -5.30172228341751
C 3.16118902928677 -1.33703481363769 -6.03061137864201
C 2.24885353839546 -2.17931083132692 -5.41237908197909
C 1.94251374985078 -2.00514246250405 -4.06930509986620
C -1.06466734629375 -1.90962907964498 -0.46283964638807S18
C -2.14042628097165 -2.32148944703151 -1.43999551554543
C -2.09547251539826 -2.49878214743011 -2.78795138177836
N -3.37306421982840 -2.77582498236602 -3.26563014563870
C -4.16860064094444 -2.76453322975583 -2.23548515114698
N -3.46245914436103 -2.49069801945172 -1.08305270612425
H 2.09808951565713 1.33013871600503 -1.88148627606805
H 2.01159256378052 0.95598637735665 0.46399236838662
H -1.93215356347306 -0.02951212640313 0.16019050901942
H -1.88660474838412 0.37065178440942 -2.14185856026298
H 1.65342265823816 -1.66944841218875 -1.50832602818283
H 3.16190646061345 -0.81714384238701 -1.28555246244494
H 3.92606301308199 0.64010113683923 -3.39598519182061
H 4.46890008072462 0.34486042129266 -5.77959739709833
H 3.40272467306569 -1.47063701375341 -7.07452204644644
H 1.77516883204692 -2.97155425080132 -5.97341904258332
H 1.23759124076948 -2.66575335784509 -3.58943335389859
H -0.11120332636699 -2.28209048494368 -0.81620709355945
H -1.24130542358017 -2.32713214172316 0.52495677242369
H -1.24136253118350 -2.43202233379424 -3.44082119631249
H -5.23809074611081 -2.94183133971965 -2.23921668377559
C -2.41709993303089 1.56237368431517 -6.29417871381452
N -2.41134033056882 1.17913091383462 -4.89237008630293
C -3.49506805506614 0.90631310537132 -4.11678093150394
C -4.91214775288887 1.15063389054633 -4.70853692953523
N -4.89750052109886 1.82220364465318 -5.98578910839818
C -3.81249597328456 2.00038445654240 -6.82290836899372
O -3.92827419190739 2.48949940442842 -7.92475500488377
O -3.41596612601344 0.50830819195693 -2.96386152811655
C -1.88565331982364 0.44261630305657 -7.23555452810267
H -1.77343727545277 2.43555035355742 -6.41385543436104
H -1.49223881147953 1.06163933990125 -4.45813967994234
H -5.78805693843355 2.09670063026023 -6.37254057561486
C -0.43734010885813 -0.00533598549686 -6.92496580247341
C -5.70370792957513 -0.19786280993492 -4.73480462900809
S -5.04078681453522 -1.42707397775902 -5.92929202091185
H -6.73458369472504 0.02062002512167 -5.00385682719349
H -5.70166085568093 -0.60971737219978 -3.72832677788577
H -4.23741410031718 -2.09694863728581 -5.01987039248546
H -5.41280336141007 1.78929804084652 -3.97361780393816
C -0.03216469619443 -1.08806654889696 -7.95649316885740
H -0.40436046975220 -0.44138285689019 -5.93312573837931
C 0.55726962930490 1.17949885275122 -6.96942325035702S19
H -1.93409046825465 0.82727430634316 -8.24824399446056
H -0.70592749363537 -1.93522825459316 -7.90493837729301
H 0.97580750187682 -1.43285916194549 -7.76776848076259
H -0.07328601242384 -0.67914853935013 -8.95892100825461
H 0.48125412128622 1.70051181178572 -7.91625855379707
H 1.56990306278718 0.81301497534995 -6.85418930068344
H 0.36567355528972 1.87988937602027 -6.16608408020875
H -2.54890035225954 -0.41157569939251 -7.17312522220129
H -3.83613164064384 -2.41410010165347 -0.14526887044718
S20
5 NMR Spectra
S21
5.1 1
H and 1
3
C NMR spectra of ANBS and DKPs 1-4
S22
Figure S 7: 1H NMR spectrum of DKP 1*TFA with 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt in D2O.
Figure S 8: 13C NMR spectrum of DKP 1*TFA with 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt in D2O.
S23
Figure S 9: 1H NMR spectrum of DKP 2 DMSO-d7.
Figure S 10: 13C NMR spectrum of DKP 2 DMSO-d7.
S24
Figure S 11: 1H NMR spectrum of DKP 3 DMSO-d7.
Figure S 12: 13C NMR spectrum of DKP 3 DMSO-d7.
S25
Figure S 13: 1H NMR spectrum of DKP 4 DMSO-d7.
Figure S 14: 13C NMR spectrum of DKP 4 DMSO-d7.
S26
Figure S 15: 1H NMR spectrum of ANBS DMSO-d7.
Figure S 16: 13C NMR spectrum of ANBS DMSO-d7.
S27
5.2 1
H HR-MAS NOE spectra of co-assembled hydrogels
For 1H HR-MAS NOESY experiments 75 µl of the warm DKP solution in D2O weretransferred to a spinner and cooled in an ice bath for 20 minutes. The hydrogel in the spinnerwas allowed to reach room temperature for 20 minutes and subsequently measured at roomtemperature.
S28
Figure S 17: 1H HR-MAS NOE spectrum of a hydrogel [1+2] (1:1) in D2O. Total DKP concentration: 56 mM (1.50wt%); Spinning frequency: 2.5 kHz; Diamonds indicate DKP 1 protons; Triangles indicate DKP 2 protons.
Figure S 18: 1H HR-MAS NOE spectrum of hydrogel [1+3] (1:1) in D2O. Total DKP concentration: 80 mM (1.94 wt%); Spinning frequency: 2.5 kHz; Diamonds indicate DKP 1 protons; Triangles indicate DKP 3 protons.
S29
Figure S 19: 1H HR-MAS NOE spectrum of hydrogel [1+4] (1:1) in D2O. Total DKP concentration: 80 mM (2.00 wt%); Spinning frequency: 4.0 kHz; Diamonds indicate DKP 1 protons; Triangles indicate DKP 4 protons.
S30
5.3 1
H NOE spectra of blended DKP solutions
As reference for 1H HR-MAS NOESY experiments, less concentrated DKP solutions in DMF-d7/D2O (1:1) were measured correspondingly.
S31
Figure S 20: 1H NOE spectrum of a DKP 1/2 (1:1) solution in DMF-d7/D2O (1:1). Total DKP concentration: 18 mM;Diamonds indicate DKP 1 protons; Triangles indicate DKP 2 protons. Circles indicate DMF protons.
Figure S 21: 1H NOE spectrum of a DKP 1/3 (1:1) solution in DMF-d7/D2O (1:1). Total DKP concentration: 18 mM;Diamonds indicate DKP 1 protons; Triangles indicate DKP 3 protons.
S32
Figure S 22: 1H NOE spectrum of a DKP 1/4 (1:1) solution in DMF-d7/D2O (1:1). Total DKP concentration: 18 mM;Diamonds indicate DKP 1 protons; Triangles indicate DKP 4 protons.
S33
6 References
[1] D. Le Nguyen, R. Seyer, A. Heitz, B. Castro, J. Chem. Soc., Perkin Trans. 1 1985, 1025.[2] A. J. Kleinsmann, B. J. Nachtsheim, Chem. Commun. 2013, 49, 7818.[3] T. Furukawa, T. Akutagawa, H. Funatani, T. Uchida, Y. Hotta, M. Niwa, Y. Takaya, Bioorg.
Med. Chem. 2012, 20, 2002.[4] A. Arcelli, C. Concilio, J. Org. Chem. 1996, 61, 1682.[5] T. C. Bruice, J. Katzhendler, L. R. Fedor, J. Am. Chem. Soc. 1968, 90, 1333.[6] a) F. Neese, WIREs Comput. Mol. Sci. 2012, 2, 73; b) F. Neese, WIREs Comput. Mol.
Sci. 2018, 8, e1327.[7] R. Sure, S. Grimme, J. Comput. Chem. 2013, 34, 1672.[8] a) K. Eichkorn, O. Treutler, H. Öhm, M. Häser, R. Ahlrichs, Chem. Phys. Lett. 1995, 240,
283; b) K. Eichkorn, F. Weigend, O. Treutler, R. Ahlrichs, Theor. Chem. Acc. 1997, 97, 119.
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