Cross-Linked Collagen Triple Helices by Oxime Ligation - Journal of

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Cross-Linked Collagen Triple Helices by Oxime Ligation Nina B. Hentzen,† Linde E. J. Smeenk,† Jagna Witek,‡ Sereina Riniker,‡ and Helma Wennemers*,† †

Laboratorium für Organische Chemie, ETH Zürich, D-CHAB, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland Laboratorium für Physikalische Chemie, ETH Zürich, D-CHAB, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland

‡

S Supporting Information *

ABSTRACT: Covalent cross-links are crucial for the folding and stability of triple-helical collagen, the most abundant protein in nature. Cross-linking is also an attractive strategy for the development of synthetic collagen-based biocompatible materials. Nature uses interchain disulfide bridges to stabilize collagen trimers. However, their implementation into synthetic collagen is difficult and requires the replacement of the canonical amino acids (4R)-hydroxyproline and proline by cysteine or homocysteine, which reduces the preorganization and thereby stability of collagen triple helices. We therefore explored alternative covalent cross-links that allow for connecting triple-helical collagen via proline residues. Here, we present collagen model peptides that are cross-linked by oxime bonds between 4-aminooxyproline (Aop) and 4-oxoacetamidoproline placed in coplanar Xaa and Yaa positions of neighboring strands. The covalently connected strands folded into hyperstable collagen triple helices (Tm ≈ 80 °C). The design of the cross-links was guided by an analysis of the conformational properties of Aop, studies on the stability and functionalization of Aop-containing collagen triple helices, and molecular dynamics simulations. The studies also show that the aminooxy group exerts a stereoelectronic effect comparable to fluorine and introduce oxime ligation as a tool for the functionalization of synthetic collagen.

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INTRODUCTION Collagen, the most abundant protein in mammals, confers mechanical stability to skin and bones.1,2 The importance of biomimetic materials for applications in tissue engineering and cell culture systems has sparked considerable interest in synthetic collagen peptides that mimic the triple-helical architecture of collagen.3,4 The formation of collagen triple helices from short, synthetically accessible peptides (20−30 mers) is, however, hampered by the entropically unfavorable assembly of three single strands.1−3 Several studies utilized noncovalent interactions to preorganize collagen model peptides (CMPs) toward triple-helix formation.5−11 An attractive and more robust alternative is covalent cross-linking of collagen strands.12,13 In early work, tripodal templates were covalently installed at the N- or C-termini to create collagen triple helices.12 These architectures have limitations since the template blocks the continued assembly of triple helices into elongated structures. Inspired by natural cystine-knots, the formation of disulfide bonds between cysteine- or homocysteine-containing CMPs has been explored.13 This strategy requires, however, the replacement of the canonical amino acids proline (Pro) and (4R)-hydroxyproline (Hyp) by cysteine or homocysteine, which reduces backbone preorganization and significantly compromises triple-helix stability.14 In addition, disulfide bonds are not stable in reducing environments, which could further limit their versatility. We therefore sought a covalent cross-linking method that leaves the fundamental architecture of the collagen triple helix intact. Collagen single strands adopt a polyproline II (PPII)-like helical structure and consist of repeating Xaa-Yaa-Gly units with © 2017 American Chemical Society

the imino acids Pro and Hyp as the most frequent residues in the Xaa and Yaa positions, respectively.1 Three strands form a triple helix that is stabilized by interstrand H-bonds between the N−H of Gly and the CO of the residue in the Xaa position. The strands coil around each other with a singleresidue stagger. As a result, Xaa, Yaa, and Gly residues from the three adjacent strands are present in each cross section along the helix axis (Figure 1a). Crystal structures of collagen triple helices show that the C(4) carbons of Pro in Xaa positions and Hyp in Yaa positions of neighboring strands are in close

Figure 1. (a) General structure of the collagen triple helix (adapted from pdb 3B0S). (b) Cross-linking of collagen single strands by oxime ligation. Received: July 18, 2017 Published: September 5, 2017 12815

DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820

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Journal of the American Chemical Society proximity.15 We therefore perceived these positions as ideal connection points. Yet, linking these residues is challenging since an appropriate covalent tether must not impose constraints on the native triple-helical architecture. In addition, the reactive groups need to be easy to introduce into CMPs and the bond-forming reaction should be robust and chemoselective. We envisioned an oxime linkage between 4‑aminooxyproline (Aop)16 and 4-oxoacetamidoproline (Alp) residues to fulfill these requirements (Figure 1b). Before creating the cross-linked architecture we needed to understand the fundamental properties of these building blocks in the context of triple helical CMPs. We therefore embarked first on studying the stereoelectronic effect of the aminooxy and oxime moieties and, second, on analyzing their influence on collagen triple-helix stability. These studies showed that aminooxy and oxime moieties have similar stereoelectronic properties to other electron-withdrawing groups (e.g., F, N3, OH) and introduce oxime ligation as a tool for functionalizing synthetic collagen. This knowledge was ultimately used to generate hyperstable triple helices that are cross-linked via oxime bonds between proline residues in adjacent strands.

Figure 2. (a) Cis and trans conformers of Ac-Pro-OMe. (b) Ktrans:cis values of (4R)- and (4S)-configured Ac-Aop-OMe and Ac-Oxp-OMe in D2O. (c) C(4)-exo and C(4)-endo puckering of proline derivatives with electron-withdrawing groups (EWG) at C(4). (d) Trans:cis ratios of the respective Hyp, Azp, and Flp derivatives. Values taken from ref 22 (Hyp, Flp) and ref 23 (Azp).

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RESULTS AND DISCUSSION Stereoelectronic Effect of Aminooxy and Oxime Groups. Whereas oxime formation has become popular for chemoselective ligations,17−20 surprisingly little is known about the conformation-directing effects of aminooxy and oxime groups. We therefore started by analyzing the influence of aminooxy and oxime groups on the trans:cis amide ratio and the pyrrolidine ring pucker of Aop. Both are important factors for the stability of collagen triple helices and critically influenced by the steric and stereoelectronic properties of substituents at C(4).1,7−11,21 The acetylated methyl esters of (4R)Aop and (4S)Aop, as well as the corresponding oxime ligation products with butanal, (4R)Oxp and (4S)Oxp (Figure 2b), were used as model systems to allow for comparison with previous studies on analogous Hyp, azidoproline (Azp), and fluoroproline (Flp) derivatives.22,23 NMR spectroscopic analysis in D2O revealed that both Ac(4R)Aop-OMe (1R) and Ac-(4R)Oxp-OMe (2R) adopt a C(4)-exo ring pucker, as evidenced by the 1H−1H coupling constants, and have trans:cis ratios of 6.7:1 around the amide bond. In contrast, the (4S)-configured derivatives Ac-(4S)AopOMe (1S) and Ac-(4S)Oxp-OMe (2S) adopt a C(4)-endo puckered conformation with trans:cis ratios of 2.2:1 and 2.1:1 (Figure 2b and c).24 These conformational properties are remarkably similar to those of corresponding (4R)- and (4S)configured Hyp, Azp, and Flp derivatives (Figure 2d).22,23 For those proline derivatives it is known that the electronwithdrawing group (EWG) exerts a stereoelectronic gauche effect. In the case of the (4R)-configured derivatives this leads to a C(4)-exo pucker and a n→π* interaction between the adjacent carbonyl moieties that stabilizes the trans amide (Figure 2c, left).25−27 In (4S)-configured derivatives, the gauche effect favors a C(4)-endo pucker, which positions the EWG and the proline carbonyl group on the same face of the pyrrolidine ring. The transannular repulsion of these two substituents results in a Ψ-angle that is not ideal for the n→π* interaction (Figure 2c, right).10 Hence, the trans-amide content is higher in the (4R)- compared to the (4S)-configured proline derivatives. Since the triple-helical structure is assembled from strands with all-trans peptide bonds, a high trans:cis ratio contributes to the entropic stabilization of collagen.1,2 The similarity of the

conformational properties of Aop and Oxp to those of Flp, Hyp, and Azp shows that the aminooxy and oxime groups exert comparable stereoelectronic effects to those of the EWGs F, N3, and OH. Notable is also that the conformational properties of the Oxp derivatives are essentially identical to those of the Aop parent compounds, indicating that the stereoelectronic effects of the aminooxy and oxime groups are equivalent. Functionalization and Thermal Stability of Collagen Triple Helices Containing Aop and Oxp. On the basis of the conformational properties of Aop and Oxp, we expected that the incorporation of these residues into collagen triple helices would result in similar thermal stabilities to those previously observed for CMPs containing other EWGs on Pro rings.7,8 For example, substitution of the canonical (4R)Hyp by (4R)Aop in the Yaa position of collagen should not affect triple-helical stability. To probe this hypothesis, we prepared the 21-mer CMP 1YR containing (4R)Aop in the Yaa position of the middle repeat unit (Table 1). Circular dichroism (CD) spectroscopic analysis showed a maximum at 225 nm, which is characteristic of collagen triple helices. Thermal denaturation using CD spectroscopy as a monitoring tool revealed a melting temperature of Tm = 46 °C (Table 1, entry 1), which is slightly higher than that previously determined for the triple helix derived from CMP 3 with the natural (4R)Hyp residue (Tm = 43 °C, Table 1, entry 9).10 To further probe the effect of Aop on collagen, we also prepared and analyzed CMPs 1XR, 1XS, and 1YS bearing (4R)Aop in the Xaa position and (4S)Aop in the Xaa or Yaa position, respectively (Table 1). CD spectra and thermal denaturation studies showed that all of these CMPs formed triple helices with relative thermal stabilities of Tm = 33−39 °C (Table 1, entries 2−4). CMP 1YS, with a (4S)Aop residue in the Yaa position, formed the least stable triple helix, as expected based on the low trans:cis ratio of (4S)Aop and the C(4)-endo pucker that is disfavored in the Yaa position (Table 1, entry 2). Next, we evaluated the derivatization of the Aop-containing CMPs and reacted 1YR, 1YS, 1XR, and 1XS with butanal in the 12816

DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820

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Journal of the American Chemical Society Table 1. Tm Values As Determined by Thermal Denaturation Studiesa of the Triple Helices Derived from CMPs Ac[ProHypGly]3-Xaa-Yaa-Gly-[ProHypGly]3-NH2 Containing Aop and Oxp in the Middle Repeat Unit

a b

entry

CMP

Xaa-Yaa-Gly

Tm (°C)a

1 2 3 4 5 6 7 8 9

1YR 1YS 1XR 1XS 2YR 2YS 2XR 2XS 3

Pro-(4R)Aop-Gly Pro-(4S)Aop-Gly (4R)Aop-Pro-Gly (4S)Aop-Pro-Gly Pro-(4R)Oxp-Gly Pro-(4S)Oxp-Gly (4R)Oxp-Pro-Gly (4S)Oxp-Pro-Gly Pro-(4R)Hyp-Gly

46 33 38 39 41 31 39 34 43b

Determined at a heating rate of 1 °C/100 s (±1 °C) at 200 μM. Value from ref 10.

presence of aniline in an aqueous solution of citric acid.17 Oxime formation proceeded quantitatively to the functionalized CMPs 2YR, 2YS, 2XR, and 2XS, as determined by HPLC analysis.28 CD spectroscopic studies showed that all of these CMPs form triple helices, and heating of the solutions revealed melting temperatures in the range of Tm = 31−41 °C (Table 1, entries 5−8). Notably, the thermal stability of the trimers derived from CMPs 1YS and 1XR hardly changed upon oxime functionalization (ΔTm ≤ 2 °C, Table 1, entries 2/6 and 3/7), but the triple helix of CMP 2YR bearing (4R)Oxp in the Yaa position is less stable (Tm = 41 °C) than the respective Aopcontaining triple helix (Tm = 46 °C) (Table 1, entries 1/5). A similarly destabilizing effect of the oxime was observed for the triple helices derived from CMPs 1XS and 2XS with (4S)Aop and (4S)Oxp, respectively, in the Xaa position (ΔTm = 5 °C, Table 1, entries 4/8). Since the stereoelectronic properties of the aminooxy and oxime groups were found to be comparable, these differences can arise only from differences in their steric bulk. Models of collagen triple helices show that the substituents point to the outside of the triple helices in case of (4S)Aop/Oxp in the Yaa and (4R)Aop/Oxp in the Xaa positions (Figures S1 and 3a, right), which is reflected by comparable thermal stabilities of trimers of 2YS and 2XR and the parent Aop-containing CMPs 1XR and 1YS. In contrast, the substituents point toward the neighboring strand in the case of (4R)Aop/Oxp in the Yaa and (4S)Aop/Oxp in the Xaa positions (Figures S2 and 3a, left), corroborating that the steric repulsion between the bulky oxime moiety and the residues of the adjacent strand disturbs the supramolecular assembly and lowers the melting temperature of the Oxp-containing trimers (2YR and 2XS) compared to those of triple helices containing the less bulky Aop (1YR and 1XS). These findings are in agreement with previous studies on collagen triple helices bearing amidoproline residues, where similar steric effects have been observed.10 Yet, the comparison of the Aop- and Oxpcontaining collagen trimers is unique since the residues differ only in their steric demand and therefore allow for a distinction between stereoelectronic and steric effects. Design of Cross-Linked Collagen Triple Helices. Encouraged by the facile introduction and derivatization of Aop in CMPs, we pursued oxime ligation to covalently connect

Figure 3. (a) Molecular models showing the relative orientations of (4S)Aop in Xaa and (4R)Alp in Yaa (left) and (4R)Aop in Xaa and (4R)Alp in Yaa (right) of neighboring strands. (b) Most populated conformation in the 100 ns MD simulations of CMPs 4SR (left) and 4RR (right). (c) Distribution of the interstrand Cα−Cα distances between coplanar residues in the MD simulations of 4SR and 4RR. The distances are given as a difference with respect to the average interstrand Cα−Cα distance in the triple helix of the unlinked reference CMP 3.

three single strands and explore the stabilizing effect on their triple-helical assembly. As a complementary residue to Aop, we chose 4-oxoacetamidoproline (Figure 1b). This proline derivative contains a glyoxylyl group that is known to react with alkoxyamines29 and is accessible from the coupling of serine to aminoproline and subsequent oxidative cleavage by sodium periodate.28,30 In accordance with the Xaa-Yaa-Gly register of collagen triple helices, we envisioned linking Aop and Alp in coplanar Xaa and Yaa positions of adjacent strands. To minimize linker strain and avoid backbone distortions, we further reasoned that the reactive groups in the Xaa and Yaa positions should point 12817

DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820

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Journal of the American Chemical Society toward each other. On the basis of crystal structures of collagen, this should best be realized with a (4S)-configured proline derivative in the Xaa position and a (4R)-substituted proline derivative in the Yaa position (Figure 3a, left).31 This design was further encouraged by the decrease in the thermal stabilities of CMPs 2XS and 2YR compared to 1XS and 1YR, which corroborates that derivatization of (4S)Aop in Xaa and (4R)Aop in Yaa by oxime ligation leads to destabilization of the triple helices due to steric interference of the oxime group with the neighboring strand. Thus, we reasoned that the proximity of these substituents at C(4) to the neighboring strand could be favorable for covalent cross-links. This left the choice of the relative positions of Alp and Aop. We ruled out incorporation of (4S)Alp in Xaa since previous studies showed that (4S)-amidoprolines in this position interfere with the interstrand H-bonding by a transannular H-bond and cause a destabilization of the triple helix.10 In contrast, a C(4)-exo puckered (4R)Alp in Yaa and a (4S)Aop residue placed in the Xaa position of the neighboring strand meet the constraints imposed by the triple-helical fold and orient the reactive aldehyde and aminooxy groups toward each other (Figure 3a, left). As a control for our design and possible alternative, we also considered a linkage between (4R)Aop in Xaa and (4R)Alp in Yaa of adjacent strands (Figure 3a, right), which should result in a more strained linker, but would point toward the outside of the helix. Molecular dynamics (MD) simulations further supported our design. Simulations using the GROMOS software package32 and the GROMOS 54A733 force field were performed on triple helices connected by oxime bonds between (4R)Alp residues in Yaa positions and (4S)Aop and (4R)Aop residues, respectively, in Xaa positions of adjacent strands. For the calculations, and later the synthesis, the cross-links were installed between the leading and middle strand as well as the middle and lagging strand (Figure 4b). Details on the parametrization of the linkers and the simulations are given in the Supporting Information. In the case of the triple helix featuring connections between (4S)Aop in Xaa and (4R)Alp in Yaa (CMP 4SR), hardly any backbone constraints compared to non-cross-linked collagen triple helices with the Pro-Hyp-Gly repeat unit (CMP 3) were predicted by the 100 ns simulations, as judged by comparison of interstrand Cα−Cα distances between coplanar residues in adjacent strands (Figure 3b, left, and 3c).34 In contrast, for the diastereoisomeric CMP 4RR, the crosslinks between (4R)Alp in Yaa and (4R)Aop in Xaa induced significant backbone distortions, resulting in the formation of a bulge between the connected strands (Figure 3b, right). This linker thus caused a significantly larger deviation of the distances between coplanar residues from the reference triple helix (Figure 3c, bottom). Interestingly, the cross-linkers did not significantly affect the relative positions of the nonconnected leading and lagging strands. Furthermore, the calculations predicted a less favorable intramolecular potential energy of CMP 4RR compared to CMP 4SR.28 Synthesis and Thermal Stability of Cross-Linked Collagen Triple Helices 4SR and 4RR. To validate our design experimentally, we prepared cross-linked CMP 4SR by connecting (4R)Alp residues in Yaa and (4S)Aop residues in Xaa positions (Figure 4). As a control, we also synthesized diastereoisomeric CMP 4RR with (4R)Aop residues in Xaa. First, CMP 5, bearing the (4R)Alp residue and serving as the leading strand, was ligated with the doubly functionalized middle strand 6S, which contains not only the reactive

Figure 4. (a) Aop- and Alp-functionalized CMPs. (b) Synthesis of cross-linked CMPs 4SR and 4RR; for details see the Supporting Information. (c) Thermal denaturation of triple helices formed by CMPs 4SR and 4RR (66 μM solutions).

aminooxy group but also an unreactive, serine-masked Alp residue. Subsequent sodium periodate mediated oxidation yielded the reactive Alp residue, and the newly formed glyoxylyl group was used for the second oxime bond formation with the (4S)Aop containing lagging strand 7S. The oxime ligations as well as the oxidation proceeded smoothly to afford the cross-linked CMP 4SR. CMP 4RR, featuring cross-links between (4R)Aop and (4R)Alp, was prepared via an analogous route from peptides 5, 6R, and 7R. Next, we evaluated the assembly state of the cross-linked CMPs. Solutions of CMPs 4RR and 4SR displayed the characteristic CD signature of a collagen triple helix. In addition, gel permeation chromatography showed only one signal at a retention time corresponding to the triple helices observed for the homotrimer of CMP 3. 28 Thermal denaturation experiments using CD spectroscopy as a 12818

DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820

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Journal of the American Chemical Society monitoring tool revealed melting temperatures of Tm > 80 °C for 4SR and Tm = 58 °C for 4RR (Figure 4c). These relative stabilities of both cross-linked peptides are significantly higher than those found for the self-assembled homotrimeric helices formed by all of the previously examined CMPs. As further references we also determined the melting temperatures of the triple helices formed by CMPs 5, 6R, 6S, 7R, and 7S and found Tm values in the range 31−39 °C (Table S1). Thus, the covalent oxime cross-links led to an increase of the relative stabilities of the triple helices by 20 to >40 °C. The difference in melting temperatures of ΔTm > 20 °C between CMPs 4SR and 4RR shows that the absolute stereochemistry of the linkage point significantly influences the thermal stability of the crosslinked triple helix. As predicted, the cross-linked triple helix with (4S)-configured Aop residues in the Xaa positions is significantly more stable compared to the isomeric trimer with a (4R)Aop residue. Thus, the unfavorable sterics of the substituent in the homotrimeric triple helix formed by CMPs 2YR and 2XS are overcome by their integration into a covalent tether.

(3) (a) Fields, G. B. Org. Biomol. Chem. 2010, 8, 1237. (b) Yu, S. M.; Li, Y.; Kim, D. Soft Matter 2011, 7, 7927. (c) Strauss, K.; Chmielewski, J. Curr. Opin. Biotechnol. 2017, 46, 34. (d) Moore, A. N.; Hartgerink, J. D. Acc. Chem. Res. 2017, 50, 714. (4) Chattopadhyay, S.; Raines, R. T. Biopolymers 2014, 101, 821. (5) (a) Tanrikulu, I. C.; Forticaux, A.; Jin, S.; Raines, R. T. Nat. Chem. 2016, 8, 1008. (b) Sarkar, B.; O’Leary, L. E. R.; Hartgerink, J. D. J. Am. Chem. Soc. 2014, 136, 14417. (c) Jalan, A. A.; Demeler, B.; Hartgerink, J. D. J. Am. Chem. Soc. 2013, 135, 6014. (d) Brodsky, B.; Thiagarajan, G.; Madhan, B.; Kar, K. Biopolymers 2008, 89, 345. (e) Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 2683. (6) (a) Zhang, Y.; Herling, M.; Chenoweth, D. M. J. Am. Chem. Soc. 2016, 138, 9751. (b) Zhang, Y.; Malamakal, R. M.; Chenoweth, D. M. J. Am. Chem. Soc. 2015, 137, 12422. (c) Dai, N.; Etzkorn, F. A. J. Am. Chem. Soc. 2009, 131, 13728. (7) (a) Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Nature 1998, 392, 666. (b) Holmgren, S. K.; Bretscher, L. E.; Taylor, K. M.; Raines, R. T. Chem. Biol. 1999, 6, 63. (c) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777. (d) Jenkins, C. L.; McCloskey, A. I.; Guezei, I. A.; Eberhardt, E. S.; Raines, R. T. Biopolymers 2005, 80, 1. (e) Kotch, F. W.; Guzei, I. A.; Raines, R. T. J. Am. Chem. Soc. 2008, 130, 2952. (f) Cadamuro, S. A.; Reichold, R.; Kusebauch, U.; Musiol, H.-J.; Renner, C.; Tavan, P.; Moroder, L. Angew. Chem., Int. Ed. 2008, 47, 2143. (g) Erdmann, R. S.; Wennemers, H. Org. Biomol. Chem. 2012, 10, 1982. (8) Erdmann, R. S.; Wennemers, H. J. Am. Chem. Soc. 2010, 132, 13957. (9) Siebler, C.; Erdmann, R. S.; Wennemers, H. Angew. Chem., Int. Ed. 2014, 53, 10340. (10) (a) Erdmann, R. S.; Wennemers, H. Angew. Chem., Int. Ed. 2011, 50, 6835. (b) Erdmann, R. S.; Wennemers, H. J. Am. Chem. Soc. 2012, 134, 17117. (11) (a) Shoulders, M. D.; Hodges, J. A.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8112. (b) Shoulders, M. D.; Satyshurb, K. A.; Forest, K. T.; Raines, R. T. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 559. (12) (a) Kwak, J.; De Capua, A.; Locardi, E.; Goodman, M. J. Am. Chem. Soc. 2002, 124, 14085. (b) Horng, J.-C.; Hawk, A. J.; Zhao, Q.; Benedict, E. S.; Burke, S. D.; Raines, R. T. Org. Lett. 2006, 8, 4735. (c) Byrne, C.; McEwan, P. A.; Emsley, J.; Fischer, P. M.; Chan, W. C. Chem. Commun. 2011, 47, 2589. (d) Melacini, G.; Feng, Y.; Goodman, M. J. Am. Chem. Soc. 1996, 118, 10359. (13) (a) Ottl, J.; Moroder, L. J. Am. Chem. Soc. 1999, 121, 653. (b) Kotch, F. W.; Raines, R. T. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3028. (c) Boulègue, C.; Musiol, H.-J.; Götz, M. G.; Renner, C.; Moroder, L. Antioxid. Redox Signaling 2008, 10, 113. (d) Boudko, S. P.; Engel, J.; Okuyama, K.; Mizuno, K.; Bächinger, H. P.; Schumacher, M. A. J. Biol. Chem. 2008, 283, 32580. (e) Tanrikulu, I. C.; Raines, R. T. J. Am. Chem. Soc. 2014, 136, 13490. (14) Persikov, A. V.; Ramshaw, J. A.; Kirkpatrick, A.; Brodsky, B. Biochemistry 2000, 39, 14960. (15) (a) Bella, B.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994, 266, 75. (b) Okuyama, K.; Miyama, K.; Mizuno, K.; Bächinger, H. P. Biopolymers 2012, 97, 607. (16) For original work on Aop, see: Liu, F.; Stephen, A. G.; Fisher, R. J.; Burke, T. R. Bioorg. Med. Chem. Lett. 2008, 18, 1096. See also: Pandey, A. K.; Naduthambi, D.; Thomas, K. M.; Zondlo, N. J. J. Am. Chem. Soc. 2013, 135, 4333. (17) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem., Int. Ed. 2006, 45, 7581. (18) For reviews, see: (a) Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Chem. - Eur. J. 2014, 20, 34. (b) Collins, J.; Xiao, Z.; Müllner, M.; Connal, L. A. Polym. Chem. 2016, 7, 3812. (c) Agten, S. M.; Dawson, P. E.; Hackeng, T. M. J. Pept. Sci. 2016, 22, 271. (19) For examples, see: (a) Schmidt, P.; Stress, C.; Gillingham, D. Chem. Sci. 2015, 6, 3329. (b) Haney, C. M.; Loch, M. T.; Horne, W. S. Chem. Commun. 2011, 47, 10915. (c) Bahta, M.; Liu, F.; Kim, S.; Stephen, A. G.; Fisher, R. J.; Burke, T. R. Nat. Protoc. 2012, 7, 686.

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CONCLUSIONS In summary, we have developed an oxime linker that covalently connects short collagen peptides between Aop and Alp proline residues placed in coplanar Xaa and Yaa positions within triple helices. This linkage enabled access to nondistorted triple helices with remarkable thermal stabilities. The study put forth the value of Aop as a handle for cross-linking and for the facile functionalization of collagen peptides. The work also highlighted the utility of this residue for the adjustment of triplehelical stabilities by the choice of the stereochemistry at C(4). Together, these findings open new opportunities for the design of functional collagen-based materials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07498. Details on the syntheses and analyses of the presented compounds as well as on the simulations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Sereina Riniker: 0000-0003-1893-4031 Helma Wennemers: 0000-0002-3075-5741 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Fonds National de la Recherche Luxembourg (FNR) for an AFR Ph.D. Fellowship for N.H. and the Swiss National Science Foundation (grants SNF 200021_159858 (H.W.) and 200021_159713 (S.R.)) for financial support.

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REFERENCES

(1) Shoulders, M. D.; Raines, R. T. Annu. Rev. Biochem. 2009, 78, 929. (2) (a) Bella, J. Biochem. J. 2016, 473, 1001. (b) Engel, J.; Bächinger, H. P. Top. Curr. Chem. 2005, 247, 7. 12819

DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820

Article

Journal of the American Chemical Society (d) Canne, L. E.; Ferré-D’Amaré, A. R.; Burley, S. K.; Kent, S. B. H. J. Am. Chem. Soc. 1995, 117, 2998. (20) (a) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523. (b) Jencks, W. P. Prog. Phys. Org. Chem. 1964, 2, 63. (21) Siebler, C.; Erdmann, R. S.; Wennemers, H. Chimia 2013, 67, 891. (22) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777. (23) Sonntag, L.-S.; Schweizer, S.; Ochsenfeld, C.; Wennemers, H. J. Am. Chem. Soc. 2006, 128, 14697. (24) In the case of the Oxp derivatives E/Z isomers in a ratio of 1.5:1 of the oxime moiety were observed. (25) (a) Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188. (b) Choudhary, A.; Gandla, D.; Krow, G. R.; Raines, R. T. J. Am. Chem. Soc. 2009, 131, 7244. (26) Newberry, R. W.; Raines, R. T. Acc. Chem. Res. 2017, 50, 1838. (27) Wilhelm, P.; Lewandowski, B.; Trapp, N.; Wennemers, H. J. Am. Chem. Soc. 2014, 136, 15829. (28) For details, see the Supporting Information. (29) El-Mahdi, O.; Melnyk, O. Bioconjugate Chem. 2013, 24, 735. (30) (a) Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992, 3, 138. (b) Haney, C. M.; Horne, W. S. Chem. - Eur. J. 2013, 19, 11342. (31) Measurements of the distances between the γ-carbons (Cγ−Cγ) of neighboring Xaa-Yaa pairs in crystal structures of CMPs reveal an average distance of ∼4.5 Å that would readily be spanned by the envisioned oxime tether (∼7 Å in an extended conformation). (32) Schmid, N.; Christ, C. D.; Christen, M.; Eichenberger, A. P.; van Gunsteren, W. F. Comput. Phys. Commun. 2012, 183, 890. (33) Schmid, N.; Eichenberger, A. P.; Choutko, A.; Riniker, S.; Winger, M.; Mark, A. E.; van Gunsteren, W. F. Eur. Biophys. J. 2011, 40, 843. (34) In the case of 4SR, both E- and Z-configured oximes are possible in the linker without inducing a bulge between the strands. In the case of 4RR, both the E- and the Z-configured oxime linker induce backbone distortions. Note that both E- and Z-configured oximes were used as starting structures.

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DOI: 10.1021/jacs.7b07498 J. Am. Chem. Soc. 2017, 139, 12815−12820