Roman S. Erdmann

Importance of Ring Puckering versus Interstrand Hydrogen Bonds for the Conformational Stability of Collagen

The fibrous protein collagen is the most abundant protein in mammals and plays a crucial role both in numerous cellular activities and as a structural protein. Understanding the factors that govern the conformational stability of collagen is therefore important. In addition, there is a growing interest in collagen-based functional materials and as a result the development of synthetic collagen that bears functionalizable groups is important. 3] Collagen is built up of single strands that form triple helices which then further assemble into bundles and fibres. The single strands consist of repeating Xaa–Yaa–Gly units with all amide bonds in trans conformation. Proline (Pro) is most often found in the Xaa position and (4R)-hydroxyproline (Hyp) in the Yaa position. Within the triple helix the three strands are held together by hydrogen bonds between the NH group of glycine (Gly) of one strand and the C=O group of Pro of the adjacent strand (Figure 1a). Crystal structures show C(4)-endo ring puckers of the Pro residues in the Xaa and C(4)-exo ring puckers of the Hyp residues in the Yaa positions (Figure 1 a). 4] For the dihedral angles Y (NiCia-Ci-Ni+1) that are responsible for the directionality of the collagen strands average values of approximately 1558 (Xaa) and approximately 1508 (Yaa) are observed. Studies with collagen model peptides (CMPs) in which the natural Pro and Hyp residues were replaced by other proline derivatives led to the conclusion that both the ring puckering and the interstrand H bonds are crucial for the conformational stability of the collagen triple helix. 6] All of the studies that address the importance of the ring puckering were performed with proline derivatives in which the trans amide conformer is significantly favored over the cis conformer in the case of C(4)-exo ring-puckered derivatives, whereas the trans conformer is less favored in derivatives with C(4)-endo ring puckers. 5] In addition, the Y angles of the C(4)-endo ring puckered derivatives examined so far are typically approximately 1808 and are not close to those in collagen. Thus, any C(4)-endo ring-puckered derivative had an unfavorable bias towards the trans amide bond and the Y angle. We have recently introduced proline derivatives, such as (4S)-acetamido proline (Acp), in which intramolecular hydrogen bonding leads to a preference for the C(4)-endo ring pucker and the trans amide conformer in aqueous solutions (Figure 1b). This is due to an enforced dihedral angle Y at approximately 1408 by the intramolecular H bond, which allows for a stabilization of the trans isomer by a n!p* interaction. This dihedral angle Y within (4S)Acp is comparable to those observed in collagen and proline derivatives such as (4R)Hyp with a C(4)-exo ring pucker. Proline derivatives such as (4S)Acp therefore allow for the first time to investigate, whether a C(4)-endo ring pucker is tolerated in the Yaa position without a concomitant unfavorable bias on the trans amide bond and the Y angle. Furthermore, the combination of the C(4)-endo ring pucker and the intramolecular H bond allows for probing whether the ring pucker or the interstrand H bonds are more important in the Xaa position for the stability of the collagen triple helix. Herein, we demonstrate that a mismatched ring pucker is tolerated, whereas the interstrand H bonds are crucial for the conformational stability of the collagen triple helix. We started our investigations by analyzing the possible effects of the incorporation of (4S)Acp in the Yaa position on the properties of the collagen triple helix (Figure 2): The ring pucker of (4S)Acp is C(4)-endo and a mismatch to that of the C(4)-exo pucker of the natural (4R)Hyp residue. Thus, if the Figure 1. a) General structure of collagen. b) Ac–(4S)Acp–OCH3 with C(4)-endo conformation by intramolecular H bonding.

Effect of sterically demanding substituents on the conformational stability of the collagen triple helix.

The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.

Switchable Proline Derivatives: Tuning the Conformational Stability of the Collagen Triple Helix by pH Changes

(4S)-Aminoproline is introduced as a pH-sensitive probe for tuning the conformational properties of peptides and proteins. The pH-triggered flip of the ring puckering and the formation/release of a transannular H bond were used to switch the formation of collagen triple helices on and off reversibly.

Conformational stability of collagen triple helices functionalized in the Yaa position by click chemistry.

Click chemistry was used to introduce moieties as sterically demanding as monosaccharides into the Yaa position of collagen model peptides. The effect of different triazolyl derivatives as well as the configuration of the functionalized proline residue on the thermal stability of the collagen triple helices was examined.

Influence of sequential modifications and carbohydrate variations in synthetic AFGP analogues on conformation and antifreeze activity.

Certain Arctic and Antarctic ectotherm species have developed strategies for survival under low temperature conditions that, among others, consist of antifreeze glycopeptides (AFGP). AFGP form a class of biological antifreeze agents that exhibit the ability to inhibit ice growth in vitro and in vivo and, hence, enable life at temperatures below the freezing point. AFGP usually consist of a varying number of (Ala-Ala-Thr)(n) units (n=4-55) with the disaccharide β-D-galactosyl-(1→3)-α-N-acetyl-D-galactosamine glycosidically attached to every threonine side chain hydroxyl group. AFGP have been shown to adopt polyproline II helical conformation. Although this pattern is highly conserved among different species, microheterogeneity concerning the amino acid composition usually occurs; for example, alanine is occasionally replaced by proline in smaller AFGP. The influence of minor and major sequence mutations on conformation and antifreeze activity of AFGP analogues was investigated by replacement of alanine by proline and glycosylated threonine by glycosylated hydroxyproline. The target compounds were prepared by using microwave-enhanced solid phase peptide synthesis. Furthermore, artificial analogues were obtained by copper-catalyzed azide-alkyne cycloaddition (CuAAC): propargyl glycosides were treated with polyproline helix II-forming peptides comprising (Pro-Azp-Pro)(n) units (n=2-4) that contained 4-azidoproline (Azp). The conformations of all analogues were examined by circular dichroism (CD). In addition, microphysical analysis was performed to provide information on their inhibitory effect on ice recrystallization.

Conformational stability of triazolyl functionalized collagen triple helices.

Functionalized collagen is attractive for the development of synthetic biomaterials. Herein we present the functionalization of azidoproline containing collagen model peptides with various alkynes using click chemistry. The influence on the stability of the collagen triple helix of the stereochemistry of the introduced triazolyl prolines (4R or 4S), the position of their incorporation (Xaa or Yaa) and the substituents attached to them are shown. The results provide a useful guide for the optimal functionalization of collagen using click chemistry.

pH‐Responsive Aminoproline‐Containing Collagen Triple Helices

(4S)- and (4R)-configured aminoproline (Amp) residues were used as pH-responsive probes to tune the thermal stability of collagen triple helices in acidic and basic environments. The different steric and stereoelectronic properties of amino versus ammonium groups lead to a switch of the ring pucker of Amp upon changing the pH. The choice of the position of Amp within collagen model peptides (CMPs) as well as the absolute configuration at C(4) of the pH-responsive probe allows for tuning of the stability of Amp-containing collagen triple helices over a broad range. Comparative quantum chemical calculations on the steric and stereoelectronic effects of amino and ammonium groups versus fluorine, hydroxy, chlorine, and methyl substituents support the experimental findings. The research also shows that substitution of the naturally occurring hydroxy group in collagen by electron-withdrawing groups with a larger hydration shell than that of the hydroxy group is not tolerated.

Synthesis of Fmoc-Pro-Hyp(TBDPS)-Gly-OHand Its Application as a Versatile Building Block forthe Preparation of Collagen Model Peptides

The efficient synthesis of the tripeptidic building block Fmoc-Pro-Hyp(TBDPS)-Gly-OH and its application for the prepa- ration of collagen model peptides (CMPs) has been achieved. The silyl ether protecting group prevents undesired side reactions during the CMP synthesis thereby facilitating purification and allowing for selective deprotection of the hydroxyproline residue without affect- ing the solid-supported CMP.