Conformational preferences and stereoelectronic effects in hydroxyproline

Hydroxyproline is the major building block of collagen which is regarded as one of the most important biomaterials [1] because of its widespread occurrence as structural protein in vertebrates [2]. Collagen makes up one third of all proteins in the human body and is an essential part of connective tissue. The skin, for instance, consists to more than 50% of collagen being responsible for skin strength and elasticity. Due to its exceptional advantageous biological characteristics, like e.g. excellent biocompatibility, collagen is widely used in biomedical applications. In particular, it is important in cosmetic surgery where it plays a major role in wound-healing applications, but it is also used, for example, as drug delivery system [1]. In addition, bio-based materials such as collagen are becoming more and more interesting for nanotechnological applications, for instance, as a mechanomutable material or as template for de novo material design.
Although collagen extracted from animals is readily available for medical purposes, it would be highly desirable to have a synthetic source in order to avoid possible complications including immunogenicity and transmission of infective diseases from animal to human [2,3]. The synthesis and design of collagen-like materials is, however, a challenging task and requires a comprehensive understanding of the collagen structure and the factors influencing its stability [3]. The thermal and mechanical stability is a complex balance between several factors, e.g. sterical influences of substituents, stereoelectronic effects, or interstrand interactions.
In the following, we present a computational study on the influence of stereoelectronic effects on the conformational properties of hydroxyproline and discuss the impact on the collagen triple-helix stability.
Collagen forms a triple helix structure which consists of three tightly coiled polyproline II-type (PPII) strands with the repeating amino acid motif Xaa-Yaa-Gly, where Xaa and Yaa are often proline (Pro) and hydroxproline (Hyp), respectively [3]. There is experimental evidence that Hyp in Yaa position has a stabilizing influence, whereas Hyp at Xaa position destabilizes the collagen triple-helix [3-5]. Furthermore, the configuration in Hyp is critical for the stability of collagenous materials [3,6,7]. A stabilizing effect is only observed when the hydroxy group is in 4R configuration (see Fig. 1), while the 4S diastereomer leads to a destabilization [3,8].

Figure 1: Structural formula of proline capped with Ac at the nitrogen and with OCH3 at the C-terminal side. X=OH, Y=H results in a 4S configuration and X=H, Y=OH in 4R configuration.

One hypothesis discussed in literature is that the stabilization of collagen is due to water mediated hydrogen bonding [9-12]. Raines et al. [3] replaced Hyp in collagen-like model peptides through fluoroproline (Flp) that is usually reluctant to form hydrogen bonds and observed similar trends in the collagen stability like for Hyp which implies that effects other than hydrogen bonding have to play a role in stabilizing the triple-helix. Experimental and theoretical studies [3,13,14] indicate that stereoelectronic effects dominate the conformation of substituted proline model compounds. In the following, we will present a work flow for analyzing the conformational preferences of hydroxyproline using MAPS software platform [15' and discuss the implications of stereoelectronic effects as structure directing element.
For analyzing the conformational preferences of Hyp, model structures for each possible combination of configuration at C4 (4R/4S) and ring conformation (exo/endo) were created using the building tools of MAPS software platform [15]. The N-terminal side has been capped with an acetyl moiety (CH3CO-) and the C=O group with -OCH3 forming a methyl ester at the C-terminal side, which has been proven to provide valuable model structures for studying conformational preferences of proline derivatives incorporated in polyproline or collagen-like helical structures [8,13]. For each model structure a conformational search has been performed using the software tool Confab [16].  Confab uses a systematic approach to explore the conformational space and Ebejer et al. [17] have demonstrated its ability to generated a diverse set of low energy conformers. The conformers obtained from the conformational search were geometry optimized at BLYP-D3/def2-TZVP [18-21] level applying Grimme's empirical D3 dispersion correction [22] using MAPS Turbomole interface.
Energy differences between the energetically most favored exo and endo isomers and trans and cis isomers, respectively, are listed in Table 1.

Table 1: Energy differences between lowest energy isomers of Ac-Hyp-OMe in kJ/mol. A negative sign of DEtrans-cis indicates that the trans isomer is preferred. For the 4R stereoisomer, the positive energy difference DEendo-exo indicates a preference for the exo ring pucker, i.e. for the gauche isomer. For the 4S stereoisomer the negative DEendo-exo implies a preference for the endo ring conformation and thus also for the gauche isomer.

The relative energies indicate a clear preference of the trans conformation between 5-9 kJ/mol which is in line with previous studies on related compounds [8,13,14]. For both diastereomers (4R and 4S), a gauche conformation between the hydroxyl and the amide group is energetically favored. For the 4S isomer, however, the preference is considerably stronger (4S: 22 kJ/mol vs. 4R: 3 kJ/mol) which can be explained by a structural analysis of the optimized structures.
The optimized geometries of 4R-Ac-Hyp-OMe and 4S-Ac-Hyp-OMe are illustrated in Fig. 2.

Figure 2: Optimized structures of 4R-Ac-Hyp-OMe and 4S-Ac-Hyp-OMe with trans conformation of amide bonds.

The energetically most favored 4R stereoisomer shows an exo conformation of the pyrrolidin ring and a trans conformation of the amide bond. In the 4S isomer, a trans conformation of the amide bond and an endo conformation of the ring is observed. This means that in both isomers, the hydroxy group at C4 and the amide group adopt a gauche conformation and that the proline ring conformation is governed by the gauche-effect [23]. A further stabilizing effect comes through the trans conformation of the amide bond. Fig. 3 illustrates that the angle between the oxygen of the acetyl group and the carbonyl group of the ester is 100° which is near an idealized so-called Buergi-Dunitz angle [24] that is known to allow for stabilizing n-p* interactions [3,16]. This is in line with the charge distribution of the molecule as indicated in the electrostatic potential map (see Fig. 3) in which the oxygen atoms of the interacting groups (O(Ac) and C=O (ester)) represent a region of high electron concentration (red) while the carbon of the ester group is accordingly positively charged.

Figure 3: Left: Optimized structure of 4R-Ac-Hyp-OMe with indicated stabilizing Buergi-Dunitz arrangement. The angle of 100° between O(Ac) and C=O (ester) is in the typical range. Right: Electrostatic potential mapped on the electronic density (Color code: red – area of low and blue – area of high electrostatic potential). 

There is evidence that the gauche-effect per-organizes the peptide backbone torsional angles in such a way that n-p* interactions are possible [3]. It should be noted that in 4R with endo conformation and in 4S with exo conformation the angle is also near 100°. However, the gauche-effect will have structure-directing influence on the backbone arrangement as observed for other proline derivatives, as well [3,13,14].
The endo conformation in the 4S diastereomer allows for the formation of an intramolecular hydrogen bond between the hydroxyl group and the carbonyl oxygen of the ester group. While in the 4R isomer the formation of an intramolecular hydrogen bond is not possible, the hydrogen bond in the 4S isomer additionally stabilizes the endo conformation over the exo conformation which explains the larger energy difference between the 4S isomers.
The systematical conformational analysis presented above provides detailed insights into the structure-directing elements in hydroxyproline. The results suggest that electronic effects such as the gauche-effect as well as intramolecular hydrogen bonding play a critical role. In natural collagen, only 4R Hyp is found, which is not able to form an intramolecular hydrogen bond in the isolated monomer unit in contrast to its 4S diastereomer. On the other hand, 4R Hyp should be more inclined to form stabilizing intermolecular hydrogen bonds with other compounds than its counterpart 4S Hyp. This could be one reason that 4R Hyp is preferred in natural collagen as it can contribute to the helix stability through favorable intermolecular interactions.

We have presented an approach for studying the conformational properties of hydroxyproline using Scienomics MAPS software platform.  The results show that the conformation of  hydroxyproline is determined by the interplay of the stereoelectronic gauche-effect and the ability of a given hydroxyproline conformation for intramolecular hydrogen bonding. The comparatively weak gauche-effect thus implicitly influences also the structural properties of collagen, whose main component is hydroxyproline. The results suggest that destabilizing effects of 4S-hydroxyproline in collagen occur presumably due to an intramolecular hydrogen bond disturbing the interstrand interactions. Though these results were obtained for the gas phase, an aqueous environment will weaken this intramolecular hydrogen bond, but not completely hamper its formation.

  1. Nair R, Sevukarajan M, Mohammed Badivaddin T, Ashok Kumar CK. Collagen based drug delivery systems: A review. Journal of Innovative Trends in Pharmaceutical Sciences 2010;1:288-304.
  2. Ramshaw JA, Werkmeister JA, Glattauer V. Collagen-based biomaterials. Biotechnol Genet Eng Rev 1996;13:335-82.
  3. Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem 2009;78:929-58.
  4. Berg RA et al. A model for the triple-helical structure of (Pro-Hyp-Gly)10 involving a cis peptide bond and inter-chain hydrogen-bonding to the hydroxyl group of hydroxyproline. Biochim Biophys Acta 1973;328:553-9.
  5. Sakakibara S et al. Synthesis of (Pro-Hyp-Gly)n of defined molecular weights. Evidence for the stabilization of collagen triple helix by hydroxypyroline. Biochim Biophys Acta 1973;303:198-202.
  6. Inouye K, Sakakibara S, Prockop DJ. Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen. Biochim Biophys Acta 1976;420:133-41.
  7. Jiravanichanun N, Nishino N, Okuyama K. Conformation of alloHyp in the Y position in the host-guest peptide with the pro-pro-gly sequence: implication of the destabilization of (Pro-alloHyp-Gly)10. Biopolymers 2006;81:225-33.
  8. Shoulders MD, Kotch FW, Choudhary A, Guzei IA, Raines RT. The aberrance of the 4S diastereomer of 4-hydroxyproline. J Am Chem Soc 2010;132:10857-65.
  9. Rao NV, Adams E. Collagen helix stabilization by hydroxyproline in (Ala-Hyp-Gly)n. Biochem Biophys Res Commun 1979;86:654-60.
  10. Suzuki E, Fraser RDB, MacRae TP. Role of hydroxyproline in the stabilization of the collagen molecule via water molecules. Int J Biol Macromolec 1980;2:54-6.
  11. Bella J, Eaton M, Brodsky ME, Berman HM. Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution. Science 1994;266:75-81.
  12. Bella J, Brodsky B, Berman HM. Hydration structure of a collagen peptide. Structure 1995;3:893-906.
  13. Sonntag LS, Schweizer S, Ochsenfeld C, Wennemers H. The "azido gauche effect"-implications for the conformation of azidoprolines. J Am Chem Soc 2006;128:14697-703.
  14. Kuemin K, Nagel YA, Schweizer S, Monnard FW, Ochsenfeld C Wennemers H. Tuning the cis/trans conformer ratio of Xaa-Pro amide bonds by intramolecular hydrogen bonds: the effect on PPII helix stability. Angew Chem Int Ed Engl 2010;49:6324-7.
  15. MAPS, Version 3.3.2, Scienomics, Paris, France, 2013.
  16. O'Boyle N, Vandermeersch T, Flynn C, Maguire A, Hutchison C. Confab - Systematic generation of diverse low-energy conformers. J Cheminform 2011;3:8.
  17. Ebejer JP, Morris GM, Deane CM. Freely Available Conformer Generation Methods: How Good Are They? J Chem Inf Model, 2012;52:1146-58.
  18. Dirac PAM. Quantum Mechanics of Many-Electron Systems. Proc R Soc Lond A 1929;123:714-33.
  19. Slater JC. A Simplification of the Hartree-Fock Method. Phys Rev 1951;81:385-90.
  20. Lee C, Yang W, Parr RG. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys Rev B 1988;37:785-9.
  21. Weigend F, Ahlrichs R. Balanced basis sets of split valence triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy. Phys Chem Chem Phys 2005;7:3297-3305.
  22. Grimme S, Antony J, Ehrlich S, Krieg H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J Chem Phys 2010;132:154104.
  23. Wolfe S. Gauche effect. Stereochemical consequences of adjacent electron pairs and polar bonds Acc Chem Res 1972;5:102-11
  24. Bürgi HB, Dunitz JD, Lehn JM, Wipff G. Stereochemistry of reaction paths at carbonyl centers. Tetrahedron 1974; 30:1563-1572.