Organic molecules as well as metal complexes may exist as several geometric isomers1 which display distinct physical properties and chemical reactivities. A molecule containing two atoms (in general, two carbons) attached together by a double bond with substituents W-Z may be found as two isomeric † C.D. dedicates this review to Professor Andrée Marquet as a mark of his admiration and gratitude. * To whom correspondence should be addressed: Tel: (33) 169 08 52 25. Fax: (33) 169 08 90 71. E-mail: christophe.dugave@cea.fr. ‡ Present address: Département de Chimie, Institut de Pharmacologie, Université de Sherbrooke, 3001, 12e Avenue nord, Sherbrooke, Québec, J1H 5N4 Canada. 2475 Chem. Rev. 2003, 103, 2475−2532

Cis-trans isomerization of organic molecules and biomolecules: implications and applications.

With its wide distribution in soft and hard connective tissues, collagen is the most abundant of animal proteins. In vitro, natural collagen can be formed into highly organized, three-dimensional scaffolds that are intrinsically biocompatible, biodegradable, nontoxic upon exogenous application, and endowed with high tensile strength. These attributes make collagen the material of choice for wound healing and tissue engineering applications. In this article, we review the structure and molecular interactions of collagen in vivo; the recent use of natural collagen in sponges, injectables, films and membranes, dressings, and skin grafts; and the on-going development of synthetic collagen mimetic peptides as pylons to anchor cytoactive agents in wound beds.

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Review collagen-based biomaterials for wound healing

Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.

Prolyl 4-hydroxylase

A polypeptide chain can adopt many conformations. Yet, the sequence of its amino acid residues directs folding to a particular native state.1 The loss of conformational entropy associated with folding destabilizes the native state. This destabilization is overcome by the hydrophobic effect, hydrogen bonds, other noncovalent interactions, and disulfide bonds.2 We have identified another force that can contribute to the conformational stability of a protein. The structure and reactivity of an organic molecule can rely on the stereochemistry of its electron pairs, bonded or nonbonded.3 Such stereoelectronic effects, which arise from the mixing of an electron pair with the antibonding σ* of an adjacent polar bond (C-X, where X ) N or O), endow nucleic acids and carbohydrates with conformational stability.4 For example, the multiple gauche effects (X-C-C-X) arising from a 2′ oxygen distinguish RNA‚RNA and RNA‚DNA duplexes from DNA‚DNA duplexes.5 The anomeric effect (X-C-X) enhances the stability of the R anomer of glycosides.6 Here, we demonstrate for the first time that a stereoelectronic effect is critical for the conformational stability of a protein. Collagen is the most abundant protein in animals.7 For example, collagen comprises one-third of the protein in humans and threefourths of the weight of human skin. The polypeptide chains of collagen are composed of approximately 300 repeats of the sequence XaaYaaGly, where Xaa is often an L-proline (Pro) residue and Yaa is often a 4(R)-hydroxy-L-proline (Hyp) residue. These chains are wound in tight triple helices, which are organized into fibrils of great tensile strength. Pro and Hyp comprise nearly one-fourth of the residues in common types of collagen.8 This prevalence of tertiary amides has dichotomous consequences for conformational stability. Pro and Hyp residues are constrained by their pyrrolidine rings, and this rigidity stabilizes triple-helical collagen.9 Yet, the trans and cis conformations of the peptide bonds to Pro and Hyp residues are of nearly equal free energy, which destabilizes collagen because all peptide bonds in triple-helical collagen are in the trans conformation (Figure 1).10 The 4(R)-hydroxyl group of the prevalent Hyp residues increases dramatically the conformational stability of collagen.11 We had shown previously that this increase arises from inductive effects.12 Can a stereoelectronic effect influence Ktrans/cis? To answer this question, we synthesized residue mimics of the form AcYaaOMe, where Yaa is Pro, Hyp, 4(S)-hydroxy-L-proline (hyp), 4(R)-fluoroL-proline (Flp), or 4(S)-fluoro-L-proline (flp).13 We chose Flp and flp because fluorine is small and electronegative and forms only weak hydrogen bonds when bound to carbon.14,15 We chose a methyl ester rather than an amide to prevent γ-turn formation, as had been observed in AcProNHMe.16 We find that the electronegativity and stereochemistry of the 4-substituent in the Yaa mimics has a significant effect on Ktrans/cis (Table 1). Compared to a flp residue, a Pro residue is twice as likely and a Flp residue is 3 times as likely to have a trans peptide bond. Does the value of Ktrans/cis have an impact on collagen stability? To answer this question, we synthesized (ProYaaGly)7 strands containing Flp or flp residues in the Yaa position.19 These strands are diastereomeric, differing only in the stereochemistry at Cγ of the Yaa residues. We found that a (ProFlpGly)7 triple helix has a Tm of 45 °C.20 In contrast, a (ProflpGly)7 triple helix has a Tm of <2 °C (Table 1). A (ProHypGly)7 triple helix has an intermediate Tm of 36 °C. Thus, both the electronegativity and

Conformational Stability of Collagen Relies on a Stereoelectronic Effect

Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids—aliphatic, aromatic, charged and polar as well as proline—is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references).

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Fluorinated amino acids: compatibility with native protein structures and effects on protein–protein interactions

The hydroxylation of proline residues in collagen increases the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res. 1994 , 44, 262−269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[β,γ-13C]d,l-proline methyl ester (1); N-acetyl-4(R)-hydroxy-l-proline [13C]methyl ester (2); and N-acetyl-4(R)-fluoro-l-proline methyl ester (3). The pKa's of the nitrogen atom in the parent amino acids decrease in the following order:  proline (10.8) > 4(R)-hydroxy-l-proline (9.68) > 4(R)-fluoro-l-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the following order:  1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater...

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Nicholas Panisik

Papers

Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability.