Masamitsu Doi

Simple and efficient syntheses of Boc- and Fmoc-protected 4(R)- and 4(S)-fluoroproline solely from 4(R)-hydroxyproline

Abstract As building blocks of collagen model peptides, Boc- and Fmoc-protected 4(R)- and 4(S)-fluoroproline, which will be widely used in peptide synthesis including solid-phase strategy, were synthesized from the readily available 4(R)-hydroxyproline in higher yield than with conventional methods. To establish the stereospecificity of the Mitsunobu reaction and the subsequent fluorination that were presumed to cause the inversion of configuration at the C-4 position of a proline derivative, the absolute configuration of one of the key products, Boc-4(S)-fluoroproline, was determined by X-ray crystallography.

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-pro-gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X‐Y‐Gly)nrepeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)‐hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)‐hydroxyproline (hyp) at the X‐position destabilizes the helix as demonstrated that the triple helix of (hyp‐Pro‐Gly)15 is less stable than that of (Pro‐Pro‐Gly)15 and that a shorter peptide (hyp‐Pro‐Gly)10 does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro‐Hyp‐Gly)4‐(hyp‐Pro‐Gly)2‐(Pro‐Hyp‐Gly)4 and analyzed its structure by X‐ray crystallography and CD spectroscopy. In the crystal, the main‐chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra‐molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac‐hyp‐NMe2 by quantum mechanical calculations.

Stabilization mechanism of triple helical structure of collagen molecules

The role of 4-hydroxyproline (Hyp) in stabilizing collagen triple helical structure has been investigated comprehensively. Recently it was emphasized that the preferential pyrrolidine ring pucker influenced by the stereoelectronic effects of substituted groups mainly affects the thermal stability of the triple helix. To examine this explanation, we synthesized and characterized (fPro R -Pro-Gly)10 and (fPro S -Pro-Gly)10. According to the results of CD and analytical ultracentrifugation, (fPro S -Pro-Gly)10 takes a triple helical structure and (fPro R -Pro-Gly)10 exists in a single chain structure, the trend of which is not consistent with the relationship between (Hyp S -Pro-Gly)10 and (Hyp R -Pro-Gly)10. In order to rationalize experimental results as a whole, we carried out DSC analyses and determined the thermodynamic parameters associated with the structural transition of these collagen model peptides. In this paper, we reported the DSC results for (Pro-Pro-Gly)10, (Pro-Hyp R -Gly)10 and (Pro-fPro R -Gly)10 as a part of this study. Based on those parameters, we concluded that Hyp and fPro stabilize the triple helix in different stabilizing mechanisms; the increased stability of (Pro-Hyp R -Gly)10 is ascribed primarily to the enthalpic effects while that of (Pro-fPro R -Gly)10 is achieved through the entropic ones.

Polymorphism of Collagen Triple Helix Revealed by 19F NMR of Model Peptide [Pro-4(R)-Hydroxyprolyl-Gly]3-[Pro-4(R)-Fluoroprolyl-Gly]-[Pro-4(R)-Hydroxyprolyl-Gly]3

We have characterized various structures of (Pro-Hyp(R)-Gly)(3)-Pro-fPro(R)-Gly-(Pro-Hyp(R)-Gly)(3) in the process of cis-trans isomerization and helix-coil transition by exploiting the sole (19)F NMR probe in 4(R)-fluoroproline (fPro(R)). Around the transition temperature (T(m)), we detected a species with a triple helical structure distinct from the ordinary one concerning the alignment of three strands. The (19)F-(19)F exchange spectroscopy showed that this misaligned and that the ordinary triple helices were interchangeable only indirectly via an extended monomer strand with all-trans peptide bonds at Pro-fPro(R), Pro-Hyp(R), and Gly-Pro in the central segment. This finding demonstrates that the helix-coil transition of collagen peptides is not described with a simple two-state model. We thus elaborated a scheme for the transition mechanism of (Pro-Hyp(R)-Gly)(n) that the most extended monomer strand can be the sole source both to the misaligned and correctly folded triple-helices. The staggered ends could help misaligned triple helices to self-assemble to higher-order structures. We have also discussed the possible relationship between the misaligned triple helix accumulating maximally at T(m) and the kinetic hysteresis associated with the helix-coil transition of collagen.

Conformational preferences of 4-chloroproline residues†

Conformational preferences of the (2S,4R)-4-chloroproline (Clp) and (2S,4S)-4-chloroproline (clp) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit solvation model, and by an X-ray diffraction study in the solid state. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-Clp-NHMe and Ac-clp-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. In water, the Clp residue has a strong preference for the up-puckered PP(II) structure, whereas the up-puckered PP(II) structure prevails a little over the down-puckered PP(II) structure for the clp residue, similar to the Pro residue. Hence, our calculated results on the puckering preference of the Clp and clp residues in water are in accord with the observed results deduced from the relative stabilities of the triple helices of the collagen model peptides. The X-ray structure of Ac-clp-NHMe was found to be the most preferred in water but that of Ac-Clp-NHMe was located as a local minimum with ΔG = 2.0 kcal/mol. In particular, the X-ray structure of Ac-Clp-NHMe was quite different from that of Ac-Clp-OMe but similar to that of Ac-Pro-NHMe. The lowest rotational barriers to the prolyl cis-trans isomerization for Ac-Clp-NHMe become nearly the same as those for Ac-Pro-NHMe in water, whereas the barriers are lower by ∼2 kcal/mol for Ac-clp-NHMe. It was found that the cis-trans isomerization may proceed through the clockwise or anticlockwise rotations for Ac-Clp-NHMe and the anticlockwise rotation for Ac-clp-NHMe and Ac-Pro-NHMe in water.