硫脯胺酸((2R)-4-thiaproline, Thp)為脯胺酸的衍生物，是以硫取代脯胺酸中吡咯烷環(pyrrolidine ring)的Cγ位而得的。在過去實驗室的研究已發現它有利於形成內環褶皺構形以及順式胜肽鍵，進而破壞PPII構形的穩定性。膠原蛋白是哺乳動物中含量最多的蛋白質，主要由(Xaa-Yaa-Gly)三聯體序列組成：其中Xaa通常為脯胺酸，Yaa通常為羥脯胺酸，二級結構上以PPII構形為主。因此在本研究中，我們將膠原蛋白模擬胜肽中的Xaa和Yaa替換為Thp並探討此替換下造成的構形及穩定性變化。研究中我們發現含有Thp的膠原蛋白肽比我們預期的更加穩定。這可能是由於環褶皺(ring pucker)在內環褶皺(endo)與外環褶皺(exo)相互轉換的容易性使得Thp的噻唑烷環(thiazolidine ring)具有高度柔韌性。此外，我們通過將Thp氧化為Thp’(N- Formyl-cysteine)及Thp’’(S,S- dioxide Thp)來製備衍生肽。我們的結果表明，Thp’在Xaa位置並不會顯著影響膠原蛋白的穩定性，但在Yaa位置確實會產生很大的影響。藉由分子建模的結果，我們認為這種現象可能是由於二面角的過度扭曲。最後，我們依Thp在Xaa位的高穩定性，另外製備了兩條置換多個Thp在Xaa的胜肽，並發現多個Thp仍會對胜肽的穩定造成一定的破壞。我們已經證明了置入Thp及其氧化衍生物對膠原蛋白摺疊熱力學和動力學的影響。實驗和模擬計算數據的結合使我們能夠對Thp、Thp’、Thp’’ 對膠原蛋白構形的影響獲得新的見解，並為膠原蛋白相關生物材料的設計提供有價值的信息。

(2R)-4-thiaproline (Thp) is a derivative of proline, replacing Cγ in the pyrrolidine ring with sulfur. It favors an endo ring pucker and a cis prolyl peptide bond destabilizing polyproline II conformation. Collagen, the most abundant protein in mammals, is mainly composed of (Xaa-Yaa-Gly) triplet sequence: where Xaa is often proline, and Yaa is frequently hydroxyproline. In this study, we substitute the Xaa and Yaa of the collagen peptides with Thp and examine the consequences of this replacement. Strikingly, we found that Thp-containing collagen peptides are more stable than we expected. It could be due to that the ease of the ring pucker interconversion makes the thiazolidine ring of Thp highly flexible. Moreover, we prepared the derivative peptides by oxidizing Thp to Thp’(N-Formyl-cysteine) or Thp’’(S,S-dioxide Thp). Our results showed that the oxidation of Thp at position Xaa does not significantly affect collagen stability, but that at position Yaa does impose a large impact. Based on the molecular modeling results, we suggested that this phenomenon could be due to the over-twist of dihedral angels. Finally, due to the high stability of Thp at position Xaa, we prepared two peptides that replaced multiple Thp at Xaa, and found that more than one Thp substitution destabilize the triple helices. We have demonstrated the consequences of incorporating Thp and its oxidative derivatives on the folding thermodynamics and kinetics of collagen triple helices. The combination of experimental and computational data allows us to gain new insights into the impact of Thp, Thp’, Thp’’ on collagen conformation and provides valuable information to the design of collagen related biomaterials.

1. Lutolf, M.; Hubbell, J., Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 2005, 23 (1), 47-55.
2. Malafaya, P. B.; Silva, G. A.; Reis, R. L., Natural–origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Adv. Drug Deliv. Rev. 2007, 59 (4-5), 207-233.
3. Nemoto, T.; Horiuchi, M.; Ishiguro, N.; Shinagawa, M., Detection methods of possible prion contaminants in collagen and gelatin. Arch. Virol. 1999, 144 (1), 177-84.
4. Brodsky, B.; Persikov, A. V., Molecular structure of the collagen triple helix. In Adv. Protein Chem., Academic Press: 2005; Vol. 70, pp 301-339.
5. Engel, J.; Bächinger, H. P., Structure, stability and folding of the collagen triple helix. In Collagen: Primer in Structure, Processing and Assembly, Brinckmann, J.; Notbohm, H.; Müller, P. K., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 2005; pp 7-33.
6. Cowan, P. M.; McGavin, S.; North, A. C., The polypeptide chain configuration of collagen. Nature 1955, 176 (4492), 1062-4.
7. Chen, Y.-S.; Chen, C.-C.; Horng, J.-C., Thermodynamic and kinetic consequences of substituting glycine at different positions in a Pro-Hyp-Gly repeat collagen model peptide. Pept. Sci. 2011, 96 (1), 60-68.
8. McKenzie, L., Cytoskeletal structural proteins. Retrieved June 22, 2022, from
http://slideplayer.com/slide/6117668/
9. Ramachandran, G. N.; Bansal, M.; Bhatnagar, R. S., A hypothesis on the role of hydroxyproline in stabilizing collagen structure. Biochim. Biophys. Acta - Protein Structure 1973, 322 (1), 166-171.
10. Privalov, P. L., Stability of proteins. Proteins which do not present a single cooperative system. Adv. Protein Chem. 1982, 35, 1-104.
11. Shoulders, M. D.; Raines, R. T., Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929-958.
12. Brodsky, B.; Ramshaw, J. A., The collagen triple-helix structure. Matrix Biol. 1997, 15 (8-9), 545-54.
13. Adzhubei, A. A.; Eisenmenger, F.; Tumanyan, V. G.; Zinke, M.; Brodzinski, S.; Esipova, N. G., Third type of secondary structure: Noncooperative mobile conformation. Protein Data Bank analysis. Biochem. Biophys. Res. Commun. 1987, 146 (3), 934-938.
14. Rath, A.; Davidson, A. R.; Deber, C. M., The structure of “unstructured” regions in peptides and proteins: Role of the polyproline II helix in protein folding and recognition*. J. Pept. Sci. 2005, 80 (2-3), 179-185.
15. Dugave, C.; Demange, L., Cis−trans isomerization of organic molecules and biomolecules:  implications and applications. Chem. Rev. 2003, 103 (7), 2475-2532.
16. Fischer, G., Chemical aspects of peptide bond isomerisation. Chem. Soc. Rev. 2000, 29 (2), 119-127.
17. Cowan, P. M.; McGavin, S., Structure of poly-L-proline. Nature 1955, 176 (4480), 501-503.
18. Adzhubei, A. A.; Sternberg, M. J. E.; Makarov, A. A., Polyproline-II helix in proteins: structure and function. J. Mol. Biol. 2013, 425 (12), 2100-2132.
19. Park, S.; Radmer, R. J.; Klein, T. E.; Pande, V. S., A new set of molecular mechanics parameters for hydroxyproline and its use in molecular dynamics simulations of collagen-like peptides. J. Comput. Chem. 2005, 26 (15), 1612-6.
20. Horng, J. C.; Raines, R. T., Stereoelectronic effects on polyproline conformation. Prot. Sci. 2006, 15 (1), 74-83.
21. Creamer, T. P.; Campbell, M. N., Determinants of the polyproline II helix from modeling studies. In Adv. Protein Chem., Academic Press: 2002; Vol. 62, pp 263-282.
22. Adzhubei, A. A.; Sternberg, M. J. E., Left-handed polyproline II helices commonly occur in globular proteins. J. Mol. Biol. 1993, 229 (2), 472-493.
23. Williamson, M. P., The structure and function of proline-rich regions in proteins. Biochem. J. 1994, 297 ( Pt 2) (Pt 2), 249-260.
24. Fischer, E., Untersuchungen uber aminosauren, polypeptide & proteine (1899-1919). Springer: 1906.
25. Improta, R.; Benzi, C.; Barone, V., Understanding the role of stereoelectronic effects in determining collagen stability. 1. A quantum mechanical study of proline, hydroxyproline, and fluoroproline dipeptide analogues in aqueous solution. J. Am. Chem. Soc. 2001, 123 (50), 12568-12577.
26. DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines, R. T.; Markley, J. L., Collagen stability: insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegative substituents on prolyl ring conformations. J. Am. Chem. Soc. 2002, 124 (11), 2497-2505.
27. Brandts, J. F.; Halvorson, H. R.; Brennan, M., Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry 1975, 14 (22), 4953-4963.
28. Choudhary, A.; Pua, K. H.; Raines, R. T., Quantum mechanical origin of the conformational preferences of 4-thiaproline and its S-oxides. Amino Acids 2011, 41 (1), 181-186.
29. Eberhardt, E. S.; Panasik, N.; Raines, R. T., Inductive effects on the energetics of prolyl peptide bond isomerization: implications for collagen folding and stability. J. Am. Chem. Soc. 1996, 118 (49), 12261-12266.
30. Ganguly, H. K.; Basu, G., Conformational landscape of substituted prolines. Biophys. Rev. 2020, 12 (1), 25-39.
31. O'Hagan, D.; Bilton, C.; Howard, J. A.; Knight, L.; Tozer, D. J., The preferred conformation of N-β-fluoroethylamides. Observation of the fluorine amide gauche effect. J. Chem. Soc., Perkin Trans. 2 2000, (4), 605-607.
32. Mooney, S. D.; Kollman, P. A.; Klein, T. E., Conformational preferences of substituted prolines in the collagen triple helix. Biopolymers 2002, 64 (2), 63-71.
33. Clegg, W.; Deboves, H. J.; Elsegood, M. R., 1-tert-butyl 2-methyl 4-R-hydroxypyrrolidine-1, 2-(2S)-dicarboxylate. Acta Crystallogr. E. 2003, 59 (12), o1987-o1989.
34. Panasik Jr, N.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T., Inductive effects on the structure of proline residues. Int. J. Pept. Protein Res. 1994, 44 (3), 262-269.
35. Pandey, A. K.; Yap, G. P.; Zondlo, N. J., (2S,4R)-4-hydroxyproline (4-nitrobenzoate): Strong induction of stereoelectronic effects via a readily synthesized proline derivative. Crystallographic observation of a correlation between torsion angle and bond length in a hyperconjugative interaction. J. Org. Chem. 2014, 79 (9), 4174-4179.
36. Ghosh, M.; Mallick, A.; Díaz, D. D., Crystal structure of (2S,4R)-2-benzyl 1-tert-butyl 4-(tosyloxy) pyrrolidine-1, 2-dicarboxylate, C24H29NO7S. Z. fur Krist. - New Cryst. Struct. 2012, 227 (3), 361-362.
37. Kotch, F. W.; Guzei, I. A.; Raines, R. T., Stabilization of the collagen triple helix by O-methylation of hydroxyproline residues. J. Am. Chem. Soc. 2008, 130 (10), 2952-2953.
38. Forbes, C. R.; Pandey, A. K.; Ganguly, H. K.; Yap, G. P.; Zondlo, N. J., 4R-and 4S-iodophenyl hydroxyproline, 4R-pentynoyl hydroxyproline, and S-propargyl-4-thiolphenylalanine: conformationally biased and tunable amino acids for bioorthogonal reactions. Org. Biomol. Chem. 2016, 14 (7), 2327-2346.
39. Szczesniak, P.; Maziarz, E.; Stecko, S.; Furman, B., Synthesis of polyhydroxylated piperidine and pyrrolidine peptidomimetics via one-pot sequential lactam reduction/Joullié–Ugi reaction. J. Org. Chem. 2015, 80 (7), 3621-3633.
40. Siebler, C.; Trapp, N.; Wennemers, H., Crystal structure of (4S)‐aminoproline: conformational insight into a pH‐responsive proline derivative. J. Pept. Sci. 2015, 21 (3), 208-211.
41. Shoulders, M. D.; Kotch, F. W.; Choudhary, A.; Guzei, I. A.; Raines, R. T., The aberrance of the 4S diastereomer of 4-hydroxyproline. J. Am. Chem. Soc. 2010, 132 (31), 10857-10865.
42. Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T., Conformational stability of collagen relies on a stereoelectronic effect. J. Am. Chem. Soc. 2001, 123 (4), 777-778.
43. Hinderaker, M. P.; Raines, R. T., An electronic effect on protein structure. Protein Sci. 2003, 12 (6), 1188-1194.
44. Hodges, J. A.; Raines, R. T., Stereoelectronic effects on collagen stability:  the dichotomy of 4-fluoroproline diastereomers. J. Am. Chem. Soc. 2003, 125 (31), 9262-9263.
45. Inouye, K.; Kobayashi, Y.; Kyogoku, Y.; Kishida, Y.; Sakakibara, S.; Prockop, D. J., Synthesis and physical properties of (hydroxyproline-proline-glycine)10: Hydroxyproline in the X-position decreases the melting temperature of the collagen triple helix. Arch. Biochem. Biophys. 1982, 219 (1), 198-203.
46. Doi, M.; Nishi, Y.; Uchiyama, S.; Nishiuchi, Y.; Nakazawa, T.; Ohkubo, T.; Kobayashi, Y., Characterization of collagen model peptides containing 4-fluoroproline; (4S-fluoroproline-Pro-Gly)10 forms a triple helix, but (4R-fluoroproline-Pro-Gly)10 does not. J. Am. Chem. Soc. 2003, 125 (33), 9922-9923.
47. Hodges, J. A.; Raines, R. T., Stereoelectronic effects on collagen stability: the dichotomy of 4-fluoroproline diastereomers. J. Am. Chem. Soc. 2003, 125 (31), 9262-9263.
48. Budisa, N.; Minks, C.; Medrano, F. J.; Lutz, J.; Huber, R.; Moroder, L., Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (2), 455-459.
49. Memmott, S. D.; Ha, Y.-s.; Dickman, M. B., Proline reverses the abnormal phenotypes of Colletotrichum trifolii associated with expression of endogenous constitutively active Ras. Appl. Environ. Microbiol. 2002, 68 (4), 1647-1651.
50. Oldach, F.; Al Toma, R.; Kuthning, A.; Caetano, T.; Mendo, S.; Budisa, N.; Süssmuth, R. D., Congeneric lantibiotics from ribosomal in vivo peptide synthesis with noncanonical amino acids. Angew. Chem. Int. Ed. 2012, 51 (2), 415-418.
51. Weber, H. U.; Fleming, J. F.; Miquel, J., Thiazolidine-4-carboxylic acid, a physiologic sulfhydryl antioxidant with potential value in geriatric medicine. Arch. Gerontol. 1982, 1 (4), 299-310.
52. De la Fuente, M.; Ferrández, M. a. D.; Del Rio, M.; Burgos, M. a. S.; Miquel, J., Enhancement of leukocyte functions in aged mice supplemented with the antioxidant thioproline. Mech. Ageing Dev. 1998, 104 (3), 213-225.
53. Roberts, J. C.; Nagasawa, H. T.; Zera, R. T.; Fricke, R. F.; Goon, D. J., Prodrugs of L-cysteine as protective agents against acetaminophen-induced hepatotoxicity. 2-(Polyhydroxyalkyl)-and 2-(polyacetoxyalkyl) thiazolidine-4 (R)-carboxylic acids. J. Med. Chem. 1987, 30 (10), 1891-1896.
54. Correa, R.; Del Rıo, M.; De La Fuente, M., Improvement of murine immune functions in vitro by thioproline. Int. Immunopharmacol. 1999, 44 (3), 281-291.
55. Chao, T.-F.; Leu, H.-B.; Huang, C.-C.; Chen, J.-W.; Chan, W.-L.; Lin, S.-J.; Chen, S.-A., Thiazolidinediones can prevent new onset atrial fibrillation in patients with non-insulin dependent diabetes. Int. J. Cardiol. 2012, 156 (2), 199-202.
56. Kumagai, H.; Mukaisho, K.-i.; Sugihara, H.; Miwa, K.; Yamamoto, G.; Hattori, T., Thioproline inhibits development of esophageal adenocarcinoma induced by gastroduodenal reflux in rats. Carcinogenesis 2004, 25 (5), 723-727.
57. Suo, M.; Mukaisho, K.-i.; Shimomura, A.; Sugihara, H.; Hattori, T., Thioproline prevents carcinogenesis in the remnant stomach induced by duodenal reflux. Cancer Lett. 2006, 237 (2), 256-262.
58. Frank, N.; Tsuda, M.; Ohgaki, H.; Frei, E.; Kato, T.; Sato, S., Detoxifying potential of thioproline against N-nitroso compounds, N-nitrosodimethylamine and N-nitrosocimetidine. Cancer Lett. 1990, 50 (3), 167-172.
59. Kurashima, Y.; Tsuda, M.; Sugimura, T., Marked formation of thiazolidine-4-carboxylic acid, an effective nitrite trapping agent in vivo, on boiling of dried shiitake mushroom (Lentinus edodes). J. Agric. Food Chem. 1990, 38 (10), 1945-1949.
60. Suvachittanont, W.; Kurashima, Y.; Esumi, H.; Tsuda, M., Formation of thiazolidine-4-carboxyiic acid (thioproline), an effective nitrite-trapping agent in human body, in Parkia speciosa seeds and other edible leguminous seeds in Thailand. Food Chem. 1996, 55 (4), 359-363.
61. Ham, Y.-H.; Jason Chan, K. K.; Chan, W., Thioproline serves as an efficient antioxidant protecting human cells from oxidative stress and improves cell viability. Chem. Res. Toxicol. 2020, 33 (7), 1815-1821.
62. Gupta, S. P.; Bahal, R., QSAR and molecular modeling studies in heterocyclic drugs. Springer-Verlag: 2006.
63. Goodman, M.; Chen, V.; Benedetti, E.; Pedone, C.; Corradini, P., Conformational aspects of polypeptide structure. XLI. Crystal structure of S-thiazolidine-4-carboxylic acid and helical structure of poly[(S)-thiazolidine-4-carboxylic acid]. Biopolymers 1972, 11 (9), 1779-1787.
64. Benedetti, E.; Christensen, A.; Gilon, C.; Fuller, W.; Goodman, M., Conformational studies of peptides. The crystal structures of N-acetyl-L-prolinamide and N-acetyl-(S)-thiazolidine-4-carboxamide. Biopolymers 1976, 15 (12), 2523-2534.
65. Kang, Y. K.; Park, H. S.; Byun, B. J., Puckering transitions of pseudoproline residues. Biopolymers 2009, 91 (6), 444-455.
66. Kang, Y. K.; Park, H. S., Conformational preferences of pseudoproline residues. J. Phys. Chem. B 2007, 111 (43), 12551-12562.
67. Lin, Y.-J.; Chang, C.-H.; Horng, J.-C., The impact of 4-thiaproline on polyproline conformation. J. Am. Chem. Soc. 2014, 118 (37), 10813-10820.
68. Clerici, F., Thiazole and thiadiazole S-oxides. In Advances in Heterocyclic Chemistry, Academic Press: 2002; Vol. 83, pp 71-115.
69. Wallace, B. A., Protein characterisation by synchrotron radiation circular dichroism spectroscopy. Q. Rev. Biophys. 2009, 42 (4), 317-70.
70. Cooper, A., Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics. Biophys. Chem. 2000, 85 (1), 25-39.
71. Cooper, A.; Johnson, C. M.; Lakey, J. H.; Nöllmann, M., Heat does not come in different colours: entropy–enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions. Biophys. Chem. 2001, 93 (2), 215-230.
72. Haynie, D. T.; Freire, E., Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry. Anal. Biochem. 1994, 216 (1), 33-41.
73. Chow, W. Y.; Forman, C. J.; Bihan, D.; Puszkarska, A. M.; Rajan, R.; Reid, D. G.; Slatter, D. A.; Colwell, L. J.; Wales, D. J.; Farndale, R. W.; Duer, M. J., Proline provides site-specific flexibility for in vivo collagen. Sci. Rep. 2018, 8 (1), 13809.