Proline Derivatives and Analogs

Introduction

Proline is a non-polar proteinogenic amino acid that forms a tertiary amide when incorporated into peptides. It does not have a hydrogen on the amide group and therefore cannot act as a hydrogen bond donor. Proline is known as a classical breaker of both the α-helical and β-sheet structures in proteins and peptides. Nevertheless, it is widely distributed in the putative transmembrane domains of many protein transporters and channels, regions believed to be α-helical.¹

Among the proteinogenic amino acids, proline plays a special role. In protein structures the planar peptide bond occurs predominantly in the trans conformation.² The proline residue restricts the conformational space of the peptide chain. However, due to the small free enthalpy difference between the cis and trans Xaa-Pro bond isomers of 2.0 kJ·mol^–1 (compared to 10.0 kJ·mol^-1 for other Xaa-non-Pro peptide bonds), there is a relatively high intrinsic probability of 30% cis conformation at RT and both cis and trans isomers are present in solution.^3,4

The cis/trans-isomerization of peptide bonds on the N-terminal side of Pro residues plays a key role in the folding process of a protein because the rotational barrier of the cis/trans-isomerization is quite high (85,0 ± 10,0 kJ·mol^–1). Therefore, this interconversion is described to be one of the limiting steps of protein folding in vitro and in vivo.⁵ In nature there is a class of enzymes, the peptidyl-prolyl-cis/trans-isomerases (PPIases). They can catalyze protein folding by accelerating the isomerization of the Xaa-Pro-bond.^6–8

Comparative studies performed with proline analogues revealed that the key step in the catalysis of the cis/trans-isomerization of a peptidyl-prolyl bond is a reduction of the double bond character of the planar, conjugated C–N amide bond. Any factor that can weaken the double bond character of the amide bond by destabilizing the planar peptide bond or shifting the hybridization of the prolyl nitrogen from sp² to sp³, is expected to accelerate the isomerization.^9,10

To understand the relationship between imide bond geometry and bioactivity of peptides,^11,12 synthetic proline analogues have been developed that provide restrictions of the Xaa-Pro imide conformation. Such proline mimetics are based on ring substitutions with alkyl and aromatic groups, incorporation of heteroatoms into the ring, or the expansion or contraction of the proline ring (Table 1). Those analogues are promising candidates for conformational studies and for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring , as well as de novo designed, linear, and cyclic peptides.

Table 1.

Proline Analog or Homolog Structures for the Restriction of the Xaa-Pro Imide Conformation

Structure	Name	IUPAC Nomenclature
	α-methyl-L-proline	(2R)-2-methyl-pyrrolidine-2-carboxylic acid
	α-benzyl-L-proline	(2R)-2-benzyl-pyrrolidine-2-carboxylic acid
	trans-4-hydroxy-L-proline	(2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid
	cis-4-hydroxy-L-proline	(2S,4S)-4-hydroxypyrrolidine-2-carboxylic acid
	trans-3-hydroxy-L-proline	(2S,3R)-3-hydroxypyrrolidine-2-carboxylic acid
	cis-3-hydroxy-L-proline	(2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid
	trans-4-amino-L-proline	(2S,4R)-4-aminopyrrolidine-2-carboxylic acid
	3,4-dehydro-DL-proline	(±)-3-pyrrolin-2-carboxylic acid
	L-2-Aziridinecarboxylic acid	(2S)-aziridine-2-carboxylic acid
	L-2-Azetidinecarboxylic acid	(2S)-azetidine-2-carboxylic acid
	L-pipecolic acid	(2S)-piperidine-2-carboxylic acid
	4-oxa-L-proline	(4S)-thiazolidine-4-carboxylic acid
	3-thia-DL-proline	thiazolidine-2-carboxylic acid
	4-thia-L-proline	(4R)-thiazolidine-4-carboxylic acid [TB1]

Several proline analogs and homologs occur in nature. Trans-3-hydroxyproline and trans-4-hydroxyproline represent constituents of common proteins as a result of post-translational hydroxylation, especially in collagens.¹³ Various 3- and 4-alkylated derivatives of proline and hydroxyproline as well as analogues with ring restrictions, such as aziridine-2-carboxylic acid and azetidine-2-carboxylic acid, and ring expansions, i.e. pipecolic acid, are found in natural products.^14,15 Derivatives such as L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, and 3,4- dehydro-DL-proline prevent pro-collagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors.¹⁶

Thiazolidine-4-carboxylic acid thiaproline has also been incorporated into collagen model compounds^17,18 and other bioactive molecules such as thrombin inhibitors,¹⁹ somatostatin,^20,21 dipeptidyl peptidase IV substrates,²² angiotensin II,²³ HIV inhibitors,²⁴ ACE inhibitors,²⁵ and oxytocin.²⁶

α-Methyl-proline is a bioactive molecule restoring normal levels of bone collagen type I synthesis.²⁷ It can be looked at as a conformationally constrained aminoisobutyric acid analog. The α-Methyl-proline residue has been inserted into morphiceptin to perform conformational studies on the bioactivity of the Xaa-Pro cis-/trans-isomers.²⁸ A α-methyl-proline containing potential dual α₄β₁ integrin antagonist has been described recently.²⁹

α-Benzyl-proline combines the conformational restrictions of a proline derivative with the electronic properties of phenylalanine. Spirolactams containing an α-benzyl-proline substructure have been synthesized as potential beta-turn mimetics.³⁰

References

1. Brandl CJ, Deber CM. 1986. Hypothesis about the function of membrane-buried proline residues in transport proteins.. Proceedings of the National Academy of Sciences. 83(4):917-921. https://doi.org/10.1073/pnas.83.4.917

2. Ramachandran G, Sasisekharan V. 1968. Conformation of Polypeptides and Proteins.283-437. https://doi.org/10.1016/s0065-3233(08)60402-7

3. Steward DE, Sarkar A, Wampler JEJ. 1990. Mol. Biol.. 254.353..

4. Weiss MS, Jabs A, Hilgenfeld R. 1998. Peptide bonds revisited. Nat Struct Mol Biol. 5(8):676-676. https://doi.org/10.1038/1368

5. Schmid FX. 1993. Prolyl Isomerase: Enzymatic Catalysis of Slow Protein-Folding Reactions. Annu. Rev. Biophys. Biomol. Struct.. 22(1):123-143. https://doi.org/10.1146/annurev.bb.22.060193.001011

6. Schmid FX, Mayr LM, Mucke M, Schonbrunner E. 1993. Prolyl Isomerases: Role in Protein Folding.25-66. https://doi.org/10.1016/s0065-3233(08)60563-x

7. Fischer G. 1984. Biomed. Biochem. Acta.. 43.4401. .

8. Lang K, Schmid FX, Fischer G. 1987. Catalysis of protein folding by prolyl isomerase. Nature. 329(6136):268-270. https://doi.org/10.1038/329268a0

9. Kern D, Schutkowski M, Drakenberg T. 1997. Rotational Barriers ofcis/transIsomerization of Proline Analogues and Their Catalysis by Cyclophilin§. J. Am. Chem. Soc.. 119(36):8403-8408. https://doi.org/10.1021/ja970606w

10. Bader RFW, Cheeseman JR, Laidig KE, Wiberg KB, Breneman C. 1990. Origin of rotation and inversion barriers. J. Am. Chem. Soc.. 112(18):6530-6536. https://doi.org/10.1021/ja00174a012

11. Yamazaki T, Ro S, Goodman M, Chung NN, Schiller PW. 1993. A topochemical approach to explain morphiceptin bioactivity. J. Med. Chem.. 36(6):708-719. https://doi.org/10.1021/jm00058a007

12. Yu W, Tasayco ML, Tung C, Wang H. NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: Cis/trans peptide isomerization. Protein Science. 9(1):20-28. https://doi.org/10.1110/ps.9.1.20

13. 1994. Guidebook to the extracellular matrix and adhesion proteins, edited by thomas kreis and ronald vale, oxford university press, 1993, 176pp, $30. Mol. Reprod. Dev.. 39(2):247-247. https://doi.org/10.1002/mrd.1080390219

14. Mauger A. 1977. In Chemistry and Biochemistry of Amino Acids, Peptide and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York.. 179

15. Wagner I, Musso H. 1983. New Naturally Occurring Amino Acids. Angew. Chem. Int. Ed. Engl.. 22(11):816-828. https://doi.org/10.1002/anie.198308161

16. Metzner L, Kalbitz J, Brandsch M. 2004. Transport of Pharmacologically Active Proline Derivatives by the Human Proton-Coupled Amino Acid Transporter hPAT1. J Pharmacol Exp Ther. 309(1):28-35. https://doi.org/10.1124/jpet.103.059014

17. Goodman M, Niu GCC, Su K. 1970. Conformational aspects of polypeptide structure. XXXI. Helical poly[(S)-thiazolidine-4-carboxylic acid] and poly[(S)-oxazolidine-4-carboxylic acid]. Theoretical results. J. Am. Chem. Soc.. 92(17):5219-5220. https://doi.org/10.1021/ja00720a038

18. Goodman M, Chen V, Benedetti E, Pedone C, Corradini P. 1972. Conformational aspects of polypeptide structure. XLI. Crystal structure ofS-thiazolidine-4-carboxylic acid and helical structure of poly[(S)-thiazolidine-4-carboxylic acid]. Biopolymers. 11(9):1779-1787. https://doi.org/10.1002/bip.1972.360110903

19. Shuman RT, Rothenberger RB, Campbell CS, Smith GF, Gifford-Moore DS, Paschal JW, Gesellchen PD. 1995. Structure-Activity Study of Tripeptide Thrombin Inhibitors Using .alpha.-Alkyl Amino Acids and Other Conformationally Constrained Amino Acid Substitutions. J. Med. Chem.. 38(22):4446-4453. https://doi.org/10.1021/jm00022a009

20. PATTARONI C, LUCIETTO P, GOODMAN M, YAMAMOTO G, VALE W, MORODER L, GAZERRO L, GÖHRING W, SCHMIED B, WÜNSCH E. Cyclic hexapeptides related to somatostatin Synthesis and biological testing. 36(5):401-417. https://doi.org/10.1111/j.1399-3011.1990.tb01300.x

21. Wünsch E. 1990. Int. J. Pept. Protein Res.. 36.418..

22. RAHFELD J, SCHUTKOWSKI M, FAUST J, NEUBERT K, BARTH A, HEINS J. 1991. Extended Investigation of the Substrate Specificity of Dipeptidyl Peptidase IV from Pig Kidney. Biological Chemistry Hoppe-Seyler. 372(1):313-318. https://doi.org/10.1515/bchm3.1991.372.1.313

23. Samanen J, Cash T, Narindray D, Brandeis E, Adams W, Weideman H, Yellin T, Regoli D. 1991. An investigation of angiotensin II agonist and antagonist analogs with 5,5-dimethylthiazolidine-4-carboxylic acid and other constrained amino acids. J. Med. Chem.. 34(10):3036-3043. https://doi.org/10.1021/jm00114a012

24. Kiso Y. 1996. Biopolymers. 40235.. https://doi.org/10.1002/(SICI)1097-0282(1996)40:2<235::AID-BIP3>3.0.CO;2-X

25. Karanewsky DS, Badia MC, Cushman DW, DeForrest JM, Dejneka T, Lee VG, Loots MJ, Petrillo EW. 1990. (Phosphinyloxy)acyl amino acid inhibitors of angiotensin converting enzyme. 2. Terminal amino acid analogs of (S)-1-[6-amino-2-[[hydroxy(4-phenylbutyl)phosphinyl]oxy]-1-oxohexyl]-L-proline. J. Med. Chem.. 33(5):1459-1469. https://doi.org/10.1021/jm00167a028

26. Einbond A, Sudol M. 1996. Towards prediction of cognate complexes between the WW domain and proline-rich ligands. 384(1):1-8. https://doi.org/10.1016/0014-5793(96)00263-3

27. Lubec G, Labudova O, Seebach D, Beck A, Hoeger H, Hermon M, Weninger M. 1995. Alpha-methyl-proline restores normal levels of bone collagen type i synthesis in ovariectomized rats. Life Sciences. 57(24):2245-2252. https://doi.org/10.1016/0024-3205(95)02217-7

28. Nelson RD. 1986. NIDA Research Monograph.101.

29. Chang LL, Truong Q, Mumford RA, Egger LA, Kidambi U, Lyons K, McCauley E, Van Riper G, Vincent S, Schmidt JA, et al. 2002. The discovery of small molecule carbamates as potent dual ?4?1/?4?7 integrin antagonists. Bioorganic & Medicinal Chemistry Letters. 12(2):159-163. https://doi.org/10.1016/s0960-894x(01)00710-7

30. Alonso E, López-Ortiz F, del Pozo C, Peralta E, Macías A, González J. 2001. Spiro?-Lactams as?-Turn Mimetics. Design, Synthesis, and NMR Conformational Analysis. J. Org. Chem.. 66(19):6333-6338. https://doi.org/10.1021/jo015714m