Int.
J . Peptide Protein Res. 40, 1992, 25-40
Unusual thionation of a cyclic hexapeptide Conformational changes and dynamics? HORST KESSLER, ARMIN GEYER, HANS MATTER and MATTHIAS KOCK
Organic Chemistry Institute, Technical University of Munich, Garching, Germany
Received 7 October 1991, accepted for publication 16 February 1992
One carbonyl oxygen of the cyclic hexapeptide cycle(-Glyl-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama’s reagent. Surprisingly it was not the C=O of Gly’ but that of PheS which was substituted and cyclo(-Gly1-Pro2-Phe3-Va14-Phe5+[ CS-NH]Phe6-) (B) was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe5 and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a cis Gly1-Pro2 bond in a BVIa-turn (minor) and a trans Gly-Pro bond in a pII‘-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a PI- and pII-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1,J were measured to verify the assumption of this fast pI/pII equilibrium within each isomer. Significant contributions to R1,-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations. Key words: conformational analysis; molecular dynamics; nuclear Overhauser effect; peptide synthesis; thiopeptide; 2D
NMR
Modification of peptide bonds has recently attracted great interest for changing structural and biological properties of peptides. Peptidomimetics offer an opportunity to modifiy biological properties of active peptides. Among them the substitution of the oxygen atom by a sulfur atom seems to be a minimal variation (“isosteric replacement” (1)). Thionation can strongly influence the backbone conformation and therefore may be helpful in the understanding of conformationally induced structure-activity relationships of bioactive compounds. In a recent publication the influence of one O/S-exchange on the conformation of cyclosporin A was described (2). Hence only the most important points will be addressed here. Sulphur is a weaker hydrogen bond acceptor than oxygen (3), the hydrogen bond is about 50 pm longer (325 to 360 pm (4)) than in amides ( 5 ) due to the larger covalent (6) and van der ‘I’ Dedicated to the memory of Juris Saulitis
Waals (7) radius. In contrast, the amide nitrogen next to the thiocarbonyl group is a stronger hydrogen donor (8). The cp, ) I conformational space in the Ramachandran diagram is drastically reduced in the vicinity of thioamides (9). The decreased C=S double bond character results in a higher barrier of rotation about the C-N bond by 2-3 kcal/mol (lo), but only in a small reduction in bond length by 1-5 pm. The cyclic hexapeptide cyclo(-Gly’-Pro2-Phe3-Va14Phe5-Phe6-) (A) was chosen for the thionation. Considering results in the literature (1 l), we expected the reaction to occur at the Gly1-Pro2bond. It turned out that the oxygen of Phe5 was substituted. Additionally an unexpected conformational change is observed for the thiopeptide. SYNTHESIS The synthesis of linear peptides with a thioamide bond in a specific position is difficult, because C-terminal 25
H. Kessler et al. elongation of a thio-dipeptide results in racemization by about 30ppm, which has been used for empirical and side reactions (12). Hence, the thiopeptides ob- prediction of the thiocarbonyl shifts in thioamides (16). tained so far by this approach only involve the fragment Hence, the position of the sulphur atom was localized -Pro$[CS-NHIGly- (13). Recently it was shown that by heteronuclear long range coupling to the thiocarbothionation can be achieved in larger peptides, such as nyl carbon with a characteristic chemical shift. The cycle(-Pro-Phe-Ile-Piz-Piz-MePhe-) (14) [ Piz = piper- analysis of the HMBCS-270 experiment (17) yields the azic acid] and cyclosporin A (2). Therefore we tried to identification of the adjacent N H and C,H protons of apply Lawesson's reagent (LR) (12) to the cyclic the thiocarbonyl group. Two experiments were run with hexapeptide cycfu(-Gly*-Pro2-Phe3-Va14-Phe5-Phe6-) semiselective excitation via a 270" Gaussian pulse (18), (A), which was synthesized by solid-phase peptide syn- for both the carbonyl and the thiocarbonyl carbons, thesis (15). Earlier reports describe the preference of leading to a complete sequencing of the molecule. The LR and similar compounds to exchange the least proton spin systems of the individual amino acids were sterically hindered carbonyl oxygen, i.e. the amide identified by COSY and TOCSY spectra. For the asbond following a Gly residue (1 1). With peptides con- signment of the proton-bearing carbon atoms the taining more sterically hindered amino acids, reaction HMQC technique with BIRD presaturation of 12CH with LR leads to mixtures of thiopeptides difficult to protons was used (1 9). The inclusion of the sulphur has separate. an effect on the chemical shifts of the proton signals of Due to the insolubility of LR in T H F at temperatures N H and C,H. Additionally, the C, reasonances of the below 35 O no reaction with A was achieved. At 38 the amino acid including the sulphur and the one following OjS exchange proceeded, but at this temperature an in the sequence were shifted by about 4-7 ppm. All inseparable mixture of products was obtained. How- proton and carbon chemical shifts are given in ever, using Yokoyama's reagent in dry THF at room Table 1. temperature one product was predominately obtained, The 'H-NMR spectrum shows two sets of signals cyclo(-Gly1-Pro2-Phe3-Va14-Phe5~[ CS-NH]Phe6-) (B). indicating the occurrence of two slowly interconverting This reagent could be used here due to better solubil- conformations by cisltruns isomerism. The 0x0-analog ity of the reagent at lower temperatures. also exists in two conformations with a ratio of 70:30 (rrans:cis).For the sulphur-analog we observed a strong temperature dependence of the cisltrans ratio, the ratio NMR MEASUREMENTS at 300 K is 55:45 (trans:cis). With a 10 K increase in The substitution of the oxygen of carbonyl groups by temperature the population of the trans isomer is raised sulphur results in a downfield shift of the carbon signal by 5 " ~ Hence, . at 330 K the population ratio is idenTABLE 1 XMR-spectroscopic paranierers of both isomers"
NH
H"
8'H)h H"
8.63 7.62 7.66 8.08 9.09
3.82,'3.61 4.15 4.52 3.72 4.8 1 5.10
-
-
1.73 1 5 4 3 32 2 94 187 3 35 3 11 3 28
1 6 2 1 13 0.73 0 54 -
8.73
3.72 4.30 4.57 3.74 4.68 4.89
Residue
H.
H
J . Peptide Protein Res. 40, 1992, 25-40
Unusual thionation of a cyclic hexapeptide Conformational changes and dynamics? HORST KESSLER, ARMIN GEYER, HANS MATTER and MATTHIAS KOCK
Organic Chemistry Institute, Technical University of Munich, Garching, Germany
Received 7 October 1991, accepted for publication 16 February 1992
One carbonyl oxygen of the cyclic hexapeptide cycle(-Glyl-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama’s reagent. Surprisingly it was not the C=O of Gly’ but that of PheS which was substituted and cyclo(-Gly1-Pro2-Phe3-Va14-Phe5+[ CS-NH]Phe6-) (B) was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe5 and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a cis Gly1-Pro2 bond in a BVIa-turn (minor) and a trans Gly-Pro bond in a pII‘-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a PI- and pII-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1,J were measured to verify the assumption of this fast pI/pII equilibrium within each isomer. Significant contributions to R1,-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations. Key words: conformational analysis; molecular dynamics; nuclear Overhauser effect; peptide synthesis; thiopeptide; 2D
NMR
Modification of peptide bonds has recently attracted great interest for changing structural and biological properties of peptides. Peptidomimetics offer an opportunity to modifiy biological properties of active peptides. Among them the substitution of the oxygen atom by a sulfur atom seems to be a minimal variation (“isosteric replacement” (1)). Thionation can strongly influence the backbone conformation and therefore may be helpful in the understanding of conformationally induced structure-activity relationships of bioactive compounds. In a recent publication the influence of one O/S-exchange on the conformation of cyclosporin A was described (2). Hence only the most important points will be addressed here. Sulphur is a weaker hydrogen bond acceptor than oxygen (3), the hydrogen bond is about 50 pm longer (325 to 360 pm (4)) than in amides ( 5 ) due to the larger covalent (6) and van der ‘I’ Dedicated to the memory of Juris Saulitis
Waals (7) radius. In contrast, the amide nitrogen next to the thiocarbonyl group is a stronger hydrogen donor (8). The cp, ) I conformational space in the Ramachandran diagram is drastically reduced in the vicinity of thioamides (9). The decreased C=S double bond character results in a higher barrier of rotation about the C-N bond by 2-3 kcal/mol (lo), but only in a small reduction in bond length by 1-5 pm. The cyclic hexapeptide cyclo(-Gly’-Pro2-Phe3-Va14Phe5-Phe6-) (A) was chosen for the thionation. Considering results in the literature (1 l), we expected the reaction to occur at the Gly1-Pro2bond. It turned out that the oxygen of Phe5 was substituted. Additionally an unexpected conformational change is observed for the thiopeptide. SYNTHESIS The synthesis of linear peptides with a thioamide bond in a specific position is difficult, because C-terminal 25
H. Kessler et al. elongation of a thio-dipeptide results in racemization by about 30ppm, which has been used for empirical and side reactions (12). Hence, the thiopeptides ob- prediction of the thiocarbonyl shifts in thioamides (16). tained so far by this approach only involve the fragment Hence, the position of the sulphur atom was localized -Pro$[CS-NHIGly- (13). Recently it was shown that by heteronuclear long range coupling to the thiocarbothionation can be achieved in larger peptides, such as nyl carbon with a characteristic chemical shift. The cycle(-Pro-Phe-Ile-Piz-Piz-MePhe-) (14) [ Piz = piper- analysis of the HMBCS-270 experiment (17) yields the azic acid] and cyclosporin A (2). Therefore we tried to identification of the adjacent N H and C,H protons of apply Lawesson's reagent (LR) (12) to the cyclic the thiocarbonyl group. Two experiments were run with hexapeptide cycfu(-Gly*-Pro2-Phe3-Va14-Phe5-Phe6-) semiselective excitation via a 270" Gaussian pulse (18), (A), which was synthesized by solid-phase peptide syn- for both the carbonyl and the thiocarbonyl carbons, thesis (15). Earlier reports describe the preference of leading to a complete sequencing of the molecule. The LR and similar compounds to exchange the least proton spin systems of the individual amino acids were sterically hindered carbonyl oxygen, i.e. the amide identified by COSY and TOCSY spectra. For the asbond following a Gly residue (1 1). With peptides con- signment of the proton-bearing carbon atoms the taining more sterically hindered amino acids, reaction HMQC technique with BIRD presaturation of 12CH with LR leads to mixtures of thiopeptides difficult to protons was used (1 9). The inclusion of the sulphur has separate. an effect on the chemical shifts of the proton signals of Due to the insolubility of LR in T H F at temperatures N H and C,H. Additionally, the C, reasonances of the below 35 O no reaction with A was achieved. At 38 the amino acid including the sulphur and the one following OjS exchange proceeded, but at this temperature an in the sequence were shifted by about 4-7 ppm. All inseparable mixture of products was obtained. How- proton and carbon chemical shifts are given in ever, using Yokoyama's reagent in dry THF at room Table 1. temperature one product was predominately obtained, The 'H-NMR spectrum shows two sets of signals cyclo(-Gly1-Pro2-Phe3-Va14-Phe5~[ CS-NH]Phe6-) (B). indicating the occurrence of two slowly interconverting This reagent could be used here due to better solubil- conformations by cisltruns isomerism. The 0x0-analog ity of the reagent at lower temperatures. also exists in two conformations with a ratio of 70:30 (rrans:cis).For the sulphur-analog we observed a strong temperature dependence of the cisltrans ratio, the ratio NMR MEASUREMENTS at 300 K is 55:45 (trans:cis). With a 10 K increase in The substitution of the oxygen of carbonyl groups by temperature the population of the trans isomer is raised sulphur results in a downfield shift of the carbon signal by 5 " ~ Hence, . at 330 K the population ratio is idenTABLE 1 XMR-spectroscopic paranierers of both isomers"
NH
H"
8'H)h H"
8.63 7.62 7.66 8.08 9.09
3.82,'3.61 4.15 4.52 3.72 4.8 1 5.10
-
-
1.73 1 5 4 3 32 2 94 187 3 35 3 11 3 28
1 6 2 1 13 0.73 0 54 -
8.73
3.72 4.30 4.57 3.74 4.68 4.89
Residue
H.
H
