Sajid, Andaleeb

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AbstractThis thesis entitled “Design and Synthesis of /-Hybrid Peptides: Identification of Novel Secondary, Super Secondary and Tertiary Structures” mainly describes the design and synthesis of peptides with heterogeneous backbone from alternating C-linked carbo-β-amino acids (-Caas) and L-Ala. The peptides containing C-terminal ‘L-Ala--Caa-L-Ala’ (--) sequence have shown unprecedented novel 9/11-helical structures. Similarly, the studies on the peptides with C-terminal ‘-Caa-L-Ala--Caa’ (--) sequence resulted in two new three residue turns, besides helix-turn motifs,  that were successfully utilized for the creation of tertiary structural motif, such as Helix-Turn-Helix. Further, the peptides made from (R)-β-Caa and D-Ala, have also shown the presence of secondary structures. An overview on peptides with homogeneous and heterogeneous backbones and their conformational propertiesA brief review is presented on -peptides, -peptides and peptides with heterogeneous backbones. The discussion encompasses the aspects of conformational analysis and protein architecture. -Peptides and most of the foldamers1 reported so far have homogeneous backbones. Peptides with heterogeneous backbones were also recently found to display well-defined folding behavior, resulting in helical and skeletal diversity. Design and synthesis of /-hybrid peptides with novel mixed helices from alternating L-Ala and (S)-C-linked carbo--amino acids [(S)--Caa]β-Amino acids are part structures of several natural products and very important components. Their importance and nonavailability from ‘chiral pool’, prompted the development of new and efficient strategies2 for their synthesis, enroute` to peptidomimetics. Seebach et al.3 and Gellman et al.4 were the first to report β-peptides with novel helical structures, which followed subsequent reports5 on a variety of novel secondary structures in β-peptides with different designs. In a further work on foldamers, peptides with heterogeneous backbone6 were reported to show novel helical patterns.The research in the recent past indicated that the attachment of carbohydrate7 to peptides improves bioavailability and water solubility, increases resistance to proteases, or enables them to enter the blood-brain barrier. Based on the structures of nikkomycins8, our group has reported the synthesis of C-linked carbo-β-amino acids9 (β-Caa), with carbohydrate moieties as side chains. Making use of these monomers, earlier our group has reported β-peptides with novel helical patterns.10The β-Caa residues were made from D-glucose. Accordingly, the Michael acceptor 2, prepared (Scheme 1) from the corresponding aldehyde 1,11 on reaction with benzyl amine12 at room temperature gave esters 3 (46%) and 4 (22%), which are epimeric at the amine stereocentre (Scheme 1).Scheme 1	Hydrogenolysis (10% Pd-C) of 3 and 4 in methanol under hydrogen atmosphere gave 5 and 6 respectively, which on reaction with (Boc)2O and Et3N in THF gave 7 (72%) and 8 (72%) respectively. Further, 7 and 8 on hydrolysis with NaOH in methanol afforded respective acids 9 (85%) and 11 (86%), while, exposure to trifluroacetic acid (TFA) in CH2Cl2 furnished the respective amine salts 10 and 12.As a part of our ongoing program on foldamers, to create skeletal and novel secondary structural diversity, peptides with heterogeneous backbone using L-Ala and -Caa were envisaged to result in /β-hybrid peptides. Based on the earlier findings from β-Caas resulting in 10/12-mixed helices, the new design with heterogeneous backbone was anticipated to result in 9/11-mixed helices.This new design to synthesize /β-hybrid peptides is based on the 1:1 alternating use of (S)-β-Caa and L-Ala (Figure 1). The peptides were prepared using standard peptide coupling13 conditions in the presence of water soluble EDCI and HOBt, with DIPEA as a base in dry CH2Cl2. Figure 1: (S)-β-Caa ((S)-C-linked carbo-β-amino acid), HCl.NH2-L-Ala-OMe (Alanine methyl ester hydrochloride) and Boc-L-Ala-acid Accordingly, the dipeptide 13, tetrapeptide 14 and hexapeptide 15 were prepared from (S)-β-Caa 7 and L-Ala by adopting segment condensation method in the presence of EDCI, HOBt and DIPEA in CH2Cl2. NMR, CD and MD studies clearly indicated the pre-	Figure 2: A) Circular dichroism spectrum of 15 in CH3OH at concentration of 0.2 mM. B) Superimposed 25 minimum energy structures of 15 (sugars are replaced with methyl groups and protons have been removed for clarity)sence of novel right-handed 9/11-mixed helical pattern in the /β-hybrid peptides 14 and 15 (Figure 2). Due to the conformational preferences, (S)-β-Caa 7 and L-Ala participated in 9- and 11-membered H-bonding respectively. Thus, the 9/11-helical pattern that was indicated in these /β-peptides is newly found and unprecedented in the literature.	Having met with success on the above new ‘design’ with (S)-β-Caa at the N-terminus, it was proposed to synthesize new /β-peptides with L-Ala at the N-terminus. Based on the above observations, the new design with L-Ala at N-terminus is anticipated to give a different helical structure such as an 11/9-helix.Accordingly, tripeptide 16, pentapeptide 17 and heptapeptide 18 were prepared from L-Ala and (S)-β-Caa 7 under standard peptide coupling conditions (EDCI, HOBt 		and DIPEA) in CH2Cl2. NMR, CD and MD studies evidently proved the presence a right-handed 11/9-mixed helical pattern in /β-hybrid peptides 16, 17 and 18. Further, this study also proved the presence of a 11/9-helix in as short as a tripeptide 16.   	Thus, the new designs on /β-hybrid peptides, resulted in a new class of peptides for the first time with robust helical structures with heterogeneous backbones, besides the report of a novel and unprecedented 9/11-mixed helical pattern. Synthesis of /β-hybrid peptides with new three residue turns and their application for Helix-Turn (HT) and Helix-Turn-Helix (HTH) motifsThe preceding discussion described the design and synthesis of /-hybrid peptides with ‘L-Ala--Caa-L-Ala’ (--) sequence at the C-terminus, from L-Ala and (S)--Caa, forming very robust 11/9-mixed helices. Further studies were undertaken on the synthesis of /-peptides having C-terminus ‘-Caa-L-Ala--Caa’ (--) three residue component. In comparison to the earlier /-peptides, in the newly designed peptides, the C-terminal (S)--Caa residue does not participate in the helix formation. It thus would be interesting to understand the implications of such a residue on the helix forming capability. 	Accordingly, tetraptide 19, hexapeptide 20 and octapeptide 21 were prepared with L-Ala (at N-terminus) and (S)-β-Caa 7, under standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2. NMR, MD and CD studies clearly indicated that /-hybrid peptides 20 and 21 fold into helix-turn super secondary structural motifs, with an 11/9-mixed helical pattern at N-terminus and 7/13-turn at C-terminus (Figure 3). This study amply indicated that the C-terminal three residue sequence has resulted on nucleation with a 11/9-helix into a novel ‘turn’ serendipitously. Figure 3: Characteristic nOes and H-bonds defining the turn in 20 (Dotted lines indicate the nOes, dark lines indicate the H-bonds in the turn region and italic represents residue number)Having successfully synthesized the /β-hybrid peptides with C-terminus ‘-Caa-L-Ala--Caa’ (--), which resulted de novo in ‘helix-turn’ motifs (super secondary structure), the attention was then focused on the synthesis of the /-hybrid peptides with (S)--Caa at the N-terminus to result in peptides with new helix-turn patterns.Accordingly, tripeptide 22, pentaptide 23 and heptapeptide 24 were prepared with  in the presence of EDCI, HOBt and DIPEA in CH2Cl2. NMR and MD studies clearly indicated that the peptides 23 and 24 with a -Caa at the N-terminus also resulted in the helix-turn motif, nucleated by a 9/11-helix (Figure 4).Figure 4: (A) Circular dichroism spectrum of 24 in CH3OH at concentration of 0.2 mM. MD structure of 24; (B) Stereoview (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity)				This study thus resulted in a new and novel three residue turn in ‘-Caa-L-Ala--Caa’. A literature survey revealed that the three-residue turns are the second most frequently observed class of hairpin forming loops after two-residue loops. Unlike the D- Figure 5: Ball and stick models of turns in: A) -helix; B) D-Pro-L-Pro-D-Ala; C) -Caa-L-Ala--CaaPro-L-Pro-D-Ala turn14 (Figure 5B), the new ‘-Caa-L-Ala--Caa’ turn (Figure 5C), is devoid of any constrained amino acids and differs in the directionality of the 13-memebered H-bond. Further, both these turns differ from the -turns15 (Figure 5A) observed in proteins.Thus, the above study resulted in the discovery of a new three residue turn motif in C-terminus ‘--’ sequence, with 7- and 13-membered H-bonded pseudo rings. Further, it is a serendipitous discovery to find the ‘--’ sequence as a turn motif upon nucleation by helix, which is the first such example of a three residue turn in linear peptides, devoid of any constrained amino acid. This study has thus resulted in new ‘helix-turn’ motif. Having successfully completed the synthesis of /β-hybrid peptides with novel ‘Helix-Turn’ (HT) motifs, the HT motifs were envisaged as an attractive option to design Helix-Turn-Helix (HTH) tertiary structures. Accordingly, peptide 25, derived by appending (S)-β-Caa at the C-terminus in 23, was investigated first. NMR and MD studies suggested that for 25, the three residue turn in the C-terminal appears to be disturbed (Figure 6). Although the signatures of the helix at the N-terminus are retained, additional H-bonding was observed between NH(5)-O(6) of furanoside ring.       Figure 6: (A) Stereoview of 25 (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity except that of residue-6) 	To understand the above results conclusively, higher oligomers 26, 27 and 28 obtained by extending 23, 20 and 24 at the C-termini with Boc-NH-β-Caa-L-Ala-β-Caa-L-Ala-OMe, respectively were studied. The NMR studies on 26, 27 and 28 though indicated the presence of stable helices at the two termini, with the characteristic H-bonding and the nOe patterns, the geometry of the ‘turn’ region differed from that in helix-turn peptides. In 26, the turn region involving the residues 4-7, displayed new nOes; NH(5)/C4H(6), NH(5)/C1H(6), NH(5)/NH(6), CH(4)/NH(6) and CH(4)/NH(7). The MD calculations provided compelling evidence for the involvement of CO(3) in a three																			centered H-bonding with NH(6) and NH(7), having 11- and 15-membered pseudo rings respectively. H-bonding was also observed between NH(5)-O(6) of the furanoside ring, further stabilizing the structure. This three residue turn, imparts an angle of about 90º between the two helices. The features described above for the peptide 26 are reiterated in 27 and 28, thus resulting in Helix-Turn-Helix tertiary structures. 	Figure 7: Characteristic nOes and H-bonds defining the turn in 26 (Dotted lines indicate the nOes, dark lines indicate the H-bonds in the turn region and italic represents residue number)Figure 8: (A) Circular dichroism spectrum of 26 in CH3OH at concentration of 0.2 mM. MD structure of 26; (B) Stereoview; (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity except that of residue-6) The H-bonding in the new ‘L-Ala--Caa--Caa’ turn (Figure 9B) differs from the ‘-Caa-L-Ala--Caa’ turn (Figure 9A), wherein ‘L-Ala--Caa--Caa’ is having a new three-centered or bifurcated H-bonding.16 Thus, the Helix-Turn-Helix (HTH) peptides 26, 27 and 28 have been identified with three centered (bifurcated) hydrogen bonding between two donar N-H groups and one acceptor C=O.Figure 9: Ball and stick models of turns in: A) -Caa-L-Ala--Caa and B) L-Ala--Caa--Caa	Synthesis of /-hybrid peptides from alternating D-Ala and (R)-C-linked Carbo--amino acids [(R)--Caa]	In the preceding discussions, the /β-hybrid peptides with heterogeneous backbone derived from L-Ala and (S)-β-Caa, having C-terminus ‘-β-’ and ‘β--β’ sequence resulted in novel right-handed 9/11-helical structures and new turns/helix-turn and helix-turn-helix motifs respectively. In a further study to explore the heterogeneous backbone peptides with helical diversity, a new design was envisaged based on the alternating use of (R)-β-Caa and D-Ala (Figure 10). 	Figure 10: (R)-β-Caa (R -C-linked carbo-β-amino acid), (D)-Alanine methyl ester hydrochloride and Boc-D-Ala-acid 	Accordingly, dipeptide 29, tripeptide 30, tetrapeptide 31, pentapeptide 32 and hexapeptides 33 were prepared having D-Ala at the N-terminus and (R)-β-Caa at the C-terminus, with the concept of alternating chirality using standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2. Similarly, new /β-hybrid peptides having N-terminus (R)-β-Caa were also prepared. Accordingly, tripeptide (35), tetrapeptide (36), pentapeptide (37) and hexapeptides (38) were prepared under the standard peptide coupling conditions. The NMR studies on these new /β-hybrid peptides from (R)-β-Caa and D-Ala indicated the presence of H-bonding by downfield shift for the amide protons. Besides, the solvent titration studies confirmed their participation in H-bonding. Further, the CD spectral data obtained for these peptides in MeOH gave proof for the presence of secondary structural elements in these /β-hybrid peptides. However, though the signatures are directing towards the presence of 9/11-mixed helical pattern, sufficient nOe data could not be obtained from NMR studies to do the MD simulation studies to conclusively prove the conformations that are present in these peptides.ConclusionThus, the work reported in the thesis resulted in the design and synthesis of a new class of /β-hybrid peptides from alternating L-Ala/(S)-β-Caa and D-Ala/(R)-β-Caa. The first design (L-Ala/(S)-β-Caa) with C-terminus -β- sequence resulted in robust 9/11-helical structures, while the peptides with C-terminal β--β sequence resulted initially in a novel three residue ‘turn’ having 7- and 13-membered H-bonding and gave Helix-Turn (HT) peptides. These HT peptides were converted into Helix-Turn-Helix peptides wherein, yet another new three residue turn (-β-β) with bifurcated (11- and 15-membered pseudo rings) H-bonding was observed. A further study on the peptides made from (R)-β-Caa and D-Ala, eventhough has shown the signatures for the presence of secondary structure (mixed helical pattern), sufficient nOe data could not be obtained to conclusively prove the secondary structure.References1.	Reviews: a) Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015. b) Gellman, S, H. Acc. Chem. Res. 1998, 31, 173. c) DeGrado, W, F.; Schneider, J. P.; Hamuro, Y. J. Pept. Res. 1999, 54, 206. d) Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med. Chem. 1999, 6, 905. e) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001, 101, 3219. f) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M-I. Curr. Med. Chem. 2002, 9, 811.2.	Reviews: a) Cole, D. C. 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