Conformational studies of C 2 symmetric peptidomimetics based on 2 , 5-anhydro sugar diacid and 2 , 5-anhydro sugar diamine scaffolds

Conformational studies of C2 symmetric peptidomimetics with identical peptide strands attached on both sides of 2,5-anhydro sugar diacid and 2,5-anhydro sugar diamine scaffolds revealed the presence of nine-membered pseudo β-turns in the sugar diacid based molecules consisting of identical intramolecular hydrogen bonds at the two ends between the AANH and sugarOH.


Introduction
We developed and studied a new class of compounds 1-8 (Figure 1), as potential HIV-protease inhibitors that were based on carbohydrate peptide hybrid structures. 1 In this strategy, identical peptide chains were attached on both sides of C 2 symmetric carbohydrate based scaffolds, leading to the formation of C 2 -symmetric peptiodomimetics. 2 Carbohydrate based molecular designs are increasingly drawing attention of chemists. 3Detailed studies on the development of new HIV-1 protease inhibitors based on C 2 symmetric scaffolds and acyclic carbohydrates have recently been reported. 4It is also being increasingly felt that small molecule protease inhibitors need to have restricted degrees of freedom which is evident from the success of cyclic urea based inhibitors. 5This prompted us to study cyclic carbohydrate based core foundations as conformationally rigid scaffolds to build a new class of molecular framework as potential protease inhibitors.
Two different types of cyclic carbohydrate frameworks of sugar diacid and sugar diamine were employed in our design.These molecules are 2,5-anhydro-D-idaric acid 9 (Idac), 1,6diamino-2,5-anhydro-1,6-dideoxy-D-iditol 10 (Idam) and 1,6-diamino-2,5-anhydro-1,6-dideoxy-D-mannitol 11 (Mdam).Attachment of identical peptide strands on both sides of these scaffolds led to the formation of peptidomimetic compounds 1-8, whose syntheses were reported earlier. 1 The conformational study of 1 was carried out in detail and revealed a C 2 symmetric structure with a nine-membered pseudo β-turn consisting of identical intramolecular hydrogen bonds at the two ends between the LeuNH and sugarOH. 6A very small temperature coefficient for the LeuNH chemical shift (∆δ/∆T = -1.2ppb.K -1 ) was observed in DMSO-d 6 .The presence of two 'cis-β-hydroxycarboxyl' moieties, the core structural motif believed to be responsible for such intramolecular H-bonds, on two sides of the tetrahydrofuran ring, nucleated identical β-turn-like structures in 1 at both ends.In this paper, we describe detailed conformational analysis of compounds 2-8 achieved by various NMR techniques, which revealed that while all these molecules had C 2 symmetric structures, only the sugar diacid based compounds 2-4, especially those with free hydroxyl groups on the sugar ring, had the propensity to form nine-membered pseudo β-turn like structures containing intramolecular hydrogen bonds between AA 2 NH → sugarOH.This supports further the earlier observations that free hydroxyl groups on sugar rings prevent furanoid sugar amino acid based short linear peptides to adopt regular β-turn structures as these hydroxyl groups themselves act as hydrogen bond acceptors.Conformational analysis of 3. Peptidomimetic 3 also displayed C 2 symmetry in DMSO-d 6 .
Various NOEs LeuNH ↔ PheNH, PheNH ↔ IdacC3H (C4H), PheNH ↔ IdacC2H (C5H) and LeuNH ↔ IdacOH were observed along with sequential NOEs AlaNH ↔ LeuCαH, LeuNH ↔ PheCαH (Figure 4).Expanded ROESY spectrum of 3 with characteristic NOE connectivities is shown in Figure 5.The temperature coefficient (∆δ/∆T) of -2.3 ppb.K -1 for leucine amide proton indicated its participation in intramolecular hydrogen bonding.The side chains of Leu and Phe appeared to take a predominant conformation about χ 1 , which is evident from J αH-βH = 9.8, J αH-β′H = 5.1 for Leu and J αH-βH = 10.4,J αH-β′H = 3.4 Hz for Phe.The structures sampled during the restrained MD calculations, 8 using distance constraints based on the ROESY cross-peak intensities 9 for 3 in DMSO-d 6 , are in agreement with the structure obtained from NMR studies.The two peptide-chains form a pseudo β-turn involving Hbond between LeuNH and IdacOH.The central portions of these structures are fairly conserved with the variations localized mainly at the Leu side-chain (Figure 6).The five-membered sugar rings take twist conformations that are in agreement with the NMR data.Conformational analysis of 5-8.Peptidomimetics 5-8 displayed perfect C 2 symmetric structures in their NMR spectra in DMSO-d 6 .However, as the temperature coefficients of all the amide protons in these molecules were very high (Table 1), they were not studied for further structural details.The ROESY spectra of 6 and 8 did not show many long range NOEs (Figure 8) and definite structural information on their structures could not be obtained.This may partly be due to the additional methylene groups attached to the sugar ring.

Discussion
The large values (7.6-8.5 Hz) of 3 J NH-αH for Phe and Leu in 3 and 4, and for Val in 2 were observed.Similarly, for the diamine peptidomimetics 5-8, the 3 J NH-αH s were large (> 8 Hz) for Phe and Leu in 5 and 7, and for Val in 6 and 8, which correspond to a value of φ in the vicinity of -120°.The existence of a complete set of sequential CαH ↔ NH NOE connectivities, wherever possible, implied the propensity of structures with large population of conformers in the β-region of φ-ψ space (φ =-180° to -60° and ψ = 60° to 180°).The side-chains of Phe, Leu and Val point to the existence of large population of a single conformation in several of these peptides.For peptides 3, 4, 5, and 7, the LeuNH ↔ LeuCβH NOEs, stronger than the LeuNH ↔ LeuCβ′H NOEs, were observed.Further LeuCαH ↔ LeuCδH and LeuNH ↔ LeuCγH NOEs were also seen.Large J αH-βH of about 9 Hz and small J αH-β′H of about 5 Hz in Phe, Leu and Val further indicated restricted rotation about χ 1 .
The observation of J H2-H3 = J H4-H5 = 3.5 Hz in 2, 3, and 4 and J H3-H4 ≈ 0 are consistent with a twist conformation of 4 3 T (where 4 and 3 refer to C3 and C4, respectively) for the five-membered ring with C3 and C4 both being exo to the respective peptide chains.

Conclusion
The anticipated nucleation of the nine-membered β-turn like ring structures at both ends of these C 2 symmetric assemblies involving identical intramolecular H-bonding, as observed in 1 between LeuNH → sugarOH, was realised in some compounds.The observation of ∆δ/∆T for LeuNH being 2.3 and 2.8 in 3 and 4, respectively, support the formation of incipient intramolecular LeuNH → sugarOH hydrogen bonds in these molecules, relatively stronger in the former.In the diamine peptidomimetics, the absence of connectivities between sugar ring and the peptide sequences, along with the disappearance of the H-bonding character, which was consistently observed in the diacid-based molecules, indicate the deviation from the designed structure.Higher degree of freedom created in Idam and Mdam by the methylene group in the sugar ring might be responsible for the absence of any ordered structure.These observations reveal the importance of the constraints imparted in these structures for obtaining the designed reverse turn mimetics.
In summary, introduction of C 2 symmetric scaffolds 9-11 helped molecules 1-8 adopt C 2 symmetric structures and thus fulfilled the primary objective of our study.Although, these compounds did not show any activity towards HIV protease inhibition up to 2 mM inhibitor concentration, 1 positioning of more appropriate residues at the P1/P1′ positions in these molecules will probably lead to substrates with improved biological activities. 10Further work is currently in progress.

Experimental Section
General Procedures.All reactions were carried out in oven or flame-dried glassware with magnetic stirring under nitrogen atmosphere using dry, freshly distilled solvents, unless otherwise noted.Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm silica gel plates with UV light, I 2 , 7% ethanolic phosphomolybdic acid-heat and 2.5% ethanolic anisaldehyde (with 1% AcOH and 3.3% conc.H 2 SO 4 )-heat as developing agents.Silica gel finer than 200 mesh was used for flash column chromatography.Yields refer to chromatographically and spectroscopically homogeneous materials unless otherwise stated.IR spectra were recorded as neat liquids or KBr pellets.NMR spectra were recorded on 200, 300 and 400 MHz spectrometers at room temperature of ~ 21 °C in CDCl 3 using tetramethylsilane as internal standard or the solvent signal as secondary standard and the chemical shifts are shown in δ scales.Multiplicities of NMR signals are designated as s (singlet), d (doublet), t (triplet), q (quartet), br (broad), m (multiplet, for unresolved lines), etc. 13 C NMR spectra were recorded with complete proton decoupling.Mass spectra were obtained under electron impact (EI), electron spray ionisation (ESI) and liquid secondary ion mass spectrometric (LSIMS) techniques.

Figure 4 .
Figure 4. Schematic representation of the proposed H-bonded structure of 3 with some of the prominent long-range NOEs seen in its ROESY spectrum.

Figure 6 .Figure 7 .
Figure 6.Ten superimposed structures sampled during MD simulation studies for 3. Structures on left and right represent views from two different angles.

Figure 8 .
Figure 8. Diagrammatic representations of some of the NOEs for 6 (left) and 8 (right) in DMSOd 6 .