Hosahudya N. Gopi

Peptide hybrids containing alpha- and beta-amino acids: Structure of a decapeptide beta-hairpin with two facing beta-phenylalanine residues

A β-hairpin conformation has been characterized in crystals of the decapeptide t-butoxycarbonyl-Leu-Val-βPhe-Val-DPro-Gly-Leu-βPhe-Val-Val-methyl ester [βPhe; (S)-β3 homophenylalanine] by x-ray diffraction. The polypeptide chain reversal is nucleated by the centrally positioned DPro-Gly segment, which adopts a type-I′ β-turn conformation. Four intramolecular cross-strand hydrogen bonds stabilize the peptide fold. The βPhe(3) and βPhe(8) residues occupy facing positions on the hairpin, with the side chains projecting on opposite faces of the β-sheet. At the site of insertion of β-residues, the polarity of the peptide units along each strand reverses, as compared with the α-peptide segments. In this analog, a small segment of a polar sheet is observed, where adjacent CO and NH groups line up in opposite directions in each strand. In the crystal, an extended β-sheet is formed by hydrogen bonding between strands of antiparallel pairs of β-hairpins. The crystallographic parameters for C65H102N10O13⋅ 3H2O are: space group P212121; a = 19.059(8) Å, b = 19.470(2) Å, c = 21.077(2) Å; Z = 4; agreement factor R1 = 9.12% for 3,984 data observed >4σ(F) and a resolution of 0.90 Å.

beta-Hairpins Generated from Hybrid Peptide Sequences Containing both alpha- and beta-Amino Acids

The incorporation of the -amino acid residues into specific positions in the strands and -turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C()C() bond () enhances the conformational repertoire in -residues. The conformational analysis of three designed peptide hairpins composed of /-hybrid segments is described: Boc-Leu-Val-Val-DPro-Phe- Leu-Val-Val-OMe (1), Boc-Leu-Val-Val-DPro-Gly-Leu-Val-Val-OMe (2), and Boc-Leu-Val-Phe-Val-DPro- Gly-Leu-Phe-Val-Val-OMe (3). 500-MHz 1H-NMR Analysis supports a preponderance of -hairpin conformation in solution for all three peptides, with critical cross-strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a -hairpin conformation with two -residues occupying facing, non-H-bonded positions in antiparallel -strands. Notably, Val(3 ) adopts a gauche conformation about the C()C() bond (65) without disturbing cross-strand H-bonding. The crystal structure of 2, together with previously published crystal structures of peptides 3 and Boc-Phe-Phe-DPro-Gly- Phe-Phe-OMe, provide an opportunity to visualize the packing of peptide sheets with local −polar segments× formed as a consequence of reversal peptide-bond orientation. The available structural evidence for hairpins suggests that -residues can be accommodated into nucleating turn segments and into both the H-bonding and non-H-bonding positions on the strands.

Click chemistry on azidoproline: high-affinity dual antagonist for HIV-1 envelope glycoprotein gp120.

Recent advances in the Cu-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes afford 1,4-disubstituted 1,2,3-triazoles with superior regioselectivity, and almost quantitative transformation under extremely mild conditions. The simple and robust features of this methodology have found application in drug discovery, bioconjugation, and material science. Herein, we report the novel use of stable and chemically accessible azidoproline within an otherwise normally constituted solid-phase-synthesized polypeptide as a platform for side-chain bioconjugation reactions through click chemistry, and its use to introduce triazole conjugates into a dual antagonist of the HIV-1 envelope protein gp120. The procedure enables rapid generation of analogues at an internal side-chain position of the antagonist. This has led to a lead inhibitor for HIV-1 infection with affinity in the nanomolar range. Acquired immunodeficiency syndrome (AIDS), the globally epidemic disease caused by HIV-1, has created an urgent need for new classes of antiviral agents. Viral infection is initiated by the binding of gp120 of HIV-1 to the CD4 antigen on the host T cell surface. The envelope glycoprotein of HIV-1 is a trimer that consists of three gp120 exterior envelope glycoproteins and gp41 transmembrane glycoproteins. The binding of gp120 to CD4 promotes a conformational change in gp120 that increases its affinity for a second host-cell receptor, one of the chemokine receptors, CCR5 and CXCR4. The interaction of gp120 with its receptors is believed to promote further conformational rearrangements in the HIV-1 envelope that drive fusion of the viral and host-cell membranes. Blockage of the interactions between gp120 and cell-surface receptors is an attractive goal for the prevention of HIV-1 infection through the inhibition of membrane fusion and viral entry. The feasibility of therapeutic efficacy with fusion inhibitors has been demonstrated recently. A promising fusion inhibitor lead is a 12-residue peptide (RINNIPWSEAMM, 1) which was discovered initially by phage library screening. Peptide 1 inhibits the interaction between gp120 and both CD4 and 17b, an antibody that recognizes an epitope that overlaps the CCR5 binding site with affinity in the micromolar range. Herein, we report that conjugation at proline 6 of peptide 1 through click chemistry leads to inhibitors with strikingly high affinity for the HIV-1 envelope and which maintain the dual inhibition of CD4 and 17b binding to the viral Env protein. The modification of proline with 4-phenyl-1,4-disubstituted 1,2,3triazole, fabricated through a [3+2] cycloaddition reaction (Scheme 1) leads to a peptide which binds to gp120 with a KD

A Crystalline β‐Hairpin Peptide Nucleated by a Type I′ Aib‐D‐Ala β‐Turn: Evidence for Cross‐Strand Aromatic Interactions

Recent progress in the design of beta-hairpin peptides[1] and beta-sheet models has been based on the ability to nucleate reverse turns of the appropriate stereochemistry. D-Pro-Gly[2] and to a lesser extent Asn-Gly[3] segments have been shown to facilitate formation of type I’ and II’ beta-turns, which are most often found at the site of sharp polypeptide chain reversal, that is, beta-hairpins in proteins.[4, 5] The prime turns, I’ and II’, can exert differing influences on the relative twist of the antiparallel strands. The I’ turn has the sense of twist that matches the twisting of adjacent beta-strands in proteins. In contrast, the II’ turn results in a more planar arrangement with the hairpin flattening to a considerable degree.[4a,e] The D-Pro-Xxx segment can in principle adopt both II’ and I’ turn conformations as si (D-Pro) values of +30 deg. and –120 deg. are energetically favorable.[1a, 5]

Crystal structure of a hydrophobic 19-residue peptide helix containing three centrally located d amino acids

The design of the synthetic 19-residue peptide Boc-Leu-Aib-Val-Ala-Leu5-Aib-Val-d-Ala-d-Leu-Leu10-Val-Phe-Val-Aib-d-Val15-Leu-Phe-Val-Val-OMe (Aib, α-aminoisobutyric acid; OMe, methyl ester) was intended to produce a crystalline peptide with independent helical and hairpin domains. The design was partially based on an octapeptide with the same sequence as residues 11–18 above, which was shown to fold into a β-hairpin in the crystal. However, the crystal structure of the present peptide provided a surprising result. The conformation is the longest characterized right-handed α-helix, with as many as three internal d residues in the sequence. The completely helical structure was also unexpected, because β-branched residues such as Val have a low propensity for helix formation in proteins. The helical peptides in the present structure assemble to form hydrophobic channels that accommodate five toluene molecules per peptide along the length of the channel. The structural results illustrate the similarity in energetics between helical and β-hairpin conformations for peptides containing Aib residues. The crystallographic parameters for C107H179N19O22·3H2O·2.5 toluenes are: space group C2, a = 34.679(3) Å, b = 12.866(1) Å, c = 31.915(3) Å, β = 96.511(8)°, V = 14,148 Å3, Z = 4, dcalc = 1.099 g/cm3, and agreement factor R1 = 10.2%.

Structural Determinants for Affinity Enhancement of a Dual Antagonist Peptide Entry Inhibitor of Human Immunodeficiency Virus Type-1

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120.

In this work, we identified a high affinity and potency metallocene‐containing triazole peptide conjugate that suppresses the interactions of HIV‐1 envelope gp120 at both its CD4 and co‐receptor binding sites. The ferrocene‐peptide conjugate, HNG‐156, was formed by an on‐resin copper‐catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl‐containing triazole conjugate HNG‐105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV‐1 gp120 due mainly to the decreased dissociation rate of the conjugate‐gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037‐08) and clade B (YU‐2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV‐1YU‐2 gp120 at both the CD4 binding site and CD4‐induced binding sites. HNG‐156 showed a close‐to nanomolar IC50 for inhibiting cell infection by HIV‐1BaL whole virus. The dual receptor site antagonist activity and potency of HNG‐156 make it a promising viral envelope inhibitor lead for developing anti‐HIV‐1 treatments. Copyright

Proteolytic stability of β-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

A set of designed internally quenched fluorescence peptide substrates has been used to probe the effects of insertion of β‐peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin α‐chain, residues 32–37. Fluorescence and mass spectral measurements demonstrate that the insertion of an β‐residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modification is also considerably reduced.

Hybrid Peptides: Direct Transformation of α/α, β-Unsaturated γ-Hybrid Peptides to α/γ-Hybrid Peptide 12-Helices

A smooth transformation of unusual planar structures of α/vinylogous hybrid peptides to ordered α/γ(4)-hybrid peptide 12-helices and the stereochemical preferences of vinylogous amino acid residues in single crystals are studied.

Copper(II) mediated facile and ultra fast peptide synthesis in methanol

A novel, ultrafast, mild and scalable amide bond formation strategy in methanol using simple thioacids and amines is described. The mechanism suggests that the coupling reactions are initially mediated by CuSO(4)·5H(2)O and subsequently catalyzed by in situ generated copper sulfide. The pure peptides were isolated in satisfactory yields in less than 5 minutes.