Synergistic n → π* and nN → π*Ar interactions in C-terminal modified prolines: effect on Xaa-Pro cis/trans equilibrium.

Carbonyl-carbonyl (CO⋯CO) n → π* interaction often coexists with a hydrogen bond (HB) or another n → π* interaction. Although the interplay between HB and n → π* interaction was previously studied, there is no systematic investigation that shows a synergistic relationship of n → π* with another noncovalent interaction. Herein, we have studied a set of proline-diacylhydrazine (Pro-DAH) molecules and observed that increase in the strength of the nN → π*Ar interaction on their DAH side strengthened the n → π* interaction on the Pro side, which was experimentally determined by measuring the Ktrans/cis of the Xaa-Pro amide bond. Overall, we describe a simple C-terminal modification strategy to stabilize the trans-Pro geometry that could be useful to stabilize PPII helices and collagen triple helices that require Pro to adopt the trans amide geometry.

Understanding the Cis-Trans Amide Bond Isomerization of N,N'-Diacylhydrazines to Develop Guidelines for A Priori Prediction of Their Most Stable Solution Conformers.

N,N'-diacylhydrazines (R1CO-NR3-NR4-COR2) are a class of small molecules with a wide range of applications in chemistry and biology. They are structurally unique in the sense that their two amide groups are connected via a N-N single bond, and as a result, these molecules can exist in eight different isomeric forms. Four of these are amide isomers [trans-trans (t-t), trans-cis (t-c), cis-trans (c-t), and cis-cis (c-c)] arising from C-N bond restricted rotation. In addition, each of these amide isomers can exist in two different isomeric forms due to N-N bond restricted rotation, especially when R3 and R4 groups are relatively bigger. Herein, we have systematically investigated the conformations of 55 N,N'-diacylhydrazines using a combination of solution NMR spectroscopy, X-ray crystallography, and density functional theory calculations. Our data suggest that when the substituents R3 and R4 on the nitrogen atoms are both hydrogens. These molecules prefer twisted trans-trans (t-t) (>90%) geometries (H-N-C═O ∼ 180°), whereas the N-alkylated and N,N'-dialkylated molecules prefer twisted trans-cis (t-c) geometries. Herein, we have analyzed the stabilization of the various isomers of these molecules in light of steric and stereoelectronic effects. We provide a guideline to a priori predict the most stable conformers of the N,N'-diacylhydrazines just by examining their substituents (R1-R4).

Deciphering the Backbone Noncovalent Interactions that Stabilize Polyproline II Conformation and Reduce cis Proline Abundance in Polyproline Tracts.

Proline (Pro) has a higher propensity to adopt cis amide geometry than the other natural amino acids, and a poly-Pro (poly-P) tract can adopt either a polyproline I (PPI, all cis amide) or a polyproline II (PPII, all trans amide) helical conformation. Recent studies have revealed a reduced abundance of cis amide geometry among the inner Pro residues of a poly-P tract. However, the forces that stabilize the polyproline helices and the reason for the higher trans amide propensity of the inner Pro residues of a poly-P tract are poorly understood. Herein, we have studied both Pro and non-Pro PPII helical sequences and identified the backbone noncovalent interactions that are crucial to the higher stability of the trans Pro-amide geometry and the preference for a PPII helical conformation. We show the presence of reciprocal CO···CO interactions that extend over the whole PPII helical region. Interestingly, the CO···CO interactions strengthen with the increase in the PPII helical chain length and the inner CO groups possess stronger CO···CO interactions, which could explain the reduced cis abundance of the inner Pro residues of a poly-P tract. We also identified a much stronger (∼0.9 kcal·mol-1) nO → σ*Cα-Cß interaction between the N-terminal CO oxygen lone pair and the antibonding orbital (σ*) of their Cα-Cß bonds. As the nO → σ*Cα-Cß interaction is possible only in the trans isomers of Pro, this interaction should be crucial for the stabilization of a PPII helix. Finally, an unusual nN(amide) → σ*C-N interaction (∼0.3 kcal·mol-1) was observed between the peptidic nitrogen lone pair (nN) and the antibonding orbital (σ*C-N) of the subsequent C-terminal peptide C-N bond. We propose a cumulative effect of these interactions in the stabilization of a PPII helix.

nN → π*Ar interactions stabilize the E-ac isomers of arylhydrazides and facilitate their SNAr autocyclizations.

We describe a novel mechanism of stabilization of the E-ac isomer of an arylhydrazide via nN → π*Ar interactions. We further show that when a leaving group (F) is present at the ortho-position of the carbonyl group of such an arylhydrazide, the nN → π*Ar interaction facilitates an SNAr autocyclization reaction to produce indazolone, an important heterocycle with biological activity. Faster autocyclization of arylhydrazide is observed when an electron withdrawing group is present in the aryl ring, which is a characteristic of SNAr reactions.

Stabilization of Azapeptides by Namide···H-Namide Hydrogen Bonds.

An unusual Namide···H-Namide hydrogen bond (HB) was previously proposed to stabilize the azapeptide ß-turns. Herein we provide experimental evidence for the Namide···H-Namide HB and show that this HB endows a stabilization of 1-3 kcal·mol-1 and enforces the trans-cis-trans (t-c-t) and cis-cis-trans (c-c-t) amide bond conformations in azapeptides and N-methyl-azapeptides, respectively. Our results indicate that these Namide···H-Namide HBs can have stabilizing contributions even in short azapeptides that cannot fold to form ß-turns.

Evidence of an nN(amide) → π*Ar Interaction in N-Alkyl-N,N'-diacylhydrazines.

1,2-Dibenzoyl-1-tert-butylhydrazine (RH-5849) and related N-alkyl-N,N'-diacylhydrazines are environmentally benign insect growth regulators. Herein, we show that an unusual nN(amide) → π*Ar interaction mediated by a hydrazide amide nitrogen atom plays a crucial role in stabilizing their biologically active trans-cis (t-c) rotameric conformations. We provide NMR and IR spectroscopic evidence for the presence of these interactions, which is also supported by X-ray crystallographic and computational studies.

Spectroscopic evidence of n → π* interactions involving carbonyl groups.

n → π* has emerged as an important noncovalent interaction that can affect the conformations of both small- and macromolecules including peptides and proteins. Carbonyl-carbonyl (COCO) n → π* interactions involving CO groups are well studied. Recent studies have shown that the COCO n → π* interactions are the most abundant secondary interactions in proteins with a frequency of 33 interactions per 100 residues and, among the various secondary interactions, n → π* interactions are expected to provide the highest enthalpic contributions to the conformational stability of globular proteins. However, n → π* interactions are relatively weak and provide an average stabilization of about 0.25 kcal mol-1 per interaction in proteins. The strongest n → π* interaction could be as strong as a moderate hydrogen bond. Therefore, it is challenging to detect and quantify these weak interactions, especially in solution in the presence of perturbation from other intermolecular interactions. Accordingly, spectroscopic investigations that can provide direct evidence of n → π* interaction are limited, and the majority of papers published in this area have relied on X-ray crystallography and/or theoretical calculations to establish the presence of this interaction. The aim of this perspective is to discuss the studies where a spectroscopic signature in support of n → π* interaction was observed. As the "n → π* interaction" is a relatively new terminology, there remains the possibility of there being earlier studies where spectroscopic evidence for n → π* interactions was obtained but it was not discussed in light of the n → π* terminology. We noticed several such studies and, as can be expected, these studies were often overlooked in the discussion of n → π* interactions in the recent literature. In this perspective, we have also discussed these studies and provided computational support for the presence of n → π* interaction.

Conformational control of N-methyl-N,N'-diacylhydrazines by noncovalent carbon bonding in solution.

In recent years, some X-ray structural and computational evidence has emerged for noncovalent carbon bonding (C-bond). However, evidence of C-bonds in solution is limited. Herein, from the conformational analyses of strategically designed N-methyl-N,N'-diacylhydrazines, we for the first time show that C-bonds can be modulated to control the conformational preferences of small molecules in solution. We show that unusual N(amide)C-X noncovalent carbon bonding interactions stabilize the trans-cis (t-c) amide bond rotamers of N-methyl-N,N'-diacylhydrazines over the expected trans-trans (t-t) rotamers.

Solid-Phase Synthesis of Hybrid 2,5-Diketopiperazines Using Acylhydrazide, Carbazate, Semicarbazide, Amino Acid, and Primary Amine Submonomers.

We report the solid-phase synthesis of N,N'-di(acylamino)-2,5-diketopiperazine, an acylhydrazide-based conformationally rigid 2,5-DKP scaffold having exocyclic N-N bonds. We also show that different combinations of acylhydrazides, carbazates, semicarbazides, amino acids, and primary amines can be used to synthesize a highly diverse collection of hybrid DKP molecules via the solid-phase submonomer synthesis route. Finally, we show incorporation of a methyl substituent in one of the carbon atoms of the DKP ring to generate chiral daa- and hybrid-DKPs without compromising the synthetic efficiency.

Relative orientation of the carbonyl groups determines the nature of orbital interactions in carbonyl-carbonyl short contacts.

Carbonyl-carbonyl (CO···CO) interactions are emerging noncovalent interactions found in many small molecules, polyesters, peptides and proteins. However, little is known about the effect of the relative orientation of the two carbonyl groups on the nature of these interactions. Herein, we first show that simple homodimers of acetone and formaldehyde can serve as models to understand the effect of relative orientations of the two carbonyl groups on the nature of CO···CO interactions. Further, from a comprehensive statistical analysis of molecules having inter- or intramolecular CO···CO interactions, we show that the molecules can be broadly categorized into six different structural motifs (I-VI). The analysis of pyramidality of the acceptor carbon atoms in these motifs and natural bond orbital (NBO) analysis suggest that the relative orientation of the two interacting carbonyl groups determines whether the orbital interaction between the two carbonyl groups would be n → π* or π → π* or a combination of both.

N, N'-Di(acylamino)-2,5-diketopiperazines: Strategic Incorporation of Reciprocal n → π* Interactions in a Druglike Scaffold.

The incorporation of the recently discovered reciprocal n → π* interactions in 2,5-diketopiperazines (DKPs) is reported to design a novel N, N'-di(acylamino)-2,5-diketopiperazine (daa-DKP) scaffold. The design, synthesis, and structural features of daa-DKPs and the effect of reciprocal n → π* interactions in their structural rigidity is discussed.

Reciprocal carbonyl-carbonyl interactions in small molecules and proteins.

Carbonyl-carbonyl n→π* interactions where a lone pair (n) of the oxygen atom of a carbonyl group is delocalized over the π* orbital of a nearby carbonyl group have attracted a lot of attention in recent years due to their ability to affect the 3D structure of small molecules, polyesters, peptides, and proteins. In this paper, we report the discovery of a "reciprocal" carbonyl-carbonyl interaction with substantial back and forth n→π* and π→π* electron delocalization between neighboring carbonyl groups. We have carried out experimental studies, analyses of crystallographic databases and theoretical calculations to show the presence of this interaction in both small molecules and proteins. In proteins, these interactions are primarily found in polyproline II (PPII) helices. As PPII are the most abundant secondary structures in unfolded proteins, we propose that these local interactions may have implications in protein folding.Carbonyl-carbonyl π* non covalent interactions affect the structure and stability of small molecules and proteins. Here, the authors carry out experimental studies, analyses of crystallographic databases and theoretical calculations to describe an additional type of carbonyl-carbonyl interaction.

Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid-azapeptoid hybrid library.

A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid-azapeptoid hybrid library tethered to an oxadiazolone 'warhead'. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.

Solid phase synthesis of 1,3,4-oxadiazin-5 (6R)-one and 1,3,4-oxadiazol-2-one scaffolds from acyl hydrazides.

Solid phase synthesis of 1,3,4-oxadiazin-5(6R)-one and 1,3,4-oxadiazol-2-one scaffolds from resin-bound acyl hydrazides is described. We demonstrate here that the reactions of resin-bound aryl or hetero-aromatic acyl hydrazides with 2-substituted-2-bromoacetic acids and 4-nitrophenyl chloroformate and subsequent treatment with DIEA lead to intramolecular cyclization reactions to produce six-membered 1,3,4-oxadiazin-5(6R)-ones and five-membered 1,3,4-oxadiazol-2-ones, respectively. We also show that acyl hydrazide-derived 1,3,4-oxadiazol-2-ones may be useful serine hydrolase inhibitors.

Submonomer synthesis of a hybrid peptoid-azapeptoid library.

We recently reported efficient conditions for the synthesis of N-azapeptoid libraries via the typical submonomer strategy of peptoid synthesis but that substitutes N-acyl hydrazides for primary amines as submonomers. Unfortunately, this approach is not applicable to the synthesis of mixed azapeptoid-peptoid libraries. When an oligomer containing an N-terminal side chain derived from an acyl hydrazide is bromoacetylated and treated with a primary amine, a chain-terminating intramolecular ring-closure to form an oxadiazinone competes with the desired displacement of the bromide by the amine. Here we overcome this limitation and demonstrate that a hybrid peptoid-azapeptoid library derived from primary amines, acyl hydrazides, carbazates, and semicarbazides can be made efficiently using standard peptoid submonomer chemistry. We find that the unwanted, chain-terminating cyclization reaction is competitive with chain extension only when aryl acyl hydrazides are present. Alkyl or heteroaromatic acyl hydrazides do not cyclize under the conditions used for peptoid-azapeptoid synthesis. We also find that carbazates and semicarbazides work well for chain extension. Using primary amines, acyl hydrazides, carbazates, and semicarbazides as submonomers, a high-quality one bead one compound library of tetramers suitable for screening against protein targets was made by split and pool synthesis.

Acyl hydrazides as peptoid sub-monomers.

The use of acyl hydrazides as peptoid sub-monomers is investigated. We demonstrate here that azapeptoids derived entirely from acyl hydrazides can be made conveniently and efficiently using standard peptoid sub-monomer chemistry. Structural studies reveal that the main chain amide bond in these molecules predominantly adopts a trans conformation. A high-quality one bead one compound library of tetramers was made by split and pool synthesis and we found that the identity of the molecule on a single bead could be determined by tandem MALDI mass spectrometry.

Synthesis, structure, spirocyclization mechanism, and glutathione peroxidase-like antioxidant activity of stable spirodiazaselenurane and spirodiazatellurane.

The first examples of stable spirodiazaselenurane and spirodiazatellurane were synthesized by oxidative spirocyclization of the corresponding diaryl selenide and telluride and were structurally characterized. X-ray crystal structures of the spirodiazaselenurane and spirodiazatellurane suggest that the structures are distorted trigonal bipyramidal (TBP) with the electronegative nitrogen atoms occupying the apical positions and two carbon atoms and the lone pair of Se/Te occupying the equatorial positions. Interestingly, the spirodiazatellurane underwent spontaneous chiral resolution during crystallization, and the absolute configurations of its enantiomers were confirmed by single-crystal X-ray analyses. A detailed mechanistic study indicates that the cyclization to spirodiazaselenurane and spirodiazatellurane occurs via selenoxide and telluroxide intermediates. The chalcogenoxides cyclize to the corresponding spiro compounds in a stepwise manner via the involvement of hydroxyl chalcogenurane intermediates, and the activation energy for the spirocyclization reaction decreases in the order S > Se > Te. In addition to the synthesis, characterization, and mechanism of cyclization, the glutathione peroxidase (GPx) mimetic activity of the newly synthesized compounds was evaluated. These studies suggest that the tellurium compounds are more effective as GPx mimics than their selenium counterparts due to the fast oxidation of the tellurium center in the presence of peroxide and the involvement of an efficient redox cycle between the telluride and telluroxide intermediate.

Theoretical investigation on the effect of different nitrogen donors on intramolecular Se...N interactions.

The effect of different donor nitrogen atoms on the strength and nature of intramolecular Se...N interactions is evaluated for organoselenium compounds having N,N-dimethylaminomethyl (dime), oxazoline (oxa) and pyridyl (py) substituents. Quantum chemical calculations on three series of compounds [2-(dime)C(6)H(4)SeX (1 a-g), 2-(oxa)C(6)H(4)SeX (2 a-g), 2-(py)C(6)H(4)SeX (3 a-g); X=Cl, Br, OH, CN, SPh, SePh, CH(3)] at the B3LYP/6-31G(d) level show that the stability of different conformers depends on the strength of intramolecular nonbonded Se...N interactions. Natural bond orbital (NBO), NBO deletion and atoms in molecules (AIM) analyses suggest that the nature of the Se...N interaction is predominantly covalent and involves nN-->sigma*(Se--X) orbital interaction. In the three series of compounds, the strength of the Se...N interaction decreases in the order 3>2>1 for a particular X, and it decreases in the order Cl>Br>OH>SPh approximately CN approximately SePh>CH(3) for all the three series 1-3. However, further analyses suggest that the differences in strength of Se...N interaction in 1-3 is predominantly determined by the distance between the Se and N atoms, which in turn is an outcome of specific structures of 1, 2 and 3, and the nature of the donor nitrogen atoms involved has very little effect on the strength of Se...N interaction. It is also observed that Se...N interaction becomes stronger in polar solvents such as CHCl(3), as indicated by the shorter r(Se...N) and higher E(Se...N) values in CHCl(3) compared to those observed in the gas phase.

Antioxidant activity of the anti-inflammatory compound ebselen: a reversible cyclization pathway via selenenic and seleninic acid intermediates.

A revised mechanism that accounts for the glutathione peroxidase (GPx)-like catalytic activity of the organoselenium compound ebselen is described. It is shown that the reaction of ebselen with H(2)O(2) yields seleninic acid as the only oxidized product. The X-ray crystal structure of the seleninic acid shows that the selenium atom is involved in a noncovalent interaction with the carbonyl oxygen atom. In the presence of excess thiol, the Se--N bond in ebselen is readily cleaved by the thiol to produce the corresponding selenenyl sulfide. The selenenyl sulfide thus produced undergoes a disproportionation in the presence of H(2)O(2) to produce the diselenide, which upon reaction with H(2)O(2), produces a mixture of selenenic and seleninic acids. The addition of thiol to the mixture containing selenenic and seleninic acids leads to the formation of the selenenyl sulfide. When the concentration of the thiol is relatively low in the reaction mixture, the selenenic acid undergoes a rapid cyclization to produce ebselen. The seleninic acid, on the other hand, reacts with the diselenide to produce ebselen as the final product. DFT calculations show that the cyclization of selenenic acids to the corresponding selenenyl amides is more favored than that of sulfenic acids to the corresponding sulfenyl amides. This indicates that the regeneration of ebselen under a variety of conditions protects the selenium moiety from irreversible inactivation, which may be responsible for the biological activities of ebselen.

Thiol cofactors for selenoenzymes and their synthetic mimics.

The importance of selenium as an essential trace element is now well recognized. In proteins, the redox-active selenium moiety is incorporated as selenocysteine (Sec), the 21st amino acid. In mammals, selenium exerts its redox activities through several selenocysteine-containing enzymes, which include glutathione peroxidase (GPx), iodothyronine deiodinase (ID), and thioredoxin reductase (TrxR). Although these enzymes have Sec in their active sites, they catalyze completely different reactions and their substrate specificity and cofactor or co-substrate systems are significantly different. The antioxidant enzyme GPx uses the tripeptide glutathione (GSH) for the catalytic reduction of hydrogen peroxide and organic peroxides, whereas the larger and more advanced mammalian TrxRs have cysteine moieties in different subunits and prefer to utilize these internal cysteines as thiol cofactors for their catalytic activity. On the other hand, the nature of in vivo cofactor for the deiodinating enzyme ID is not known, although the use of thiols as reducing agents has been well-documented. Recent studies suggest that molecular recognition and effective binding of the thiol cofactors at the active site of the selenoenzymes and their mimics play crucial roles in the catalytic activity. The aim of this perspective is to present an overview of the thiol cofactor systems used by different selenoenzymes and their mimics.