Cis Amide Research Articles

A cis Amide Bond Surrogate Incorporating 1,2,4‐Triazole.

AbstractFor Abstract see ChemInform Abstract in Full Text.

A cis amide bond surrogate incorporating 1,2,4-triazole.

A novel cis amide bond surrogate incorporating 1,2,4-triazole was designed and synthesized by the reaction of a thionotripeptide, formic hydrazide, and mercury(II) acetate. This method of surrogate formation was also applicable to a cyclic thionopeptide.

Binding selectivity of conformationally restricted analogues of (-)-indolactam-V to the C1 domains of protein kinase C isozymes.

Two conformationally restricted analogues of (-)-indolactam-V (1) (cis and trans amides) were examined for their binding selectivity to the synthetic C1 peptides of all protein kinase C (PKC) isozymes. Although the binding constants of the cis amide-restricted analogue (2) were equal to those of 1, the trans amide-restricted analogue (3) bound significantly only to the novel PKC (delta, epsilon, eta, theta) isozymes.

Conformational analysis of the eight-membered ring of the oxidized cysteinyl-cysteine unit implicated in nicotinic acetylcholine receptor ligand recognition.

Nicotinic acetylcholine receptors (nAChRs) are membrane-bound, pentameric ligand-gated ion channels associated with a variety of human disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, and pain. Most known nAChRs contain an unusual eight-membered disulfide-containing cysteinyl-cysteine ring, ox-[Cys-Cys], as does the soluble acetylcholine binding protein (AChBP) found in the snail Lymnaea stagnalis. The cysteinyl-cysteine ring is located in a region implicated in ligand binding, and conformational changes involving this ring may be important for modulation of nAChR function. We have studied the preferred conformations of Ac-ox-[Cys-Cys]-NH2 by NMR in water and computationally by Monte Carlo simulations using the OPLS-AA force field and GB/SA water model. ox-[Cys-Cys] adopts four distinct low-energy conformers at slightly above 0 degrees C in water. Two populations are dependent on the peptide omega2 dihedral angle, with the trans amide favored over the cis amide by a ratio of ca. 60:40. Two ox-[Cys-Cys] conformers with a cis amide bond (C+ and C-) differ from each other primarily by variation of the chi3 dihedral angle, which defines the orientation of the helicity about the S-S bond (+/- 90 degrees ). Two trans amide conformers have the same S-S helicity (chi3 approximately -90 degrees ), but are distinguished by a backbone rotation about phi2 and psi1 (T- and T'-). The ratio of T-/T'-/C+/C- is 47:15:29:9. The orientation of the pendant moieties from the eight-membered ring is more compact for the major trans conformer (T-) than for the extended conformations adopted by T'-, C+, and C-. These conformational preferences are also observed in tetrapeptide and undecapeptide fragments of the human alpha7 subtype of the nAChR that contains the ox-[Cys-Cys] unit. Conformer T- is nearly identical to the conformation seen in the X-ray structure of ox-[Cys(187)-Cys(188)] found in the unliganded AChBP, and is a Type VIII beta-turn.

Combined quantum chemical and RRKM modeling of the main fragmentation pathways of protonated GGG. I. Cis‐trans isomerization around protonated amide bonds

AbstractDetailed analysis of the possible fragmentation channels of protonated GGG suggests that a pre‐dissociation cis‐trans isomerization of the N‐terminal amide bond has to take place when y1 ions are formed on the ‘diketopiperazine’ pathway. Quantum chemical calculations were performed in order to determine the effect of different isomerization states (trans‐trans, cis‐trans, trans‐cis, and cis‐cis) on the energetics of protonation of GGG at the most important protonation sites including the terminal amino group and amide oxygen and nitrogen atoms. These calculations indicate that cis‐trans isomerization is energetically feasible for protonated GGG and the relative energy of the most stable such species that contain a cis amide bond is at a few kcal/mol calculated with respect to the trans‐trans global minimum. Analysis of the possible pathways for the cis‐trans isomerization suggests that this process involves species protonated at the nitrogen of the N‐terminal amide bond. Detailed discussion of the fate of such species indicates that cis‐trans isomerization of the N‐terminal amide bond of protonated GGG is kinetically controlled while a low‐energy pathway connecting the most stable trans‐trans and cis‐trans species no doubt exists. Copyright © 2001 John Wiley & Sons, Ltd.

Using pentafluorophenyl as a Lewis acid to stabilize a cis secondary amide conformation.

[reaction: see text]. N-(pyrimidin-2-yl)pentafluorobenzamide adopts a cis amide bond in the solid state with a pyrimidyl nitrogen pointing toward the center of the perfluorophenyl ring. In solution, the compound is a mixture of cis and trans rotamers. The conformational equilibrium is strongly solvent dependent, and the cis rotamer is entropically favored. It is proposed that the entropic driving force is decreased solvation of the two aryl groups when in the cis conformation.

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six [Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa(1)]oxytocin, [dPen(1)]oxytocin, [5-t-BuPro(7)]oxytocin, [Mpa(1), 5-t-BuPro(7)]oxytocin and [dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro [Pen(6)]oxytocin, [Pen(6), 5-t-BuPro(7)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in [Pen(6)]oxytocin and had no influence on the binding affinity of [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relative to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen(6)]oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.

Infrared Spectroscopy of H-Bonded Bridges Stretched across the cis-Amide Group: II. Ammonia and Mixed Ammonia/Water Bridges

Clusters of two model cis amides, oxindole and 3,4-dihydro-2(1H)-quinolinone, containing one and two ammonia molecules have been studied in the IR hydride stretch region using resonant ion-dip IR spectroscopy. The spectra confirm that ammonia is able to form hydrogen-bonded bridges across the adjacent amide N−H and CO sites in a manner very similar to that of water. Such bridged structures require that ammonia assume the role of a hydrogen bond donor. Further similarities of the hydrogen bonding capabilities of ammonia and water have been revealed by investigations of ternary clusters containing an amide, one ammonia, and one water molecule. Experimentally, two species are observed having IR spectra consistent with a hydrogen-bonded bridge structure. The two species differ only in the relative positions of the ammonia and water molecules within the bridge. These experimental results are well supported by optimized structures, vibrational frequencies, and IR intensities calculated using density functional ...

A new prearranged tripodant ligand N, N′, N″-trimethyl- N, N′, N″-tris(3-pyridyl)-1,3,5-benzene tricarboxamide is easily obtained via the N-methyl amide effect

The N-methyl amide cis generating effect has been utilised to create a new prearranged tripodant ligand in two synthetic steps from benzene-1,3,5-tricarboxylic acid. Crystals of the ligand itself and of complexes with metal salts such as silver(I) triflate, copper(I) and copper(II) chloride and ruthenium(II) chloride have been prepared and the crystal structures solved by analysis of X-ray diffraction data.

Structural Features in the Seven-Membered vs. Fourteen-Membered Ring Cyclization of Hydroxyamido Ketals Derived from β-Amino Alcohols

Hydroxyamido ketals 1 and 3−12, prepared from β-amino alcohols, are obtained either as a single form with a trans (1 and 3−8) or cis (9) conformation in the R = H series or as two cis and trans rotamers in the R ≠ H series (10−12). − The cyclization of these compounds was studied under acidic conditions (PTSA) and over a well defined range of concentrations (c = 0.1−0.2 M). While cis amide 9 and mixtures of cis and trans rotamers 10−12 resulted practically exclusively in the formation of seven-membered ring lactams 22−25, trans compounds 1 and 3−8 afforded fourteen-membered ring bislactams 2, 13−18, the formation of which involves a dimerization. Macrocycles were obtained as three diastereomers in the chiral series 2, 13−16 and as two isomers in the achiral series 17 and 18. Nineteen macrocyclic bislactams have thus been synthesized.

Peptoid oligomers with alpha-chiral, aromatic side chains: effects of chain length on secondary structure.

Oligomeric N-substituted glycines or "peptoids" with alpha-chiral, aromatic side chains can adopt stable helices in organic or aqueous solution, despite their lack of backbone chirality and their inability to form intrachain hydrogen bonds. Helical ordering appears to be stabilized by avoidance of steric clash as well as by electrostatic repulsion between backbone carbonyls and pi clouds of aromatic rings in the side chains. Interestingly, these peptoid helices exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have utilized CD to systematically study the effects of oligomer length, concentration, and temperature on the chiral secondary structure of organosoluble peptoid homooligomers ranging from 3 to 20 (R)-N-(1-phenylethyl)glycine (Nrpe) monomers in length. We find that a striking evolution in CD spectral features occurs for Nrpe oligomers between 4 and 12 residues in length, which we attribute to a chain length-dependent population of alternate structured conformers having cis versus trans amide bonds. No significant changes are observed in CD spectra of oligomers between 13 and 20 monomers in length, suggesting a minimal chain length of about 13 residues for the formation of stable poly(Nrpe) helices. Moreover, no dependence of circular dichroism on concentration is observed for an Nrpe hexamer, providing evidence that these helices remain monomeric in solution. In light of these new data, we discuss chain length-related factors that stabilize organosoluble peptoid helices of this class, which are important for the design of helical, biomimetic peptoids sharing this structural motif.

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.

Solution 13C NMR spectroscopy of polyamide homopolymers (nylons 6, 11, 12, 66, 69, 610 and 612) and several commercial copolymers

Well-resolved 13C NMR spectra of nylons 6, 11, 12, 66, 69, 610, and 612 dissolved in 4:1 TFE/CDCl3 allowed complete assignment of all major backbone peaks. In addition, several low intensity peaks were identified representing cis amide groups, acid end-groups and amine end-groups. Several peaks associated with the trans amide units in the spectra of each polyamide homopolymer were identified as uniquely characteristic of that polymer. Using these unique peaks, all the polyamides studied could be distinguished from each other as homopolymers and as repeat units in copolymers. For example, three unknown polyamides (supplied by a colleague) were identified as nylon 6, nylon 66 and nylon 610 using only solution 13C NMR spectroscopy. Based on these results, several commercial polymers were examined. Using solution 13C NMR spectroscopy, the copolymer compositions, molecular weights, end-group and cis amide contents, plus the amount of residual ε-caprolactam were all determined. For example, Spiderwire Super Mono® fishing line was found to have a number average molecular weight of 25,333g/mol and to contain 82mol% nylon 6 repeat units and 18mol% nylon 66 repeat units. The residual ε-caprolactam, acid end-group and amine end-group contents were calculated as 1.25, 0.61 and 0.0mol%, respectively. The cis amide contents were calculated to be 1.2mol% for nylon 6 units and 0.61mol% for nylon 66 segments.

Solution 13C NMR Characterization of Nylon 66: Quantitation of Cis Amide Conformers, Acid and Amine End Groups, and Cyclic Unimers

Using a 2,2,2-trifluoroethanol/deuteriochloroform solvent mixture (TFE/CDCl3), well-resolved solution 13C NMR spectra of nylon 66 samples were obtained that showed several low-intensity peaks not seen in other, more acidic solvents. These low-intensity peaks were correlated to the methylene carbons near cis amide conformers, acid and amine end groups, and within cyclic oligomers. On the basis of relative peak intensities, the quantitative contents of these components for a typical sample of nylon 66 were determined to be 1.1 mol % cis amide conformers, 1.0 mol % acid end groups, and 0.5 mol % amine end groups. A number-average molecular weight of 30 177 g/mol was calculated from end group concentrations, which is a typical value for commercial nylon 66 samples. The chemical shift values of methylene carbons contained in end groups and in cyclic oligomers were found to be pH sensitive and therefore dependent on the relative concentrations of acid and amine end groups present in each sample. Understanding t...

A study of the relationship between biological activity and prolyl amide isomer geometry in oxytocin using 5-tert-butylproline to augment the Cys(6)-Pro(7) amide cis-isomer population.

Three [5-t-BuPro(7)]oxytocin analogues were synthesized by substituting (2S,5R)-5-tert-butylproline for proline in oxytocin, [Mpa(1)]oxytocin, and [dPen(1)]oxytocin. Relative to oxytocin, [5-t-BuPro(7)]oxytocin and [Mpa(1),5-t-BuPro(7)]oxytocin exhibited strongly reduced binding affinity to the receptor; however, both peptides maintained the pharmacophore characteristics responsible for signal transfer evoking the same maximal response as oxytocin in the single-dose procedure and exhibiting partial agonistic activity in the cumulative dose-response procedure. Although [dPen(1)]oxytocin exhibited inhibitory as well as partial agonistic activity, [dPen(1),5-t-BuPro(7)]oxytocin exhibited only inhibitory potency with a similar in vitro pA(2) value of 7.50 in the absence of magnesium. In the presence of magnesium, [dPen(1), 5-t-BuPro(7)]oxytocin exhibited stronger inhibitory potency than [dPen(1)]oxytocin and no partial agonism. Assignment of the proton signals for the 5-tert-butylprolyl amide cis- and trans-isomers by two-dimensional NMR experiments in water indicated that the Cys(6)-Pro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured respectively at 35%, 33%, and 20% in the 5-tert-butylproline(7) analogues of oxytocin, [Mpa(1)]oxytocin and [dPen(1)]oxytocin. Although caution must be taken when relating the increase in cis-isomer population with an influence on biological activity in [5-t-BuPro(7)]oxytocin analogues, the synthesis and evaluation of analogues 1-3 have provided additional evidence that can be used to support the hypothesis that the prolyl amide cis-isomer may favor antagonism and the trans-isomer is necessary for agonist activity.

An examination of the steric effects of 5-tert-butylproline on the conformation of polyproline and the cooperative nature of type II to type I helical interconversion.

The influence of steric effects on the helical geometry and the interconversion of type II to type I polyproline in water was examined by the synthesis and analysis of proline dimers and hexamers containing up to three (2S,5R)-5-tert-butylproline residues. In the dimers, the bulky 5-tert-butyl substituent was found to exert a significant influence on the local prolyl amide geometry such that the predominant trans-isomer in N-(acetyl)prolyl-prolinamide (1) was converted to 63% cis isomer in N-(acetyl)prolyl-5-tert-butylprolinamide (2) as measured by (1)H-nmr spectroscopy. Similarly, the presence of a 5-tert-butyl group on the C-terminal residue in the polyproline hexamer Ac-Pro(5)-t-BuPro-NH(2) (4) produced a local 5-tert-butylprolyl amide population containing 61% cis isomer in water. In spite of the presence of a local prolyl cis amide geometry, the downstream prolyl amides in 4 remained in the trans isomer as determined by NOESY spectroscopy. Conformational analysis by (13)C-nmr and CD spectroscopy indicated that Ac-Pro(6)-NH(2) (3) adopted the all-trans amide polyproline type II helix in water. As the amount of 5-tert-butylproline increased from one to three residues in hexamers 4-6, a gradual destabilization of the polyproline type II helical geometry was observed by CD spectroscopy in water; however, no spectrum was obtained, indicative of a complete conversion to a polyproline type I helix. The implications of these results are discussed with respect to the previously proposed theoretical mechanisms for the helical interconversion of polyproline, which has been suggested to occur by either a cooperative C- to N-terminal isomerization of the prolyl amide bonds or via a conformational intermediate composed of dispersed sequences of prolyl amide cis and trans isomers.

New pyrazole containing bicarboxylic α-amino acids: mimics of the cis amide bond

A series of novel optically active α-amino acids, containing a pyrazole ring, which can be regarded as building blocks for peptidomimetics, have been prepared starting from readily available α-amino acids. The synthetic strategy employed allows the regio- and stereoselective preparation of 1,3- or 1,5-substituted pyrazolyl rings. The pyrazole containing peptidomimetics can be considered as analogues of peptides with a cis amide bond.

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.

Non-proline Cis peptide bonds in proteins

In a non-redundant set of 571 proteins from the Brookhaven Protein Data Base, a total of 43 non-proline cis peptide bonds were identified. Average geometrical parameters of the well-defined cis peptide bonds in proteins determined at high resolution show that some parameters, most notably the bond angle at the amide bond nitrogen, deviate significantly from the corresponding one in the trans conformation. Since the same feature was observed in cis amide bonds in small molecule structures found in the Cambridge Structural Data Base, a new set of parameters for the refinement of protein structures containing non-Pro cis peptide bonds is proposed. A striking preference was observed for main-chain dihedral angles of the residues involved in cis peptide bonds. All residues N-terminal and most residues C-terminal to a non-Pro cis peptide bond (except Gly) are located in the β-region of a φ/ψ plot. Also, all of the few C-terminal residues (except Gly) located in the α-region of the φ/ψ plot constitute the start of an α-helix in the respective structure. In the majority of cases, an intimate side-chain/side-chain interaction was observed between the flanking residues, often involving aromatic side-chains. Interestingly, most of the cases found occur in functionally important regions such as close to the active site of proteins. It is intriguing that many of the proteins containing non-proline cis peptide bonds are carbohydrate-binding or processing proteins. The occurrence of these unusual peptide bonds is significantly more frequent in structures determined at high resolution than in structures determined at medium and low resolution, suggesting that these bonds may be more abundant than previously thought. On the basis of our experience with the structure determination of coagulation factor XIII, we developed an algorithm for the identification of possibly overlooked cis peptide bonds that exploits the deviations of geometrical parameters from ideality. A few likely candidates based on our algorithm have been identified and are discussed.

Alkyl 3-Position Substituents Retard the Isomerization of Prolyl and Hydroxyprolyl Amides in Water

The influence of alkyl 3-position substituents on the rate of amide isomerization N-terminal to proline and hydroxyproline has been explored via the synthesis and analysis of (2S)-N-(acetyl)proline N‘-methylamide (1), (2S,4R)- and (2S,4S)-N-acetyl-4-hydroxyproline N‘-methylamides 2 and 3, and their respective 3,3-dimethyl analogues 4−6. The relative populations of the amide cis and trans isomers as well as the rates for cis-to-trans and trans-to-cis isomerization of 1−6 in water were ascertained by NMR spectroscopy and magnetization transfer experiments. The relative populations of free C-terminal and hydrogen-bonded amides in the γ-turn conformation were also estimated by integrating the N−H stretch absorbances in the FT-IR spectra of 1 and 4 in CHCl3. In addition, the structure of the amide trans isomer of (2S,4S)-N-acetyl-3,3-dimethyl-4-hydroxyproline N‘-methylamide (6) was determined in the solid state by X-ray crystallographic analysis. In prolyl peptides 1−6, the 3,3-dimethyl and hydroxyl substituents had little effect on the amide isomer equilibrium. A dramatic decrease in the rate of cis-to-trans amide isomerization was observed for N-acetyl-3,3-dimethylproline N‘-methylamide (4), which exhibited a kct nearly 7-fold slower than that of 1. Similar effects of the 3,3-dimethyl substituents were observed, albeit to a lesser degree, in the cases of the hydroxyprolyl peptides. The FT-IR data for 4 and X-ray data for 6 both demonstrated that the 3,3-dimethyl substituents restricted the proline ψ dihedral angle and prevented the formation of a γ-turn conformation, having a seven-membered hydrogen bond between the C-terminal amide NH and N-terminal amide carbonyl. Furthermore, restriction of the ψ dihedral angle by the methyl groups was observed in systematic computational conformational analyses of 1−6, in which the ψ and ω dihedral angles were rotated at 30° intervals and the energies of the local minima were determined. Retardation of the rate of cis-to-trans amide isomerization in the dimethyl analogues may be attributed to steric interactions favoring a ψ dihedral angle at which the C-terminal amide carbonyl destabilizes the transition state through Coulomb repulsion of either the developing nitrogen lone pair or the carbonyl oxygen of the pyramidalized N-terminal amide. The consequences of 3-alkyl and 4-hydroxyl substituents on the rate of proline amide isomerization in water, which was observed to decrease in the order 1 ≈ 3 > 2 > 6 > 5 > 4, may result from influences on the ψ dihedral angle geometry, inductive effects, and intramolecular hydrogen bonding.