Exo Conformation Research Articles

Synthesis of Thiazolidin-4-Ones Derivatives, Evaluation of Conformation in Solution, Theoretical Isomerization Reaction Paths and Discovery of Potential Biological Targets.

Thiazolin-4-ones and their derivatives represent important heterocyclic scaffolds with various applications in medicinal chemistry. For that reason, the synthesis of two 5-substituted thiazolidin-4-one derivatives was performed. Their structure assignment was conducted by NMR experiments (2D-COSY, 2D-NOESY, 2D-HSQC and 2D-HMBC) and conformational analysis was conducted through Density Functional Theory calculations and 2D-NOESY. Conformational analysis showed that these two molecules adopt exo conformation. Their global minimum structures have two double bonds (C=N, C=C) in Z conformation and the third double (C=N) in E. Our DFT results are in agreement with the 2D-NMR measurements. Furthermore, the reaction isomerization paths were studied via DFT to check the stability of the conformers. Finally, some potential targets were found through the SwissADME platform and docking experiments were performed. Both compounds bind strongly to five macromolecules (triazoloquinazolines, mglur3, Jak3, Danio rerio HDAC6 CD2, acetylcholinesterase) and via SwissADME it was found that these two molecules obey Lipinski's Rule of Five.

Molecular structure and selective theophylline complexation by conformational change of diethyl N,N'-(1,3-phenylene)dicarbamate.

The receptor ability of diethyl N,N'-(1,3-phenylene)dicarbamate (1) to form host-guest complexes with theophylline (TEO) and caffeine (CAF) by mechanochemistry was evaluated. The formation of the 1-TEO complex (C12H16N2O4·C7H8N4O2) was preferred and involves the conformational change of one of the ethyl carbamate groups of 1 from the endo conformation to the exo conformation to allow the formation of intermolecular interactions. The formation of an N-H...O=C hydrogen bond between 1 and TEO triggers the conformational change of 1. CAF molecules are unable to form an N-H...O=C hydrogen bond with 1, making the conformational change and, therefore, the formation of the complex impossible. Conformational change and selective binding were monitored by IR spectroscopy, solid-state 13C nuclear magnetic resonance and single-crystal X-ray diffraction. The 1-TEO complex was characterized by IR spectroscopy, solid-state 13C nuclear magnetic resonance, powder X-ray diffraction and single-crystal X-ray diffraction.

Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG)n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα fromHomo sapiens ADAR1 to a locked "A-type" version of the (CpG)3 construct (LNA (CpG)3) where the sugar pucker is locked into the C3'-endo/C2'-exo conformation, which prevents the duplex from adopting the alternating C2'/C3'-endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that ZαADAR1 binds to the LNA (CpG)3 using a similar interface as for Z-form binding, with a dissociation constant (KD) of ∼4 μM. In contrast to Z-DNA/Z-RNA, where two ZαADAR1 bind to every 6 bp stretch, our data suggests that ZαADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because ZαADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.

Conformation matters: siRNAs with antisense strands with 5'-(E)-vinyl-phosphonate-α-L-LNA elicit stronger RNAi-mediated gene silencing than those with 5'-(E)-vinyl-phosphonate-LNA.

Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, (E)-vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3'-exo (south) conformation, than when the antisense strand was modified with 5'-VP-LNA, which adopts a C3'-endo (north) pucker. These data underscore the critical role of conformation of nucleotides in RNA interference.

Synthesis, Structural, and Conformational Analysis of 4′‐C‐Alkyl‐2′‐O‐Ethyl‐Uridine Modified Nucleosides

AbstractSugar modifications have attracted much attention due to their potential structural and functional influence on therapeutic nucleic acids. Herein, we report the synthesis of dual modified 4′‐C‐azidomethyl‐2′‐O‐ethyl‐uridine (4′‐AzM‐2′‐OEt−U) and 4′‐C‐aminomethyl‐2′‐O‐ethyl‐uridine (4′‐AM‐2′‐OEt−U) nucleosides using linear multi‐step synthesis (16 linear steps). Additionally, we report an alternative route for the synthesis of 2′‐O‐ethyl‐uridine nucleoside which has been achieved in three steps with an overall yield of 40 %. X‐ray structure of 4′‐AzM‐2′‐OEt−U illustrates that the nucleoside adopts C2′‐endo (South) conformation having a DNA‐type glycosidic bond (χ) angle of −116.01°. Computational studies revealed the C2′‐endo and C4′‐exo conformations for 4′‐AzM‐2′‐OEt−U and 4′‐AM‐2′‐OEt−U free nucleosides, respectively. The C4′‐exo conformation in 4′‐AM‐2′‐OEt−U free nucleoside is collectively stabilized by various non‐covalent interactions between positively charged aminomethyl and 2′,3′‐hydroxyl groups. Insights into the structural and conformational analysis of dual sugar modified nucleosides and oligonucleotides will be helpful in the rational design of modified nucleosides and therapeutic oligonucleotides.

Anhydrides of Arylfuran and Arylpyran Pseudoacids: Formation and Structures; C–O Bond Lengths Trends in Pseudo o-Formylbenzoic Acid Derivatives

Three methods for producing anhydrides of arylfuran and arylpyran pseudoacids were explored. These included thermal dehydration, phosgene or thionly chloride activation and decomposition, and dicyclohexylcarbodiimide activation and coupling. Derivatives of the cyclic forms of o-formylbenzoic acid, o-acetylbenzoic acid, 2-carboxyphenylacetaldehyde and of 4,4-dimethyl-3,4-dihydro-3-hydroxy-[1H]-isobenzopyran-1-one were formed including dipseudoanhydides and normal-pseudo anhydrides. Crystal and molecular structures for meso and (R,R/S,S)-bis(1[3H]-isobenzofuranone-3-yl)ether, (R,R/S,S)-bis(3-methyl-1[3H]-isobenzofuranone-3yl)ether, meso (3,4-dihydro-[1H]-isobenzopyran-1-one-3-yl)ether, 3-benzoyloxy-1[3H]-isobenzofuranone, 3-benzoyloxy-3-methyl-1[3H]isobenzofuranone, 3-(4′-nitrobenzoyloxy)-4,4-dimethyl-3,4-dihydro-[1H]-isobenzopyran-1-one, and (1[3H]-isobenzofuranone-3-yl)(4,4,dimethyl-3,4-dihydro-[1H]-isobenzopyran-1-one-3-yl)ether are reported. Endocyclic pseudoacyl C–O bonds are always longer than the exocyclic pseudoacyl C–O bonds. It is possible to refine the previously established C–O bond length dependencies on the pKa (of the conjugate acids) of the leaving groups for 3-substituted 1-[3H]-isobenzofuranones. Of six dipseudoanhydrides studied, conformations are found with exocyclic C–O(ether) linkages synclinal to the endocyclic C–O and away from the ring (exo conformation) in two meso structures, two of three RR/SS forms and in a chiral unsymmetrical form. An endo conformation is observed in one of the RR/SS forms. In three normal-pseudo anhydrides, both endo and exo conformations are observed. Synthetic methods for formation of anhydrides of several arylfuran and arylpyran pseudoacids are described, and the pseudoacyl C–O bond length trends are determined for leaving groups spanning over 30 pKa units.

Tetrathiafulvalene Dimer Merged with a Binuclear Ring of Sn and Sb: Synthesis and Molecular Structures Induced by Heteroatoms

In this study, we describe the synthesis and characterization of dimeric tetrathiafulvalene (TTF) derivatives that are doubly bridged by Sn (1) and Sb (2a) atoms as a new class of organic‐inorganic hybrid compounds able to provide enhanced dimensionality in the solid state. The antimony heterocyclic framework was constructed by a simple Sn–Sb metal exchange reaction of a 1,4‐distannine moiety with p‐tolyl antimony dichloride (p‐tolSbCl2). Compound 2a was obtained in cis and trans forms as a stable configurational isomer. From X‐ray crystallographic analysis, there were three conformational isomers in which the tolyl rings were directed in the endo or exo direction. Crystal packing revealed a supramolecular three‐dimensional network through intermolecular short contacts of Sb···S and Sb···π. These packing forces gave a trans‐2aexo–exo conformation in the crystal, although the theoretical calculations predict its instability compared with other conformers. Cyclic voltammetry (CV) measurements of 1 and cis/trans‐2a revealed two pairs of reversible redox processes corresponding to the formation of TTF/TTF·+ and TTF·+/TTF2+. Furthermore, CV of cis/trans‐2a also revealed the reduction waves derived from the reduction of SbIII. Dicationic and tetracationic species were prepared by chemical oxidation, and their electronic structures were also investigated by electronic spectroscopy.

Comparative analysis of inosine-substituted duplex DNA by circular dichroism and X-ray crystallography

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from ‘electrostatic’ or ‘base stacking’ influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I⋅C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I⋅C and two A⋅T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1′-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2′-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I⋅C pairs has a geometry similar to that of the reference duplex with eight G⋅C and two A⋅T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.

Reactivity of 7‐Azanorbornenes in Bioorthogonal Inverse Electron‐Demand Diels–Alder Reactions

In the preparation of multicomponent compounds, the accumulation of components through sequential click reactions is an attractive strategy. In this work we examined the reactivity of various N‐substituted 7‐azanorbornenes in inverse electron‐demand Diels–Alder (iEDDA) reactions with tetrazines to explore the potential of 7‐azanorbornenes as clickable hub molecules. The iEDDA reaction of 7‐azanorbornene is expected to proceed faster when the nitrogen atom at the 7‐position is substituted with an electron‐donating substituent. Contrary to this expectation, the electron‐donating alkyl‐bearing derivative reacted much more slowly than those bearing electron‐withdrawing acyl groups. The results of DFT calculations indicate that the reaction rates correlate well with an increase in sp2 character of the 7‐nitrogen atoms: The ease of conversion of the more stable exo conformer into the more reactive endo conformer may lower the activation energy of the first rate‐determining hetero‐Diels–Alder step. Indeed, the reaction rates of N‐acylated 7‐azanorbornenes, which have a more planar nitrogen atom, were found superior to those of other derivatives and comparable to those of norbornenes. Finally, we successfully labeled a tetrazine on a protein surface by fluorophore‐conjugated 7‐azanorbornene in the presence of other proteins.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance.

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

Lead(II) Binding in Natural and Artificial Proteins.

This article describes recent attempts to understand the biological chemistry of lead using a synthetic biology approach. Lead binds to a variety of different biomolecules ranging from enzymes to regulatory and signaling proteins to bone matrix. We have focused on the interactions of this element in thiolate-rich sites that are found in metalloregulatory proteins such as Pbr, Znt, and CadC and in enzymes such as δ-aminolevulinic acid dehydratase (ALAD). In these proteins, Pb(II) is often found as a homoleptic and hemidirectic Pb(II)(SR)3- complex. Using first principles of biophysics, we have developed relatively short peptides that can associate into three-stranded coiled coils (3SCCs), in which a cysteine group is incorporated into the hydrophobic core to generate a (cysteine)3 binding site. We describe how lead may be sequestered into these sites, the characteristic spectral features may be observed for such systems and we provide crystallographic insight on metal binding. The Pb(II)(SR)3- that is revealed within these α-helical assemblies forms a trigonal pyramidal structure (having an endo orientation) with distinct conformations than are also found in natural proteins (having an exo conformation). This structural insight, combined with 207Pb NMR spectroscopy, suggests that while Pb(II) prefers hemidirected Pb(II)(SR)3- scaffolds regardless of the protein fold, the way this is achieved within α-helical systems is different than in β-sheet or loop regions of proteins. These interactions between metal coordination preference and protein structural preference undoubtedly are exploited in natural systems to allow for protein conformation changes that define function. Thus, using a design approach that separates the numerous factors that lead to stable natural proteins allows us to extract fundamental concepts on how metals behave in biological systems.

A Computational Re-examination of the Criegee Intermediate-Sulfur Dioxide Reaction.

The atmospheric oxidation of sulfur dioxide by the parent and dimethyl Criegee intermediates (CIs) may be an important source of sulfuric acid aerosol, which has a large impact on radiative forcing and therefore upon climate. A number of computational studies have considered how the CH2OOS(O)O heteroozonide (HOZ) adduct formed in the CI + SO2 reaction converts SO2 to SO3. In this work we use the CBS-QB3 quantum chemical method along with equation-of-motion spin-flip CCSD(dT) and MCG3 theories to reveal new details regarding the formation and decomposition of the endo and exo conformers of the HOZ. Although ∼75% of the parent CI + SO2 reaction is initiated by formation of the exo HOZ, hyperconjugation preferentially stabilizes many of the endo intermediates and transition structures by 1-5 kcal mol(-1). Our quantum chemical calculations, in conjunction with statistical rate theory models, predict a rate coefficient for the parent CI + SO2 reaction of 3.68 × 10(-11) cm(3) molecule(-1) s(-1), in good agreement with recent experimental measurements. RRKM/master equation simulations based on our quantum chemical data predict a prompt carbonyl + SO3 yield of >95% for the reaction of both the parent and dimethyl CI with SO2. The existence of concerted cycloreversion transition structures 10-15 kcal mol(-1) higher in energy than the HOZ accounts for most of the predicted SO3 formation.

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix.

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

Investigation and conformational analysis of fluorinated nucleoside antibiotics targeting siderophore biosynthesis.

Antibiotic resistance represents one of the greatest threats to public health. The adenylation inhibitor 5'-O-[N-(salicyl)sulfamoyl]adenosine (SAL-AMS) is the archetype for a new class of nucleoside antibiotics that target iron acquisition in pathogenic microorganisms and is especially effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. Strategic incorporation of fluorine at the 2' and 3' positions of the nucleoside was performed by direct fluorination to enhance activity and improve drug disposition properties. The resulting SAL-AMS analogues were comprehensively assessed for biochemical potency, whole-cell antitubercular activity, and in vivo pharmacokinetic parameters. Conformational analysis suggested a strong preference of fluorinated sugar rings for either a 2'-endo, 3'-exo (South), or a 3'-endo,2'-exo (North) conformation. The structure-activity relationships revealed a strong conformational bias for the C3'-endo conformation to maintain potent biochemical and whole-cell activity, whereas improved pharmacokinetic properties were associated with the C2'-endo conformation.

Comparing spectroscopic and electrochemical properties of complexes of type Cp’M(η3-C3H5)(CO)2 (Cp’ = Cp, Ind, Flu): A complementary experimental and DFT study

A series of allyl complexes of the general formula [Cp'Mo(η3-C3H5)(CO)2], where Cp' = Cp, CpMe, Cp*, Ind, IndMe, IndMe2, Flu, and three tungsten analogues, has been prepared and characterized by 1H, 13C, and 95Mo NMR, cyclic voltammetry, and the structure of [IndMo(η3-C3H5)(CO)2] was determined by X-ray single crystal analysis. Two conformers, corresponding to the two extreme orientations of the allyl ligand (endo and exo), have been identified in solution by 1H and 95Mo NMR for all the complexes, except for [FluMo(η3-C3H5)(CO)2], which only presents an exo conformation in solution.A 95Mo NMR investigation also shows the influence of electron donor capability of the Cp' ligands on the 95Mo chemical shifts of the Mo center. He I and He II photoelectron spectra probe the electron richness of the metal center and the electronic structure of the complexes. Cyclic voltammetry studies show one oxidation process, which is only reversible for the Cp derivatives. DFT calculations provided a reliable way to determine ionization energies, and reflected very well the trends of the other properties, from the 95Mo NMR chemical shifts, to vibration frequencies, and oxidation potentials.

2'-Fluorination of tricyclo-DNA controls furanose conformation and increases RNA affinity.

The synthesis of 2'-fluoro tricyclo-DNA pyrimidine nucleosides with fluorine in the ribo-configuration and their incorporation into oligodeoxynucleotides was accomplished. Unlike the parent tc-T nucleoside, the 2'F-RNA-tc-T unit occurs in the 2'-exo conformation in the crystal. Specifically, F-RNA-tc-T was found to stabilize duplexes with RNA by +2 to +4 °C in Tm/mod. F-RNA-tc-nucleosides mix well with the DNA backbone and thus open up possibilities of using shorter and mixed-(DNA/tc-DNA) backbone oligonucleotides for therapeutic applications.

Synthesis and properties of 6'-fluoro-tricyclo-DNA.

The synthesis of the two fluorinated tricyclic nucleosides 6'-F-tc-T and 6'-F-tc-5(Me)C, as well as the corresponding building blocks for oligonucleotide assembly, was accomplished. An X-ray analysis of N(4)-benzoylated 6'-F-tc-(5Me)C reavealed a 2'-exo (north) conformation of the furanose ring, characterizing it as an RNA mimic. In contrast to observations in the bicyclo-DNA series, no short contact between the fluorine atom and the H6 of the base, reminiscent of a nonclassical F···H hydrogen bond, could be observed. Tm measurements of modified oligodeoxynucleotides with complementary RNA showed slightly sequence-dependent duplex stabilization profiles with maximum ΔTm/mod values of +4.5 °C for 6'-F-tc-(5Me)C and +1 °C for 6'-F-tc-T. In comparison with parent tc-modified oligonucleotides, no relevant changes in Tm were detected, attributing the fluorine substituent a neutral role in RNA affinity. A structural analysis of duplexes with DNA and RNA by CD-spectroscopy revealed a shift from B- to A-type conformation induced by the 6'-F-tc-nucleosides. This is not a specific "fluorine effect", as the same is also observed for the parent tc-modifications. The two fluorinated tc-nucleosides were also incorporated into a pure tricyclo-DNA backbone and showed no discrimination in Tm with complementary RNA, demonstrating that 6'-F substitution is also compatible within fully modified tc-oligonucleotides.

A New Family of Conformationally Constrained Bicyclic Diarylprolinol Silyl Ethers as Organocatalysts

AbstractSimple chemical manipulations of trans‐4‐L‐hydroxy proline allow the access to a new family of bicyclic silyl ether organocatalysts that display some remarkable features. Apart from being extremely stable to hydrolytic conditions and possessing excellent catalytic performances, the rigidity of the bicyclic structure imposes a synclinal endo disposition of the bulky substituents with respect to the pyrrolidine ring, opposed to the more stable synclinal exo conformations of Jørgensen–Hayashi catalysts.

Pentacyclo[8.2.1.14,7.02,9.03,8]tetradeca-5,11-diene

The title compound, C14H16, was prepared through [2 + 2] cyclo­addition of norbornadiene. There are two independent mol­ecules in the asymmetric unit: each is centrosymmetric with the centroid of the four-membered ring located about an inversion center. Each mol­ecule possesses an exo–trans–exo conformation.

Conformational Analysis of Fluorinated Pyrrolidines Using 19F–1H Scalar Couplings and Heteronuclear NOEs

A series of 3-fluoropyrrolidines have been studied to investigate the influence of the stereoelectronic fluorine gauche effect on ring conformations in the solid state by single-crystal X-ray analysis and in solution phase by NMR spectroscopy. As part of these studies 1D (19)F-(1)H heteronuclear NOE (HOESY) experiments have been optimised for applications to small molecules and are described in detail. These have been employed to estimate (19)F-(1)H internuclear distances and were combined with vicinal (3)J(F,H) and (3)J(H,H) scalar coupling constants to analyse the ring conformations. Where possible the derived solution-phase structural data have been compared with those of the crystalline state. The results demonstrate the influence of the gauche effect in stabilising C(γ)-exo conformations of the fluorinated pyrrolidines. It was further shown that when steric interactions were also present, this conformational bias was diminished and the contribution of the alternative C(γ)-endo conformation was seen to increase in solution at lower sample temperatures.