Chemoenzymatic synthesis of (S)-hexafluoroleucine and (S)-tetrafluoroleucine

We have developed a short chemoenzymatic synthesis for both (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl) and (S)-5,5,5',5'-tetrafluoroleucine (Qfl) on gram scale. Qfl was incorporated into a peptide using standard solid-phase peptide synthesis protocols to measure its helix propensity. The helix propensity for Qfl is 0.68 kcal.mol-1 more favorable compared to Hfl.     

Stabilizing interactions between aromatic and basic side chains in alpha-helical peptides and proteins. Tyrosine effects on helix circular dichroism

Here we investigate the structures and energetics of interactions between aromatic (Phe or Tyr) and basic (Lys or Arg) amino acids in alpha-helices. Side chain interaction energies are measured using helical peptides, by quantifying their helicities with circular dichroism at 222 nm and interpreting the results with Lifson-Roig-based helix/coil theory. A difficulty in working with Tyr is that the aromatic ring perturbs the CD spectrum, giving an incorrect helicity. We calculated the effect of Tyr on the CD at 222 nm by deriving the intensities of the bands directly from the electronic and magnetic transition dipole moments through the rotational strengths corresponding to each excited state of the polypeptide. This gives an improved value of the helix preference of Tyr (from 0.48 to 0.35) and a correction to the helicity for the peptides containing Tyr. We find that Phe-Lys, Lys-Phe, Phe-Arg, Arg-Phe, and Tyr-Lys are all stabilizing by -0.10 to -0.18 kcal.mol-1 when placed i, i + 4 on the surface of a helix in aqueous solution, despite the great difference in polarity between these residues. Interactions between these side chains have previously been attributed to cation-pi bonds. A survey of protein structures shows that they are in fact predominantly hydrophobic interactions between the CH2 groups of Lys or Arg and the aromatic rings.     

Design of a functional membrane protein by engineering a heme-binding site in glycophorin A

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Out of the 32 residues comprising the transmembrane helix of GpA, five amino acids were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis, and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 +/- 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Moreover, ME1 catalyzes the hydrogen peroxide dependent oxidation of organic substrates such as TMB (2,2',5,5'-tetramethyl-benzidine). This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.     

Transesterification and amide cis-trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc-Asp(Dpa)-OBzl

The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions.     

The first structural characterization and determination of the isomerization activation parameters of a chiral phosphatitanocene

The activation parameters (delta G++298 = 11.5 (+/- 1.0) kcal mol-1, delta H++ = 16.3 (+/- 3.0) kcal mol-1, delta S++ = 16 (+/- 11) cal mol-1 K-1) have been determined for the rac to meso isomerization of a phosphametallocene, bis(3,4-dimethyl-2-phenylphospholyl)titanium dichloride, 2, which has been structurally characterized.     

Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines.

A heptamer composed of C5-(1-propynyl) pyrimidines (Y(p)'s) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.; Froehler, B. C. Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5'-dCCUCCUU-3':3'-rGAGGAGGAAAU-5', have been measured by UV melting experiments. For nucleotides tested, an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly, except that addition of a 5' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y(p)-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5'-dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3':3'-rGAGIAGGAAAU-5', destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex. This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.     

Aggregation and C-N rotation of the lithium salt of N,N-dimethyldiphenylacetamide

[formula: see text] Two methyl 1H NMR signals for the Li salt of N,N-dimethyldiphenylacetamide are observed at low temperature and assigned to the monomer and dimer. From line shape analysis, the dimerization constant (K1,2) is 40 +/- 10 M-1 at 200 K (delta G degree = 1.5 kcal mol-1, delta H degree = 0.8 kcal mol-1, delta S degree = 12 eu) and the activation parameters are delta H++ = 5.5 kcal mol-1 and delta S++ = -18 eu. The C-N bond rotation is too fast to observe on the NMR time scale, indicating a rotation barrier of less than 10 kcal mol-1.     

Characterization of an NH-pi interaction in Co(III) ternary complexes with aromatic amino acids

The NH-pi interaction has been detected in the crystal structures of Co(III) ternary complexes with N,N-bis(carboxymethyl)-(S)-phenylalanine (BCMPA) and aromatic amino acids including (S)-phenylalanine ((S)-Phe), (R)-phenylalanine ((R)-Phe), and (S)-tryptophan ((S)-Trp)). Additionally, this interaction has been studied in solution for Co(III) ternary complexes with BCMPA or NTA (NTA = nitrilotriacetic acid) and several amino acids (AA) by means of electronic absorption, circular dichroism (CD), and (1)H NMR spectroscopies. The CD intensities of the Co(III) complexes with aromatic amino acids measured in the d-d region ( approximately 20.5 x 10(3) cm(-)(1)) are significantly decreased in ethanol solutions relative to water. Analogous complexes with aliphatic amino acids do not exhibit this solvent effect. The (1)H NMR spectra of the Co(III) complexes with aromatic amino acids measured in DMSO-d(6) exhibit upfield shifts of the NH peaks compared with those with aliphatic amino acids, which suggest a shielding effect due to the aromaticity. The upshift values coincide with those experimentally evaluated from the crystal structures. The magnitude of the upfield shifts agrees well with Hammett's rule, indicating that the increase of pi-electron densities on the aromatic rings leads attractive NH-pi interaction that exerts a larger shielding effect for the NH protons. In ligand-substitution reactions of the carbonatocobalt(III) complexes with amino acids, the yields of those with aromatic amino acids are higher than the yields obtained for complexes with aliphatic amino acids. This observation is discussed in connection with the important contribution of the NH-pi interaction as one of the promotion factors in the reaction.     

Exploiting the right side of the Ramachandran plot: substitution of glycines by D-alanine can significantly increase protein stability

A major goal of protein engineering is the enhancement of protein stability. Here we demonstrate a rational method for enhancing the stability of globular proteins by targeting glycine residues which adopt conformations with Phi > 0. Replacement of such a glycine by d-alanine can lead to a significant increase in stability. The approach is tested at three sites in two model proteins. NMR and CD indicated that the substitutions do not alter the structure. Replacement of glycine-24 of the N-terminal domain of L9 (NTL9) with d-Ala results in an increase in stability of 1.3 kcal mol-1, while replacement of glycine-34 of NTL9 leads to an increase of 1.9 kcal mol-1. Replacement of glycine-331 of the UBA domain with d-Ala leads to an increase in stability of 0.6 kcal mol-1.     

Dynamic 1H NMR study of the barrier to rotation about the C-N bond in primary carbamates and its solvent dependence

The dynamic 1H NMR study of some primary carbamates in the solvents CDCl3 and CD3COCD3 between 183 and 298 K is reported. The free energies of activation, thus obtained (12.4 to 14.3 kcal mol-1), were attributed to the conformational isomerization about the N-C bond. These barriers to rotation show solvent dependence in contrast to the tertiary analogues and are lower in free energy by ca. 2-3 kcal mol-1.     

Selective anion encapsulation by a metalated cryptophane with a pi-acidic interior

Metalation of the exterior arene faces of the molecular capsule (+/-)-cryptophane-E with [Cp*Ru]+ moieties results in a pi-acidic cavity capable of encapsulating anions. The [CF3SO3]- and [SbF6]- salts have been crystallographically characterized and demonstrate the encapsulation of these anions by the metalated cryptophane. 1H and 19F NMR spectroscopy establish the binding of anions in NO2CD3 solution and reveal the relative affinity of the cavity for different anions (KX-/KOTf-): [BF4]- approximately 0, [PF6]- = 1.18, [CF3SO3]- identical with 1, [SbF6]- = 0.30. Variable temperature rate studies reveal the activation barrier for triflate encapsulation to be DeltaG298K = 18.0(8) kcal.mol-1 (DeltaH = 17.5(4) kcal.mol-1 and DeltaS = 2(1) cal.mol-1.K-1).     

Bimetallo-radical carbon-hydrogen bond activation of methanol and methane

Carbon-hydrogen bond cleavage reactions of CH3OH and CH4 by a dirhodium(II) diporphyrin complex with a m-xylyl tether (.Rh(m-xylyl)Rh.(1)) are reported. Kinetic-mechanistic studies show that the substrate reactions are bimolecular and occur through the use of two Rh(II) centers in the molecular unit of 1. Second-order rate constants (T = 296 K) for the reactions of 1 with methanol (k(CH3OH) = 1.45 x 10-2 M-1 s-1) and methane (k(CH4) = 0.105 M-1 s-1) show a clear kinetic preference for the methane activation process. The methanol and methane reactions with 1 have large kinetic isotope effects (k(CH3OH)/k(CD3OD) = 9.7 +/- 0.8, k(CH4)/k(CD4) = 10.8 +/- 1.0, T = 296 K), consistent with a rate-limiting step of C-H bond homolysis through a linear transition state. Activation parameters for reaction of 1 with methanol (DeltaH = 15.6 +/- 1.0 kcal mol-1; DeltaS = -14 +/- 5 cal K-1 mol-1) and methane (DeltaH = 9.8 +/- 0.5 kcal mol-1; DeltaS = -30 +/- 3 cal K-1 mol-1) are reported.     

Structure, conformation, stereodynamics, dimer formation, and absolute configuration of axially chiral atropisomers of hindered biphenyl carbinols

NMR spectra of biphenyl derivatives bearing a single CR2OH substituent in the ortho position indicate that they exist as sp (more stable) and ap (less stable) conformers, due to the restricted rotation about the Ar-CR2OH bond. When R = Et (compound 2) the corresponding rotation barrier was determined (7.5 kcal mol-1) by line shape simulation of the low-temperature NMR spectra. Introduction of the prochiral i-Pr group in the position 3' of a biphenyl with the CMe2OH substituent in the position 2 (4) allowed the determination of the enantiomerization barrier (due to the Ar-Ar bond rotation) for the stereolabile axially chiral atropisomers (13.95 kcal mol(-1)). DFT computations of these barriers were all in agreement with the experiments. Biphenyls bearing two CR2OH groups in the 2,2' positions were found to exist as configurationally stable atropisomers: when R = Me (7) they were separated by enantioselective HPLC and the absolute configuration assigned on the basis of the corresponding CD spectra. In solution, compounds 6 (R = H) and 7 (R = Me) were found to originate a dimer, due to H-bond interactions between two enantiomers. In the case of 7, the free energy of activation (9.5 kcal mol-1) for the exchange of the monomer with the dimer could be measured, for the first time, by dynamic NMR. The conformational preferences, predicted by computations for the biphenyls with two CR2OH substituents in the 2,2' positions, were confirmed by X-ray diffraction in the case of R = H (6), R = Me (7), and R = i-Pr (9).     

Thermochemistry of methyl and ethyl nitro, RNO2, and nitrite, RONO, organic compounds

Computational quantum theory is employed to determine the thermochemical properties of n-alkyl nitro and nitrite compounds: methyl and ethyl nitrites, CH3ONO and C2H5ONO, plus nitromethane and nitroethane, CH3NO2 and C2H5NO2, at 298.15 K using multilevel G3, CBS-QB3, and CBS-APNO composite methods employing both atomization and isodesmic reaction analysis. Structures and enthalpies of the corresponding aci-tautomers are also determined. The enthalpies of formation for the most stable conformers of methyl and ethyl nitrites at 298 K are determined to be -15.64 +/- 0.10 kcal mol-1 (-65.44 +/- 0.42 kJ mol-1) and -23.58 +/- 0.12 kcal mol-1 (-98.32 +/- 0.58 kJ mol-1), respectively. DeltafHo(298 K) of nitroalkanes are correspondingly evaluated at -17.67 +/- 0.27 kcal mol-1 (-74.1 +/- 1.12 kJ mol-1) and -25.06 +/- 0.07 kcal mol-1 (-121.2 +/- 0.29 kJ mol-1) for CH3NO2 and C2H5NO2. Enthalpies of formation for the aci-tautomers are calculated as -3.45 +/- 0.44 kcal mol-1 (-14.43 +/- 0.11 kJ mol-1) for aci-nitromethane and -14.25 +/- 0.44 kcal mol-1 (-59.95 +/- 1.84 kJ mol-1) for the aci-nitroethane isomers, respectively. Data are evaluated against experimental and computational values in the literature with recommendations. A set of thermal correction parameters to atomic (H, C, N, O) enthalpies at 0 K is developed, to enable a direct calculation of species enthalpy of formation at 298.15 K, using atomization reaction and computation outputs.     

Negative-ion photoelectron spectroscopy, gas-phase acidity, and thermochemistry of the peroxyl radicals CH(3)OO and CH(3)CH(2)OO

Methyl, methyl-d(3), and ethyl hydroperoxide anions (CH(3)OO(-), CD(3)OO(-), and CH(3)CH(2)OO(-)) have been prepared by deprotonation of their respective hydroperoxides in a stream of helium buffer gas. Photodetachment with 364 nm (3.408 eV) radiation was used to measure the adiabatic electron affinities: EA[CH(3)OO, X(2)A' '] = 1.161 +/- 0.005 eV, EA[CD(3)OO, X(2)A' '] = 1.154 +/- 0.004 eV, and EA[CH(3)CH(2)OO, X(2)A' '] = 1.186 +/- 0.004 eV. The photoelectron spectra yield values for the term energies: Delta E(X(2)A' '-A (2)A')[CH(3)OO] = 0.914 +/- 0.005 eV, Delta E(X(2)A' '-A (2)A')[CD(3)OO] = 0.913 +/- 0.004 eV, and Delta E(X(2)A' '-A (2)A')[CH(3)CH(2)OO] = 0.938 +/- 0.004 eV. A localized RO-O stretching mode was observed near 1100 cm(-1) for the ground state of all three radicals, and low-frequency R-O-O bending modes are also reported. Proton-transfer kinetics of the hydroperoxides have been measured in a tandem flowing afterglow-selected ion flow tube (FA-SIFT) to determine the gas-phase acidity of the parent hydroperoxides: Delta(acid)G(298)(CH(3)OOH) = 367.6 +/- 0.7 kcal mol(-1), Delta(acid)G(298)(CD(3)OOH) = 367.9 +/- 0.9 kcal mol(-1), and Delta(acid)G(298)(CH(3)CH(2)OOH) = 363.9 +/- 2.0 kcal mol(-1). From these acidities we have derived the enthalpies of deprotonation: Delta(acid)H(298)(CH(3)OOH) = 374.6 +/- 1.0 kcal mol(-1), Delta(acid)H(298)(CD(3)OOH) = 374.9 +/- 1.1 kcal mol(-1), and Delta(acid)H(298)(CH(3)CH(2)OOH) = 371.0 +/- 2.2 kcal mol(-1). Use of the negative-ion acidity/EA cycle provides the ROO-H bond enthalpies: DH(298)(CH(3)OO-H) = 87.8 +/- 1.0 kcal mol(-1), DH(298)(CD(3)OO-H) = 87.9 +/- 1.1 kcal mol(-1), and DH(298)(CH(3)CH(2)OO-H) = 84.8 +/- 2.2 kcal mol(-1). We review the thermochemistry of the peroxyl radicals, CH(3)OO and CH(3)CH(2)OO. Using experimental bond enthalpies, DH(298)(ROO-H), and CBS/APNO ab initio electronic structure calculations for the energies of the corresponding hydroperoxides, we derive the heats of formation of the peroxyl radicals. The "electron affinity/acidity/CBS" cycle yields Delta(f)H(298)[CH(3)OO] = 4.8 +/- 1.2 kcal mol(-1) and Delta(f)H(298)[CH(3)CH(2)OO] = -6.8 +/- 2.3 kcal mol(-1).     

A new enantiomerization mechanism for tripodal penta-coordinate Zn(II)(nta) complexes. Theoretical clarification of the observed 1H NMR spectrum

Based on DFT calculations (RB3LYP/LANL2DZp), the unexpected single-line 1H NMR spectrum of Zn(II)(nta), nta = 2,2',2'-nitrilotriacetate, can be ascribed to a non-dissociative enantiomerization process (deltadeltadelta<=>lambdalambdalambda) from C3viaC3v to C3 symmetry. The energy barrier is rather low and depends to a lesser extent on the nature of the co-ligand in [Zn(nta)X]2- (X: H-, CH3- NH2-, OCH3-, F-, Cl-, Br-, I-) and [Zn(nta)Y]- (Y: NCH, CO, N2, O(CH3)2), but more so on the overall charge of the complex. The energy barrier for enantiomerization of [Zn(nta)X]2- is between 5.7 and 6.7 kcal mol-1, and for [Zn(nta)Y]- between 2.2 and 3.1 kcal mol-1.     

Computational design of a self-assembling β-peptide oligomer

The first computationally designed self-assembling oligomer consisting of exclusively β-amino acids (βAAs) is presented. The packing of a β-3(14) helix into coiled-coils of varying stoichiometries as a function of amino acid sequence is examined. β-Peptides with hVal repeating every third residue in the sequence appeared to have a strong propensity to pack into hexameric bundles. The designed sequence was synthesized and characterized with CD spectroscopy, NMR, and analytical ultracentrifugation, suggesting that the peptide adopts a well-folded hexameric structure.     

Unimolecular reactions of chemically activated CF2BrCF2CH3 and CF2BrCF2CD3: evidence for 1,2-FBr interchange

Vibrationally excited CF2BrCF2CH3 and CF2BrCF2CD3 molecules were prepared with 96 kcal mol-1 energy at room temperature by the recombination of CF2BrCF2 and CH3 (CD3) radicals. The observed unimolecular reactions are 1,2-BrF interchange to give CF3CFBrCH3 (CD3) molecules and 2,3-FH (FD) elimination; the rate constants are 2.2 x 10(5) (1.5 x 10(5)) s(-1) and 2.0 x 105 (0.75 x 10(5)) s(-1), respectively. The CF3CFBrCH3 (CD3) molecules rapidly, relative to the reverse reaction, eliminate HBr or DBr to give the observed product CF3CF=CH2 (CD2). Density functional theory at the B3PW91/6-311+G(2d,p) level was used to obtain vibrational frequencies and moments of inertia of the molecule and transition states for subsequent calculations of statistical rate constants for CF2BrCF2CH3 and CF2BrCF2CD3. Matching experimental and calculated rate constants gave threshold energies of 62 and 66 kcal mol-1 for 1,2-BrF interchange and 2,3-FH elimination, respectively. The BrF interchange reaction is compared to ClF interchange from CF2ClCF2CH3 and CF2ClCHFCH3.     

Conformational studies by dynamic NMR. 79. Dimesityl sulfine revisited: detection of the helical antipodes and determination of their enantiomerization pathways.

By means of low-temperature NMR spectra, it is demonstrated that dimesityl sulfine (Mes2C=SO) adopts in solution the same chiral propeller conformation (C1 symmetry) determined by X-ray diffraction in the crystalline state. With the help of MM calculations, it has been also shown that a correlated rotation (cog wheel effect) of the two mesityl rings reverses the molecular helicity according to an enantiomerization process entailing a one-ring flip pathway with delta G++ = 5.9 kcal mol-1 and a two-ring flip pathway with delta G++ = 13.8 kcal mol-1. On the contrary the Z- and E-isomers of mesityl phenyl sulfine (MesPhC=SO) adopt essentially achiral conformations (Cs symmetry), having the Ph-CSO rotation barriers equal to 5.2 and 5.8 kcal mol-1, respectively, and the mesityl-CSO rotation barriers equal to 21.3 and 15.1 kcal mol-1, respectively.     

Cyclic 1,3-dipoles or acyclic phosphonium ylides? Electronic characterization of "Montréalones"

The structural features of a recently introduced class of 1,3-dipolar reagents have been computed by density functional theory and ab initio methods. The reagents are formally derived from Münchnones by replacement of the C O group with a PR3 unit. The parent species (PR3 = PH3) shows a long P...O interaction (2.55 A at the B3LYP/6-31+G(d) level), together with a nonplanar ring, and is best described as a weakly chelated acylamino-phosphonium ylide. The corresponding acyclic form, in which the P...O interaction is absent, is predicted to be 2-3 kcal mol-1 higher in enthalpy. Variation of the phosphorus substituents exerts a marked effect on the P...O distance, with electron-withdrawing groups favoring a covalent interaction [P...O 1.97 A for PR3 = PPh(catechyl)] and electron-donating groups favoring a weak interaction [P...O 3.92 A for PR3 = PPh3]. However, this variation has little effect on the relative energies of the cyclic and acyclic forms. The barriers for concerted cycloadditions with ethylene are 22.8 kcal mol-1 (PH3), 31.7 kcal mol-1 (PPh3), and 16.2 kcal mol-1 [PPh(catechyl) with axial O], which correspond with experimental observations and follow the same trend as the energies required to distort the dipole to the TS geometry.