Discrimination in resolving systems. V. Pseudoephedrine - fluoromandelic acid diastereomers

Resolution of the isomeric 2'-, 3'-, and 4'-fluoromandelic acids with (+)-(1S;2S)-pseudoephedrine in 95% ethanol produces both well and poorly discriminating, hydrated and unsolvated binary salts. Seven observed diastereomeric phases are represented by five crystal structure types including three of the four types observed in the pseudoephedrine mandelates. Type a: monoclinic hemihydrate less-soluble (L) (R)-3'-fluoromandelate and more-soluble (M) (R)-4'-fluoromandelate (I); type b: orthorhombic unsolvated M (S)-2'-fluoromandelate; type c: orthorhombic unsolvated L (R)-2'-fluoromandelate; type d: orthorhombic dihydrate M (S)-3'-fluoromandelate and L (S)-4'-fluoromandelate; type e: monoclinic unsolvated M (R)-4'-fluoromandelate (II). Largest (15-fold) discriminating solubilities in 95% ethanol are found between the diastereomers with 2'-fluoromandelic acid, 50% more than in the corresponding ephedrine system. Principle interionic interactions are hydrogen-bonds between protonated secondary ammonium ions and carboxylates. Infinite chains of these are found in type c, with a four-atom repeating unit H-N(+)-H.O(-C(-)-O) [C(2)(1)(4)], and in types b and d, with a six-atom repeating unit H-N(+)-H.O-C(-)-O [C(2)(2)(6)]. Water of crystallization intervenes in the chains of type a but not of type d hydrated salts, according with higher average dehydration temperatures in the former. Hydrated salts in general are excessively soluble in 95% ethanol.     

Discrimination in resolving systems: ephedrine-mandelic acid.

Resolution of mandelic acid with (-)-(1R,2S)-ephedrine in water and ethanol produces intermediate diastereomeric salts with greatly disparate solubilities and melting points. Single crystal X-ray analysis of the less (L) and more (M) soluble (-)-ephedrinium mandelates (I, II) shows crystal structures which are isosteric, each crystallizing in the monoclinic system, space group C2. Protonated ephedrines occupy the same relative positions in the L- and M-salts, and mandelates are in the same general locations. Hydrogen bonds link alternating protonated ephedrine nitrogens and mandelate carboxylate oxygens in each salt forming columns of ions. The helical H-bonded chain winds down the crystallographic 2-fold screw axis. Additional H-bonds form between 2-fold related mandelates in the L-salt. Mixed crystals, containing both mandelate isomers, (2R)- and (2S)-mandelates, are obtained from the resolving system partly depleted of the L-salt. A specimen with nearly equal amounts of the mandelates (III) is also isosteric with the commensurate structures. I (294K), L-salt: a = 18.160(7), b = 6.538(2), c = 13.898(4) A, beta = 92.02(3) degrees, V = 1649.1(9) A3; IIa (294K), M-salt: a = 17.978(11), b = 7.164(4), c = 13.574(6)A, beta = 96.41(4) degrees, V = 1737.3(16) A3; IIb (223K), M-salt: a = 17.805(8), b = 7.115(2), c = 13.50(5) A, beta = 96.89(3) degrees, V = 1697.9(15) A3; III (294K), mixed-salt: a = 18.184(22), b = 6.792(7), c = 13.808(19) A, beta = 93.74(10) degrees, V = 1701.7(35) A3.     

Discrimination in resolving systems II: Ephedrine-substituted mandelic acids

Binary diastereomeric (-) (1R,2S)-ephedrine salts of various mandelic acids obtained from 95% ethanol show considerable differences in solubility. Structures and some properties of the less-soluble (L) and more-soluble (M) solid phases of (-)-ephedrine with unsubstituted mandelic acid, 2-, 3-, and 4-monosubstituted halo (F, Cl, Br) mandelic acids, and 3- and 4-methylmandelic acids have been determined. Salts were found to be binary, without solvent of crystallization, and composed of double-layered arrays of alternating anions and cations linked by H-bonds normal to the layers. H-bonding links charged donors and acceptors usually along a crystallographic 2-fold screw axis. A striking discrimination is evident in that the (2R)-mandelate salts typically display a compact four-atom chain as the H-bonding repeating unit [⁺N—H…O(—C^?—O)…H-N′, C₂¹(4)] while the (2S)-mandelate salts adopt a more dimensionally variable six-atom chain repeating unit [⁺N—H…O—C^?—O…H—N′, C₂²(6)]. Two distinct packing schemes display the shorter H-bonding chain of the (2R)-mandelates which always occurs with ephedrinium ions in the fully extended conformation. Slightly greater packing efficiency and H-bonding energies of the (2R)-mandelate salts correlates with increased fusion points, lower solubilities (95% ethanol), and higher heats of fusion relative to the phase adopted by their diastereoisomers. In contrast, (2S)-mandelate salts exhibit considerably more structural variability involving all three major ephedrinium conformations, and at least four distinct packing motifs. Mandelates with larger 3′-substituents (Cl, Br, methyl) show similar property discriminations, but these occur with an opposing trend, that is, between phases in which the less-soluble salts contain (2S)-mandelates. Salts with 2-bromomandelate do not show property disparities and their structures are dissimilar to the other phases. © 1995 Wiley-Liss, Inc.     

Reexamination of a canonical model for plant organ biomass partitioning

A previously proposed "canonical" model for the scaling relations among leaf, stem, and root biomass (M(L), M(S), and M(R), respectively) asserts that the proportional relations M(L) ∝ M(S)(3/4) ∝ M(R)(3/4) and M(S) ∝ M(R) hold across seed plant species. This model is scrutinized by determining whether the scaling relations between M(L), M(S), and M(R) vs. basal stem diameter D(S) and between M(L), M(S), and M(R) vs. plant height h are logically consistent with previously predicted scaling exponents. For example, if M(L) is observed to scale as the 2-power of D(S) and the model asserts that M(L) ∝ M(S)(3/4), then M(S) must scale as the 8/3-power of D(S) if the model is valid. Using a large data base for species with self-supporting stems, statistical support was found for most such comparisons between predicted and observed scaling relationships. However, this judgement is predicated on (1) the assertion that the scaling exponents for M(R) with respect to D(S) (or h) are numerically "deflated" due to a systematic underestimate of fine and small root biomass and (2) the stringent protocol used to calculate the 95% confidence intervals of scaling exponents, which favors rejection of the model. In light of these features, the "canonical" model is logically consistent with the new scaling relations reported here. Therefore, the model is judged valid within the context of this evaluation.     

Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.     

Optical resolution by preferential crystallization of 4-methylpiperidinium hydrogen (RS)-phenylsuccinate

Gibbs energy of racemate formation, binary melting point diagram, and ternary solubility diagram suggested that 4-piperidinium hydrogen (RS)-phenylsuccinate [(RS)-4-MP salt] exists in a conglomerate. Appropriate conditions were explored on the basis of free energy of critical nucleation in a supersaturated solution to resolve efficiently (RS)-4-MP salt by preferential crystallization. Successive preferential crystallization of (RS)-4-MP salt in ethanol at 20°C gave (R)- and (S)-4-MP salts of 90–94% optical purities. Optically pure (R)- and (S)-phenylsuccinic acids were obtained by recrystallization of the (R)- and (S)-4-MP salts, followed by treatment of the salts purified with hydrochloric acid. © 1994 Wiley-Liss, Inc.     

Hydroxylation-induced stabilization of the collagen triple helix. Acetyl-(glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)(10)-NH(2) forms a highly stable triple helix

The collagen triple helix is one of the most abundant protein motifs in animals. The structural motif of collagen is the triple helix formed by the repeated sequence of -Gly-Xaa-Yaa-. Previous reports showed that H-(Pro-4(R)Hyp-Gly)(10)-OH (where '4(R)Hyp' is (2S,4R)-4-hydroxyproline) forms a trimeric structure, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not form a triple helix. Compared with H-(Pro-Pro-Gly)(10)-OH, the melting temperature of H-(Pro-4(R)Hyp-Gly)(10)-OH is higher, suggesting that 4(R)Hyp in the Yaa position has a stabilizing effect. The inability of triple helix formation of H-(4(R)Hyp-Pro-Gly)(10)-OH has been explained by a stereoelectronic effect, but the details are unknown. In this study, we synthesized a peptide that contains 4(R)Hyp in both the Xaa and the Yaa positions, that is, Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) and compared it to Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2), and Ac-(Gly-4(R)Hyp-Pro)(10)-NH(2). Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) showed a polyproline II-like circular dichroic spectrum in water. The thermal transition temperatures measured by circular dichroism and differential scanning calorimetry were slightly higher than the values measured for Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2) under the same conditions. For Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), the calorimetric and the van't Hoff transition enthalpy DeltaH were significantly smaller than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). We postulate that the denatured states of the two peptides are significantly different, with Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forming a more polyproline II-like structure instead of a random coil. Two-dimensional nuclear Overhauser effect spectroscopy suggests that the triple helical structure of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) is more flexible than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). This is confirmed by the kinetics of amide (1)H exchange with solvent deuterium of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), which is faster than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). The higher transition temperature of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), can be explained by the higher trans/cis ratio of the Gly-4(R)Hyp peptide bonds than that of the Gly-Pro bonds, and this ratio compensates for the weaker interchain hydrogen bonds.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the number of SC-MC hydrogen bonds to the total number of polar residues is close to 1:2, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor ('i') and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterized by specific combinations of backbone and side-chain torsion angles. (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms O(gamma)(i) and O(i-4). More than half the examples of such hydrogen bonds are found at the middle of alpha-helices rather than at their ends. The most favoured motif of these examples is alpha(R)alpha(R)alpha(R)alpha(R)(g(-)). (b) These residues also show great preference to form hydrogen bonds between O(gamma)(i) and O(i-3), which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by alpha(R)alpha(R)alpha(R)alpha(R)(g(+)) is most preferred in these cases. (c) The Ser, Thr and Glu are the most frequently found residues participating in intra-residue hydrogen bonds (between the side-chain and main-chain of the same residue) which are characterized by specific motifs of the form beta(g(+)) for Ser/Thr residues and alpha(R)(g(-)g(+)t) for Glu/Gln. (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterized by the motifs beta (tt')alphaR and beta(t)alpha(R), respectively. These hydrogen bonded segments, referred to as Asx turns, are known to provide stability to type I and type I' beta-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of alpha-helices, which are characterized by the beta(g(+))alpha(R)alpha(R) motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein.     

NMR study on hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides: experimental evidence of hydrogen bonding and chemical exchange interactions in κ/μ oligosaccharides

The hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides have been studied by NMR spectroscopy in 85% H(2)O/15% acetone-d(6). Hydration and hydrogen bonding interactions in di- (κ), tetra- (κκ), hexa (κκκ), and octa- (κκκκ) κ-oligosaccharides and hexa- (κμκ), octa- (κμμκ), and deca- (κμμμκ) κ/μ-oligosaccharides have been investigated by measuring the chemical shifts, temperature coefficients, and chemical exchange of the hydroxy protons. These NMR parameters indicate that no strong and persistent intramolecular hydrogen bonds involving hydroxy protons stabilize the structure of κ-carrageenan oligosaccharides in aqueous solution. In the κ/μ-oligosaccharides, the presence of chemical exchange between OH3 of α-d-Gal-6-sulfate (D6S) and OH2 of β-d-Gal-4-sulfate (G4S) across the β-d-Gal-4-S-(1→4)-α-d-Gal-6-S linkage reveals the existence of a weak hydrogen bond interaction between the two hydroxyl groups. The smaller temperature coefficients of OH2_D6S and OH3_D6S indicate reduced hydration, interpreted as spatial proximity to the 4-sulfate group and O5 ring oxygen of the neighboring G4S residues, respectively. These first experimental results on the conformation of κ/μ-carrageenan oligosaccharides shine light on the potential role of "kinks" in the properties of the three-dimensional carrageenan gel network.     

Discrimination in resolving systems. III: Pseudoephedrine-mandelic acid

(+)-(1S;2S)-Pseudoephedrine and racemic mandelic acid form three distinct diastereomeric salts from solutions in 95% ethanol. The least-soluble phase, a hemihydrate, contains the (2R)-mandelate. A salt phase of intermediate solubility is the unsolvated double salt, containing both the (2R)- and the (2S)-mandelate. The most-soluble salt phase contains the (2S)-mandelate. Mandelate configuration and order of solubility (based on the heats of fusion) is inverted from that found in the same system synthesized from chiral base and acid, and then crystallized from benzene solution. The (2R)-mandelate hemihydrate (−H₂O at 349.5K, mp 391K), monoclinic, P2₁, a = 6.788(5), b = 29.415(35), c = 9.488(10)Å, β = 108.91(8)°, Z = 4 (2 ion-pairs/asymmetric unit). Intermediate double salt (2S)- and (2R)-mandelate, mp 377.6K, anorthic, P1, a = 7.758(4), b = 9.966(5), c = 13.366(6)Å, α = 72.99(4), β = 79.98(4), γ = 70.51(4)°, Z = 1 (2 ion-pairs/asymmetric unit). The (2S)-mandelate (mp 386.2K), orthorhombic, P2₁2₁2₁, a = 7.079(6), b = 13.443(10), c = 18.820(14)Å, Z = 4 is identical to a salt made from a combination of enantiomeric moieties from benzene solution. While differing from ephedrine mandelates in configuration at one center, solubilities of pseudoephedrine mandelates in 95% ethanol are much larger. A comparison of molecular structure (non-polar and H-bonding) regions of pseudoephedrine and ephedrine mandelates shows similarities and differences that are tentatively linked to crystal properties. This study reemphasizes the necessity for consistency in solvent use in resolution and in phase identification and comparison because the phases produced are frequently dependent upon the solvent. Chirality 10:325–337, 1998. © 1998 Wiley-Liss, Inc.     

Biomimetic oxidation of ibuprofen with hydrogen peroxide catalysed by horseradish peroxidase (HRP) and 5,10,15,20-tetrakis-(2',6'-dichloro-3'-sulphonatophenyl)porphyrinatoiro n(III) and manganese(III) hydrates in AOT reverse micelles

The oxidation of ibuprofen with H2O2 catalysed by Horseradish peroxidase (HRP), Cl8TPPS4Fe(III)(OH2)2 and Cl8TPPS4Mn(III)(OH2)2 in AOT reverse micelles gives 2-(4'-isobutyl-phenyl)ethanol (5) and p-isobutyl acetophenone (6) in moderate yields. The reaction of ibuprofen (2) with H2O2 catalysed by HRP form carbon radicals by the oxidative decarboxylation, which on reaction with molecular oxygen to form hydroperoxy intermediate, responsible for the formation of the products 5 and 6. The yields of different oxidation products depend on the pH, the water to surfactant ratio (Wo), concentration of Cl8TPPS4Fe(III)(OH2)2 and Cl8TPPS4Mn(III)(OH2)2 and amount of molecular oxygen present in AOT reverse micelles. The formation of 2-(4'-isobutyl phenyl)ethanol (5) may be explained by the hydrogen abstraction from ibuprofen by high valent oxo-manganese(IV) radical cation, followed by decarboxylation and subsequent recombination of either free hydroxy radical or hydroxy iron(III)/manganese(III) porphyrins. The over-oxidation of 5 with high valent oxo-manganese, Mn(IV)radical cation intermediate form 6 in AOT reverse micelles by abstraction and recombination mechanism.     

Crystal structure and aqueous solubility of ammonium D-glucarate

Ammonium D-glucarate, NH(4)(C(6)H(9)O(8)) [ammonium D-saccharate, NH(4)-SAC], has been synthesized, and its crystal structure solved by single-crystal X-ray diffraction methods. NH(4)-SAC crystallizes in the monoclinic space group P2(1) (#4) with cell parameters a = 4.8350(4) Angstroms, b = 11.0477(8) Angstroms, c = 16.7268(12) Angstroms, beta = 90.973(1) degrees, V = 894.34(12) Angstroms(3), Z = 3. The structure was refined by full-matrix least-squares on F(2) yielding final R-values (all data) R1 = 0.0353 and R(w)2 = 0.0870. The structure consists of alternating (NH(4))(+) and (C(6)H(11)O(6))(-) layers parallel to the bc plane. An extended network of N-H...O(SAC) and O(SAC)-H...O(SAC) hydrogen bonds provide the 3-D connectivity. The aqueous solubility (S(w)) has been shown to be pH independent at ambient conditions within the range 4.5 < pH < 10 with S(w) = 2.19 M/L, whose value is about a factor of two lower than that of the ammonium isosaccharate analogue.     

New asymmetric AB(n)-shaped amphiphilic poly(ethylene glycol)-b-[poly(l-lactide)](n) (n = 2, 4, 8) bridged with dendritic ester linkages: I. Syntheses and their characterization

This study presents new investigations on chemical syntheses and characterization of new asymmetric AB(n)-shaped amphiphilic diblock methoxy poly(ethylene glycol)-b-[poly(l-lactide)](n), MPEG-b-(PLLA)(n) (n = 2, 4, and 8), bridged with dendritic ester linkages. First, a new series of A(OH)(n)-shaped hydroxy end-capped MPEG-(OH)(2), MPEG-(OH)(4), and MPEG-(OH)(8) bearing corresponding one- to three-generation dendritic ester moieties were efficiently derived from the starting MPEG (M(n) = 2 KDa) and 2,2'-bis(hydroxymethyl)propionic acid (Bis-HMPA) via ester coupling and a facile hydroxy protection-deprotection cycle, and then, chemical structures of these functional MPEG-(OH)(n) were characterized by nuclear magnetic resonance spectrometry (NMR) and MALDI-FTMS. Subsequently, by employing these MPEG-(OH)(n) as functional macroinitiators, new asymmetric AB(n)()-shaped amphiphilic MPEG-b-(PLLA)(2) S1, MPEG-b-(PLLA)(4) S2, and MPEG-b-(PLLA)(8) S3 bridged with dendritic Bis-HMPA ester linkages of L1-L3 as well as linear structural MPEG-b-PLLA references (R1-R3) were synthesized through the SnOct(2)-catalyzed ring-opening polymerization (ROP) of l-lactide at 130 degrees C in m-xylene solution, and their structures were further examined by NMR and gel permeation chromatography (GPC). It was demonstrated that the functional MPEG-(OH)(n) efficiently initiated the ROP of LLA, finally leading to successful formation of the AB(n)-shaped amphiphilic MPEG-b-(PLLA)(n) (n = 2, 4, and 8) with each PLLA arm weight close to 2 KDa and very narrow molecular weight distribution. Moreover, thermal history, crystallization, and spherulite morphologies were studied by means of differential scanning calorimeter (DSC), thermal gravimetric analyzer (TGA), and polarized microscope (POM) for these new structural amphiphilic S1-S3 as well as the linear R1-R3, intriguingly indicating a strong molecular architecture dependence of segmental crystallizability, spherulite morphology, and apparent crystal growth rate. Due to the favorable biodegradability and biocompatibility of the PLLA and MPEG, these results may therefore create new possibilities for these novel structural AB(n)-shaped amphiphilic MPEG-b-(PLLA)(n) as potential biomaterials.     

Molecular recognition in purine-rich internal loops: thermodynamic,structural, and dynamic consequences of purine for adenine substitutions in 5'(rGGCAAGCCU)2

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A.A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5'(rGGCAAGCCU)(2), to give 5'(rGGCPAGCCU)(2), 5'(rGGCAPGCCU)(2), and 5'(rGGCPPGCCU)(2). The 5'(rGGCAAGCCU)(2) duplex has sheared A(anti).A(anti) (A.A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969-14977]. In comparison to 5'(rGGCAAGCCU)(2), replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P.A noncanonical pairs of the 5'(rGGCPAGCCU)(2) duplex have a sheared anti-anti structure (P.A trans Hoogsteen/Sugar-edge) with P4.A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5'(rGGCAAGCCU)(2). In contrast, the A.P pairs of the 5'(rGGCAPGCCU)(2) duplex have a face-to-face conformation (A.P trans Watson-Crick/Watson-Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5'(rGGCPPGCCU)(2) are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.     

Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.     

Conformational response of tartaric acid to derivatization: role of 1,3-dipole-dipole interactions

The four-carbon chain in (R,R)-tartaric acid derivatives is predominantly antiperiplanar (trans) in the acid, its salts, esters, and NH-amides, while (-)-synclinal (gauche) conformer is the most abundant in N,N'-tetraalkyltartramides. Trialkylsilylation or tert-butylation of the hydroxy groups at C2 and C3 does not appear to affect the conformational preference of NH-tartramides, but it does change the conformational equilibrium in the case of tartrates (toward (-)-gauche) and N,N'-tetraalkyltartramides (toward trans), as judged from the NMR data. X-ray diffraction data point to the stabilizing role of antiparallel dipole-dipole interactions due to the 1,3-CO/CH bonds. These interactions can be found in the trans and (-)-gauche conformers but are not possible for the (+)-gauche conformers of (R,R)-tartaric acid derivatives. This rationalizes small proportion of (+)-gauche conformers in tartaric acid derivatives and points to a significance of 1,3-dipole-dipole interactions. The conformation around the C1-C2 (and C3-C4) bond is different in tartrates (O-C-C=O, syn) and tartramides (O-C-C=O, anti); the CD data (n-pi* band) show that O-silylation or O-tert-butylation brings about conformational changes around the C1-C2 bond in the case of N,N'-tetraalkyldiamides only.     

Reactions of site-differentiated [Fe4S4]2+, 1+ clusters with sulfonium cations: reactivity analogues of biotin synthase and other members of the S-adenosylmethionine enzyme family

The first examples of reduced 3:1 site-differentiated Fe(4)S(4) clusters have been synthesized as [Fe(4)S(4)(LS(3))(SR')](3-) (R=Et, Ph) by chemical reduction of previously reported [Fe(4)S(4)(LS(3))(SR')](2-) clusters, and isolated as NBu(4)(+) salts. The reduced clusters were characterized by electrochemistry and EPR, 1H NMR, and M?ssbauer spectroscopies. The reaction of oxidized clusters with the sulfonium ions [PhMeSCH(2)R](+) (R=COPh, p-C(6)H(4)CN) in acetonitrile results in electrophilic attack on coordinated thiolate and production of PhSMe and R'SCH(2)R when the reaction occurs at the unique cluster site. The reactions of reduced clusters with these substrates were examined in relation to the reductive cleavage of the cofactor S-adenosylmethionine, the first step in the catalytic cycle of biotin synthase. Product analysis indicated a approximately 4:1 ratio of reductive cleavage to electrophilic attack. The cleavage products are PhSMe, R'SCH(2)R, and RCH(3) for both clusters, and also PhMeS=CHR and RCH(2)CH(2)R from secondary reactions when the sulfonium cation is [PhMeSCH(2)COPh](+) and [PhMeSCH(2)-p-C(6)H(4)CN](+), respectively. Reaction schemes for reductive cleavage based on product distributions are presented. These results parallel those previously reported for homoleptic [Fe(4)S(4)(SR')(4)](2-,3-) clusters and demonstrate that site-differentiated clusters sustain a high percentage of reductive cleavage, a necessary result in the context of biotin synthase activity preceding an investigation of the mode of binding of sulfonium substrates and inhibitors at the unique iron site. [LS(3)=1,3,5-tris[(4,6-dimethyl-3-mercaptophenyl)thio]-2,4,6-tris(p-tolylthio)benzene(3-)].     

Crystal structure and site-specific mutagenesis of pterin-bound human phenylalanine hydroxylase

The crystal structure of the dimeric catalytic domain (residues 118-424) of human PheOH (hPheOH), cocrystallized with the oxidized form of the cofactor (7,8-dihydro-L-biopterin, BH(2)), has been determined at 2.0 A resolution. The pterin binds in the second coordination sphere of the catalytic iron (the C4a atom is 6.1 A away), and interacts through several hydrogen bonds to two water molecules coordinated to the iron, as well as to the main chain carbonyl oxygens of Ala322, Gly247, and Leu249 and the main chain amide of Leu249. Some important conformational changes are seen in the active site upon pterin binding. The loop between residues 245 and 250 moves in the direction of the iron, and thus allows for several important hydrogen bonds to the pterin ring to be formed. The pterin cofactor is in an ideal orientation for dioxygen to bind in a bridging position between the iron and the pterin. The pterin ring forms an aromatic pi-stacking interaction with Phe254, and Tyr325 contributes to the positioning of the pterin ring and its dihydroxypropyl side chain by hydrophobic interactions. Of particular interest in the hPheOH x BH(2) binary complex structure is the finding that Glu286 hydrogen bonds to one of the water molecules coordinated to the iron as well as to a water molecule which hydrogen bonds to N3 of the pterin ring. Site-specific mutations of Glu286 (E286A and E286Q), Phe254 (F254A and F254L), and Tyr325 (Y325F) have confirmed the important contribution of Glu286 and Phe254 to the normal positioning of the pterin cofactor and catalytic activity of hPheOH. Tyr325 also contributes to the correct positioning of the pterin, but has no direct function in the catalytic reaction, in agreement with the results obtained with rat TyrOH [Daubner, S. C., and Fitzpatrick, P. F. (1998) Biochemistry 37, 16440-16444]. Superposition of the binary hPheOH.BH(2) complex onto the crystal structure of the ligand-free rat PheOH (which contains the regulatory and catalytic domains) [Kobe, B., Jennings, I. G., House, C. M., Michell, B. J., Goodwill, K. E., Santarsiero, B. D., Stevens, R. C., Cotton, R. G. H., and Kemp, B. E. (1999) Nat. Struct. Biol. 6, 442-448] reveals that the C2'-hydroxyl group of BH(2) is sufficiently close to form hydrogen bonds to Ser23 in the regulatory domain. Similar interactions are seen with the hPheOH.adrenaline complex and Ser23. These interactions suggest a structural explanation for the specific regulatory properties of the dihydroxypropyl side chain of BH(4) (negative effector) in the full-length enzyme in terms of phosphorylation of Ser16 and activation by L-Phe.     

1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.