Investigating diproline segments in proteins: occurrences, conformation and classification

The covalent linkage between the side‐chain and the backbone nitrogen atom of proline leads to the formation of the five‐membered pyrrolidine ring and hence restriction of the backbone torsional angle ? to values of ?60 °± 30° for the L ‐proline. Diproline segments constitute a chain fragment with considerably reduced conformational choices. In the current study, the conformational states for the diproline segment ( ^L Pro‐ ^L Pro) found in proteins has been investigated with an emphasis on the cis and trans states for the Pro‐Pro peptide bond. The occurrence of diproline segments in turns and other secondary structures has been studied and compared to that of Xaa‐Pro‐Yaa segments in proteins which gives us a better understanding on the restriction imposed on other residues by the diproline segment and the single proline residue. The study indicates that P_II–P_II and P_II–α are the most favorable conformational states for the diproline segment. The analysis on Xaa‐Pro‐Yaa sequences reveals that the Xaa‐Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The present study may lead to a better understanding of the behavior of proline occurring in diproline segments which can facilitate various designed diproline‐based synthetic templates for biological and structural studies. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 54–64, 2012.     

Tuning the beta-turn segment in designed peptide beta-hairpins: construction of a stable type I' beta-turn nucleus and hairpin-helix transition promoting segments

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu-Val-Val-OMe ((D)Xxx = (D)Ala, 3a;(D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-(D)Ala-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD(3)OH and DMSO-d(6). However, in CDCl(3) evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl(3) and a significant population of both helices and hairpins in CD(3)OH and DMSO-d(6). The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central beta-turn permits generation of models for robust beta-hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions.     

Structural features of the human salivary mucin, MUC7

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (～25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model.     

Investigating proline puckering states in diproline segments in proteins

In the current study, the puckering states of the Proline ring occurring in diproline segments (^LPro‐^LPro) in proteins has been investigated with a segregation made on the basis of cis and trans states for the Pro‐Pro peptide bond and the conformational states for the diproline segment to investigate the effects of conformation of the diproline segment on the corresponding puckering state of the Proline ring in the segment if any. The value of the endocyclic ring torsional angles of the pyrrolidine ring has been used for calculating and visualizing various puckering states using a proposed new sign convention (+/?) nomenclature. The results have been compared to that obtained in a previous study on peptides from this group. In this study, quite interestingly, the Planar (G) conformation that was present in 14.3% of the cases in peptides, appears to be nearly a rare conformation in the case of proteins (1.9%). The present study indicates that the (C^γ‐exo/C^γ‐exo), (C^γ‐exo/Twisted C^γ‐exo‐C^β‐endo) and (Twisted C^γ‐endo‐C^β‐exo/Twisted C^γ‐endo‐C^β‐exo) categories are the most preferred combinations. For Proline rings in proteins, the states C^γ‐exo, Twisted C^γ‐exo‐C^β‐endo and Twisted C^γ‐endo‐C^β‐exo are the most preferred states. Within diproline segments, the pyrrolidine ring conformations do not show a strong co‐relation to the backbone conformation in which they are observed. It is likely that five‐membered rings have a considerable plasticity of structure and are readily deformed to accommodate a variety of energetically preferred backbone conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 605–610, 2013.     

Ultrafast folding and molecular dynamics of a linear hydrophobic β-hairpin

The first computational study of the folding and dynamics of a hydrophobic β-hairpin containing a central heterochiral diproline segment is reported. Linear hydrophobic sequences containing centrally positioned diproline motifs, heterochiral (DL/LD) and homochiral (LL/DD)), are investigated for their ability to form β-hairpins. Heterochiral diproline motifs (LD/DL) reveal the formation of stable β-hairpins with the backbone adopting β-turn conformation and the formation of backbone hydrogen bonds with antiparallel cross-strand registry, whereas the homochiral diproline (LL/DD) containing sequences tend to adopt PP_II helix conformation. The competition between the β-turn formation and the backbone H-bond ladder of the antiparallel β-strands in heterochiral diproline containing sequences is employed to validate the hypothesis that β-turn formation precedes inter-strand registry in the folding of a β-hairpin (“zipper” mechanism). The observation of noncanonical hydrogen bonds leads to a folded β-hairpin-like conformation and points to the existence of relatively stable transition state intermediates, between the unfolded (extended) and folded (β-hairpin) states. The MD simulations are in excellent agreement with the experimental studies on the model system and constitute the very first computational investigation of the folding and dynamics of a completely hydrophobic synthetic β-hairpin containing heterogeneous residues of mixed chirality.     

Chain length effects on helix-hairpin distribution in short peptides with Aib-DAla and Aib-Aib segments

The Aib-D Ala dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and Boc-Aib-Xxx-NHMe (4, 8), where Xxx=DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7), and Boc-Aib-Aib-NHMe (8) helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3 OH. The β-turn conformations (type-I'/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc-Aib-D Ala-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 3(10)-helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc-Val-Aib-DAla-Leu-NHMe (3) adopts a novel α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of an NOE between Val (1) NH?HNCH3 (5) in CD3OH suggests, that the solid state conformation is maintained in methanol solutions.     

Two conformational states of Turkey ovomucoid third domain at low pH: three-dimensional structures,internal dynamics,and interconversion kinetics and thermodynamics

Turkey ovomucoid third domain (OMTKY3) is shown to exist at low pH as two distinctly folded, interconverting conformations. Activation parameters were determined for the transition, and these were of the type reported previously for cis/trans isomerizations of prolyl peptide bonds. Multidimensional, multinuclear NMR spectroscopy was used to determine the three-dimensional structure of each of the two states of P(5)-Pro(14)Asp OMTKY3 at pH 2.5 and 25 degrees C, under conditions where the two states have equal populations with interchange rates of 0.25 s(-1). The results showed that the two states differ by cis/trans isomerization of the P(8)-Tyr(11)-P(7)-Pro(12) peptide bond, which is cis in the conformer dominant at neutral pH and trans in the conformer appearing at low pH. The major structural differences were found to be in the region of the reactive site loop. The core of the protein, including the antiparallel beta-sheet and a alpha-helix, is preserved in both structures. The state with the cis peptide bond is similar to previously reported structures of OMTKY3 determined by NMR spectroscopy and X-ray crystallography. The cis-to-trans transition results in the relocation of the aromatic ring of P(8)-Tyr(11), disrupts many interactions between the alpha-helix and the reactive-site loop, and leads to more open spacing between this loop and the alpha-helix. In addition, the configurations of two of the three disulfide bonds, P(11)-Cys(8)- P(20)'-Cys(38), and P(3)-Cys(16)- P(17)'-Cys(35), are altered such that the C(alpha)-C(alpha) distances for each disulfide bridge are longer by approximately 1 A in the trans state than in the cis. Mutations at P(1)-Leu(18), P(6)-Lys(13), and P(5)-Pro(14) influence the position of the cis <= => trans equilibrium. In P(1)-Leu(18)Xxx OMTKY3 mutants, the trans state is more favored by P(1)-Gly(18) than by Ala(18) or Leu(18); in P(6)-Lys(13)Xxx OMTKY3 mutants, the trans state is more favored by P(6)-Glu(13) and P(6)-Asp(13) than Lys(13) or His(13). Stabilization of the trans state in P(5)-Pro(14)Xxx OMTKY3 mutants follows the series Xxx = Gly > Asp > Glu > Ala approximately equal His > Pro. In comparing the state with the trans peptide bond to that with the cis, the pK(a) values of P(12)-Asp(7) and P(1)'-Glu(19) are higher and those of P(9)-Glu(10) and P(25)'-Glu(43) are lower. The pK(a) values of other titrating groups in the molecule are similar in both conformational states. These pK(a) changes underlie the pH dependence of the conformational equilibrium and can be explained in part by observed structural differences. (15)N transverse relaxation results indicate that residues P(6)-Lys(13)-P(3)-Cys(16) in the trans state undergo a dynamic process on the microsecond-millisecond time scale not present in the cis state.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective somatostatin derivatives

Three cyclic disulfide analogs related to somatostatin, D-Phe(1)-cyclo(Cys(2)-Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-Xxx(7))-Thr(8)- NH2 (where Xxx = L-Pen 1; L-Cys 3; or D-Pen 4) were examined in DMSO-d6 by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a beta II' turn solution conformation for the core tetrapeptide -Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.     

Homologs of 1,2,5-hexahydro-3-one-1H-1,4-diazepine (DAP) as novel dipeptidomimetics and molecular scaffolds: efficient preparation of synthons

Structurally constraint dipeptidomimetics represent an important class of conformationally rigid dipeptide surrogates and molecular scaffolds, which are frequently employed in peptide-based structure-activity relationships (SAR) and construction of combinatorial libraries. We report on the design of an improved and general synthetic procedure to prepare synthons related to the trisubstituted 1,2,5-hexahydro-3-one-1H-1,4-diazepines [DAP(Xxx)(alpha7)] (DAP: 1,2,5-hexahydro-3-one-1H-1,4-diazepine; DAP(Xxx)(alpha7): the homologous series of DAP in which alpha refers to the location of the chiral carbon in the i(th) amino acid, Xxx represents the three letter notation for the i-1 amino acids, and 7 denotes the number of atoms in the ring) and their higher homologs [DAP(Xxx)(alphaN)] [Xxx = Phe, Asp(beta-OcHex) (cHex: cyclohexyl), and Arg(N(G)-Tos] (Tos: p-toluenesulfonyl); N = 8-10]. These dipetidomimetic structures are generated by reductive alkylation-mediated Calpha(i)-to-N(i-1) bridging between a Calpha (i)-(CH(2))(i-1)(n)-COSEt (n = 1-4) and H(2)N-C(i-1)HR-CO(2)Fm (Fm: 9-fluorenylmethyl) followed by H(2)N(i)-to-C(i-1)-CO(2)H lactam formation. We also describe the preparation of blocked N-Ac-[DAP(Phe)(alphaN)]-CONMe(2) (N = 8-10), which serve as model systems for detailed conformational analysis reported in the accompanying article.     

Purification,characterization, and crystallization of alliinase from garlic

Glycosylated dimeric alliinase (EC 4.4.1.4) was purified to homogeneity from its natural source, garlic. With 660 units/mg, the specific enzymatic activity of the pure enzyme is the highest reported to date. Based on both CD spectroscopy data and sequence-derived secondary structure prediction, the alpha-helix content of alliinase was estimated to be about 30%. Comparisons of all available amino acid sequences of alliinases revealed a common cysteine pattern of the type C-x18-19-C-x-C-x2-C-x5-C-x6-C in the N-terminal part of the sequences. This pattern is conserved in alliinases but absent in other pyridoxal 5'-phosphate-dependent enzymes. It suggests the presence of an epidermal growth factor-like domain in the three-dimensional structures of alliinases, making them unique among the various families of pyridoxal 5'-phosphate-dependent enzymes. Well-ordered three-dimensional crystals of garlic alliinase were obtained in four different forms. The best diffraction was observed with crystal form IV (space group P2(1)2(1)2(1), a=68.4, b=101.1, c=155.7 A) grown from an ammonium sulfate solution. These crystals diffract to at least 1.5 A resolution at a synchrotron source and are suitable for structure determination.     

Conformational characteristics of dimeric subunits of RNA from energy minimization studies. Mixed sugar-puckered ApG, ApU, CpG, and CpU.

Following the procedure described in the preceding article, the low energy conformations located for the four dimeric subunits of RNA, ApG, ApU, CpG, and CpU are presented. The A-RNA type and Watson-Crick type helical conformations and a number of different kinds of loop promoting ones were identified as low energy in all the units. The 3E-3E and 3E-2E pucker sequences are found to be more or less equally preferred; the 2E-2E sequence is occasionally preferred, while the 2E-3E is highly prohibited in all the units. A conformation similar to the one observed in the drug-dinucleoside monophosphate complex crystals becomes a low energy case only for the CpG unit. The low energy conformations obtained for the four model units were used to assess the stability of the conformational states of the dinucleotide segments in the four crystal models of the tRNAPhe molecule. Information on the occurrence of the less preferred sugar-pucker sequences in the various loop regions in the tRNAPhe molecule has been obtained. A detailed comparison of the conformational characteristics of DNA and RNA subunits at the dimeric level is presented on the basis of the results.     

Influence of the nature and substitution of chiral 2,3-epoxy alcohol derivatives on the enantiomeric elution order on chiralcel OD column

A number of 2,3-epoxy alcohol derivatives (1–16), obtained either as racemates or through the Sharpless asymmetric epoxidation reaction, were studied on a Chiralcel OD column. Nearly all compounds exhibit good enantioselective resolution on this chiral support. The order of elution of enantiomers is reversed between nerol and geraniol compounds. For 2,3-epoxy alcohols bearing a remote alkoxy (or silyloxy) group, the order of the enantiomeric elution alternates with the number n (n = 1–3) of methylenic groups present between the epoxide ring and the terminal OR (R = p − BrBn or OSitBuPh₂) functionality. In the case of trans 2,3-epoxy alcohols for the same number n, the order of elution is reversed when changing the terminal group −OSi to −OR. The latter group greatly improves the separation of the two enantiomers. Chirality 10:804–807, 1998. © 1998 Wiley-Liss, Inc.     

Enantiomeric Polyketides from the Starfish‐Derived Symbiotic Fungus Penicillium sp. GGF16‐1‐2

One new racemic mixture, penicilliode A ( 1 ) and four pairs of enantiomeric polyketides, penicilliode B and C ( 2 and 3 ) and coniochaetone B and C ( 4 and 5 ), were obtained from the starfish‐derived symbiotic fungus Penicillium sp. GGF16‐1‐2. Interestingly, the strain GGF16‐1‐2 can produce enantiomers. The absolute configuration of 1 was determined by X‐ray diffraction (XRD) analysis, and the absolute configurations of 2 – 4 were determined by the optical rotation (OR) values and electronic circular dichroism (ECD) calculations. Compounds 1 – 5 were firstly isolated from the marine‐derived fungus Penicillium as racemates, and 2 – 5 were separated by HPLC with a chiral stationary phase. All the compounds were evaluated for their antibacterial, cytotoxic and inhibitory activities against PDE4D2.     

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxy-2-arylacetic acids

A series of structurally related 2-aryloxy-2-arylacetic acids (1-3, 5-16) together with a thioisostere derivative (4) have been synthesized and characterized by GC-MS and 1H NMR. The designed compounds were analyzed on a Penicillin G Acylase chiral stationary phase (PGA-CSP) and the influence of the structure variations on retention and enantioselectivity was investigated. The chromatographic study includes the direct separation of the enantiomers of the synthesized compounds and the determination of the elution order of selected racemic mixtures. 10 out of 16 racemates were separated; high chromatographic enantioseparation factors (alpha > 2) were achieved for some compounds. For the enantiomers of four compounds whose absolute configuration was known (1, 3, 12, 16), the elution order was R:S with the exception of 2-(4-chloro-phenoxy)phenylacetic acid (1), for which the elution order was reversed. Preliminary molecular modeling studies suggest that both polar and charge-transfer interactions as well as steric effects play an important role in determining the retention factors and the enantioselectivities observed.     

Direct resolution of propranolol and bupranolol by thin-layer chromatography using cellulose derivatives as stationary phase

Cellulose triphenylcarbamate derivatives have been used as stationary phases for resolution of the enantiomers of the β-blockers propranolol and bupranolol by TLC. The derivatives examined were: cellulose trisphenylacarbamate (1), cellulose tris(2,3-dichlorophenyl carbamate) (2), cellulose tris(2,4-dichlorophenyl carbamate) (3), cellulose tris(2,6-dichlorophenyl carbamate) (4), cellulose tris (2,3-dimethylphenyl carbamate) (5), cellulose tris(3,4-dichlorophenyl carbamate) (6), cellulose tris(3,5-dichlorophenyl carbamate) (7), and cellulose tris(3,5-dimethylphenyl carbamate) (8). A variety of mobile phases were used to achieve useful separations and the effects of solvent polarity are also discussed. The best resolution of rac-propranolol was obtained on CSP 8 (R_fR = 0.26, R_fS = 0.06, α = 4.33) in mobile phase hexane:propan-2-ol (80:20 v/v). The best resolution of rac-bupranolol was obtained on CSP 5 (R_fR = 0.29, R_fS = 0.09, α = 3.22) in mobile phase hexane:propan-2-ol (80:20 v/v). These results demonstrated the potential of cellulose triphenylcarbamates as chiral stationary phases in TLC and indicate that this is potentially a useful method for the direct, simple, and rapid (within 30 min) resolution of racemates in the analytical control of enantiomeric purity. Physical aspects such as problems in cracking of the CSP, adhesion to plate, and interference of spot detection due to triphenylcarbamate chromphores are also discussed, along with the method employed to overcome them. Chirality 9:139–144, 1997. © 1997 Wiley-Liss, Inc.     

Approach to studying the structure-activity relationship of native peptides. I. Structural organization of the opioid peptide Tyr-Glu-Gly-Phe-Met-Arg-Gly-Leu and its artificial analogs]

Theoretical conformational analysis was carried out for the octapeptide Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Gly7-Leu8. Possible structure of the opioid peptide under physiological conditions may be described by a set of low-energy conformations belonging to 14 different forms of the backbone. The solution of the "reverse conformational problem" for the opioid peptide enables one to predict the modified amino acid sequences (Ala2, D-Ala2, Ala3, D-Ala3, Ala7, D-Ala7, MeMet5, MeArg6-analogues) which may assume one of the low-energy states of the native hormone. The influence of the solute was not taken into account in our calculations.     

Muscarone: An Enigma Resolved?

WITH the exception of muscarone, the potencies of the optical enantiomers of stimulants of the cholinergic post-ganglionic parasympathetic (muscarinic) nervous junction differ greatly, typically by about 200:1 (refs. 1 and 2). Furthermore, the absolute configuration of all the more potent enantiomers is the same³, that of L(+)-muscarine. The ratio of activities for muscarone, however, is only about 3:1 (refs. 1 and 2) and the more potent enantiomer is the D(?). Various rationalizations of this exception have been proposed in the absence of any conformational information. We have determined the conformation of muscarone in crystals of the iodide and suggest an alternative explanation of the activities of the enantiomers which fits the proposed structure-activity relationships of muscarinic^{3, 4} and nicotinic⁵ agonists of cholinergic nervous systems.     

Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting, fully reduced and CO-bound fully reduced states are isomorphous with each other.

Fully reduced and CO-bound fully reduced forms of cytochrome c oxidase from beef heart muscle were crystallized in the presence of sodium ascorbate under N2 or CO atmosphere. Hexagonal bipyramidal and tetragonal crystals were obtained for both forms depending on buffer species. The hexagonal bipyramidal crystals, as large as 0.6 mm in the largest dimension, diffracted X-rays at 7 A resolution, showing an identical space group and cell dimension, P6(2) or P6(4) and a = b = 209 A, c = 283 A, respectively. These parameters coincide with those for crystals of the fully oxidized resting enzyme. This result suggests that a large conformational change, like a subunit arrangement, is not induced by the redox change and/or binding of CO (and possibly O2) to heme a3.     

Chiral investigation of midodrine, a long-acting alpha-adrenergic stimulating agent

Midodrine hydrochloride is a peripheral alpha(1)-adrenoreceptor agonist that induces venous and arterial vasoconstriction. Midodrine, after oral or intravenous administration, undergoes enzymatic hydrolysis and releases deglymidodrine, a pharmacologically active metabolite. Midodrine and deglymidodrine have a chiral carbon in the 2-position. To investigate the bioactivity of racemates and enantiomers of the drug and metabolite, three chromatographic chiral stationary phases, Chiralcel OD-H, Chiralcel OD-R, and alpha(1)-AGP, were evaluated for enantiomeric resolution. Good enantioseparation of midodrine racemate was obtained using the Chiralcel OD-H column. This stationary phase was then used to collect separately the midodrine enantiomers. By alkaline hydrolysis of rac-midodrine and each separated enantiomer, rac-deglymidodrine and its enantiomers were prepared. The control of the enantiomeric purity was carried out by alpha(1)-AGP stationary phase, while the hydrolysis of rac-midodrine and its enantiomers was controlled by capillary electrophoresis using trimethyl-beta-cyclodextrin as chiral selector. The pharmacological activity of the two racemates and the two enantiomeric pairs was tested in vitro on a strip of rabbit descending thoracic aorta. The tests continued that the activity of the drug and metabolite is due only to the (-)-enantiomer because neither of the (+)-enantiomers is active.