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ABSTRACT: Pseudopeptide analogues of the delta opioid antagonists H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic-Phe-OH (TIP) containing a reduced peptide bond between the Tic2 and Phe3 residues were synthesized. The two compounds, H-Tyr-Tic psi [CH2NH]Phe-Phe-OH (TIPP [psi]) and H-Tyr-Tic psi-[CH2NH]Phe-OH (TIP [psi]), were tested in mu-, delta-, and kappa-receptor-selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. In comparison with their respective parent peptides, both pseudopeptide analogues showed increased delta antagonist potency in the MVD assay, higher delta receptor affinity and further improved delta receptor selectivity. The more potent compound, TIPP [psi], displayed subnanomolar delta receptor affinity and in direct comparisons with other selective delta ligands was shown to have unprecedented delta specificity (Ki mu/Ki delta = 10,500). Furthermore, this compound turned out to be highly stable against enzymatic degradation and, unlike other delta antagonists, showed no mu or kappa antagonist properties. TIPP [psi] is likely to find wide use as a pharmacological tool in opioid research.
Journal of Medicinal Chemistry 11/1993; 36(21):3182-7. · 5.25 Impact Factor
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ABSTRACT: A series of glycoconjugates, in which [Met5]enkephalin or [D-Ala2,Met5]enkephalin have been linked through an ester bond to the HO-6 of various D-glycopyranose moieties, were synthesized by classical solution methods. The biological activities of these compounds were determined on selective pharmacological preparations: guinea pig ileum and mouse vas deferens for opioid activity, and two mouse cell lines, fibroblasts L929 and melanoma B16BL6, to study the influence on growth processes. The results reported in this study demonstrate the differential effect of the carbohydrate part in enkephalin-related glycoconjugates on receptor recognition. In addition, synthesized neo-glycopeptides stimulate growth of the examined mouse cell lines, whereas parent peptide demonstrated some growth inhibitory properties. Full growth curves showed a dose-dependent effect at concentrations of 10(-7) to 10(-10) M.
International journal of peptide and protein research 05/1993; 41(4):399-404.
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International journal of peptide and protein research 04/1993; 41(3):313-6.
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ABSTRACT: A topochemical model to explain the bioactivity of morphiceptin (Tyr1-Pro2-Phe3-Pro4-NH2) was developed by taking account of accessible conformations around rotatable bonds which define relative spatial arrangements of opioid pharmacophores, the amine and phenolic groups of tyrosine and the aromatic ring of phenylalanine, necessary for receptor recognition. For this purpose, 1H-NMR measurements and computer simulations were extensively carried out on 10 stereoisomeric analogs related to morphiceptin: Tyr-Pro-(L and D)-Phe- (L and D)-Pro-NH2; Tyr-Pro-(L and D)-(NMe)Phe-(L and D)-Pro-NH2; Tyr-(NMe)Ala-Phe-D-Pro-NH2; and Tyr-Ala-Phe-D-Pro-NH2. These analogs are structurally close to one another but display various opiate potencies from highly active to inactive. The conformation of each rotatable bond has been specifically identified by measuring accessible space for the analogs, in which the difference in composition is observed in the specific site affecting only the conformation around the target bond. The most interesting characteristic of the model is a requirement of a cis amide bond linking residues 1 and 2. The model also requires the side chains in a trans conformation (chi 1 = 180 degrees) for the Tyr and Phe residues. The distances between the three pharmacophores, d1 (Tyr N to Tyr OH), d2 (Tyr N to the center of the aromatic ring of the third residue), and d3 (Tyr OH to the center of the aromatic ring of the third residue), were found to be approximately 8, approximately 7, and approximately 11-13 A, respectively. This model should aid in pharmaceutical design of peptide and nonpeptide ligands with opioid potencies.
Journal of Medicinal Chemistry 04/1993; 36(6):708-19. · 5.25 Impact Factor
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NIDA research monograph 02/1993; 134:238-52.
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ABSTRACT: The recently discovered highly specific delta opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) was radiolabelled by catalytic tritiation of its precursor [Tyr (3', 5'-I2)1]TIPP. The tritiated ligand labelled rat brain membrane binding sites with a Kd value of 0.64 nM and a Bmax of 82 fmol/mg protein. Binding competition experiments with various unlabelled mu-, delta- and kappa-opioid receptor ligands resulted in mu/delta and kappa/delta selectivity ratios of 600 and 9100, respectively, and thus confirmed the previously established high delta specificity of TIPP. Because of its high delta receptor affinity, extraordinary delta selectivity and low non-specific binding, [3H]TIPP represents an excellent new radioligand for the study of delta opioid receptor interactions.
Life Sciences 02/1993; 53(5):PL57-62. · 2.53 Impact Factor
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ABSTRACT: Opioid peptide analogs consisting entirely of aromatic amino acid residues and containing conformationally restricted phenylalanine derivatives in position 2 of the peptide sequence were synthesized and pharmacologically characterized in vitro. Both diastereoisomers of H-Tyr-(D or L)-NMePhe-Phe-Phe-NH2 (NMePhe is N alpha-methylphenylalanine) were mu-receptor-selective, were full agonists in the mu-receptor-representative guinea pig ileum assay, and were partial agonists in the mouse vas deferens assay, with the L-NMePhe2 analog displaying somewhat higher intrinsic activity than the D-NMePhe2 analog. Further conformational restriction at position 2 in the sequence, as achieved through substitution of D- or L-tetrahydro-3-isoquinoline carboxylic acid (Tic), produced a configuration-dependent differential effect on receptor selectivity and intrinsic activity, leading to a potent mu-selective mu agonist (the D-Tic2 analog) with increased intrinsic activity in the mouse vas deferens assay and to a potent delta-selective delta antagonist (the L-Tic2 analog). These results demonstrate that imposition of conformational constraints in a peptide not only may alter receptor selectivity but also may decrease, totally abolish, or even enhance intrinsic activity. The tetrapeptide H-Tyr-Tic-Phe-Phe-NH2 was a moderately potent full agonist in the guinea pig ileum assay and, thus, represents a compound with mixed mu-agonist/delta-antagonist properties. The corresponding peptide with a free C-terminal carboxyl group H-Tyr-Tic-Phe-Phe-OH showed high delta-receptor affinity (Ki delta = 1.2 nM), unprecedented delta selectivity (Ki mu/Ki delta = 1410), high potency as delta antagonist (Ke = 3-8 nM against various delta agonists in the mouse vas deferens assay) and, unlike other delta antagonists, had no mu-antagonist properties. The tripeptides H-Tyr-Tic-Phe-OH and H-Tyr-Tic-Phe-NH2 were also delta antagonists.
Proceedings of the National Academy of Sciences 01/1993; 89(24):11871-5. · 9.68 Impact Factor
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ABSTRACT: Conformationally restricted deltorphin analogues were synthesized either through incorporation of cyclic phenylalanine analogues in position 2 or 3 of the peptide sequence or through various side chain-to-side chain cyclizations. Compounds were tested in mu-, delta-, and kappa-receptor selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. Replacement of Phe3 in [D-Ala2]deltorphin I with 2-aminoindan-2-carboxylic acid (Aic) or L- or D-2-aminotetralin-2-carboxylic acid (Atc) resulted in agonist compounds which retained the high delta receptor selectivity of the parent peptide. Substitution of a tetrahydroisoquinoline-3-carboxylic acid (Tic) residue in the 2-position of [D-Ala2]deltorphin I and of [Phe4,Nle6]deltorphin produced a partial delta agonist, H-Tyr-Tic-Phe-Asp-Val-Val-Gly-NH2, and a pure delta antagonist, H-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH2, respectively. The latter antagonist displayed high delta selectivity (Ki mu/Ki delta = 502) and was a potent antagonist against selective delta agonists in the MVD assay (Ke congruent to 10 nM). Various [D-Ala2]-deltorphin I analogues cyclized between the side chains of Orn (or Lys) and Asp (or Glu) residues substituted in positions 2 and 4, 4 and 7, and 2 and 7 were essentially nonselective. Comparison with corresponding N-terminal tetrapeptide analogues revealed that the C-terminal tripeptide segment in the deltorphin heptapeptides made a crucial contribution to delta affinity and delta selectivity in the case of the agonist peptides but not in the case of the antagonist.
Journal of Medicinal Chemistry 11/1992; 35(21):3956-61. · 5.25 Impact Factor
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ABSTRACT: A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.
Biochemistry 06/1992; 31(18):4487-93. · 3.42 Impact Factor
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ABSTRACT: As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.
International journal of peptide and protein research 03/1992; 39(2):145-60.
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ABSTRACT: Three N-glycoconjugates of the general formula H-Tyr-Gly-Gly-Phe-Leu-NH-R (R = carbohydrate residue) were synthesized in order to determine the influence of some carbohydrate molecules (6-amino-6-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-glucopyranose, beta-D-glucopyranosylamine) on the biological activity, conformation, and stability of the opioid pentapeptide [Leu5]enkephalin. For the preparation of this compound different methods of peptide synthesis (active ester and mixed anhydride) were investigated. In comparison with [Leu5]enkephalin, all three N-glycoconjugates showed higher potency in the guinea pig ileum assay and lower potency in the mouse vas deferens assay, indicating a decrease in delta opioid receptor selectivity.
International journal of peptide and protein research 02/1992; 39(1):12-7.
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ABSTRACT: In an effort to determine the effect of side chain conformational restriction on opioid receptor selectivity, the cyclic phenylalanine analogues 2-aminoindan-2-carboxylic acid (Aic), 2-aminotetralin-2-carboxylic acid (Atc), and tetrahydroisoquinoline-3-carboxylic acid (Tic) were substituted for Phe in the potent cyclic opioid peptide analogue H-Tyr-D-Orn-Phe-Glu-NH2, which lacks significant opioid receptor selectivity. Compounds were tested in mu- and delta-opioid receptor representative binding assays and bioassays in vitro. The analogue H-Tyr-D-Orn-Aic-Glu-NH2 was found to be a potent agonist with high preference of mu receptors over delta receptors. Opening of the five-membered ring of Aic in the latter peptide, as achieved through substitution of C alpha-methylphenylalanine or o-methylphenylalanine, resulted in only slightly selective compounds, indicating that the high mu selectivity of the Aic analogue is exclusively the consequence of the imposed side chain conformational restriction. Both diastereoisomers of H-Tyr-D-Orn-(D,L)-Atc-Glu-NH2 were highly mu-selective and, in contrast to the weak affinity observed with the D-Phe3 analogue as compared to the L-Phe3 analogue, both had similar potency. Thus, stereospecificity was lost as a consequence of side chain conformational restriction. Further structure-activity data obtained with analogues containing L- or D-homophenylalanine (Hfe) or 3-(1'-naphthyl)alanine (Nap) in place of Phe3 and consideration of geometric interrelationships between Nap and the L and D isomers of Atc, Hfe, and Phe led to the proposal that the D-Phe3 and the D-Atc3 analogue may have different modes of binding to the receptor. The very low potency observed with H-Tyr-D-Orn-N alpha MePhe-Glu-NH2 (N alpha MePhe = N alpha-methylphenylalanine) and H-Tyr-D-Orn-Tic-Glu-NH2 indicated that N alpha-alkylation at the 3-position is detrimental to activity.
Journal of Medicinal Chemistry 11/1991; 34(10):3125-32. · 5.25 Impact Factor
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ABSTRACT: A theoretical conformational analysis (molecular mechanics study) of the delta opioid receptor-selective enkephalin analog H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) was performed, based on the use of the SYBYL software. The study led to the identification of several conformers that were significantly lower in energy than previously reported candidate conformers of DPDPE which, for comparative purposes, were also minimized by using the standard SYBYL force field. The results revealed a considerable degree of conformational flexibility of the DPDPE molecule, and suggested that incorporation of further conformational constraints into this enkephalin analog will be necessary in order to elucidate its receptor-bound conformation.
Journal of Computer-Aided Molecular Design 09/1991; 5(4):293-302. · 3.39 Impact Factor
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ABSTRACT: In order to study structure-activity relationships of enkephalin-related analogues, we report the biological activity and conformational analysis of four 14-membered cyclic enkephalin analogues with beta-(1-naphthyl) alanine in place of phenylalanine at the fourth position, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-(L and D)-Leu]. The L-beta Nal(1)-containing analogues display higher activity at both the mu and delta receptors than the corresponding analogues with the L-Phe residue. In contrast to the linear enkephalins, the cyclic analogues with the D-beta Nal(1) residue are also active at the mu receptor since the relative spatial arrangement of functional groups required for biological activity is achieved by the constrained nature of the cyclic molecules. A comparison of the findings from the conformational analysis and biological assays establishes that relatively extended structures, in which the two aromatic side chains are oriented in opposite directions with a approximately 14 A separation, is required for activity at the mu receptor. On the other hand, folded conformations with nearly parallel orientations and a close proximity (less than 10A) of the aromatic rings of the Tyr and beta Nal(1) residues are required for activity at the delta receptor. It should be noted that the overall structures and thus the biological profiles of the 14-membered cyclic enkephalin analogues are strongly dependent on the conformation of the second residue. The folded conformations with parallel orientation of the two aromatic side chains of Tyr-c[D-A2bu-Gly-L-beta Nal(1)-D-Leu] is stabilized by an interaction between the Tyr phenolic OH proton and beta Nal(1) C*O groups. This analogue, which shows the highest activity at both the mu and delta receptors among the four stereoisomers studied, displays an increase of the fraction of the side-chain chi 1 = t conformer for the beta Nal(1) residue. It is concluded that the incorporation of the D-Leu residue at the fifth position increases the relative fraction of the folded conformations with parallel orientation of the aromatic side chains, and hence enhances activity at the delta receptor as compared to the corresponding L-Leu containing analogue.
Biopolymers 07/1991; 31(7):877-98. · 2.87 Impact Factor
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ABSTRACT: We report the synthesis, biological activity, and conformational analysis of tetrapeptide analogs related to [Val4]morphiceptin and [D-Val4]morphiceptin in which the proline at the second position has been replaced with cis-2-aminocyclopentane carboxylic acid (cis-2-Ac5c). Since the cis-2-Ac5c residue contains a normal amide, only the trans form has been observed about the amide bond between the first and second residues. The cis-2-Ac5c is a beta amino acid with two chiral centers resulting in two possible configurational isomers, namely (1S, 2R) and 1R, 2S) forms. The analogs containing the (1S, 2R)-Ac5c residue show activity at the mu-receptor but are inactive at the delta-receptor, resulting in a high selectivity for the mu-receptor. The (1R,2S)-Ac5c containing analogs are completely inactive at both the mu- and delta-receptors. The conformational analysis indicates that the separation of the aromatic rings of the tyrosine and phenylalanine residues, as expressed by the center-to-center distance, is 10.1-12.7 A for the preferred conformations of the bioactive analogs containing the (1S,2R)-Ac5c residue while a range of 4.8-7.0 A is observed for the preferred conformations of the inactive analogs with the (IR,2S)-Ac5c residue. A comparison of the findings from the conformational analysis and biological assays establishes the fact that a relatively large separation of the two aromatic side chains is required for the mu-opioid receptor activity of these molecules. Since the tetrapeptide amides studied in this investigation show similar biological profiles to those of the morphiceptin-related analogs, we have compared the preferred conformations estimated for the cis-2-Ac5c containing analogs with those of morphiceptin. One of the low energy conformations calculated for morphiceptin with the cis form about the tyrosine and proline residues has considerable topological similarity with the bioactive analogs containing the (1S,2R)-Ac5c residue, indicating that the cis from about these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline at the second position.
International journal of peptide and protein research 06/1991; 37(5):364-81.
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ABSTRACT: In a preliminary communication we reported [(Tetrahedron Lett. 31, 619 (1990)] that acetyl hypofluorite can be used efficiently to introduce fluorine regiospecifically (ortho to OH) into the phenolic ring of tyrosine-containing peptides. This procedure has been applied to the fluorination of a number of mu-selective opioid peptides derived from dermorphin. While the procedure can be used even when the side chains of Arg, Lys, and Tyr are left unprotected, the sulfoxide of a Met(O)-containing analogue was oxidized to sulfone faster than fluorination of the phenolic ring. This method can also be used when the peptide is attached to Merrifield resin. Thus, Tyr(3-F)-D-Ala-Phe-Gly-NH2 and Tyr(3-F)-D-Arg-Phe-Lys-NH2 (F-DALDA) have been prepared, purified, and characterized. Affinities of these fluorinated peptides for both mu- and delta- opioid receptors are reduced (two- to nine-fold) relative to their nonfluorinated analogues, but their selectivity for mu-opioid receptors is not significantly altered. Similarly, the in vitro biological potencies (GPI and MVD assays) of the fluorinated analogues are reduced (two- to seven-fold) relative to their nonfluorinated parent peptides. Thus, F-DALDA, which has high affinity (Ki mu = 15.2 nM) and selectivity (Ki delta/Ki mu = 5390) for mu-opioid receptors, has potential use in biochemical studies which utilize 19F or 18F- labeled compounds.
International journal of peptide and protein research 06/1991; 37(5):430-9.
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ABSTRACT: A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the Förster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)
International journal of peptide and protein research 05/1991; 37(4):257-67.
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P W Schiller
NIDA research monograph 02/1991; 112:180-97.
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NIDA research monograph 02/1991; 112:218-38.
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P W Schiller
Progress in Medicinal Chemistry 02/1991; 28:301-40.
