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A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.

Thirteen Schiff base salts in the class of compounds represented by retinal pyrrolidinium perchlorate were prepared (most of them for the first time), fully characterized analytically and spectroscopically, and examined in the 30–100 GHz range with respect to their putative ability to absorb radar signals. While some signal loss was noted with salts having I3− counterions, the compounds of this class show negligible potential as components of radar-absorbing coating systems.

Described herein are the stereoselective syntheses of the (24 R , 24 S ) and (25 R , 25 S ) isomers of 5β-cholestane-3α,7α,12α,24,25-pentols and 5β-cholestane-3α,7α,12α,25,26-pentols by using a modified osmium-catalyzed Sharpless asymmetric dihydroxylation process. Also presented herein are the results of lanthanide-induced CD Cotton effect measurements and 1 H- and 13 C-nuclear magnetic resonance studies of (24 R , 24 S ) and (25 R , 25 S )-5β-cholestanepentols and their derivatives. These compounds were required to study the biosynthesis of cholic acid from cholesterol.

The NMR shift reagent Europium (III)-tris-(1,1,1,2,2,3,3)-heptafluoro-7,7-dimethyl-4,6-octanedione [Eu(fod)3) complexes efficiently with chiral 2- and 3-hydroxycarboxylic acids, esters and ethers in chloroform. These complexes exhibit characteristic circular dichroism (CD) spectral pattern in the 350–250 nm region. A fast and simple procedure (also in micro scale) has been worked out which utilizes the signs of these CD bands for the determination of the absolute configuration at the C-2 or C-3 carbon in situ. In D(R)-series1, a negative CD band is observed at around 310 nm and a positive one in the 290 nm region. The CD spectra of the Eu complexes of L(S)-enantiomers are mirror images of those of D(R)-ones. An empirical rule has been proposed.

The configuration at C-25 in 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol isolated from the bile and feces of patients with cerebrotendinous xanthomtosis (CTX) was determined from the lanthanide-induced circular dichroism (CD) Cotton effects and 13C-NMR measurements. Under anhydrous conditions, CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol in the presence of Eu(fod)3 exhibited a large induced negative Cotton effect at 320 nm. On the basis of the empirical rule (primary-tertiary-alpha-diols) in which R compounds have positive Cotton effects and S compounds have negative Cotton effects at 320 nm, it was concluded that 25,26-pentol has the 1,2,glycol structure with C-25 having the S-configuration. This assignment was based upon comparison with model compounds, 25(R and S),26-dihydroxy cholesterols and 25(R and S),26-dihydroxy cholecalciferols whose single-crystal X-ray structure and 13C-NMR studies have been performed. It is suggested that these data may be helpful to clarify the stereospecificity of the hydroxylation of the terminal methyl group of the cholesterol side chain in CTX.

Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.

Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.

2-Methoxy-2,4-diphenyl-3(2H)furanone (MDPF) and 4-phenylspiro[furan 2(3H),1′-phthalan]-3,3′-dione (fluorescamine, Fluram®) react efficiently with primary amino groups to form pyrrolinone type (N-substituted 3,5-diphenyl-5-hydroxy-2-pyrrolin-4-ones) chromophores with UV absorption maxima at 270–280 nm and 380–390 nm regions. In Circular Dichroism (CD) measurements, in ethanol, the synthesized MDPF derivatives of twenty-four L-, and D-amino acids showed three characteristic Cotton effects: first at 400–370 nm, second at 335–315 nm, and third at 290–270 nm displaying in L-series (+)-, (−)-, (+)-, signs. Chromophoric reactions of MDPF and fluorescamine with α-amino acids, their esters and dipeptides can be performed in test tubes. CD spectra are obtained from the resulting reaction mixtures in situ. Investigations showed that in configurational studies of fluorescamine derivatives only the second and third Cotton effects should be used following the empirical rule: in L-series the second CD band at 340–315 nm is negative, whereas the third one at 290–270 nm is positive. For the MDPF condensation compounds with dipeptides, only the sign of the third Cotton effect is reliable. A pyrrolinone chirality rule is proposed. The experimental parameters, solvent, and pH effects on the sign of the Cotton effects are discussed. The main advantage of these chromophoric reactions is their simplicity, reliability and the fact that they can he carried out under mild conditions within minutes.

Primary and secondary amines react reproducibly with fluorescamine to form pyrrolinones or aminoenone type chromophores with long wavelength absorption maxima in the 375–410 and 310–320 nm regions, respectively. A simple procedure has been worked out for the simultaneous colorimetric determination of primary and secondary amines.

1,3-Dimethyl-1,2-dihydro-2-imino-4(3H)pyrimidinone (1,3-dimethylisocytosine) was prepared by methylation of 2-amino-3-methyl-4(3H)pyrimidinone and was degraded in alkaline solution to a mixture of 3-methyl-2-methylamino-4(3H)pyrimidinone, 1,3-dimethyl-2,4-(1H,3H)pyrimid-indione, 2-methylamino-l-methyl-4(1H)pyrimidinone, 1-methyl-2,4-(1H,3H)pyrimidindione and 3-methyl-2,4-(1H,3H)pyrimidindione. Thiation of the title compound gave both 1,2-dihydro-1,3-dimethyl-2-thio-4(3H)pyrimidinone and 1,3-dimethyl-2,4-(1H,3H)pyrimidinedithione. The title compound is a tautomerically fixed pyrimidine and its uv spectra were compared with related compounds, notably 3-methyl-2-dimethylarnino-4(3H)pyrimidinone which is also tautomerically fixed.

The urine and feces of a patient with the rare inherited lipid storage disease, sitosterolemia and xanthomatosis, were analyzed. Substantial quantities of C26-bile alcohol, 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol along with 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24R,25-pentol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol were found. The structure of the C26-bile alcohol was confirmed by direct comparison (gas-liquid chromatography-mass spectrometry and thin-layer chromatography) with a standard sample synthesized from cholic acid. The configurational assignment at C-24 was determined by lanthanide-induced circular dichroism Cotton effect measurements. The increased excretion of these C26- and C27-bile alcohols suggests an abnormality of bile acid biosynthesis in this disease.

Structures have been determined for a potent analogue of vasoactive intestinal peptide (VIP), Ac-[Lys12, Lys14, Nle17, Val26, Thr28]VIP (VIP'), in methanol/water solutions. In CD studies, both VIP and VIP' were helical in methanol/water, with the percentage of alpha-helix increasing with percentage methanol. The pH had little effect on the structure. Complete 1H NMR assignments were made for VIP' in 25% methanol at pH 4 and 6 and in 50% methanol at pH 6, using two-dimensional COSY, NOESY, and relay-COSY experiments. There were no widespread changes in chemical shifts between the samples at pH 4 and 6; however, widespread changes were observed between the samples in 25% and 50% methanol. Complete sets of NOEs were obtained for VIP' in 25% methanol, pH 4, and in 50% methanol, pH 6. These NOEs were converted into distance constraints and applied in molecular dynamics and energy minimization calculations using the program CHARMM. A set of low-energy structures was obtained for VIP' in each solvent system. In 25% methanol, VIP' has two helical segments at residues 9-17 and 23-28. The remainder of the structure is not well determined. In 50% methanol, residues 8-26 form a regular, well-defined alpha-helix and residues 5-8 form a type III beta-turn. The remaining residues are not ordered. These structural assessments agree with the CD data. In the lowest energy structure in 50% methanol, the side chains of Asp3, Phe6, Thr7, and Tyr10 are clustered together--these residues are conserved throughout the family of peptide hormones homologous to VIP.

Fluorescamine reacts efficiently with primary amines to form pyrrolinones with long wavelength absorption at 390–410nm. A procedure has been worked out which allows the use of this reaction for the colorimetric assay of primary amines.

Fluorescamine reacts efficiently with primary and secondary amino acids to form fluorescent pyrrolinone and nonfluorescent aminoenone type chromophores, respectively. A procedure is described for quantitative colorimetric determination at one fixed wavelength of the full array of natural amino acids, including proline and its derivatives. Simultaneous colorimetric-fluorometric analysis enables distinction between primary and secondary amino acids and permits their quantitative analysis with fluorescamine over a broad concentration range. These methods can also be applied for peptide analyses.

The absolute configurations of the C27 pentahydroxy bile alcohols present in bile and feces of two patients with cerebrotendinous xanthomatosis (CTX) were determined by circular dichroism (CD) spectroscopy. The CD spectra of 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol in the presence of Eu(fod)3 [tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-dionato) europium (III)] exhibited a negative Cotton effect and was assigned to 24R absolute configuration. Conversely, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol showed a strong positive Cotton effect and was assigned the 24S configuration. These assignments were based upon comparison with a model compound, 5-cholestene-3beta,24(R),25-triol, whose single-crystal X-ray structure has been determined. The importance of these data is to establish a structural mechanism for the conversion of 5beta-cholestane-3alpha,7alpha,12alpha,24S,25-pentol rather than 5beta-cholestane-3alpha,7alpha,12alpha,24R,25-pentol into cholic acid in man as well as in animals.

The synthesis and X-ray structural determination of 3-O-[(bis-morpholino)phosphinyl]-5,6-O-isopropylidene- l -ascorbate (9) are described. Acid-catalyzed hydrolysis of 9 afforded the 2-O-phosphate 6. Definitive structural proof of 6 is based on a study of the pH profile of its UV spectra as compared with those of ascorbic esters, 2 and 9 (Figs. 1–3).

The synthesis and some chemical reactions of various iodonitroimidazoles and methods of distinguishing between the 4- and 5- nitro derivatives by UV and pK measurements are described.

The S-configuration of the alkaloids cryptostylines I, II, and III was assigned on the basis of a single crystal X-ray analysis of unnatural cryptostyline II hydrobromide (5.HBr: orthorhombic, P212121 a = 10·162, b = 12·352, c = 16·456 A) and confirmed by application of the aromatic chirality method. Further evidence for the configurational assignment was provided by the CD spectra of the monophenolic derivatives of unnatural cryptostyline II (14).

Standard samples of pure (+)-salutaridine and (−)-thebaine required to study the mammalian origin of morphine, were prepared from (+)-6-demethylsalutaridine by published procedures and were characterized by CD spectra and physical data. Reductive N-methylation of (−)-northebaine afforded (−)-thebaine, and when 13C-labeled formalin was used, (−)-thebaine with a 13C label on the N-methyl carbon atom resulted. The latter represents a model procedure to prepare ultimately N-14CH3-labeled (−)-thebaine and 14C-labeled congeners.

Chiral α-amino acids react with 2-methoxy - 2,4 - diphenyl - 3 (2 H ) - furanone 1 to afford N -substituted 3,5 - diphenyl - 5 - hydroxy - 2 - pyrrolin - 4 - ones 2 . The characteristic cotton effects given by these chromophoric derivatives provide a means for the determination of the absolute configuration of the parent amino acids. The longest wavelength(first) extremum in the chiroptical spectra (ORD and CD) of the l -amino acid derivatives is always positive, while it is negative for the d -amino acid derivatives.

The aS,7S-absolute configuration of (−)-colchicine (1) and (−)-N-acetylcolchinol methyl ether (3, NCME) suggested on the basis of 1H NMR data and negative Cotton effects at about 260 nm (EtOH) is firmly established by an X-ray analysis of urea 5, a compound derived from 3. Binding of these compounds to tubulin requires an aS-configuration of the biaryl system.

The complete stereostructure of the new antibiotic Ro 22-5417 has been established as 3-[(3S,5S)-7-oxo-1-aza-4-oxabicyclo[3.2.O]hept-3-yl]-L-alanine. This result together with the synthesis of an (3R,5R)-L-analog allowed us to postulate that clavams require the R-configuration at the ring juncture for beta-lactamase inhibitory activity, while the opposite S-stereochemistry is essential for antifungal activity.

The isolation of biologically important peptides from the thymus gland has been studied extensively in the last few years. Several thymic peptides have been shown to play certain roles in T-cell maturation (White, 1980; Trainin et al., 1980a, b; Goldstein and Lau, 1980; Bach and Goldstein, 1980). Thymosin α1, a highly acidic Nα-acetyl octacosapeptide, isolated from calf thymus gland (Goldstein et al., 1977) and characterized by sequence analysis (Low and Goldstein, 1979), has been reported to exhibit biological activities involved in the development of thymus-dependent lymphocytes (T cells) (Goldstein et al., 1977). Thymosin a1 has been synthesized by classical procedures in solution (Wang et al., 1979; Birr and Stollenwerk, 1979) and by solid-phase methods (Wong and Merrifield, 1980; Wang et al., 1980; Colombo, 1981).

Fluorescamine is a useful reagent for the fluorometric assay of primary amines. The extent of the reaction between fluorescamine and primary amines, as well as the fluorescence intensities of the resulting fluorophors depend on pH, solvent composition and reagent concentration. Optimum values for these variables further depend on the amine under study. The influence of these parameters on the fluorogenic reaction of representative amines, and on their fluorophoric derivatives has been investigated, and the results are reported here.