Tritiation of Peptides to High Specific Radioactivity. Part 1. Synthesis and Biological Properties of [13-(3H4)Norvaline]-α-MSH and of [2,23-Bis((3H2)tyrosine)]ACTH(1–24)

α-MSH and ACTH(1–24) were tritiated to high specific radioactivity (> 100 Ci/mmol) using a new tritiation apparatus with which the tritiation reaction can be performed at slightly elevated pressure. This allows short reaction times with the least possible damage to the molecule. The starting compounds for the tritiation were [13-propargylglycine]α-MSH and [2,23-Bis(3′,5′-diiodotyrosine)]ACTH(1–24). Both tritiations were quantitative and yielded products of high purity, full biological activity, and with a specific radioactivity of 115 Ci/mmol and 100 Ci/mmol, respectively.     

Divergent Melanophore-Dispersing and Tyrosinase-Stimulating Activity of Synthetic Leucine9-α-melanotropin

Leucine⁹-α-melanotropin ([Leu⁹]α-MSH) was synthesized in homogeneous solution by a fragment condensation approach, and it was assayed for its melanophore-dispersing and its tyrosinase-stimulating activity with a reflectometric in vitro frog skin assay and with cultured mouse melanoma cells, respectively. In both assay systems, parallel log dose-response curves were obtained for ([Leu⁹)]α-MSH and α-MSH; however, in the frog skin assay the activity of the title compound was 1 middot; 10¹⁰ Units/mmol, i.e. 25% of the activity of α-MSH, whereas its tyrosinasestimulating potency was only 1% compared to α-MSH (EC₅₀= 2.5 · 10^?7M ). This indicates a major difference in the recognition/stimulation process of the receptors of the two cell types.     

Synthesis of [D-alanine, 4'-azido-3',5'-ditritio-L-phenylalanine, norvaline4[alpha-melanotropin as a 'photoaffinity probe' for hormone-receptor interactions (author's transl)]

Synthesis of [D -alanine¹, 4′-azido-3′, 5′-ditritio-L -phenylalanine², norvaline⁴]α-melanotropin as a ‘photoaffinity probe’ for hormone-receptor interactions. The synthesis of an α-MSH derivative containing 4′-azido-3′,5′-ditritio-L -phenylalanine is described: Ac · D -Ala-Pap(³H₂)-Ser-Nva-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂. This hormone analogue is being used for specific photoaffinity labelling of receptor molecules. The synthesis was performed in a way to minimize the number of radioactive steps and to introduce the radio-active and the photoaffinity label exclusively into position 2. The dipeptide N(α)-acetyl-D -alanyl- (4′-amino-3′,5′-diiodo)-L -phenylalanine was tritriated and transformed into the azido compound, N(α)-acetyl-D -alanyl-(4′-azido-3′,5′-ditritio)-L -phenylalanine which was then condensed with H · Ser-Nva-Glu(OtBu)-His-Phe-Arg-Trp-Gly-Lys(BOC)-Pro-Val · NH₂ to the tridecapeptide. The α-MSH analog displayed a specific activity of 11 Ci/mmol, and a biological activity of about 4 · 10⁹ U/mmol (10% of α-MSH).     

Melanotropin Receptors II. Synthesis and Biological Activity of α-Melanotropin/Tobacco Mosaic Virus Disulfide Conjugates

Asymmetric disulfide conjugates of mercaptosuccinyl tobacco mosaic virus (TMV ～ SH) with N^α-desacetyl-N^α-5-(mercaptovaleryl)-α-melanotropin were prepared via the S-sulfoderivative of the peptide. The conjugates, TMV ～ S? S ～ α-MSH(n), contained up to n = 330 disulfide-linked peptide molecules/virion. Similarly, fluorescent conjugates, Rh(m) ～ TMV ～ S? S ～ α-MSH(n) were prepared, containing m ≈? 200 rhodamine molecules linked to the virions by thiourea bridges. Such conjugates were designed to study α-MSH receptor localization and dynamics (mainly internalization), because the carrier virions which served to enhance specific receptor binding and as fluorescent or radioactive markers may be detached from the neuropeptides at will by reduction. Reduction occurred in solution and on the cell surface, but not in the cytoplasm, thus allowing detection of internalized agonist-receptor complexes. The conjugates were superpotent agonists for tyrosinase stimulation in Cloudman S-91 melanoma cell cultures, but were inactive for cyclic AMP accumulation. Their rather rapid internalization and the influence of reducing agents and other agonists on their biologic activity suggest a close connection between receptor location and biologic response as well as the presence of essential receptor HS-groups.     

Mass spectra of some α-substituted phenyl-α,α′-dimethoxyl ketones and their derivatives

The mass spectra of some α-substituted phenyl-α,α′-dimethoxyl ketones (compounds 1) and their 2,4-dinitrophenylhydrazones (compounds 2) and semicarbazones (compounds 3) have been studied. The characteristic fragments at m/z (M ? 73) from compounds 1, m/z (M ? 253) from compounds 2 and m/z (M ? 130) from compounds 3 are abundant and proposed to be [ArCROCH₃]⁺. Fragmentations yielding [M⁺ ? 49] from compounds 2 are abnormal and probably involve the methoxyl and nitro groups. The intense peak at m/z 130 due to [CH₃OCH₂CNNHCONH₂]⁺ from compounds 3 corresponds to α-cleavage of the molecular ion. Some other fragments from these new compounds are interpreted in this paper.     

5-(α-Fluorovinyl)tryptamines and 5-(α-Fluorovinyl)-3-(N-methyl-1′,2′,5′,6′,-tetrahydropyridin-3′- and -4′-yl)indoles—5-(α-Fluorovinyl)tryptamines

5-(α-Fluorovinyl)tryptamines 4a, 4b and 5-(α-fluorovinyl)-3-(N-methyl-1′,2′,5′,6′-tetrahydropyridin-3′- and -4′-yl) indoles 5a, 5b were synthesized using 5-(α-fluorovinyl)indole ( 7 ). The target compounds are bioisosteres of 5-carboxyamido substituted tryptamines and their tetrahydropyridyl analogs.     

Synthese von radioaktiv markierten Bromacetyl- und Diazoacetyl-α-melanotropin-Derivaten zum Studium von kovalenten Hormon-Makromolekül-Komplexen

Synthesis of radioactive α-melanotropin derivatives containing a bromoacetyl or diazoacetyl group for studies of covalent hormone-macromolecule complexes α-Melanotropin derivatives and fragments covalently bound to human serum albumin through their N-terminal end exhibit almost the same biological activity as the corresponding free N^α-acetylated peptides [6]. The preparation of such complexes requires derivatives with a ‘reactive’ N-terminal acetyl group. We describe here the synthesis of three α-melanotropin fragments and of two specifically tritiated α-melanotropin derivatives containing N^α-bromoacetyl or N^α-diazoacetyl groups: BrCH₂CO · Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂, BrCH₂CO · Glu-His-Phe-Arg-Trp-Gly · OH, BrCH₂CO · Trp-Gly-Lys-Pro-Val middot; NH₂, BrCH₂CO · D -Ala-Tyr(³H₂)-Gly-Nva-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂, and N₂ = CHCO · Gly-Tyr(³H₂)-Ser-Nva-Glu-His-Phe-Arg-Trp-Gly- Lys-Pro-Val · NH₂. The latter two analogues displayed a specific radioactivity of about 20 and 36 Ci/mmol, and a biological activity of 2 · 10⁹ and 6 · 10⁹ U/mmol respectively. They are also being used for affinity and photoaffinity labelling of receptor molecules and antibody combining sites.     

Synthesis and biological properties of p-azidophenylalanine13-β-melanotropin,a potent photoaffinity label for MSH receptors

p-Azidophenylalanine¹³-α-melantropin ([Pap¹³]-α-MSH) was synthesized in homogeneous solution by the fragment condensation method, and its biological activity was determined in three different assay systems. The pigment-dispersing activity relative to α-MSH was 65%, measured with melanophores of Rana pipiens or of Xenopus laevis tadpoles. The tyrosinase-stimulating activity was 50%, determined with cultured mouse melanoma cells. UV. irradiation of solutions containing ≤10^?4_M[Pap¹³]-α-MSH at 338 nm (intensity: 10^?3 W · cm^?2) led to complete photolysis of the photolabel within <20 min. Under these conditions [Pap¹³]-α-MSH was covalently inserted into MSH-receptors which produced a longlasting pigment dispersion in Xenopus melanphores (see [3]). The extent of this prolonged stimulation depended on the hormone concentration used during photolysis. 1.8·10^?9_M [Pap¹³]-α-MSH which produced a full initial response failed to prolong the effect, whereas 1.2·10^?8_M hormone caused irreversible stimulation. It appears that only about 10% of the initially occupied receptors were covalently labelled because the log dose response curve was shifted to ～ 10x higher concentration after a 200 min wash period: EC₅₀ immediately after photolysis was 6 · 10^?10_M; after 200 min EC₅₀ increased to ～8·10^?9_M.     

Lipophilic Functionalized Cobyrinic-Acid Derivatives. Part 1. Cobester α-monoacids (α,α,α,β,β,β-hexamethyl α-hydrogen Coα, Coβ-dicyanocobyrinates)

The four α,α,α, β,β,β,-hexamethyl α-hydrogen Coα, Coβ-dicyanocobyrinates 2b, d–f , with a free b-, d-, e-, and f-propionic-acid function, respectively, were prepared by partial hydrolysis of heptamethyl Coα, Coβ-dicyanocobyrinate (cobester; 1 ) in aqueous sulfuric acid. The cobester monoacids 2b, d–f were obtained as a ca. 1:1:1:1 mixture which was separated. The monoacids were purified by chromatography and isolated in crystalline form. The position of the free propionic-acid function was determined by an extensive analysis of 2b, d–f using 2D-NMR techniques; an analysis of the C,H-coupling network topology resulted in an alternative assignment strategy for cobyrinic-acid derivatives, based on pattern recognition. Additional information on the structure of the most polar of the four hexamethyl cobyrinates, of the b-isomer 2b , was also obtained in the solid state from a single-crystal X-ray analysis. Earlier structural assignments based on 1D-NMR spectra of the corresponding regioisomeric monoamides 3b, d–f (obtained from crystalline samples of the monoacids 2b, d–f ) were confirmed by the present investigations.     

Chemical Synthesis and Biological Activity of the Dogfish (Squalus acanthias) α-melanotropins I and II,and of related peptides

The purpose of this investigation is to provide synthetic proof for the structure of dogfish (Squalus acanthias) α-MSH and to investigate the consequences of the presence of methionine in position 13 and the lack of the N-terminal acetyl group in this hormone. Because of the facile oxidation of methionine (13) during handling or storage, a number of specifically oxidized hormones (Met⁴ and Met¹³) as well as tripeptides belonging to the C-terminal second message sequence were investigated. All the products were prepared by classical methods in homogeneous solution; intermediate and end products were extensively purified and characterized (Tables 3 and 4). The assays for melanotropic activity were performed in vitro with the modified reflectometric assay using the skin of Rana pipiens. It is concluded that the structures assigned to dogfish α-MSH I and II are correct and that the isolated samples contain slight quantities of [13-methionine (S-oxide)]dogfish α-MSH and [4, 13-bis-methionine (S-oxide)]dogfish α-MSH. The peptides with methionine in position 13 are as active in this assay as those containing valine. This also holds for the ? NH₂/? OH interchange distinguishing dogfish α-MSH I from dogfish α-MSH II. However, the lack of the N-terminal acetyl group strongly reduces the biological activity. Its introduction into dogfish α-MSH I results in a product that is equipotent with mammalian α-MSH. These and other conclusions are discussed in detail.     

Angiotensin-II Analogues. I: Synthesis and incorporation of the halogenated amino acids 3-(4′-iodophenyl)alanine, 3-(3′,5′-dibromo-4′-chlorophenyl)alanine, 3-(3′,4′,5′-tribromophenyl)alanine,and 3-(2′,3′,4′,5′,6′-pentabromophenyl)alanine

The synthesis of the polyhalogenated phenylalanines Phe(3′,4′,5′-Br₃) ( 3 ), Phe(3′,5′-Br₂-4′-Cl) ( 4 ) and DL -Phe (2′,3′,4′,5′,6′-Br₅) ( 9 ) is described. The trihalogenated phenylalanines 3 and 4 are obtained stereospecifically from Phe(4′-NH₂) by electrophilic bromination followed by Sandmeyer reaction. The most hydrophobic amino acid 9 is synthesized from pentabromobenzyl bromide and a glycine analogue by phase-transfer catalysis. With the amino acids 4, 9 , Phe(4′-I) and D -Phe, analogues of [1-sarcosin]angiotensin II ([Sar¹]AT) are produced for structure-activity studies and tritium incorporation. The diastereomeric pentabromo peptides L - and D - 13 are separated by HPLC. and identified by catalytic dehalogenation and comparison to [Sar¹]AT ( 10 ) and [Sar¹, D -Phe⁸]AT ( 14 ).     

Pterin Chemistry. Part 92.. Loading experiments with 6α,β-tetrahydro-L-[3′-2H1] biopterin

6α,β-Tetrahydro-L -[3′-²H₁]biopterin ([3′-²H₁]- 1 ) was administered orally to two primapterinuric patients in order to investigate the biosynthetic pathway of 7-substituted pterins in humans. L -Primapterin ( 2 ) and L -biopterin were isolated from urine after loading and measured by GC/MS. L -Biopterin and L -primapterin were labelled with ²H to an equal extent. From this result, one can conclude that L -primapterin is formed from tetrahydro-L -biopterin, very probably via an intramolecular rearrangement.     

Carbon-13 NMR and conformational analysis of meso- and dl-α,α′-disubstituted succinic acids

meso-and dl-Diastereomers of a number of α,α′-disubstituted succinic acids have been shown to give different ¹³C NMR chemical shifts. The results can be satisfactorily explained on the basis of their conformational analyses. A discussion of the observed chemical shifts is presented, and the preferred conformation for each of several compounds is predicted on the basis of these chemical shifts.     

Reaction of Cα,O-dilithiooximes with functionalized carbonyl compounds. Part 2 . Reaction with α-chloroketones and α,β-unsaturated aldehydes and ketones

The reaction of Cα,O-Dilithiooximes 2 and α-chloroketones afforded 5-(hydroxymethyl)-Δ²-soxazolines 4 . α,β-Unsaturated aldehydes and ketones reacted with 2 to give the corresponding acyclic 1,2-addition products 5 . The latter were cyclized with phosphorus pentoxide to 5-vinyl-Δ²-isoxazolines 6 .     

Polyesters,polycarbonate, and polyurethanes from a novel monomer: α,α,α′,α′-tetramethyl-1,4-tetrafluorobenzenedimethanol

The novel diol monomer, α,α,α′,α′-tetramethyl-1,4-tetrafluorobenzenedimethanol, has been synthesized by a convenient route which involves the addition of acetone to 1,4-dilithiotetrafluorobenzene and can be purified by washing with hexanes. It does not directly undergo condensation polymerizations with diacid chlorides. Its disodium salt, prepared by its reaction with sodium hydride, similarly fails to undergo such polymerizations readily. However, the dilithium salt, prepared in situ by the reaction of the title diol with 2 equiv of n-butyllithium in tetrahydrofuran, is suitable for the preparation of various classes of condensation polymers. Four polyesters and one polycarbonate derived from the reactions of the dilithium salt of the diol with adipoly dichloride, sebacoyl dichloride, isophthaloyl dichloride, terephthaloyl dichloride, and phosgene and two polyurethanes derived from its reactions with tolylene-2,4-diisocyanate and methylene-di-1,4-phenyl diisocyanate were prepared. Each was fully characterized by GPC, NMR, IR, and UV-visible spectroscopies, and the results of these studies are reported herein. © 1993 John Wiley & Sons, Inc.     

Thermische Lactonisierung von Estern γ-Brom-α, β-ungesättigter Carbonsäuren zu Δα-Butenoliden. Direkte γ-Bromierung von α, β-ungesättigten Säuren

By heating with iron powder at 120–150° some γ-bromo-α, β-unsaturated carboxylic methyl esters, and, less smothly, the corresponding acids, were lactonized to Δ^7alpha;-butenolides with elimination of methyl bromide. The following conversions have thus been made: methyl γ-bromocrotonate ( 1c ) and the corresponding acid ( 1d ) to Δ^α-butenolide ( 8a ), methyl γ-bromotiglate ( 3c ) and the corresponding acid ( 3d ) to α-methyl-Δ^α-butenolide ( 8b ), a mixture of methyl trans- and cis-γ-bromosenecioate ( 7c and 7e ) and a mixture of the corresponding acids ( 7d and 7f ) to β-methyl-Δ^α-butenolide ( 8c ). The procedure did not work with methyl trans-γ-bromo-Δ^α-pentenoate ( 5c ) nor with its acid ( 5d ). Most of the γ-bromo-α, β-unsaturated carboxylic esters ( 1c, 7c, 7e and 5c ) are available by direct N-bromosuccinimide bromination of the α, β-unsaturated esters 1a, 7a and 5a ; methyl γ-bromotiglate ( 3c ) is obtained from both methyl tiglate ( 3a ) and methyl angelate ( 4a ), but has to be separated from a structural isomer. The γ-bromo-α, β-unsaturated esters are shown by NMR. to have the indicated configurations which are independent of the configuration of the α, β-unsaturated esters used; the bromination always leads to the more stable configuration, usually the one with the bromine-carrying carbon anti to the carboxylic ester group; an exception is methyl γ-bromo-senecioate, for which the two isomers (cis, 7e , and trans, 7d ) have about the same stability. The N-bromosuccinimide bromination of the α,β-unsaturated carboxylic acids 1b , 3b , 4b , 5b and 7b is shown to give results entirely analogous to those with the corresponding esters. In this way γ-bromocrotonic acid ( 1 d ), γ-bromotiglic acid ( 3 d ), trans- and cis-γ-bromosenecioic acid ( 7d and 7f ) as well as trans-γ-bromo-Δ^α-pentenoic acid ( 5d ) have been prepared. Iron powder seems to catalyze the lactonization by facilitating both the elimination of methyl bromide (or, less smoothly, hydrogen bromide) and the rotation about the double bond. α-Methyl-Δ^α-butenolide ( 8b ) was converted to 1-benzyl-( 9a ), 1-cyclohexyl-( 9b ), and 1-(4′-picoly1)-3-methyl-Δ^α-pyrrolin-2-one ( 9 c ) by heating at 180° with benzylamine, cyclohexylamine, and 4-picolylamine. The butenolide 8b showed cytostatic and even cytocidal activity; in preliminary tests, no carcinogenicity was observed. Both 8b and 9c exhibited little toxicity.     

Synthesis of Some Novel Phenyl Substituted Oxothiazolidin- 1’’,3’’,4’’-thiadiazolo-[3’,4’-b]thiazoline of 3β-Substituted Cholestane

Three title compounds 4a—4c have been synthesized by the cyclodehydration of 1’-benzylidine-4’-(3β-substituted-5α-cholestane-6-yl)thiosemicarbazones 2a—2c with thioglycolic acid followed by the treatment with cold conc. H2SO4 in dioxane. The compounds 2a—2c were prepared by condensation of 3β-substituted-5α-cholestan- 6-one-thiosemicarbazones 1a—1c with benzaldehyde. These thiosemicarbazones 1a—1c were obtained by the reaction of corresponding 3β-substituted-5α-cholestan-6-ones with thiosemicarbazide in the presence of few drops of conc. HCl in methanol. The structures of the products have been established on the basis of their elemental, analytical and spectral data.     

Synthese von diastereo- und enantio-selektiv deuterierten β,ε-, β,β-, β,γ- und γ,γ-Carotinen

Synthesis of Diastereo- and Enantioselectively Deuterated β,ε-, β,β-, β,γ- and γ,γ-Carotenes We describe the synthesis of (1′R, 6′S)-[16′, 16′, 16′-²H₃]-β, εcarotene, (1R, 1′R)-[16, 16, 16, 16′, 16′, 16′-²H₆]-β, β-carotene, (1′R, 6′S)-[16′, 16′, 16′-²H₃]-γ, γ-carotene and (1R, 1′R, 6S, 6′S)-[16, 16, 16, 16′, 16′, 16′-²H₆]-γ, γ-carotene by a multistep degradation of (4R, 5S, 10S)-[18, 18, 18-²H₃]-didehydroabietane to optically active deuterated β-, ε- and γ-C₁₁-endgroups and subsequent building up according to schemes \documentclass{article}\pagestyle{empty}\begin{document}${\rm C}_{11} \to {\rm C}_{14}^{C_{\mathop {26}\limits_ \to }} \to {\rm C}_{40} $\end{document} and C₁₁ → C₁₄; C₁₄+C₁₂+C₁₄→C₄₀. NMR.- and chiroptical data allow the identification of the geminal methyl groups in all these compounds. The optical activity of all-(E)-[²H₆]-β,β-carotene, which is solely due to the isotopically different substituent not directly attached to the chiral centres, is demonstrated by a significant CD.-effect at low temperature. Therefore, if an enzymatic cyclization of [17, 17, 17, 17′, 17′, 17′-²H₆]lycopine can be achieved, the steric course of the cyclization step would be derivable from NMR.- and CD.-spectra with very small samples of the isolated cyclic carotenes. A general scheme for the possible course of the cyclization steps is presented.     

α,α,α′,α′-Tetraaryl-1,3-dioxolane-4,5-dimethanols (TADDOLs) for Resolutions of Alcohols and as Chiral Solvating Agents in NMR Spectroscopy

The use of α,α,α′,α′ -tetraaryl-1,3-dioxolane-4,5-dimethanols ( = TADDOLs;1) as chiral NMR shift reagents (¹H, ¹³C, ¹⁹F) is described. In many cases, the ratio of enantiomeric alcohols and amines can be determined under standard conditions of measurement (CDCl₃ as solvent, room temperature). The preparation and use of a new type of TADDOL, the tetrakis(dimethylamino) derivative 1d , is described. Menthol, octan-2-ol, and oct-1-yn-3-ol are partially resolved by crystallization of clathrates with 1c and 1d .     

Synthesis of α-2′-deoxynucleosides

α-Thymidine (4) was synthesized from thymidine (1) in 3 steps in 36% overall yield without using chro-matography and with the possibility of increasing the yield to 85% by reusing the remaining α,β-mixture. 1-(2-Deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl)thymine (3) was further converted to 1-(2-deoxy-α-D-erythro-pentofuranosyl)-5-methylcytosine (5) .