Biosynthese in vitro d'iodothyronines a partir du diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine

In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) (equimolecular in tyrosyl rings).Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction.Synthesis of thyroid hormones from Tyr(I)₂-Tyr(I)₂ is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)₂).A mechanism of iodothyronine formation via Tyr(I)₂ - Tyr(I)₂ and different from the one occuring for Tyr(I)₂ is suggested.

Résumé

Une étude comparative de deux types de synthèse in vitro d'iodothyronines a été faite à partir de la 3,5-diiodotyrosine Tyr(I)₂ et à partir d'un dipeptide iodé: le diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) dans des conditions équimoléculaires en noyaux tyrosyl.Les incubations sont effectuées en présence de coupes de thyroïdes de rat en milieu de survie ou en présence de fraction microsomale thyroïdienne.La synthèse d'hormones thyroïdienes à partir du Tyr(I)₂-Tyr(I)₂ est plus rapide et plus importante qu'à partir de la Tyr(I)₂.Un mécanisme de synthèse des iodothyronines à partir du Tyr(I)₂-Tyr(I)₂ différent de celui intervenant pour la Tyr(I)₂ est proposé.     

In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (author's transl)]

A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)2-Tyr(I)2) (equimolecular in tyrosyl rings). Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction. Synthesis of thyroid hormones from Tyr(I)2-Tyr(I)2 is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)2). A mechanism of iodothyronine formation via Tyr(I)2-Tyr(I)2 and different from the one occuring for Tyr(I)2 is suggested.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: II. Role of the pyridoxal phosphate and of the peroxidase

In this paper several details of the in vitro pathway of synthesis of hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, have been investigated.

1. 1. We showed by incubations of I₂Tyr-I₂Tyr with a tautomerase that a phenylpyruvic form of the dipeptide did not occur.

2. 2. The reaction required pyridoxal phosphate: this coenzyme acts as an activator of the molecule.

3. 3. The peroxidase is involved in the reaction, since in the absence of a hydrogen peroxidase-generationg system there is no synthesis of iodothyronines when I₂Tyr-I₂Tyr is used as a precursor.

The significance of these findings is discussed and in particular it is concluded that such a mechanism cannot be transposed completely to conditions in vivo.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

α-D-mannosidase and α-D-galactosidase from protein bodies of Lupinus angustifolius cotyledons

Two forms of p-nitrophenyl α-D-mannosidase and p-nitrophenyl α-D-galactosidase were purified from the protein bodies of mature Lupinus angustifolius seeds. A MW of 300 000 was calculated for both α-mannosidase A and B with K_m = 1.92 and 2.70 mM and activation energies of 10.9 and 10.8 kcal/mol, respectively. α-Galactosidase I and II had MWs of 70800 and 17000 with K_m = 0.282 and 0.556 mM and activation energies 17.7 and 11.5 kcal/mol, respectively. The enzymes had acid pH optima and were inhibited by various metal ions, carbohydrates and glycoproteins. They were able to release free sugar from several putative natural substrate oligosaccharides and the Lupinus storage glycoprotein, α-conglutin.     

The synthesis and cytotoxic activity of 1,3,4-tri-O-acetyl-2,6-dideoxy-L-arabino- and -L-lyxo-hexopyranose

Methyl 2,6-dideoxy-α-L-arabino-hexopyranoside (6) was prepared from L-rhamnose in five steps. Hydrolysis of6 with 50% aqueous acetic acid gave 2,6-dideoxy-L-arabino-hexopyranose. Treatment of 3,4-di-O-acetyl-L-rhamnal with acetic acid in the presence of acetic anhydride and 2% sulfuric acid afforded 1,2,3-tri-O-acetyl-2,6-dideoxy-L-arabino-hexopyranose in 65% yield. Selective benzoylation and subsequent mesylation of 6 afforded methyl 3-O-benzoyl-2,6-dideoxy-4-O-mesyl-α-L-arabino-hexopyranoside, which was treated with sodium benzoate and sodium azide in hexamethylphosphoric triamide to give the corresponding 3,4-dibenzoyl 9 and 4-azido 11 analogs. Hydrogenation and N-acetylation of 11 afforded the 4-acetamido derivative 12. Deprotection of 9 and 12 gave 2,6-dideoxy-L-lyxo-hexopyranose and 4-acetamido-2,4,6-trideoxy-L-lyxo-hexopyranose, which were characterized as their peracetates. The free and corresponding peracetylated derivatives were assayed for their ability to inhibit the growth of P388 leukemia cells in culture. Although the free sugars did not inhibit the replication of these tumor cells under the conditions employed, their peracetylated derivatives demonstrated significant activity.     

Synthesis and some reactions of 3,5-di-O-methyl-d-glucose and -fructose

Hydrolysis of 1,2-O-isopropylidene-3,5-di-O-methyl-α-d-glucofuranose by strong acid yielded 3,5-di-O-methyl-d-glucofuranose (6) and its 1,6-anhydride (10). The mechanism of the reaction giving 10 is discussed. On treatment with a catalytic amount of sodium methoxide, 1,2,6-tri-O-acetyl-3,5-di-O-methyl-d-glucofuranose (8) gives the 6-O-acetyl derivative, whereas complete deacetylation, and subsequent isomerization to the d-fructose derivative 16, takes place in the presence of 0.1m sodium methoxide. The structure of 16 was proved both chemically and spectroscopically. Reduction of 6 or 8 with a borohydride afforded 3,5-di-O-methyl-d-glucitol.²     

Synthesis and characterization of propyl O-β-d-galactopyranosyl-(1→4)-O-β-d-galactopyranosyl-(1→4)-α-d-galactopyranoside

Allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl-α-d- galactopyranoside was O-deallylated to give the 1-hydroxy derivative, and this was converted into the corresponding 1-O-(N-phenylcarbamoyl) derivative, treatment of which with dry HCl produced the α-d-galactopyranosyl chloride. This was converted into the corresponding 2,2,2-trifluoroethanesulfonate, which was coupled to allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give crystalline allyl 4-O-[4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di- O-benzyl-β-d-galactopyranosyl]-2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside (15) in 85% yield, no trace of the α anomer being found. The trisaccharide derivative 15 was de-esterified with 2% KCN in 95% ethanol, and the product O-debenzylated with H₂-Pd, to give the unprotected trisaccharide. Alternative sequences are discussed.     

Spontaneous reactions of the irreversible β-D-galactosidase inhibitor 2,6-anhydro-1-deoxy-1-diazo-D-glycero-L-manno-heptitol

2,6-Anhydro-1-deoxy-1-diazo-D-glycero-L-manno-heptitol (2) decomposes in 0.01M methanolic sodium methoxide with a half-life of approx. 18 min. Decomposition in aqueous solution is too rapid for spectrophotometric measurement. Seven products could be identified in methanolic and aqueous reaction mixtures. 2,6-Anhydro-1-deoxy-D-galacto-hept-1-enitol (6), 2,7-anhydro-1-deoxy-β-D-galacto-heptulopyranose (10), and 4-O-vinyl-D-lyxose (12) are products of rapid intramolecular reactions. The major portion consists of the direct solvolysis products 2,6-anhydro-1-O-methyl-D-glycero-L-manno-heptitol (3) and 2,6-anhydro-D-glycero-L-manno-heptitol (5).     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

Crystal structure at 87 K of sodium α-l-guluronate dihydrate

X-Ray data collected at 87 K showed crystals of sodium α-l-guluronate dihydrate (C₆H₉O₇Na · 2 H₂O) to be orthorhombic, P2₁2₁2₁ with a = 7.591(2), b = 18.884(5), c = 6.842(2) Å, and Z = 4. The structure was solved by direct methods, and full-matrix least-squares refinement based on 1587 F_o yielded R = 0.043 and R_w = 0.033. The structure analysis indicates partial anomeric disorder with α:β ～90:10. The guluronate ring has the ¹C₄(l) conformation. Sodium binds two translation-equivalent guluronate units and one water molecule in a primary five-fold coordination. The complexing oxygen functions, which include all axial hydroxyl groups and one carboxylate oxygen atom in the guluronate ring, describe a distorted trigonal bipyramid. A prominent feature of the crystal structure is the stacks of sodium atoms and guluronate residues in alternating sequence along the c axis. The stacks are held together by an intricate system of hydrogen bonds involving all oxygen atoms in the structure. The water molecules play an important role in this system both as hydrogen donors and acceptors.     

The deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talo- and -β-D-allo-furanoside with nitrous acid

Deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talofuranoside (6) with sodium nitrite in 90% acetic acid at ≈0° gave methyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (8a) and methyl 6-deoxy-2,3-O-isopropylidene-β-D-allofuranoside (9a) (combined yield, 12.3%), the corresponding 5-acetates 8b (2.9%) and 9b (26.4%), and the unsaturated sugar methyl 5,6-dideoxy-2,3-O-isopropylidene-β-D-ribo-hex-5-enofuranoside (10) (43.5%). Similar deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-β-D-allofuranoside (7) gave 8a and 9a (combined yield, 20.4%), 8b (12.5%), 9b (25.8%), 10 (7.7%), and the rearranged products 6-deoxy-2,3-O-isopropylidene-5-O-methyl-L-talofuranose (13a, 17.5%) and the corresponding 1-acetate 13b (14.1%). A synthesis of 13a was accomplished by successive methylation and debenzylation of the conveniently prepared benzyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (15b). Differences between the two sets of deamination products can be rationalized by assuming that the carbonium-ion intermediate reacts in the initial conformation assumed, before significant interconversion to other conformations occurs.     

Syntheses of 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-d-glucopyranose (n-acetylmaltosamine) and 2-acetamido-2-deoxy-4-O-β-d-glucopyranosyl-α-d-glucopyranose

Condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-benzyl-1-O-(N-methyl)acetimidoyl-β-D-glucopyranose gave benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside which was catalytically hydrogenolysed to crystalline 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-D-glucopyranose (N-acetylmaltosamine). In an alternative route, the aforementioned imidate was condensed with 2-acetamido-3-O-acetyl-1,6-anhydro-2-deoxy-β-D-glucopyranose, and the resulting disaccharide was catalytically hydrogenolysed, acetylated, and acetolysed to give 2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-α-D-glucopyranose Deacetylation gave N-acetylmaltosamine. The synthesis of 2-acetamido-2-deoxy-4-O-β-D-glucopyranosyl-α-D-glucopyranose involved condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in the presence of mercuric bromide, followed by deacetylation and catalytic hydrogenolysis of the condensation product.     

Kristallstruktur von 2,3,4-tri-O-acetyl-β-D- xylopyranosylchlorid

Although 2,3,4-tri-O-acetyl-β-D-xylopyranosyl chloride is shown by n.m.r. data to be 80 percent in the ¹C₄ conformation in chloroform solution, it crystallizes in the normal ⁴C₁ conformation as shown by a three-dimensional, X-ray structure analysis. The crystals are orthorhombic, space group P2₁2₁2₁. The phase problem was solved by the heavy-atom method. The parameters were refined to an R-value of 0.039 for 1101 structure factors. With the chlorine atom being in equatorial position in the ⁴C₁ conformation, the C-1O-6 bond is not shortened and the C-1Cl-1 bond is not lengthened. These results are in agreement with comparable values for cis-2,3-dichloro-1,4-dioxane.     

A β-citryl-L-glutamate-hydrolysing enzyme in rat testes

An enzyme responsible for the deacylation of β-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored $\text{N-}\text{formyl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamine}$ in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-β-alanine deacetylase (EC 3.5.1.21), and various peptidases.     

The crystal structure of 2-deoxy-β-d-arabino-hexopyranose at −150°

2-Deoxy-β-d-arabino-hexopyranose, C₆H₁₂O₅, is orthorhombic, P2₁2₁2₁, with cell dimensions at ?150° [20°], a = 6.484(2) [6.510(3)], b = 10.364(2) [10.427(4)], c = 11.134(3) [11.153(5)] Å, V = 748.2 [757.1] ų, Z = 4, D_x = 1.457 [1.440], and D_m = [1.455] g.cm^?3. The intensities of 1269 reflections were measured by using MoKα radiation. The structure was solved by direct methods, and refined by full-matrix least-squares, with anisotropic, thermal parameters for the carbon and oxygen atoms, and isotropic parameters for the hydrogen atoms. The pyranose has the ⁴C₁(d) conformation, with puckering parameters Q = 0.563 Å, θ = 3.9°, and ? = 350.3°. The departure from ideality is very small, and less than that in β-d-glucopyranose, Q = 0.584 Å and θ = 6.9°. The β-glycosidic, CO bond is short, 1.383(4) Å, and the OCOH torsion angle is ?87°, consistent with the anomeric effect. The hydrogen-bonding scheme consists of infinite chains, with side chains terminating at a ring-oxygen atom.     

Synthesis of 3-amino-2,3,6-trideoxy-d-ribo- and -l-lyxo-hexofuranoses suitable for nucleoside synthesis

N-Acetylepidaunosamine (3-acetamido-2,3,6-trideoxy-d-ribo-hexopyranose) was converted into the diethyl dithioacetal and this was cyclized with HgCi₂, HgO, and MeOH, to give methyl 3-acetamido-2,3,6-trideoxy-α- and -β-d-ribo-hexofuranoside (4 and 5). These anomers were acetylated or (p-nitrobenzoyl)ated, and the esters were subjected to acetolysis, to afford 3-acetamido-1,5-di-O-acetyl-2,3,6-trideoxy-d-ribo-hexofuranose and 3-acetamido-1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-d-ribo-hexofuranose, respectively. Alternatively, compounds 4 and 5 were hydrolyzed to the free bases with barium hydroxide, and these were converted into the trifluoroacetamido derivatives which, on (p-nitrobenzoyl)ation and acetolysis, afforded 1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-d-ribo-hexofuranose. To prepare the corresponding daunosamine derivative, 2,3,6-trideoxy-3-(trifluoroacetamido)-l-lyxo-hexopyranose was converted into the diethyl dithioacetal, and this was cyclized in the same way, to afford methyl 2,3,6-trideoxy-3-(trifluoroacetamido)-α- and -β-l-lyxo-hexofuranoside. On (p-nitrobenzoyl)ation and acetolysis, both afforded 1-O-acetyl-2,3,6-trideoxy-5-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexofuranose.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Selenium derivatives of L-arabinose, D-ribose,and D-xylose

Attempted cyclization of 2,3,4-tri-O-methyl-5-seleno-L-arabinose dimethyl acetal in acidic solution gave the corresponding diselenide. Intramolecular attack by the selenobenzyl group at C-5 of 5-O-p-tolylsulfonyl-L-arabinose dibenzyl diseleno-acetal resulted in the formation of benzyl 1,5-diseleno-L-arabinopyranoside. Similarly, 2,3,5-tri-O-methyl-4-O-p-tolylsulfonyl-D-xylose dibenzyl diselenoacetal gave benzyl 2,3,5-tri-O-methyl-1,4-diseleno-L-arabinofuranoside, and 2,3,4-tri-O-acetyl-5-O-p-tolylsulfonyl-D-xylose (or ribose) dibenzyl diselenoacetal gave benzyl 2,3,4-tri-O-acetyl-1,5-diseleno-D-xylo- (or ribo-)pyranoside. The glycosylic benzylseleno group was removed from the pyranoside with mercuric acetate, but attempted deacetylation of the product led to decomposition and not to the expected 5-seleno-D-xylopyranose.