CVI. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Synthesis of two nonanucleotides and a heptanucleotide corresponding to nucleotide sequences 22 to 30, 41 to 49 and 28 to 34

Two nonadeoxynucleotides with the sequences, d-C-T-A-A-G-G-G-A-G (nonanucleotide-I) and d-T-C-T-C-C-G-G-T-T (nonanucleotide-II), and a heptadeoxynucleotide having the sequence, d-A-G-A-G-T-C-T, have been chemically synthesized. These polynucleotides represent, respectively, the nucleotide sequences 22 to 30, 41 to 49, and 28 to 34 of the gene for yeast alanine transfer RNA (Fig. 1). The synthetic steps used in the synthesis of the nonanucleotide-I were: the condensation of the protected nucleoside, d-MMTr-C^An, with the protected nucleotide, d-pT-OAc, to give the dinucleotide, d-MMTr-C^AnpT; the condensation of the dinucleotide with d-pA^Bz-OAc to give the trinucleotide, d-MMTr-C^AnpTpA^Bz; the condensation of the latter with the dinucleotide, d-pA^BzpG^1B-OAc, to give the pentanucleotide, d-MMTr-C^AnpTpA^BzpA^BzpG^1B; the condensation of this pentanucleotide with d-pG^1BpG^1B-OAc to give the protected heptanucleotide, d-MMTr-C^AnpTpA^BzpA^BzpG^1BpG^1BpG^1B, and finally, the condensation of this heptanucleotide with the dinucleotide, d-pA^BzpG^1B-OAc, to give the protected nonanucleotide, d-MMTr-C^AnpTpA^BzpA^BzpG^1BpG^1BpG^1BpA^BzpG^1B. The steps used in the synthesis of the nonanucleotide-II were: the condensation of d-MMTr-T with the tetranucleotide, d-pC^AnpTpC^AnpC^An-OAc, to give the pentanucleotide, d-MMTr-TpC^AnpTpC^AnpC^An; the condensation of the latter with the dinucleotide, d-pG^1BpG^1B-OAc, to give the heptanucleotide, d-MMTr-TpC^An-pTpC^AnpC^AnpG^1BpG^1B, and finally, the condensation of the heptanucleotide with the dinucleotide, d-pTpT-OAc, to give the protected deoxynonanucleotide, d-MMTr-TpC^AnpTpC^AnpC^AnpG^1BpG^1BpTpT. For the synthesis of the heptanucleotide, A-G-A-G-T-C-T, the 5′-monocyanoethyl tetranucleotide, d-CEpA^Bz-pG^1BpA^BzpG^1B, was condensed with the trinucleotide, d-pTpC^AnpT-OAc, to give the protected heptanucleotide, d-pA^BzpG^1BpA^BzpG^1BpTpC^AnpT. After removal of the N-protecting groups, the completely deprotected nonanucleotides, as well as the intermediate oligonucleotides and the heptanucleotide, d-A-G-A-G-T-C-T, were purified further by a combination of paper and column chromatography.     

Gel filtration of acylated oligonucleotides

The behavior of a number of acylated deoxyribooligonucleotides in gel permeation chromatography has been studied and dependence on factors other than molecular size has been noted. Retardation has been observed to increase as follows: pT < d-pA^Bz < d-pC^An < d-pG^iBu, and this order is reflected in the elution parameters of derived oligomers. Certain nucleotide derivatives—notably d-pC^An and its relatives—were eluted in two peaks.     

CXII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized polydeoxynucleotides to form the DNA duplex representing nucleotide sequence 1 to 20

The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-³²P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-³²P or ³³P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-³²P or ³³P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide.     

Mitochondrial biogenesis in Neurospora crassa. I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An ^-) indicate single nuclear gene inheritance (An ^-/An ⁺). An ⁺ and An ^- are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An ⁺ mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.     

The attachment of poly(ribitol phosphate) to lipoteichoic acid carrier

2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (21) and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (22), 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(glycine ethyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(phenylalanine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine were synthesized by condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with the appropriate protected amino acids and tri- and tetra-peptides. The amino acid sequences of 21 and 22 correspond to the protected amino acid sequences 34–37 and 34–38 of ribonuclease B that are adjacent to the carbohydrate-protein linkage.     

Selective cleavage of O-acyl protecting groups from blocked deoxyribotides

Selective hydrolysis of the 3′-O-acetyl groups in the 5′-P-cyanoethyl-blocked deoxyribonucleotides, commonly used in oligomer synthesis by the diester method, can be achieved by mild hydrolysis with aqueous ammonia in pyridine for a brief period of time. The 3′-O-isobutyryl group is much more resistant. Conditions for 3′-unblocking of the four commonly used protected monomers as well as a model oligomer, d(CNEtpbzA-ibuG)Ac, are described.     

Spectroscopic properties of oligonucleotides excised from the anticodon region of phenylalanine tRNA from yeast

Ultraviolet absorption and static fluorescence properties of hexanucleotide (Gm-A-A-Y-A-ψp) and a dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-ψ-m⁵C-U-Gp) excised from the anticodon region of phenylalanine tRNA from yeast have been studied with respect to temperature, pH, ionic strength, and Mg²⁺ concentration. At low temperature these oligomers have a largely stacked structure. Only the melting data of the dodecanucleotide in absence of Mg²⁺ fit a two-state model. From the different melting behavior of the oligonucleotides after excision of base Y, a rodlike structure of the hexanucleotide produced by stacking interactions can be concluded. The Y fluorescence increase produced by Mg²⁺ has been used to evaluate the binding equilibria between Mg²⁺ and the oligonucleotides. One strong binding site per oligonucleotide and a greater number of weak binding sites have been found. The fluorescence of the free base Y is not influenced by Mg²⁺. The dodecanucleotide enhances the ethidium fluorescence to the same extent as tRNA^Phe and produces comparable shifts in the excitation and emission spectra. Therefore a double helical structure for this oligomer under the assay conditions is suggested. Only weak binding of ethidium to the hexanucleotide is observed, indicating that intercalation of the dye into its structure is not favored. The data show the decisive role of the nucleobase Y in maintaining a rigid stacked structure of the anticodon nucleotides. This structure is stabilized by high ionic strength, Mg²⁺, and ethidium.     

Analysis of permethylated hexopyranosyl-2-acetamido-2-deoxyhexitols by g.l.c.-m.s.

The major product obtained on acetonation of d-mannose with a 2-molar excess of isopropenyl methyl (or ethyl) ether is 4,6-O-isopropylidene-α-d-mannopyranose (3a), the product of kinetic acetonation: a larger excess of the reagent leads, to the 2,3:4,6-diisopropylidene acetal (6). The course of the reaction and side-products formed were examined in detail. The 1,2,3-triacetate of 3a was deacetonated to give α-d-mannopyranose 1,2,3-triacetate; similar reactions were performed on the β anomers. The 1-acetate of the diacetal 6 could be selectively deacetonated to give 1-O-acetyl-2,3-O-isopropylidene-α-d-mannopyranose. The reactions provide access to protected derivatives of d-mannose, and partially acylated derivatives, having modes of substitution different from those obtainable by classical acetonation procedures conducted under conditions of thermodynamic control.     

Functional comparison of versatile carbohydrate esterases from families CE1, CE6 and CE16 on acetyl-4-O-methylglucuronoxylan and acetyl-galactoglucomannan

BackgroundThe backbone structure of many hemicelluloses is acetylated, which presents a challenge when the objective is to convert corresponding polysaccharides to fermentable sugars or else recover hemicelluloses for biomaterial applications. Carbohydrate esterases (CE) can be harnessed to overcome these challenges.MethodsEnzymes from different CE families, AnAcXE (CE1), OsAcXE (CE6), and MtAcE (CE16) were compared based on action and position preference towards acetyl-4-O-methylglucuronoxylan (MGX) and acetyl-galactoglucomannan (GGM). To determine corresponding positional preferences, the relative rate of acetyl group released by each enzyme was analyzed by real time ¹H NMR.ResultsAnAcXE (CE1) showed lowest specific activity towards MGX, where OsAcXE (CE6) and MtAcE were approximately four times more active than AnAcXE (CE1). MtAcE (CE16) was further distinguished by demonstrating 100 times higher activity on GGM compared to AnAcXE (CE1) and OsAcXE (CE6), and five times higher activity on GGM than MGX. Following 24 h incubation, all enzymes removed between 78 and 93% of total acetyl content from MGX and GGM, where MtAcE performed best on both substrates.Major conclusionsConsidering action on MGX, all esterases showed preference for doubly substituted xylopyranosyl residues (2,3-O-acetyl-Xylp). Considering action on GGM, OsAcXE (CE6) preferentially targeted 2-O-acetyl-mannopyranosyl residues (2-O-acetyl-Manp) whereas AnAcXE (CE1) demonstrated highest activity towards 3-O-acetyl-Manp positions; regiopreference of MtAcE (CE16) on GGM was less clear.General significanceThe current comparative analysis identifies options to control the position of acetyl group release at initial stages of reaction, and enzyme combinations likely to accelerate deacetylation of major hemicellulose sources.     

On the biosynthesis of cerebrosides containing non-hydroxy acids. 1. Mass spectrometric evidence for the psychosine pathway

[1, 1, 1′, 2′, 3′, 4′, 5′, 6′, 6′-²H₉]1-O-(β-galactopyranosyl) DL-sphinganine and [4, 5-³H₂]1-O-(β-D-galactopyranosyl) D-sphinganine^∗ were prepared, and the conversion to cerebroside of a mixture of these compounds was studied with rat brain microsomes. The product was characterized by thin layer radiochromatography in several solvent systems and, as the trimethylsilyl ether derivative, by gas-liquid chromatography — mass spectrometry. The mass spectrometric analyses conclusively showed that the glycosidic bond of the substrate remained intact during the transformation to cerebroside.     

The chemical synthesis of glucosaminylphosphatidylglycerol. Comparison with a new phospholipid isolated from Bacillus megaterium

1. We describe the synthesis of a glucosamine derivative of phosphatidylglycerol having the same structure as that of the natural compound isolated from Bacillus megaterium. 2. 2-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-d-glucopyranosyl)-3-O-benzyl-1-iodo-sn-glycerol was prepared by a Königs–Knorr condensation between 3-O-benzyl-1-toluene-p-sulphonyl-sn-glycerol and 3,4,6-tri-O-acetyl-1-bromo-2-deoxy-2-phthalimido-d-glucopyranose followed by replacement of the toluene-p-sulphonyl group with iodine. The iodide was treated with the silver salt of 2-isolauroyl-1-oleoyl-sn-glycerol 3-(monobenzyl hydrogen phosphate) to form the fully protected phosphoglycolipid. 3. Removal of benzyl protecting groups by catalytic hydrogenolysis, phthaloyl group with hydrazine and acetyl groups with pH10 buffer furnished 2-O-(2-amino-2-deoxy-d-glucopyranosyl)-1-(2-isolauroyl-1-stearoyl-sn-glycero-3-phosphoryl)-sn-glycerol. 4. The synthetic and natural compounds appeared identical when compared by chromatography and by identification of hydrolysis products from chemical and enzymic degradations.     

Synthesis of three oligosaccharides that form part of the complex type of carbohydrate moiety of glycoproteins

Silver trifluoromethanesulfonate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-phthalimido-β-d-glucopyranosyl bromide with benzyl 3,6-di-O-benzyl-α-d-mannopyranoside and benzyl 3,4-di-O-benzyl-α-d-mannopyranoside gave the protected 2,4- and 2,6-linked trisaccharides in yields of 54 and 32%, respectively. After exchanging the 2-deoxy-2-phthalimido groups for 2-acetamido-2-deoxy groups and de-blocking, the trisaccharides 2,4-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-mannose and 2,6-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-mannose were obtained. Similar condensation of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d-glucopyranosyl bromide with benzyl 3,4-di-O-benzyl-α-d-mannopyranoside gave a pentasaccharide derivative in 52% yield. After transformations analogous to those applied to the trisaccharides, 2,6-di-O-[β-d-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)]-d-mannose was obtained.     

Ketodeoxyhexosylpurines. Synthesis and properties of keto derivatives of 7-(beta-L-fucopyranosyl)-theophylline

Catalytic fusion of 1,2,3,4-tetra-O-acetyl-L-fucose with theophylline gave 7-(2,3,4-tri-O-acetyl-6-deoxy-β-L-galactopyranosyl)theophylline (1) which was deacetylated with sodium methoxide to give 7-(6-deoxy-β-L-galactopyranosyl)theophylline (2), further transformed by selective condensation with acetone into 7-(6-deoxy-3,4-O-isopropylidene-β-L-galactopyranosyl)theophylline (3). Oxidation of 3 employing a modified Pfitzner-Moffatt procedure led to 7-(6-deoxy-3,4-O-isopropylidene-β-L-lyxo-hexopyranosulosyl)theophylline (5). However, treatment of 3 with dimethyl sulfoxide-acetic anhydride according to the procedure used for deoxy hexoses gave only the 2′-O-acetyl analog 4. Treatment of 5 with alkali showed it to be more stable than 2′-ketouridine or 2′-ketocytidine. Finally, in vivo biological assays showed that 7-(6-deoxy-β-L-lyxo-hexopyranosulosyl)theophylline (7) inhibits cellular growth, whereas the nucleoside 2 is inactive before oxidation.     

The relationship of molecular weight, and sulfate content and distribution to anticoagulant activity of heparin preparations

The crystalline intermediate 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranosyl azide (5), obtained by condensation of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl bromide with either 2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranosyl azide or its 6-O-triphenylmethyl derivative, was reduced in the presence of Adams' catalyst to give a disaccharide amine. Condensation with 1-benzyl N-(benzyloxycarbonyl)-L-aspartate afforded crystalline 2-acetamido-6-O-(2-acetamido-3,4 6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-acetyl-1-N-[1-benzyl N-(benzyloxycarbonyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine (9). Catalytic hydrogenation in the presence of palladium-on-charcoal was followed by saponification to give 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-1-N-(L-aspart-4-oyl)-2-deoxy-β-D-glucopyranosylamine (11) in crystalline form. From the mother liquors of the reduction of 5, a further crystalline product was isolated, to which was assigned a bisglycosylamine structure (12).     

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.     

Synthesis of a branched d-mannopentaoside and a branched d-mannohexaoside: Models of the outer chain of the glycan of soybean agglutinin

Synthetic routes are described to the d-mannopentaoside methyl 3-O-(3,6-di-O-α-d-mannopyranosyl-α-d-mannopyranosyl)-6-O-α-d-mannopyranosyl-α-d-mannopyranoside, and the d-mannohexaoside methyl 3-O-(3,6-di-O-α-d-mannopyranosyl-α-d-mannopyranosyl)-6-O-(2-O-α-d-mannopyranosyl-α-d-mannopyranosyl)-α- d-mannopyranoside, formed in a regio- and stereo-controlled way by employing the properly protected d-mannobioside methyl 2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-α-d-mannopyranosyl)-α-d-mannopyranoside and d-mannotrioside methyl 2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-α-d-mannopyranosyl)-6-O-(3,4,6-tri-O-benzyl-α-d- mannopyranosyl)-α-d-mannopyranoside as key intermediates.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

The synthesis,and study of the β-elimination reaction,of di- and tri-peptides having a 3-O(2-acetamido-3,4,6-trio-O-acetyl-2-deoxy-β-D-glucopyranosyl)-L-serine residue

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyra-nosyl chloride was condensed with the N-(benzyloxycarbonyl) derivatives of, respectively, L-seryl-glycine ethyl, L-seryl-L-alanine methyl, L-seryl-L-phenylalanine methyl, and L-seryl-L-aspartic dibenzyl esters to give (3-O-GlcpNAc-CbzN-L-Ser)-GlyOEt (8), (3-O-GlcpNAc-CbzN-L-Ser)-L-AlaOMe (9), (3-O-GlcpNAc-CbzN-L-Ser)-L-PheOMe (10), and (3-O-GlcpNAc-CbzN-L-Ser)-L-Asp(diOBzl) (11), respectively; O-(2-acetamido-3,4,6-tri-O-acetyl-β-D-glucopyranosy-l)-N-(benzyloxycarbonyl)-L-serine methyl ester was deblocked by treatment with hydrobromic acid in glacial acetic acid, followed by triethylamine, to give a glycoamino acid that was condensed with the N-(benzyloxycarbonyl) derivatives of the p-nitrophenyl ester of glycine, L-alanine, and L-proline, respectively, to give CbzNGly-(3-O)-Glcp NAc-L-SerOMe) (17), CbzN-L-Ala-(3-O-GlcpNAc-L-SerOMe), and CbzN-L-Pro-(3-O-GlcpNAc-L-SerOMe), respectively. Similarly, the glycopeptide resulting from 8 was condensed with the activated esters of glycine, L-alanine, L-phenylalanine, L-proline, and L-serine, respectively, to give CbzNGly-(3-OGlcpNAc-L-Ser)-GlyOEt, CbzN-L-Ala-(3-O-GlcpNAc-L-Ser)-GlyOEt, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-GlyOEt, and CbzN-L-Ser-(3-O-GlcpNAc-L-Ser)-GlyOEt, respectively; that from 9, with the p-nitrophenyl esters of glycine¹,L-alanine, L-phenylalanine, L-proline, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-AlaOMe, CbzN-L-Ala(3-O-GlcpNAc-L-Ser)-L-AlaOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L]-AlaOMe, CbzN-L-Pro-(3-O-GlcpNAc-L-Ser)-L-AlaOMe, and CbzN-L-Leu-(3-O-GlcpNAc- L-Ser)-L-AlaOMe, respectively; that from 10, with the derivatives of glycine, L-alanine, L-phenylalanine, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-PheOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-PheOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-PheOMe, and CbzN-L-Leu-(3-O-GlcpNAc-L-Ser)-L-PheOMe, respectively; and that from 11, with the derivatives of glycine, L-alanine, L-phenylalanine, L-proline, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Ala-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Pro-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), and CbzN-L-Leu-(3-O-GlcpNAc-L-Ser)-L-Asp-(diOBzl), respectively. O-(2-Acetamido-3,4,5-tri-O-acetyl-2-deoxy-β-D-gluco-pyranosyl)-N-(benzyloxycarbonyl)- L-asparaginylglycyl-L-serine methyl ester (20) was synthesized by treating the free amine of 17 with the p-nitrophenyl ester of N-(benzyloxycarbonyl)-L-asparagine. 2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbo-nyl)-L-aspart-1-oyl-(glycyl-L-serine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (41) was synthesized by the condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbo-nyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with glycyl-L-serine methyl ester. Attempts to transfer the 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl group from the hydroxyl group of L-serine in 20 to the amido group of L-asparagine, to give 41, were unsuccessful. The β-elimination of some of the glycodi- and glycotri-peptides was studied.     

A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3′-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2′-O-methyl RNA and LNA-2′-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2′-O-methyladenosine with 2′-O-methyl-2,6-diaminopurine riboside (D^M) or LNA-2,6-diaminopurine riboside (D^L) increases the thermodynamic stability (ΔΔG°₃₇) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37°C. D-A and D-G but not D-C mismatches formed by D^M or D^L generally destabilize 2′-O-methyl RNA/RNA and LNA-2′-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2′-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays.     

Isolation and structural analyses of positional isomers of 61,6m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-α-d-mannopyranosyl-cyclomaltooctaose (n=2, 3, 4, and 6)

Eight positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (γCD) (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD (n=2, 3, 4, and 6) in a mixture of products from γCD and d-mannose by condensation reaction of α-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-γCD were determined by LC–MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying α-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.