New effective method for the synthesis of oligonucleotides via phosphotriester intermediates

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Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP [28][29][30][31][32][33][34][35][36] Pulmonary surfactant is a lipid-rich material secreted as tightly packed lamellae into the extracellular alveolar fluid layer (1). Within the alveolar space, surfactant lipids are found in a number of different structural forms, including lamellar bodies, tubular myelin, and various vesicular structures (2). Although the lipid compositions of the surfactant structures are similar, the physical properties, particularly the ability ofthe lipoprotein complexes to form a surface film, are quite different (3). Specific proteins appear to influence the structure and surface activity of surfactant-lipid complexes (4-10).The predominant surfactant-associated protein is the glycoprotein SP 28-36 (28-36 kDa) characterized by a collagenlike NH2-terminal domain and variable N-linked glycosylation of the COOH-terminal region (11-13). SP 28-36 is water soluble but readily associates with phospholipids (PLs) (14). In the presence of calcium, SP 28-36 causes PL aggregation and increases the rate of adsorption of surfactant lipids to an air-fluid interface (6,8,14). A second group of very hydrophobic proteins has been identified in lamellar bodies isolated from lung homogenate and in surfactant isolated from the bronchoalveolar wash (4, 9, 10, 15-17). Very little is known about the homogeneity, structure, or function ofthis group of hydrophobic proteins, but it has been reported that at least one of these proteins enhances PL surface film formation (9,10,18 (20). The surfactant in water (-16 mg of PL per ml, 2 mg of protein per ml) was extracted in 1-butanol (1:50, vol/vol) at room temperature (21). The surfactant/butanol mixture was spun twice at 10,000 X gav for 20 min to sediment the butanolinsoluble protein (94% of the total). The butanol supernatant was dried by rotary evaporation and the residue was resuspended in chloroform/methanol/0.1 M HCl, 1:1:0.5, vol/vol). A small amount of insoluble material was removed by centrifugation and the supernatant, containing 30 mg ofPL in 1 ml, was applied to a 1 cm x 45 cm column of Sephadex and eluted at 4 ml/hr with the same solvent at 40C. The eluted fractions, 0.5 ml, were assayed for protein (22) in the presence of 1% NaDodSO4, phosphorus (23) for the calculation of the PL content, and cholesterol (24). An aliquot of each fraction containing protein was analyzed by NaDodSO4/PAGE (25) and silver staining (26). 66The publication costs of this article were defrayed in part by page charge payment. This article...

Section: Resultsmentioning

confidence: 65%

mentioning

confidence: 99%

See 1 more Smart Citation

Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption.

Hawgood¹,

Benson²,

Schilling³

et al. 1987

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237

“…A mixture of six 32-residue oligonucleotides, 5' AGGC-GCTTCAGGGAGCTGGGGAAGCAGGGGAT 3 phosphotriester method (16,17) and purified by PAGE. Oligonucleotides were phosphorylated with T4 polynucleotide kinase and [y-32P]ATP (18).…”

Section: Methodsmentioning

confidence: 99%

Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.

Warr¹,

Hawgood²,

Buckley³

et al. 1987

152

Pulmonary surfactant is a lipid-protein complex that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. A group of hydrophobic surfactant-associated proteins has been shown to be essential for rapid surface film formation by surfactant phospholipids. We have purified a hydrophobic surfactant protein of :5 kDa that we term SP5 from bronchopulmonary lavage fluid from a patient with alveolar proteinosis and shown that it promotes rapid surface film formation by simple mixtures of phospholipids. We have derived the full amino acid sequence of human SP5 from the nucleotide sequence of cDNAs identified with oligonucleotide probes based on the NH2-terminal sequence of SP5. SP5 isolated from surfactant is a fragment of a much larger precursor protein (21 kDa). The precursor contains an extremely hydrophobic region of 34 amino acids that comprises most of the mature SP5. This hydrophobicity explains the unusual solubility characteristics of SP5 and the fact that it is lipid-associated when isolated from lung.Pulmonary surfactant is a phospholipid-protein complex that lowers surface tension at the air-liquid interface in the alveolus (1). The lipid composition of surfactant has been studied in detail (2), and the major lipid components by weight are dipalmitoylphosphatidylcholine, monoenoic phosphatidylcholine, and phosphatidylglycerol. Four surfactant protein species have been identified in canine and human bronchoalveolar lavage. The most abundant species is a glycoprotein with a collagen-like 4). This protein has been shown to enhance uptake of surfactant lipids into alveolar type II cells (5) and inhibit secretion of surface-active material from these cells (6). The three other surfactant proteins, SP5 (5 kDa), SP8 (8 kDa), and SP18 (18 kDa), are very hydrophobic and have proved difficult to purify to homogeneity. The NH2-terminal amino acid sequences of the canine hydrophobic proteins have been determined, and it was shown that SP5 and SP8 share a common NH2 terminus (7). The complete amino acid sequence of SP18 deduced from canine and human cDNA sequences does not contain any region corresponding to the NH2 terminus of SP5 or SP8 (7,8).Various groups have studied the ability of the surfactant proteins to enhance surface activity of phospholipids in vitro using a surface balance. It has been shown that the small molecular weight hydrophobic proteins isolated from bovine surfactant enhance surface film formation by phospholipids (9-12). We have shown that mixtures of SP5, -8, and -18 or that SP18 alone, isolated from canine surfactant, stimulated phospholipid surface film formation (7). Unfortunately, detailed comparison of data between groups is difficult due to different methods of isolation and characterization of surfactant proteins.In this article we report the effect of purified human SP5 on the surface activity of simple phospholipid mixtures and the amino acid sequence of the precursor of SP5 derived from the sequences of near full-leng...

“…The hexanucleotides were synthesized by using the phosphotriester method. The coupling reagent used in most of these syntheses was mesitylene sulfonyl chloride in conjunction with N-methylimidazole (8). The dinucleotides C'(Dmt)p(CH3)G'(tBuPh2Si) and G'(Dmt)p(CH3)C'(tBuPh2Si) were synthesized by coupling the 5'-protected nucleoside phosphonate with the 3'-silyl-protected nucleoside (C', N-benzyl-2'-deoxycytidine; G', N-isobutyl-2'-deoxyguanosine).…”

mentioning

confidence: 99%

B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.

Callahan¹,

Han²,

Watt³

et al. 1986

The simulation of the B-Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the Rp stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the Sp stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral Sp oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group ofguanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the Rp stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the Sp stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral Sp oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B-Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate oxygens and the N-2 amino group of guanine.Pohl and Jovin (1) showed that the alternating copolymer poly[d(C-G)] underwent a salt-induced conformational change. The new species thus formed was left-handed Z-