Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Pyrano chalcones and a flavone from Neoraputia magnifica and their Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase-inhibitory activities

The fruits of Neoraptua magnifica var. magnifica afforded three new flavonoids: 2'-hydroxy-4,4',-dimethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, 2'-hydroxy-3,4,4'-trimethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, and 3',4'-methylenedioxy-5,7-dimethoxyflavone which were identified on the basis of spectroscopic methods. The known flavonoids 2'-hydroxy-3,4,4',5-tetramethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone, 3',4'-methylenedioxy-5,6,7-trimethoxyflavone, 3',4'-methylenedioxy-5',5,6,7-tetramethoxyflavone, 3',4',5',5,7-pentamethoxyflavanone and 3',4',5'5,7-pentamethoxyflavone were also identified. The latter flavone was the most active as glyceraldehyde-3-phosphate dehydrogenase-inhibitor.     

Periodicities in coding and noncoding regions of the genes

Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns. The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes. Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2). The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions. (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3). (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3). The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses.     

Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.     

Synthesis and separation of diastereomers of uridine 2',3'-cyclic boranophosphate

The first boron-containing 2',3'-cyclic phosphate-modified analogue, uridine 2',3'-cyclic boranophosphate (2',3'-cyclic-UMPB), was synthesized. 5'-O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2',3'-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2',3'-cyclic-UMPB in high yield. The two diastereomers of 2',3'-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2',3'-cyclic phosphorothioate (2',3'-cyclic-UMPS) via H-phosphonate was also described.     

Synthesis and bioactivities of nitronyl nitroxide and RGD containing pseudopeptides

1-(1',3'-dioxyl-4',4',5',5'-tetramethyldihydroimidazol-2'-yl)-phenyl-4-yloxylacetic acid (3), and 1-(1',3'-dioxyl-4',4',5',5'-tetramethyldihydroimidazol-2'-yl)-phenyl-4-yloxylacetyl-RGDS (13), -RGDV (14), -RGDF (15) were synthesized. The ESR measurement gave the same spectroscopy for 3 and 13-15. The NO scavenging tests in vitro, anti-platelet aggregation tests in vitro and the anti-thrombosis assay in vivo indicated that introducing 3 into the N-terminal of RGDS, RGDV and RGDF the corresponding bioactivities for both of 3 and RGD peptides can be remained completely. The present combinations provided a beneficial strategy for simultaneous scavenging NO and anti-thrombosis, and for the use of spin label of RGD peptides in the conformational researches.     

Stimulation of cholinergic receptors and changes in cyclic nucleotide levels in the cerebral cortex of the guinea pig

The effect of Atropine and Physostigmine on 3'5' AMP and 3'5' GMP content was investigated in slices of guinea-pig cerebral cortex maintained at rest or electrically-stimulated. Atropine and Physostigmine did not modify either the basal content or the electrically-evoked increase of 3'5' AMP and 3'5' GMP. On the contrary, Betanechol 25 micro M significantly increased 3'5' GMP and 3'5' AMP content in slices kept at rest. The effect was abolished by Atropine 1,5 x 10(-7) M and d-tubocurarine 7 x 10(-6) M, respectively.     

Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica

The tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone and 5,7,4',5',7',3',4'-heptahydroxy[3-8'] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone) showed much stronger activity (IC50 2.5 microM) than that of kojic acid (IC50 9.1 microM) when L-tyrosine was used as the substrate.     

Flavones and phenylpropenoids in the surface exudate of Psiadia punctulata

Three flavones, 5,7-dihydroxy-2',3',4',5'-tetramethoxyflavone, 5,4'-dihydroxy-7,2',3',5'-tetramethoxyflavone, and 5,7,4'-trihydroxy-2',3',5'-trimethoxyflavone were isolated from the leaf exudate of Psiadia punctulata, together with the previously reported 5-hydroxy-7,2',3',4',5'-pentamethoxyflavone and 5,7,3'-trihydroxy-2',4',5'-trimethoxyflavone. The two phenylpropenoids, Z-docosyl-p-coumarate and E-docosyl-p-coumarate were also isolated. The structures were determined on the basis of spectroscopic evidence.     

Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Effect of cyclic nucleotides and isopropylnoradrenaline on oxygen tension and absorption]

Isopropylnoradrenaline (ISO), 3',5'-AMP and dibutyryl-3',5'-AMP decreased the oxygen tension (pO2) in the liver and the spleen and increased the body oxygen consumption (VO2). Time dynamics of these two effects was closely correlated for ISO and 3',5'-AMP. An increase of heat output was not accompanied by any significant changes in the respiration coefficient. Pempidine and dihydroergotamine failed to prevent 3',5'-AMP effects; inderal somewhat decreased these effects. Apparently, the catecholamine influence upon pO2 was a result of the VO2 increase through 3'5'-AMP effects are largely direct, but they include the in vivo and beta-receptor component; 2',3'-AMP decreased pO2 and VO2.     

Mutants of ETS domain PU.1 and GGAA/T recognition: free energies and kinetics.

The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5'-GGAA-3' and 5'-GGAA/T-3', respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5'-GGAA-3' containing oligonucleotide caused by a faster release of the protein from the DNA (k(off) tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5'-GGAA-3' and 5'-GGAT-3' oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5'-GGAA-3' (delta deltaG((A231S-N236Y) - (N236Y)) = -1.2 kcal mol confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5'-GGAA-3' and 5'-GGAT-3' sequences, while A231S mutation modulates the degree of specificity toward 5'-GGAA-3' and 5'GGAT-3' sequences. This study explains why wild-type PU.1 does not recognize 5'-GGAT-3' sequences and in addition broadens our understanding of 5'-GGAA/T-3' recognition by ETS protein family members.     

Biochemical effects of pure isomers of hexachlorobiphenyl: fatty livers and cell structure.

The effects of a single oral dose (50 mg/kg body wt.) of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB for a period of 72 h have been studied in the male rat. Only 3,4,5,3',4',5'-HCB caused necrosis of thymocytes. 3,4,5,3',4',5'-HCB caused marked pathological changes in the liver with less marked effects being caused by 2,4,5,2',4',5'-HCB and 2,3,5,2',3',5'-HCB. Total lipid content was increased by all the isomers studied, but 3,4,5,3',4',5'-HCB had more pronounced effect on total lipid content. Lipid accumulation was pericentral in the livers obtained from rats treated with 3,4,5,3',4',5'-HCB, but midzonal in the liver obtained from the rats treated with the other two isomers. Analysis of various lipid fractions showed that triacylglycerols were increased seven-fold only by 3,4,5,3',4',5'-HCB, while phospholipids were increased slightly by 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB. Only 3,4,5,3',4',5'-HCB increased the level of total and esterified cholesterol. These results show that the fatty livers caused by 3,4,5,3',4',5'-HCB were qualitatively and quantitatively different from those caused by the other two isomers at the same dose. For the first time a hexachlorobiphenyl unchlorinated in the para position, 2,3,5,2',3',5'-HCB has been shown to be a specific inducer of cytochrome P-450. The effects of 2,3,5,2',3',5'-HCB on cell structure and phospholipid content were quantitatively similar to those caused by 2,4,5,2',4',5'-HCB. Thus, chlorination at para (4,4') positions in chlorobiphenyls is not necessarily required for biological activity. It is hypothesized that net stereoelectronic properties of the isomers or resistance to metabolism may be the underlying factor in determining structure-activity relationships.     

Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.     

Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'.     

Isoflavanones and their O-glycosides from Desmodium styracifolium

Two isoflavanones (5,7-dihydroxy-2',3',4'-trimethoxy-isoflavanone and 5,7-dihydroxy-2'-methoxy-3',4'-methylenedioxy-isoflavanone), four isoflavanone O-glycosides (5,7-dihydroxy-2',3',4'-trimethoxy-isoflavanone 7-O-beta-glucopyranoside, 5,7-dihydroxy-2'-methoxy-3',4'-methylenedioxy-isoflavanone 7-O-beta-glucopyranoside, 5,7-dihydroxy-2',4'-dimethoxy-isoflavanone 7-O-beta-glucopyranoside, and 5,7,4'-trihydroxy-2',3'-dimethoxy-isoflavanone 7-O-beta-glucopyranoside), and a coumaronochromone (3,5,7,4'-tetrahydroxy-coumaronochromone), along with 25 known compounds, were isolated from the aerial parts of Desmodium styracifolium. This is for the first time isoflavanone O-glycosides were isolated from a natural source.     

Synthesis and characterization of oligodeoxyribonucleotides containing terminal phosphates. NMR, UV spectroscopic and thermodynamic analysis of duplex formation of [d(pGGAATTCC)]2, [d(GGAATTCCp)]2 and [d(pGGAATTCCp)]2.

Derivatives of the oligomer [d(GGAATTCC)]2 with 5' (5'-P), 3' (3'-P) and both 5' and 3' (5',3'-P2) terminal phosphate groups have been synthesized and studied by temperature dependent UV and NMR spectroscopic methods. Thermodynamic studies of the helix to strand transition indicate that addition of 3' phosphate groups has very little effect on the delta G degree for helix formation at 37 degrees C while addition of 5' phosphate groups adds approximately -0.5 kcal/mole to the delta G degree for duplex formation. The helix stabilization by 5' phosphate groups occurs at salt concentrations of 0.1 M and above, and is primarily enthalpic in origin. Tm studies as a function of ionic strength also indicate that the oligomers fall into two groups with the parent and 3'-P derivatives being similar but less stable than the 5'-P and 5',3'-P2 derivatives. Imino proton and 31P NMR studies also divide the oligomers into these same two groups based on spectral comparisons and temperature induced chemical shift and linewidth changes. 31P NMR analysis suggests that addition of 5' phosphate groups results in a small change in phosphodiester torsional angles in the g,t to g,g direction, indicating improved base stacking at the 5' end of the modified oligomer. No such changes are seen at the 3' end of the oligomer on adding 3' phosphate groups.