The role of metaphosphate in the activation of the nucleotide by TPS and DCC in the oligonucleotide synthesis.

The course of the activation of 3'-acetylthymidine 5'-phosphate by TPS and DCC were followed up by 31P FT nmr. The fact that the "metaphosphate" (delta-5.1) first becomes detectable only at later stage of the activation and does coexist with pyrophosphate and triphosphate suggests that the pyridinium derivative of "metaphosphate" is most probably not directly formed from the hypothetical mixed anhydride or active "pseudourea" right at the beginning of the reaction of pdTac with TPS or DCC, but rather formed at later stage of the activation reaction from the degradation of the pyro- and triphosphates by the activating agent. The mixed anhydride or the active "pseudourea" is most possibly the active key intermediate.     

113CD-1H spin-spin couplings in homonuclear 1H correlated spectroscopy of metallothionein. Identification of the cysteine 1H spin systems

Heteronuclear spin-spin couplings between 113Cd and C beta protons of the metal-bound cysteines were observed in phase-sensitive, double-quantum filtered, homonuclear two-dimensional correlated (COSY) 1H NMR spectra of 113Cd-metallothionein-2 from rabbit liver. Comparison of 113Cd- and 112Cd-metallothionein-2 spectra revealed that 19 1H spin systems show heteronuclear couplings to at least one 113Cd and were thus identified as 19 of the 20 cysteines in this protein. From a detailed analysis of the manifestations of heteronuclear couplings in the homonuclear 1H COSY spectra, two cysteines could be identified as 'bridging cysteines', with spin-spin couplings to two different 113Cd nuclei. The observed 113Cd-1H coupling constants vary between less than or equal to 5 Hz and 80 Hz.     

The reactivity of phosphomono-and phosphodiester groups in oligonucleotides.

The rate constants were estimated by phosphorus NMR spectroscopy for the reactions of alcohols (Tr-dT, 2-cyanoethanol) in pyridine with the main types of the reactive phosphorylating intermediates formed by treatment of pdT-Ac, pdTpdT-Ac, Tr-dTpdT-Ac, Tr-dTpdTpdT-Ac with 2, 4, 6-triisopropylbenzene-sulfonyl chloride (TPS): 1) B type derivatives with phosphomono ester (PME) group converted to a phosphoryl pyridinium residue; 2) C type derivatives with PME and phosphodiester (PDE) groups converted to trisubstituted pyrophosphate; 3) D type derivatives with PDE groups converted to tetrasubstituted pyrophosphate. The two latter types are partially present as cyclic intramolecular pyrophosphates Ci and Di. The reactivity of the intermediates decrease in the series B greater than Ci approximately Di greater than C approximately D. The Ci derivative of pdTpdT-Ac when obtained in dimethylformamide was found to be rather stable to hydrolysis and could be separated from the other dinucleotide derivatives by ion-exchange chromatography. The Arrhenius parameters of all steps of the conversion of PME group of pdT-Ac to B derivative and of the reaction of TPS with PDE group of dinucleoside phosphate Tr-dTpdT-Ac were measured.     

Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2''-S-cycloadenosine.

A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9.     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.     

5,6,7,8-Tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring

It is suggested from analysis of proton spin-spin coupling constants that the tetrahydropyrazine ring of tetrahydrofolate is a roughly equal mixture of two half-chair conformations, one with the C-6 proton axial and the other with the C-6 proton equatorial. The chemical shifts and spin-spin coupling constants for the carbon-bound protons of (+/-)-L-, (-)-L-, and (-)-L-[6-2H] 5,6,7,8-tetrahydrofolate were measured at 25 degrees and at 300 MHZ. The resonances corresponding to the two C-7 protons in the deuterated compound constituted an AB quartet with JAB of 12 Hz and chemical shift difference of 92 Hz or 0.307 ppm; the C-7 protons are proposed to be a geminally coupled axial-equatorial pair whose rapid equilibration does not result in equivalence due to the adjacent chiral center at C-6. The spin-spin splitting in the C-7 resonances were 3.0 and 6.6 Hz for the low field and high field resonances, respectively, reflecting coupling to the C-6 proton. These coupling constants reflect the conformational equilibrium. The resonances assignable to C-9 protons are nearly equivalent in the 6-2H compound, but exhibit the resonances corresponding to a complex spin system in the 6-H compound.     

Synthesis and structure determination of [8(-13)C]guanosine 5'-diphosphate

A combined chemical and enzymatic synthesis of [8(-13)C]guanosine 5'-diphosphate (GDP) from H13COOH is described. About 35 mg nucleotide was obtained from 500 mg H13COOH. Analysis of the [8(-13)C]GDP by negative ion fast atom bombardment mass spectrometry and by 13C NMR confirmed that one atom of 13C was incorporated at the 8-position of the guanine ring at 90 +/- 10% enrichment. The chemical shift of the C(8) was 140.2 ppm downfield from internal trimethylsilylpropionate at neutral pH and room temperature, with a spin-spin coupling 1J(13C(8)-H(8] of 217 Hz and a 3J(13C(8)-H(1'] of 3.9 Hz.     

The mechanism of stabilization of the structure of nuclease-T by binding of ligands.

The rate of unfolding of Nuclease-T at pH 8,20 degrees was determined as a function of concentration of the ligands deoxythymidine 3',5'-diphosphate (pdTp) and Ca2+ on the basis of the rate of exchange between free fragment, Nuclease-T(50-149) and labeled fragment, Nuclease-T-(50-149) incorporated in the structure of nuclease-T (Taniuchi, H. (1973) J. Biol. Chem. 248, 5164-5174). The rate constant of unfolding of unliganded Nuclease-T' was 4.6 times 10-4s-1. Those of Nuclease-T' bound with pdTp, with Ca2+, and with both pdtp and Ca2+ were 9.0 times 10-5, 1.6 times 10-4, and 2.2 times 10-5s-1, respectively. The association constants of pdTp and Ca2+ with Nuclease-T' were found to be 1.0 times 10-4 and 2.0 times 10-2 m-1, respectively. Those of pdTp with Nuclease-T' plus Ca2+ and of Ca2+ with Nuclease-T' plus pdTp were 4 times 10-5 and 1.4 times 10-4M-1, respectively. The calculation of free energy change on the basis of the association constants shows that the magnitude of negative free energy change involved in the binding of either of the two ligands increases by approximately 2 kcal when the other ligand is already bound. There is a correlation between the free energy change and the specifically coupled with the cooperative interacions operating throught the three-dimensional structure resulting in strengthening of the interactions throughtout the structure, including those with the ligands, without a large change in conformation.     

Reaction of a nucleoside 2, 4-dinitrophenyl phosphate with fluoride; a convenient method for the preparation of the nucleoside phosphorfluoridate.

Examination of the reaction of 2, 4-dinitrofluorobenzene with thymidine-5' phosphate in detail reveals that the initial product is the 2, 4-dinitrophenyl ester. This reacts with fluoride to produce thymidine-5' phosphorfluoridate. This second reaction provides the basis for the conversion of preformed thymidine-5' 2, 4-dinitrophenyl phosphate to thymidine-5' phosphorofluoridate.     

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.     

Comparison of conformational features of staphylococcal nuclease in ternary complexes with pdTp, pdGp, and nitrophenyl-pdTp.

The conformations of wild-type staphylococcal nuclease (SNase) in the ternary complexes with thymidine 3',5'-bisphosphate (pdTp), 2'-deoxyguanine 3',5'-bisphosphate (pdGp), and thymidine 3'-phosphate 5'-(p-nitrophenylphosphate) (NpdTp) with Ca2+ were examined by two-dimensional NMR NOESY and ROESY experiments. The results of these experiments indicate that the conformational features of the SNase are quite similar in the three ternary complexes. This suggests that the conformational features of SNase, in these ternary complexes, are not strongly dependent on whether the 5'-phosphate is a mono- or diester. This is in contrast to our prior studies on substitutions of active site charged amino acids which indicated that the conformational features of SNase in the ternary complex are quite sensitive to substitutions for active site charged amino acids (Hibler et al., 1987; Wilde et al., 1988; Pourmotabbed et al., 1990). The similarity of the SNase conformational features in the ternary complexes with pdTp and pdGp indicates that the features of the nucleotide bound at the active site are not strong determinants of the enzyme conformation in the ternary complexes. These conclusions are in general agreement with the results on pdApdT ternary complexes with SNase which suggested that it is the conformational features of the bound nucleic acid which determine the differences in catalysis observed for SNase with different substrates (Weber et al., 1991), more so than the conformational features of the enzyme.     

Conjugation of resveratrol with RGD and KGD derivatives

The reaction between Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) derivatives with 3,4',5-trihydroxy-trans-stilbene (resveratrol) was investigated. Knowing that resveratrol, RGD as well as KGD analogues inhibit human platelet aggregation in vitro, it was tempting for us to examine whether their coupling products present enhanced biological activity. Here, we report on the synthesis and identification of these coupling products. The N-protected peptides were synthesized by solid phase technique, using the 2-chlorotrityl-chloride resin, by the method of carbodiimides. Coupling reactions with resveratrol took place in solution using N,N-dicyclohexylcarbodiimide (DCC) as coupling reagent and 4-dimethylaminopyridine (DMAP) as catalyst. The reaction products were purified by reversed phase HPLC and identified by ESI-MS. The mono-esterified resveratrol derivative was the main (or only) reaction product, whereas the di- and the tri-ester (to a less extent) formation was noticed in some cases.     

Chiral recognition in dipeptides containing 1-aminocyclopropane carboxylic acid or alpha-aminoisobutyric acid: NMR studies in solution.

Protected dipeptides containing 1-aminocyclopropane carboxylic acid (Ac3c) or alpha-aminoisobutyric acid (Aib) residues at the C-terminus and Phe, Val or Ala residues at the N-terminus displayed different proton NMR spectra for the pure enantiomers and the racemic mixtures in deuterochloroform (CDCl3) solution. An unequal mixture of enantiomers showed two sets of resonances (NMR nonequivalence), one corresponding to major and the other to minor enantiomer. The NMR nonequivalence was originated by the presence of the C-terminal Ac3c or Aib residues, which have been known for their unique spatial preferences in avoiding an extended (C5) conformation. When a C5 conformation favoring residue such as glycine was incorporated in place of Ac3c or Aib, negligible NMR nonequivalence was observed. The magnitude of the NMR nonequivalence depended on the side chain as well as on the protecting groups at N-terminus alpha-amino acid. For the same peptide, the magnitude of nonequivalence increased with increasing solution concentration and/or with decreasing the solution temperature. The NMR nonequivalence disappeared in polar solvent-like deuterated dimethylsulfoxide (DMSO-d6). A preference for hetero-chiral recognition leading to dimeric association under fast exchange conditions had been invoked to explain the observed phenomenon. The dipeptides thus prepared could well serve as 'model peptides' for the evaluation of any preparative methods.     

Coherence transfer in deoxyribose sugars produced by isotropic mixing: an improved intraresidue assignment strategy for the two-dimensional NMR spectra of DNA

Two-dimensional isotropic mixing spectroscopy has been used to confirm assignments of the deoxyribose sugar protons in the H NMR spectrum of the DNA oligonucleotides d(CGCGTTTTCGCG) and [d(GCCGTGGCCACGGC)]2. The broad-band decoupling sequence MLEV-16 was applied during the mixing period to induce isotropic coupling within the spin systems, resulting in net transfer of coherence throughout the coupled spin networks. Nearly all 1', 2', 2', 3', and 4' protons of a given nucleotide could be identified on the basis of through-bond scalar connectivities. In addition, in the hairpin, a number of connectivities to 5'/5' protons were found. The dependence of cross-peak intensity on the length of radio-frequency irradiation for several different coherence transfer orders is presented, and implications for optimization are discussed.     

An improved synthetic route to the beta-hydroxyethyl esters of 5''-nucleotides.

A simple method is described for the preparation of the beta-hydroxyethyl esters of nucleoside 5'-phosphates by treatment of the appropriate 2',3'-isopropylidene nucleoside with 2-chloro-2-oxo-1,3-dioxaphospholane. Unambigous structural assignments were based on 13C nmr spectroscopy. Chemical shifts and 13C-31P spin-spin coupling constants are discussed.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramyl-pentapeptide translocase.

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by phospho-N-acetylmuramyl (MurN Ac)-pentapeptide translocase (UDP-MurNAc-Ala-gamma DGlu-Lys-DAla-DAla undecaprenyl phosphate phospho-MurNAc-pentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30 degrees C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of N-oxyl-4',4'-dimethyloxazolidine derivatives of 12- and 16-ketostearic acid intercalated into membranes from this organism define the lower (T1 = 16--22 degrees C) and upper (Th = 30 degrees C) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurNAc-penetapeptide translocase has a significant effect on the catalytic activity of this enzyme.     

The mechanism of the chemical synthesis of oligonucleotides and its synthetic consequences.

The data obtained mainly by pulsed NMR spectroscopy on phosphorus nuclei on the mechanism of the internucleotide phosphodiester (PDE) group formation are summarised. With arylsulphonyl chloride as condensing reagent monomeric nucleotide derivative B (nucleoside metaphosphate or its pyridinium adduct) is the highly reactive intermediate. In the presence of PDE groups in nucleoside or nucleotide component the significantly less reactive derivatives with trisubstituted pyrophosphoryl residues are formed both with arylsulphonyl chloride and dicyclohexylcarbodiimide (DCC). The reactive B form of nucleotide component may be obtained using greater excess of arylsulphonyl chloride with simultaneous convertion of PDE groups to tetrasubstituted pyrophosphates amenable to side reactions. The convertion of PDE groups to easily hydrolysable dicyclohexylurea derivatives by reaction with DCC is proposed to reversible blocking of PDE groups of nucleoside component. The B type derivatives of mononucleotides or oligonucleotides with blocked PDE groups seems to be the best nucleotide components.     

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.