3'-modified oligonucleotides and their conjugates.

Solid-phase synthesis of 3'-aminoalkyl, 3'-thioalkyl, and 3'-polyethyleneglycol are described. The first two are important as specific points of attachment for a large variety of reporter groups; the latter is important because of its increased cell membrane permeability, which aids in the development of antisense drugs. These protocols cover details of the synthetic organic chemistry needed to make each solid support, as well as DNA synthesis, workup, characterization, and conjugation.

A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5'-Tamra DNA.

5-Carboxy Tamra 1 was conjugated to 4-hydroxypiperidine with BOP and N-methylmorpholine, and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl tetraisopropylphosphorodiamidite to give 5-[N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite)]-Tamra carboxamide 3. Solutions of 3 were coupled onto the 5'-hydroxyl of solid-phase-supported DNA fragments with standard amidite coupling techniques. Cleavage and deprotection with aqueous tert-butylamine cocktail gave 5-Tamra-functionalized DNA as well as an additional compound without the Tamra chromophore. A mass spectrum of this product showed the incorporation of tert-butylamine. The extra product was completely suppressed by including a 5 min acetylation step after coupling. A model study of 3 coupled onto thymidine-functionalized CPG showed similar results. NMR and mass spectra of cleaved products confirmed the addition of tert-butylamine to the minor product. Coupling a Tamra active ester onto T CPG which was previously coupled with N-(4-methoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) 4 produced the same major Tamra-bearing product, which coeluted on reverse phase HPLC with the major product generated with 3.

A phosphate bound universal linker for DNA synthesis.

A uridine-based linker immobilized onto polystyrene beads at the 5' terminus via a phosphodiester group and then used as a universal DNA synthesis support gives post synthesis DNA cleavage in 8 hrs or less without alkali metal salts. DNA produced with the new support was analyzed by HPLC, MALDI mass spectroscopy and PAGE. Each analysis showed DNA of equivalent quality to that produced with standard CPG supports, without contaminating materials resulting from linker or support backbone decomposition.

Versatile linker chemistry for synthesis of 3'-modified DNA.

A general method is described for the solid phase supported synthesis of DNA containing 3'-terminal phosphodiesters modified with linkers bearing either amino, thiol, or hydroxyl groups. These products are all made from a common intermediate, obtained by the reaction of trimellitic anhydride chloride with aminopropyl CPG. The anhydride-derivatized support was then reacted with three appropriate bifunctional spacers, giving DMT-protected hydroxyl solid supports bearing the masked functionality as an ester, amide, or thioester. DNA synthesis was then performed, followed by ammonia cleavage and deprotection, giving the hydroxyl-, amino-, or thiol-functionalized DNA 3'-phosphate diesters, respectively. Test mononucleotides synthesized with each of the new supports were identical with control mononucleotides made with 5'-immobilized nucleosides and alkylhydroxyl, alkylamino, and alkylthio phosphoramidites. The new supports were then used to prepare several 3'-modified oligonucleotides, which were characterized by gel electrophoresis, HPLC, and MALDI mass spectroscopy. The amino- and thiol-functionalized DNAs were conjugated with chromophores, and purification of these products was facilitated by use of reversed phase cartridges.

A new universal linker for solid phase DNA synthesis.

A method is described as an alternative to the use of nucleoside pre-functionalized supports for DNA synthesis. The procedure should allow the generation of 3'-OH terminal moieties of any natural or modified DNA fragment using a single derivatized solid support material. The method utilizes 1-O-(4,4' dimethoxytrityl)-2- O-succinoyl-3-N-allyloxycarbonylpropane immobilized on amino-propyl CPG followed by subsequent coupling of unit phosphoramidites. Work up is accomplished by removal of the 3-N-allyloxycarbonyl group [Pd(0) at 50 degrees C for 15 min] followed by cleavage under very mild conditions (aqueous TEAA/NH3 buffer pH 10, room temperature) to release the desired product. The mechanism is believed to involve nucleophilic attack of the linker-derived amino group on the 3'-phosphate triester, followed by elimination of the desired product. DNA synthesis with the new support and with classical nucleotide synthesis supports have been performed, and the products shown to be identical. Further proof of product integrity was given by MALDI mass spectral studies and the efficacy of DNA primers made with the new support in PCR amplification.

Design, synthesis, and evaluation of latent alkylating agents activated by glutathione S-transferase.

In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)-activated latent alkylating agent gamma-glutamyl-alpha-amino-beta-[[[2-[[bis[bis(2-chloroethyl)amino]ph osp horyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs.

Salt-induced immobilizations of DNA oligonucleotides on an epoxide-activated high-performance liquid chromatographic affinity support.

Synthetic oligonucleotides, possessing a recognition sequence for the transcription factor NF-kappa B, were immobilized onto an epoxide-activated hydroxyethylmethacrylate HPLC affinity support in the presence of high concentrations of potassium phosphate. The extent of immobilization increased with salt concentration in a manner analogous to that which has been reported for salt-induced immobilizations of proteins. High immobilization efficiencies were achieved, and at 2.7 M potassium phosphate, 85-90% of the DNA initially present in the reaction mixture was immobilized. Reactions were examined for double stranded DNA, one strand of which was modified with a 5'-mercaptohexyl spacer arm, and for each of the strands comprising the duplex. For double stranded immobilizations, about 85% of the non-modified strand (the d22 strand) was released from the support under melting conditions, suggesting that d22 exhibited low reactivity when organized as the duplex. For immobilizations of single stranded DNA, mild acid hydrolysis of the products was used to provide information concerning the mode of attachment. For reactions of the d22 strand alone, only about 60% each of guanine and adenine were recovered from the immobilized oligonucleotide following mild acid hydrolysis. This suggests that when d22 is immobilized as the single strand, significant attachment occurs through the purine bases, in contrast with the low reactivity exhibited by d22 in the duplex. Purified p50 protein, the DNA binding element of NF-kappa B, and nuclear extracts from phorbol ester-stimulated HeLa cells were injected onto a column packed with the double stranded product. In both cases p50 was retained on the column and was recovered upon elution with a salt gradient.

p-Nitrobenzyl side-chain protection for solid-phase synthesis.

A solid-phase supported peptide synthesis (SPPS) strategy using p-nitrobenzyl (pNB) esters, thioethers and carbamates for side-chain protection is described. The synthesis of Fmoc p-nitrobenzyl side-chain protected amino acids of lysine, cysteine, glutamic acid and aspartic acid are synthesized and incorporated into the synthesis of tetramers by standard Fmoc methodology using Wang polystyrene resins. Deprotection is carried out on resin in mildly acidic reducing conditions, using a solution of DMF, SnCl2, phenol and HOAc. The yellow by-products associated with the deprotection of the pNB protecting group are then removed by treatment with a solution of benzene sulfinic acid in DMF. The methodology is successfully extended in the synthesis of a peptide with multiple pNB protecting groups.

Modulation of detoxification gene expression in human colon HT29 cells by glutathione-S-transferase inhibitors.

We investigated the effects of glutathione-S-transferase (GST) inhibitor treatment on human colon HT29 cell mRNA levels of dihydrodiol dehydrogenase (DDH), glyoxalase I, and gamma-glutamylcysteine synthetase. Time- and concentration-dependent increases in both DDH and gamma-glutamylcysteine synthetase mRNAs resulted from treatment with ethacrynic acid, ethacrynic acid/glutathione conjugate, and T.199 (gamma-glutamyl-S-(benzyl)-cysteinyl-R(-)-phenyl glycine diethyl ester), a selective GST pi inhibitor. In contrast, glutathione analogue GST alpha- and GST mu-selective inhibitors did not induce expression of these genes. Treatment with ethacrynic acid or T.199 had no effect on the mRNA levels of the glutathione-dependent glyoxalase I gene. Pretreatment of cells with buthionine-DL-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor and glutathione depleter, coupled with ethacrynic acid, ethacrynic acid/glutathione conjugate, or T.199 resulted in greater levels of gamma-glutamylcysteine synthetase and DDH induction compared with single treatments. Treatment with buthionine-DL-sulfoximine alone resulted in modest increases in both gamma-glutamylcysteine synthetase and DDH expression. Analyses of DDH induction by both differential Northern hybridization with specific oligonucleotides as probes and reverse transcriptase-polymerase chain reaction amplification of products, followed by diagnostic restriction digestion with endonucleases, showed that ethacrynic acid induced multiple DDH transcripts in HT29 cells and human HepG2 and SKHep1 hepatoma cells. Possible induction mechanisms include the alteration of sulfhydryl status by the electrophilic properties of EA or by elevations of endogenously generated oxidative stress via transient removal of GST pi from the cytosolic GST pool.

Mutagenesis using trinucleotide beta-cyanoethyl phosphoramidites.

There is no easy way to selectively introduce mixtures of codon triplets into mutagenesis libraries. Solid-phase-supported DNA synthesis using successive coupling of mixtures of mononucleotides can be made to supply 32 codons, which gives redundancies in coding for 20 natural amino acids, as well as an often unwanted stop codon. Resin-splitting methods have been described, but the representation of all permutations is limited by mechanical factors for a large library, and the method is experimentally cumbersome. To demonstrate a third, improved method, the 3'-cyanoethyl phosphoramidite codon triplets dATA, dCTT, dATC, dATG and dAGC were made by solution-phase methods, with protecting groups fully compatible with modern automated phosphoramidite DNA synthesis chemistry. The reagents were then used to synthesize a 54-mer DNA fragment, wherein 15 internal base pairs were randomized by coupling a mixture of the five codons five times. The fragment was amplified as a cDNA pool, which was subcloned into a phagemid vector, and 16 randomly selected recombinants from this mini-library were sequenced. These clones showed random incorporation of the proper transcribed codon sequences at the correct location. Other functional tests involving the trinucleotide phosphoramidites showed modest (ca. 70%) coupling efficiencies and structural integrity of the DNA produced.

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.

Glutathione-S-transferase activates novel alkylating agents.

Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis.

Glutathione-S-transferase (GST) isozyme-selective inhibitors were designed by an empirically guided strategy. In the first phase, literature data were used to select C-terminal modifications which generated maximum variation in the catalytic efficiency (Vmax/Km) for glutathione (GSH) analogs used as substrates with different rat GSTs. Also, on the basis of literature data, the sulfhydryl group was functionalized with a selection of alkyl and aryl groups to maximize potential isozyme specificity. Affinity chromatography sorbents were prepared from these which showed isozyme selectivity for both rat tissue and recombinant human GST isozymes. Some of these compounds also showed selective inhibition of GST activity in catalysis of the reaction of 1-chloro-2,4-dinitrobenzene with GSH. In the second phase, electronic effects were explored through synthesis of an isostructural series of S-benzyl GSH ligands with different substituents on the aromatic ring. GST isozyme specificity for these ligands, measured by binding to derivatized sorbents, varied substantially, with hydrophobic substituents favoring the human GST M1a isozyme and electronegative moieties favoring GST P1. In the third phase, information obtained from testing both series of compounds was combined and used to prepare GSH analogs with chemical features responsible for isozyme specificity at both the C-terminus and the sulfur. This approach gave two new compounds which showed improved potency while still maintaining selectivity in the inhibition of GSTs. A detailed discussion of the logic used in the selection of functional groups for maximum potency and selectivity is included.

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.

Construction of affinity sorbents utilizing glutathione analogs.

Solution-phase N-fluorenylmethoxycarbonyl (Fmoc) mediated peptide synthesis has been adapted to the synthesis of glutathione (gamma-glutamyl-cysteinyl-glycine) analogs. A protecting group strategy has been devised in which all of the masking groups are removed with mild base. This allows for the synthesis of acid-sensitive materials and lessens concerns about the alkylation at sulfur by carbocations known to be present in the trifluoroacetic acid mixtures usually employed for deprotection of peptides made by the Fmoc methodology. A series of structurally varied glutathione analogs were prepared by modifying the peptide in two ways. The first involved C-terminal substitution for glycine by one of several different amino acids. The second involved substitution of one of five alkyl or aryl groups onto the cysteine sulfhydryl. The complete set of all combinations would yield 48 reagents, of which 25 have actually been synthesized. Following confirmation of the structures by FAB mass spectrometry, the peptides were immobilize by conjugation to epoxyfunctionalized Sepharose at pH 11-12. The amount and identity of immobilized peptide was assayed by amino acid analysis of acid-hydrolyzed resin. One of the tripeptides was purified by ion-exchange and preparative HPLC.

Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.

A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.

Enzymatic and NMR analysis of oligoribonucleotides synthesized with 2'-tert-butyldimethylsilyl protected cyanoethylphosphoramidite monomers.

The regioisomeric integrity of the internucleotide phosphate linkage in synthetic RNA using 2'-tert-butyldimethylsilyl protection was examined using enzymatic and NMR techniques. Two sets of DNA-RNA hybrid nonamers, T3XT5 and T5XT3 (where X = rA, rC, rG and U) and the tetramer AGCU were analyzed. Enzyme catalyzed hydrolysis of the nonamers with ribonuclease T2 showed that the linkage at the ribonucleotide was the desired 3'-5'. A control nonamer with a 2'-5' linkage was subjected to the enzyme, and showed no cleavage. High-resolution proton NMR of the tetramer also gave a favorable comparison with the same molecule obtained by non-chemical means.