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In the interest of pursuing the synthesis of 2′‐O‐carbamoylmethyl‐modified oligonucleotides from building blocks already containing the modification, we have performed studies on the chemical and enzymatic stability of a 2′‐O‐carbamoylmethyl‐modified dinucleotide. The dinucleotide was subjected to ammonolysis and other basic conditions such as treatment with ethylenediamine and sodium hydroxide at different temperatures. The amide of the carbamoylmethyl group partially hydrolyzed under standard deprotection conditions for oligonucleotide synthesis (conc. aq. NH3at 55 °C). However, under several other conditions, including saturated NH3in methanol, the amide remained intact. Treatment of the dinucleotide with Phosphodiesterase I from Crotalus adamanteusvenom and Phosphodiesterase II from Bovine spleen, showed that the carbamoylmethyl moiety gives substantial protection of the phosphodiester towards enzymatic degradation.

Fmoc-histidine derivatives protected with 2,6-dimethoxybenzoyl (2,6-Dmbz) units, either on the τ- (1) or on the π-position (2) of the imidazole residue, have been prepared and applied to the synthesis of the dipeptide Fmoc-His(2,6-Dmbz)-Val-OMe, with high coupling efficiencies (99 and 95%, respectively) and low racemization (0.3 and 0.1%, respectively), as ascertained by HPLC analysis on the fully protected dipeptides. In addition, the hexapeptide H-Ala-Ser-Val-His-Val-Phe-OH (I) has been synthesized on a solid support employing the novel building blocks 1 and 2 as well as commercially available Fmoc-His(τ-Trt)-OH (3). The crude deprotected products were analyzed by reverse-phase HPLC and mass spectrometry, which indicated that when the τ-protected derivatives 1 and 3 were employed the products were of comparable quality (93−94% major peak). The hexapeptide synthesized with the π-protected histidine 2 was contaminated with a product corresponding to 2,6-Dmbz-His-Val-Phe-OH (12), which was probably formed through the intramolecular acyl transfer of a 2,6-Dmbz unit from the π-nitrogen atom to the α-amino group of the N-terminal histidine unit upon removal of the Fmoc group. This side reaction was insignificant (less than 0.5%) when using the τ-protected derivative 1. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Studies of the stabilities of a series of N-acylimidazoles towards acidic and basic conditions of potential usefulness for the removal of common temporary protection in peptide and oligonucleotide synthesis are presented. N-Acylimidazoles with a variety of substituents in the acyl component were prepared and treated with 3% trifluoroacetic acid (TFA) in chloroform and with 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in N,N-dimethylformamide (DMF), the extent of their degradation being determined by proton NMR. N-(2,4,6-Trimethylbenzoyl)imidazole (1) and N-(2,6-dimethoxybenzoyl)imidazole (2) remained unaffected under the above acidic and basic conditions after 4 d and 2 d, respectively. In addition, 1 and 2 were resistant to treatment with a solution of 2% piperidine/2% DBU in DMF for 24 h. Under ammonolytic conditions, 2 was rapidly cleaved (less than 1 h), whereas 1 was 64% degraded after 48 h, as ascertained by reversed-phase HPLC. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

A novel, N-(pyrrolidinyl-2-methyl)glycine-based (Pmg-based) PNA is introduced. The synthesis of the backbone was accomplished in good yield, starting from prolinol. Thymine (S)- and (R)-Pmg and adenine- and cytosine-derived (R)-Pmg monomers were prepared. Five different fragments − two with either the (R) or the (S) isomer of the thymine Pmg monomer, two oligomers with two consecutive (R)- or (S)-thymine Pmg units, and fully modified (R)-Pmg decamer − were assembled on a solid support. UV thermal melting experiments with complementary DNA and RNA were performed in order to determine the effects of conformational restriction, steric hindrance, and chirality on the duplex stability. It was found that the (R)-Pmg-containing PNAs bound better to DNA and RNA than those containing the (S)-Pmg isomer and that both (S)- and (R)-Pmg-containing PNAs preferentially bound to complementary RNA with a selectivity higher than that of 2-(aminoethyl)glycine (Aeg) PNA. However, even (R)-Pmg-containing oligomers showed poorer duplex stability than nonmodified Aeg-PNA, while no detectable binding either to the DNA or to the RNA was observed with the fully modified (R)-Pmg decamer. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

A novel, N-(pyrrolidinyl-2-methyl)glycine-based (Pmg-based) PNA is introduced. The synthesis of the backbone was accomplished in good yield, starting from prolinol. Thymine (S)- and (R)-Pmg and adenine- and cytosine-derived (R)-Pmg monomers were prepared. Five different fragments − two with either the (R) or the (S) isomer of the thymine Pmg monomer, two oligomers with two consecutive (R)- or (S)-thymine Pmg units, and fully modified (R)-Pmg decamer − were assembled on a solid support. UV thermal melting experiments with complementary DNA and RNA were performed in order to determine the effects of conformational restriction, steric hindrance, and chirality on the duplex stability. It was found that the (R)-Pmg-containing PNAs bound better to DNA and RNA than those containing the (S)-Pmg isomer and that both (S)- and (R)-Pmg-containing PNAs preferentially bound to complementary RNA with a selectivity higher than that of 2-(aminoethyl)glycine (Aeg) PNA. However, even (R)-Pmg-containing oligomers showed poorer duplex stability than nonmodified Aeg-PNA, while no detectable binding either to the DNA or to the RNA was observed with the fully modified (R)-Pmg decamer. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

The introduction of a urea bond linking a protected diethylenetriamine (DETA) unit and the terminal amino group of a resin-bound peptide nucleic acid (PNA) decamer gave access to a PNA – DETA adduct (shown here), which hydrolyzed the target 25-mer RNA rapidly and sequence specifically.

Die Einführung einer Harnstoffgruppe zur Verknüpfung einer geschützten Diethylentriamin(DETA)-Einheit mit der terminalen Aminogruppe eines harzgebundenen Peptidnucleinsäure(PNA)-Decamers führte zu einem PNA-DETA-Addukt (siehe Bild), das ein RNA-25mer rasch und sequenzspezifisch hydrolysiert.

AbstractAutomated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.

A convenient route to the preparation of 4-methoxy-1-oxido-pyridine-2-methyl N-2-(4,4′-dimethoxytrityloxy)ethyl- N-thymin-1-yl-aminomethylphosphonate ( 1a, T ∗) and the corresponding N4-benzoylcytosin-1-yl derivative 1b(C ∗) is reported. These PPNA monomers proved to be suitable building blocks in a solid-support synthesis of the tetradecameric fragment (C ∗T ∗T ∗T ∗C ∗T ∗T ∗T ∗T ∗C ∗T ∗C ∗T ∗)dT.

An automated online solid-phase synthesis of Ac-cac ct T ∗GG TC t ∗ac ct-Gly-OH 1using standard DNA and appropriately protected PNA building blocks ( 2–5) is described. This chimera forms stable duplexes with complementary DNA and RNA.

Condensation of a properly protected uridylylated nucleopeptide with a heptadecapeptide gives after one-step deprotection the target nucleopeptide H-Gly-Ala-Tyr(5′pU)-Thr-Gly-Leu-Pro-Asn-Lys-Lys-Pro-Asn-Val-Pro-Thr-Ile-Arg-Thr-Ala-Lys-Val-Gln-OH.

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Before Integration of the human immunodeficiency virus (HIV) DNA, two nucleotides are removed from the 3′ ends of the viral DNA by the integrase (IN) protein. We studied the chemistry of this reaction, and found that IN mediates site-specific hydrolysis of a phosphodiester bond, resulting in release of a dinucleotide. A class of alcohols (including glycerol, 1,2-propanediol, but not 1,3-propanediol) can also act as nucleophile In this reaction, and likewise the alcoholic amino acids L-serine and L-threonine can be covalently linked to the dinucleotide. No evidence was found for a covalent linkage between the IN protein and this dinucleotide, suggesting that IN directs a single nucleophilic attack of water at the specific phosphodiester bond.

We have previously shown that nuclear DNA of bloodstream form Trypanosoma brucei contains a novel base β-glucosyl-hydroxymethyluracil, called J. Base J is enriched in minichromosome fractions but not in the minichromosome internal repeats, suggesting the association of J with telomeric DNA. To test whether J is present in the long telomeric (GGGTTA)n repeat arrays, which are 2–26 kb in T.brucei, we have purified these arrays both by hybrid selection and by isolating 2–26 kb fragments from DNA digested with multiple restriction enzymes. We find that in purified telomeric repeats ∼13% of T is replaced by J, compared to 0.8% in total DNA, and we estimate that ∼50% of the total J is in these repeats. Highly purified complementary strands of the repeats were obtained by alkaline CsCl equilibrium centrifugation. In the (TAACCC)n strand 14% of T was replaced by J. In the (GGGTTA)n strand ∼36% of the second T was replaced by J; the first T was not detectably replaced. Modified bases have not been found in telomeric repeats before. How the bulky base J affects telomere function and structure in bloodstream form trypanosomes remains to be determined.