Dimethoxytrityl Removal in Organic Medium: Efficient Oligonucleotide Synthesis Without Chlorinated Solvents

Abstract

Oligonucleotides are finding widespread utility in various applications in diagnostics and molecular biology and as therapeutic agents. In standard synthesis of such oligonucleotides through phosphoramidite coupling, removal of the typical acid-labile 4,4′-dimethoxytrityl 5′-protecting group (DMTr), from the support-bound oligonucleotide plays a crucial role in each synthesis cycle in achieving high product yield and oligonucleotide quality. Although several reagents have been developed for this purpose, many have limited applicability to automated oligonucleotide synthesis on solid supports. The most commonly used reagents today are dilute solutions (2–15%) of an organic acid, typically trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5) in dichloromethane. The high volatility (boiling point 40 °C) of dichloromethane and its high toxicity and carcinogenicity pose a hazard for personnel and the environment. In addition, as oligonucleotide synthesizers are now available to allow syntheses of up to 0.5 mole scale, the quantities of chlorinated waste generated have become quite large. In this context we became interested in replacing dichloromethane as deblocking reagent solvent with a less harmful solvent while preserving product yield and quality. We now report that it is not necessary to use halogenated solvents such as dichloromethane in the deblocking step of automated oligonucleotide synthesis in order to obtain high yields of high quality oligonucleotide product.     

A novel solid support for synthesis of oligonucleotide 3'-phosphorothioate monoesters

A new reagent immobilized on solid support allowing for solid-phase synthesis of oligonucleotides with a 3'-terminal phosphorothioate monoester is described. The support is compatible with phosphoramidite chemistry for automated oligonucleotide synthesis. Final deprotection with ammonia under standard conditions leads to oligonucleotide 3'-terminal phosphorothioate.     

Design and synthesis of chiral peptidic nucleic acids

Summary Due to the increasing interest in the use of oligonucleotide analogues as antisense and antigene drugs, we designed a chiral analogue constituted of a peptidic frame bearing nucleobases in suitable positions (C-PNA). We recently reported the synthesis of four nonnatural α-amino acids with the DNA bases in the lateral chain. In this paper we present an improved synthesis of the Fmoc monomers and their polymerisation to polypeptidic oligonucleotide analogues using a modification of the standard protocol for solid phase peptide synthesis.     

Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl.

The synthesis of protected nucleoside phosphoramidites bearing various markers such as biotinyl, dinitrophenyl, dansyl and pyrenyl groups are reported. These labelled deoxynucleosides phosphoramidites were used for solid phase oligonucleotide synthesis in the same way than the usual protected phosphoramidities without any change in the synthetic cycle and the deprotection step. The new labelled building blocks described herein have been used in conjunction with the labile base protected phosphoramidites ('PAC phosphoramidites') which allowed mild ammonia deprotection, especially recommended for the dinitrophenyl-labelled oligonucleotides. Multiple labelling (i.e. 10 to 20 biotins) can be efficiently and easily performed, on the same oligonucleotide which results in an increase of sensitivity. The polylabelled oligonucleotides are chemically well defined and gave increased signal and low background coloration for in situ hybridisation. The modified oligonucleotides can still be kinased in the normal way as the reporter groups are on the heterocycles.     

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.     

New insights into the de novo gene synthesis using the automatic kinetics switch approach

Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6 kbp from 1 nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656 bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74 kbp from 1 nM oligonucleotide.     

Synthesis and application of a novel, crystalline phosphoramidite monomer with thiol terminus, suitable for the synthesis of DNA conjugates

A new, crystalline 5'-thiol modifier phosphoramidite monomer (3), suitable for DNA synthesis, has been prepared. This monomer has been built into an oligonucleotide using the standard protocol. After cleavage, purification and removal of the trityl group with Ag(+), a free 5'-thiol terminal oligonucleotide (15) has been obtained which was subsequently coupled to a cysteine derivative via a disulfide bridge to afford conjugate 16.     

PCR-generated padlock probes detect single nucleotide variation in genomic DNA

Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5′- and 3′-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.     

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use.     

STEP-BY-STEP CONTROL BY MALDI-TOF MS OF AN OLIGONUCLEOTIDE SYNTHESIS ON STANDARD CPG SOLID-SUPPORT

MALDI-TOF mass spectrometry was used to monitor DNA solid-phase synthesis on long-chain alkylamine controlled-pore glass (LCAA-CPG) with a standard succinyl linker between the solid support and the growing oligonucleotide.     

Step-by-step control by MALDI-TOF MS of an oligonucleotide synthesis on standard CPG solid-support

MALDI-TOF mass spectrometry was used to monitor DNA solid-phase synthesis on long-chain alkylamine controlled-pore glass (LCAA-CPG) with a standard succinyl linker between the solid support and the growing oligonucleotide.     

Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.     

Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis.

Fully protected CPG-immobilized monomer, dimer and trimer oligonucleotides were used to study depurination during the chemical synthesis of oligonucleotides. Disappearance of the oligonucleotide during acid exposure time relative to an internal thymidine standard not subject to depurination was monitored by reverse phase HPLC analysis. Depurination half-times obtained for dichloroacetic acid (DCA) and trichloroacetic acid (TCA) in methylene chloride were found to be 3% DCA >> 15% DCA > 3% TCA. In order to understand the implications of depurination during DNA synthesis, the detritylation kinetics of model compounds DMT-dG-pT dimer and DMT-[17mer] mixed-base sequence were also measured. These results improve our ability to properly balance the contradictory goals of obtaining maximum detritylation with minimum depurination in oligonucleotide synthesis.     

Reagents for the preparation of two oligonucleotides per synthesis (TOPS).

In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.     

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy.     

Preactivated carboxyl linker for the rapid conjugation of alkylamines to oligonucleotides on solid support

A C10 linker phosphoramidite reagent terminated with a succinimidyl-activated carboxyl group was prepared and used to couple to the 5'-end of an oligonucleotide synthesized on a solid support. The succinimidyl-activated carboxyl functionality can be used for rapid conjugation of amines to oligonucleotides on solid support or it can be hydrolyzed to form a carboxylic acid functionality. The activated linker was successfully used for conjugation of several primary and secondary aliphatic amine derivatives (including biotin and fluorescein cadaverine) onto a solid support-bound 12-mer DNA oligonucleotide at scales ranging from 0.15 to 1.0 micromol. The overall yields of the conjugation products after AMA deprotection and cleavage from the solid support ranged from 43 to 75% of the total oligonucleotide product. This value is significant, as it includes oligonucleotide synthesis, coupling of the linker, and conjugation of the amine. In addition, the entire process of oligonucleotide synthesis, linker coupling, amine conjugation, deprotection, and cleavage of the oligonucleotide from solid support can be accomplished in 1 day.     

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 μm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays.     

Synthesis and hybridization properties of oligonucleotides containing 2'-O-modified ribonucleotides.

A versatile, general way is described for the introduction of different functional groups into oligonucleotides by means of a simple linker at the 2'-position of the sugar. Nucleotide building blocks carrying lipophilic, intercalating or tertiary amino groups can be placed deliberately at any desired position of oligonucleotides by standard automated oligonucleotide synthesis. Thermal denaturation studies with these oligonucleotides reveal the following general trends: i) Modification with lipophilic n-octyl groups has little if any effect on duplex stability; a destabilizing (lipophilic) substituent is better tolerated at or near the ends than in the middle of the oligo. ii) An intercalating substituent (2-aminoanthraquinone) substantially increases duplex stability. iii) N,N-Dimethyl amino residues also increase duplex stability though to a smaller extent than intercalating residues. iv) Modifications at the 5'-end have a more pronounced influence on the TM than the corresponding 3'-modifications. v) Oligonucleotides modified in such a way show little or no loss in sequence specificity.