Oligo Synthesis : CEPs

bases affecting duplex stAbility

C-5 methyl pyrimidine nucleosides are known to stabilize duplexes relative to the non-methylated bases. Therefore, enhanced binding can be achieved using 5-methyl-dC in place of dC, duplex melting temperature being increased by 1.3°. Improved stacking in this case is believed to be brought about by elimination of water molecules from the duplex. 2,6-Diaminopurine 2"-deoxyriboside (2-amino-dA) forms an additional hydrogen bond with Thymidine, thereby leading to duplex stabilization with a melting temperature increase of 3°. Our 2-amino-dA monomer exhibits fast and effective deprotection in ammonium hydroxide and it is stabilized to depurination during synthesis. Sequences with high GC content may contain mismatches and still hybridize because of the high stability of the G-C base pair. The N4-ethyl analogue of dC (N4-Et-dC) hybridizes specifically to natural dG but the stability of the base pair is reduced to about the level of an AT base pair.

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DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

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Glen Report volume 26 number 1 May 2014

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MSDS

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One of the earliest minor bases introduced by Glen Research was 5-Me-dC-CE Phosphoramidite. 5-Me-dC has always been interesting to researchers because it stabilizes duplexes relative to dC by 1.5° per insertion. This desirable effect is attributed to the hydrophobic nature of the C5 methyl group which helps exclude water from the duplex. Antisense and diagnostic developers have long made use of 5-Me-dC as a simple and relatively inexpensive substitution for dC.

More recently, 5-Me-dC has generated interest in its own right in the burgeoning field of epigenetics. Methylation of dC usually occurs in areas with high concentration of CG bases (CpG islands) and the transformation of dC to 5-Me-dC is mediated by specialized DNA methyltransferase (DNMT) enzymes. Once methylated, 5-Me-dC can then be oxidized to form, consecutively, 5-hydroxymethyl-dC, 5-formyl-dC, and 5-carboxy-dC, all modified nucleosides of immense interest to epigenetics researchers.

Our existing 5-Me-dC monomer (1) with the N4-amine protected with a benzoyl group has served the research community well for decades. However, this monomer is not compatible with deprotection using AMA. As shown in Figure 2, deprotection of a simple oligo containing benzoyl protected 5-Me-dC leads to around 7% of the N4-Me mutation caused by displacement of benzamide by methylamine. In addition, the benzoyl protecting group is not compatible with UltraMild deprotection since it is not removed by potassium carbonate in methanol.

To remedy these shortcomings, we are introducing the acetyl-protected 5-Me-dC monomer, Ac-5-Me-dC-CE Phosphoramidite (2). This monomer is fully compatible with AMA deprotection and none of the N4-Me mutation is observed on deprotection, as shown in Figure 2. The N4-acetyl protecting group is also completely removed under the conditions of UltraMild deprotection.

We are happy to offer the acetyl-protected monomer in addition to its older benzoyl-protected cousin.

Historically, we have offered a CPG support for the benzoyl-protected 5-Me-dC. However, the acetyl version is fully compatible with AMA deprotection and, therefore, a universal support like Glen UnySupport can be used.

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