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5-Formyl-dC (III) CE-Phosphoramidite

5-Formyl-dC (III) CE-Phosphoramidite

Phosphoramidite used to incorporate a 5-formyl-modified deoxycytidine into an oligonucleotide for epigenetic studies.
  • Useful in epigenetic pathway studies.
  • In DNA, cytidine is methylated to form 5-mdC. This is oxidised to 5-hydroxymethyl-dC, then to 5-formyl-dC, then to 5-carboxy-dC.
  • Both 5-carboxy-dC and 5-formyl-dC can be converted back to dC via thymidine DNA glycosylase mediated base excision repair.
  • 5-Formy-dC III has been designed to meet all of the requirements to prepare an oligo containing all of the methylated variants.
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Product information

Epigenetics is the study of heritable silencing of genes where there is no change to the coding sequence. Interest in this area has grown significantly over the past few years particularly looking at changes induced and sustained by non-coding RNA gene silencing, histone modification and DNA methylation of cytidine in CpG islands.(1) At Biosearch Technologies, we offer a range of modifiers for this purpose. In particular we have amidites of 5-hydroxy-dC, 5-hydroxy-dU, 5-hydroxymethyl-dU, 5-hydroxymethyl-dC, 5-carboxy-dC, 5-formyl-dC, 5-hydroxymethyl-dC II and 5-formyl-dC III for use in the study of oxidative damage and repair, methylation and epigenetics.

Oxidised pyrimidines such as 5-hydroxy dU and 5-hydroxy dC are derived from dC via oxidative metabolic processes, UV or ionising radiation to form 5-HO-dC which spontaneously undergoes deamination to form 5-HO-dU.

Although there are repair mechanisms to convert 5-HO pyrimidines back to dC,(2) the fact that they are observed in cellular DNA at consistent levels suggests that these repair mechanisms are inefficient,(3) at least in certain cell types. Oligonucleotides modified with 5-hydroxy dU or 5-hydroxy dC are useful in understanding such processes.

The presence of either 5-HO-dU or 5-HO-dC can both lead to mutations resulting from their ability to mismatch with A and A/C respectively hence where the repair mechanism fails, such mutations can be permanently incorporated into the resulting gene. 5-Hydroxymethyl-dU (5-hmdU) is also a result of oxidative process or ionizing radiation but in this case dT is modified.(4) It is also possible that 5-hmdU is formed by deamination of 5-hmdC but Müller and Carell recently showed that this does not contribute to the steady state levels of hmdU in mouse embryonic stem cells, but that dT is a substrate for ten eleven translocation enzymes (Tet) leading to the formation of 5-hmdU.(5) Hence, hmdU is an important reagent for the study of both oxidative processes and epigenetics.

Once incorporated into an oligonucleotide, these modifiers represent the various products in the biochemical pathway of the modification of dC. In DNA, cytidine is methylated by a DNA methyl transferase catalysed reaction with S-adenosylmethionine to form 5-mdC. This is oxidised by Tet enzymes to 5-hydroxymethyl-dC which is further oxidised to 5-formyldC, which in turn is further oxidised to 5-carboxy-dC. Both 5-carboxy-dC and 5-formyl-dC can be converted back to dC via thymidine DNA glycosylase mediated base excision repair.(6). The first generation hmdC phosphoramidite was fairly very well accepted but requires fairly harsh deprotection conditions. Therefore, a second generation building block (5-Hydroxymethyl-dC II) was developed that is compatible with UltraMild deprotection.(7) 5-Formy-dC III has been designed to meet all of the requirements to prepare an oligo containing all of the methylated variants.(8)

Ref:

  1. Epigenetics in human disease and prospects for epigenetic therapy, G. Egger, G. Liang, A. Aparicio and P.A. Jones. Nature, 429, 457-463, 2004.
  2. Base excision repair in a network of defence and tolerance, H. Nilsen and H.E. Krokan, Carcinogenesis, 22, 987-998, 2001.
  3. Endogenous oxidative damage of deoxycytidine in DNA, J.R. Wagner, H. Chia-Chieh and B.N. Ames, Proc. Nat. Acad. Sci., 89, 3380-3384, 1992.
  4. Oxidative damage to DNA: formation, measurement, and biological significance, J. Cadet, M. Berger, T. Douki and J.-L. Ravanat, Rev. Physiol. Biochem. Pharmacol., 131, 1-87, 1997.
  5. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA, T. Pfaffeneder, F. Spada, M. Wagner, C. Brandmayr, S.K. Laube, D. Eisen, M. Truss, J. Steinbacher, B. Hackner, O. Kotljarova, D. Schuermann, S. Michalakis, O. Kosmatchev, S. Schiesser, B. Steigenberger, N. Raddaoui, G. Kashiwazaki, U. Müller, C.G. Spruijt, M. Vermeulen, H. Leonhardt, P. Schär, M. Müller and T. Carell, Nat. Chem. Biol., 10 (7), 574-81, 2014.
  6. Tet enzymes, TDG and the dynamics of DNA methylation, R.M. Kholi and Y. Zhang, Nature, 502, 472-479, 2013.
  7. Efficient Synthesis of 5-Hydroxymethylcytosine Containing DNA, M. Münzel, D. Globisch, C. Trindler and T. Carell, Org. Lett., 12, 5671–5673, 2010.
  8. Synthesis of a DNA promoter segment containing all four epigenetic nucleosides: 5-methyl-, 5-hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine, A.S. Schröder, J. Steinbacher, B. Steigenberger, F.A. Gnerlich, S. Schiesser, T. Pfaffeneder and T. Carell, Angewandte Chemie-International Edition, 53, 315-318, 2014.

Applicable Products

LK2541 5-Hydroxy-dU-CE Phosphoramidite
LK2542 5-Hydroxymethyl-dU-CE Phosphoramidite (hmdU)
LK2543 5-Hydroxy-dC-CE Phosphoramidite
LK2544 5-Hydroxymethyl-dC-CE Phosphoramidite (hmdC)
LK2545 5-Carboxy-dC-CE Phosphoramidite
LK2546 5-Formyl-dC-CE Phosphoramidite
LK2547 5-Hydroxymethyl-dC II-CE Phosphoramidite
LK2548 5-Formyl-dC III-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK2541 C41H49N4O10P 788.83 306.17 3.17 6.34 12.68
LK2542 C42H51N4O10P 802.86 320.19 3.11 6.23 12.46
LK2543 C53H56N5O10P 954.03 305.18 2.62 5.24 10.48
LK2544 C50H57N6O9P 917.00 319.21 2.73 5.45 10.91
LK2545 C49H56N5O10P 905.97 333.19 2.76 5.52 11.04
LK2546 C47H58N5O12P 915.96 317.19 (Formyl), 349.23 (Diol) 2.73 5.46 10.92
LK2547 C41H48N5O9P 785.82 319.21 3.18 6.36 12.73
LK2548 C51H60N5O11P 950.02 317.19 (Formyl), 375.27 (Acetal) 2.63 5.26 10.53

Coupling

A coupling time of 25-60s is recommended for all amidites, except for LK2548 which should be coupled for 180s.

Cleavage & Deprotection

Recommendations are specific to each product, but will also need to take into account the requirements of other bases and modifications in the oligo.

LK2541 - Use Fast or UltraMILD protection on the nucleobases (plus UltraMILD Cap A, LK4210). Cleave and deprotect with ammonium hydroxide at RT for 24h.

LK2542 - Use Fast or UltraMILD protection on the nucleobases (plus UltraMILD Cap A, LK4210). Cleave and deprotect with AMA at 65ºC for 10min (although this is not compatible with Bz-dC), or use ammonium hydroxide at RT for 2h, then deprotect the nucleobases as required.

LK2543 - Use UltraMILD protection on the nucleobases (plus UltraMILD Cap A, LK4210). Cleave and deprotect with 0.05M Potassium carbonate in methanol at RT for 4h, or ammonium hydroxide at RT for 2h.

LK2544 - Any nucleobase protection can be used, however the product is not compatible with AMA or UltraMILD deprotection methods. Cleavage and deprotection is with 30% ammonium hydroxide at 75ºC for 17h.

LK2545 - Use a combination of iBu-dG and Ac-dC nucleobase protection, or UltraMILD (plus UltraMILD Cap A, LK4210). Cleave and deprotect with 0.4M NaOH in MeOH/water 4:1 (v/v) at RT for 17h.

LK2546 - Any nucleobase protection can be used, dependent on the deprotection strategy required. Typically, use ammonium hydroxide at 55ºC for 17h, AMA at 65ºC for 10 min, or 0.4 M NaOH in MeOH/water 4:1 (v/v) at RT for 17h. Then oxidise with 50mM sodium periodate at 4ºC for 30min, and desalt (G25).

LK2547 - Either utilise iBu-dG/Ac-dC base protection and deprotect with 0.4M NaOH in MeOH/water 4:1 at RT for 17h, or use UltraMILD deprotection (plus UltraMILD Cap A, LK4210) and deprotect with 0.05M potassium carbonate in methanol at RT for 4h.

LK2548 - Either utilise dmf-dG/Ac-dC base protection and deprotect with 30% ammonium hydroxide at RT for 17h and immediately evaporate, or employ iBu-dG/Ac-dC or UltraMILD (plus UltraMILD Cap A, LK4210) and deprotect with 0.4 M NaOH in MeOH/water 4:1 (v/v) at RT for 17h, desalt (G25) and evaporate. Subsequently, remove the acetal protecting group using 80% acetic acid in water at 20ºC for 6h.

Storage & Stability

All products are stored dry in a freezer at -10 to -30°C, or short term at 2-8ºC (except LK2548 where -10 to -30°C is required). Stability in solution is 2-3 days, except for LK2546 and LK2548 which must be used with 1-2 days.

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