Solid-Phase Supports for Oligonucleotide Synthesis

Pon, Richard T.

doi:10.1385/0-89603-281-7:465

Cited by 23 publications

(10 citation statements)

References 1 publication

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The appropiate 3'-(hydrogen succinate) of the 2'-deoxycytidine derivative 14 exhibited a high sensitivity of the dimethoxytrityl residue towards even small amounts of acid and could, therefore, not be isolated by chromatographic means. Therefore, its support had to be built up in the reverse manner, forming first the LCAMA-CPG-derived succinamic acid 22 according to Pons protocol [29], followed by an EDC-promoted (…”

Section: Methodsmentioning

confidence: 99%

Nucleotides, Part LXI, Phthaloyl Strategy: A New Concept of Oligonucleotide Synthesis

Beier

Pfleiderer

1999

HCA

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Nucleotides, Part LXI, Phthaloyl Strategy: A New Concept of Oligonucleotide Synthesis

Beier

Pfleiderer

1999

HCA

View full text Add to dashboard Cite

“…[6] Figure 3 shows HPLC chromatogram data of incomplete dephosphorylation of the US and UL ODNs (panels A and B, respectively) where the linker arms are still attached to the 3 end of each ODN. US-linker has a shorter retention time at 3.82 minutes compared with the UL-linker at 4.78 minutes, due to its less hydrophobic methyl side group.…”

Section: Odn Universal Linker Dephosphorylationmentioning

confidence: 99%

“…Furthermore, the universal linker allows for a wide variety of specialized monomers to be coupled at the 3 end that cannot be achieved with standard support. What also makes the universal linker unique is that once it is cleaved from the support it must go through a rate-limiting process of dephosphorylation or phosphate intramolecular cyclization [6] in order to dissociate completely from the ODN, generating a 3 hydroxyl. Incomplete dephosphorylation, for example, results in a linker-bound ODN, which inhibits biological activity; applications such as PCR require that primers maintain this hydroxyl group for polymerase recognition and strand elongation.…”

Section: Introductionmentioning

confidence: 99%

“…[6] Typically, the end-user manually pipettes NH 4 OH into each individual column/well; after which, the samples are capped off to prevent loss of ammonia through evaporation. With a minimum two-day turnaround, this entails cleavage from the support (1-2 hours at room temperature, nucleoside deprotection (17 hours at 55 • C) and lyophilization of final product (4 hours) to remove residual ammonia.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Gas-Phase Cleavage and Dephosphorylation of Universal Linker-Bound Oligodeoxynucleotides

Jensen

Anderson

Davis

2010

Nucleosides, Nucleotides & Nucleic Acids

View full text Add to dashboard Cite

While base-specific support is commonly used for single-column oligodeoxynucleotide synthesis, the universal linker is critical for high-throughput synthesis of potentially thousands of samples in a single run. Here, we report conditions for cleavage and complete dephosphorylation of two commercial universal linkers, UnySupport and UnyLinker, processed in the gas phase (NH(3)) using our custom device. First, we compared the average yield of T10mers over time (15, 30, 60, 120, and 240 minutes, 40 psi, 80°C and 90°C). For samples processed with water added prior to incubation, we discovered a substantial increase in yield compared to those left dry (up to 55%). This was also the case for samples subjected to increases in chamber pressure (10, 20, 30 and 40 psi, 120 minutes, 80°C and 90°C). Next, we compared the effects of increased temperature, pressure and incubation times on the rates of dephosphorylation. We found the optimum conditions to be either 10 psi, 120 minutes at 80°C or 60 minutes at 90°C; in both cases, water added to columns prior to incubation had a substantial effect on rate of reaction as well as overall yield compared with those left dry. Finally, performance between the two linkers was similar enough to conclude each fulfills the desired requirements for mainstream, high-throughput oligodeoxynucleotide cleavage/deprotection and dephosphorylation in the gas phase.

show abstract

“…1 or 2) to an insoluble amino derivatized support via a dicarboxylic acid linker arm. 1 The required amide linkage can be produced using a number of different carbodiimide [2][3][4][5][6][7] or other coupling reagents 8,9 . However, the need for synthetic oligonucleotides in either large numbers (millions/yr for research) or large scales (tonnes/yr for therapeutics) also requires that new methods be developed to avoid environmentally unfriendly materials, such as pyridine or halogenated solvents, and to minimize cost, especially consumption of expensive nucleosides.…”

mentioning

confidence: 99%

Efficient and Rapid Coupling of Nucleosides to Amino Derivatized Solid-Phase Supports

Pon¹,

Yu²

1999

Synlett

Self Cite

View full text Add to dashboard Cite

High loading efficiency (up to 96%) and rapid coupling (5 min) of nucleosides to amino solid-phase supports can be obtained using HBTU and DMAP as coupling reagents in acetonitrile. Pyridine or halogenated solvents can be eliminated and the method is amenable to both automation and large-scales.Solid-phase oligonucleotide synthesis begins with covalent attachment of a nucleoside (i.e. 1 or 2) to an insoluble amino derivatized support via a dicarboxylic acid linker arm. 1 The required amide linkage can be produced using a number of different carbodiimide 2-7 or other coupling reagents 8,9 . However, the need for synthetic oligonucleotides in either large numbers (millions/yr for research) or large scales (tonnes/yr for therapeutics) also requires that new methods be developed to avoid environmentally unfriendly materials, such as pyridine or halogenated solvents, and to minimize cost, especially consumption of expensive nucleosides. Unfortunately, most coupling procedures fail to satisfy these criteria, since a large excess (typically 3-10 fold) of nucleoside in either pyridine or halogenated solvents is commonly required. One study, by Bhongle and Tang 3 , has considered these requirements. They reduced the pyridine concentration to just 5% and increased the nucleoside incorporation efficiency to 85-90%. However, this method was slow and required overnight coupling.Our laboratory has been developing new linker arms 10 , reusable supports 11 , and rapid coupling methods 12 to improve the efficiency of oligonucleotide synthesis. One of our goals is a coupling method, which meets all of the above requirements, and is fast enough to allow automated "in situ" nucleoside derivatization. This will allow high throughput synthesizers to use inexpensive "universal" amino supports. Previously, we found that a variety of common uronium or phosphonium peptide synthesis reagents could be used to rapidly couple nucleosides to solid-phase supports. However, the nucleoside incorporation efficiency was poor and a large excess (~ 4-fold) of nucleoside was required 12 . Since then, we have greatly improved nucleoside incorporation efficiency (up to 96%) and further reduced coupling times.We began by repeating the conditions, reported by Bhongle and Tang, for maximum nucleoside incorporation efficiency using DIC and HOBT 3 with the long chain alkylamine controlled pore glass (LCAA-CPG) support (101 µmol NH 2 groups/g of support) used in our laboratory. Thus, nucleoside 1a (0.1 mmol) was coupled overnight with DIC/HOBT to LCAA-CPG (1 g) to produce a nucleoside loading of 69 µmol/g (loadings were determined by trityl analysis with an accuracy of ± 5% 1 ). Although the 70% nucleoside incorporation was less than the 80-90% efficiency reported by Bhongle and Tang 3 , this was probably due to differences between the CPG supports used. This result was used as a benchmark for our subsequent coupling conditions. We found that we could obtain the same result, with the same amount and concentration of nucleoside, in only five minutes instead of...

show abstract

Solid-Phase Supports for Oligonucleotide Synthesis

Cited by 23 publications

References 1 publication

Nucleotides, Part LXI, Phthaloyl Strategy: A New Concept of Oligonucleotide Synthesis

Nucleotides, Part LXI, Phthaloyl Strategy: A New Concept of Oligonucleotide Synthesis

Gas-Phase Cleavage and Dephosphorylation of Universal Linker-Bound Oligodeoxynucleotides

Efficient and Rapid Coupling of Nucleosides to Amino Derivatized Solid-Phase Supports

Contact Info

Product

Resources

About