Glen UnySUPPORT

Our original Universal Support (20-5000) has been discontinued since complete dephosphorylation using ammonium hydroxide, AMA and anhydrous methylamine gas takes longer than most companies wish to allocate. A recent development has been the use of a support based on a molecule which is “conformationally preorganized” to accelerate the dephosphorylation reaction.^1,2 By using a rigid bicyclic molecule on the support, it was hoped that the molecule’s conformation would facilitate the formation of the cyclic phosphate transition state, thereby stimulating the rate of dephosphorylation. It was found that the rate of elimination is markedly faster than the original Universal Support.

The structure of Glen UnySupport is shown below. The N-phenyl version, developed at Isis Pharmaceuticals as UnyLinker™, is available from several companies for large scale oligo synthesis. Glen UnySupport is the N-methyl version, which is preferred for high throughput oligonucleotide synthesis since methylamine rather than aniline is formed on deprotection. We are happy to offer Glen UnySupport in a variety of popular formats under license from Isis Pharmaceuticals.

UNIVERSAL SUPPORT

Traditional procedures in oligonucleotide synthesis require that the solid support contains the first nucleoside which is destined to become the nucleoside at the 3’-terminus of the synthetic oligonucleotide. This situation therefore requires that an inventory of all four regular nucleoside supports must be maintained. At the same time, oligonucleotides with unusual nucleosides, available as phosphoramidites but not as supports, at the 3’-terminus can not be readily prepared. However, the most worrisome aspect of this situation is the potential for a mistake to be made in the selection of the column containing the 3’-nucleoside. This potential for error may be fairly low in regular column-type synthesizers, but it is especially significant in the new generation of parallel synthesizers where 96, 192 wells or even more may contain all four supports in a defined grid.

A universal support for preparing regular oligonucleotides must allow the elimination, during the cleavage and deprotection steps, of the terminal phosphodiester linkage along with the group originally attached to the support.

A support which has the potential to be universal for the production of oligonucleotides containing a 3’-hydroxyl group has been described.1 The base-mediated elimination of the terminal phosphodiester group must proceed promptly under conditions comparable with routine deprotection strategies and some suggestions are shown to the left.

Universal Support II / III

The key step in the use of any universal support in oligonucleotide synthesis is the dephosphorylation of the 3’-phosphate group to form the desired 3’-hydroxyl group. Azhayev1,2 has excelled in the investigation of neighboring group assistance in the dephosphorylation reaction. Amide groups may be considered to be weak N-H acids and can display basic properties in ammonium hydroxide or aqueous methylamine. (±)-3-Amino-1,2-propanediol was used to form a novel universal support. In our original US II support, a succinate linker attaches the 3-amino group to the support and the 2-OH is protected with a base-labile group to set up an amide assisted elimination in mildly basic conditions. In this way, the dephos­­phor­ylation reaction would eliminate the desired 3’-OH oligonucleotide into solution and the product of any ß-elimination competing side reaction would remain bound to the support. A further improvement has been achieved by using a carbamate group to connect the universal linker to the support, now called Universal Support III. The structures of the two supports are shown below right. Because the universal linker is unchanged and the succinate or carbamate groups remain attached to the support, we use the same catalog numbers for US II and III. Using Universal Support II or III, an oligo yield of > 80% can be achieved on CPG supports and > 95% on polymeric supports, with purity equivalent to the same oligo prepared normally.

Conditions for Cleavage and Deprotection are outlined in the table opposite. Universal Support II/III has been shown to generate oligonucleotides with the same efficacy in polymerase extension reactions as regular oligos. Despite the mild elimination reaction, oligonucleotides up to 75mer in length can be prepared routinely without loss of oligo during the synthesis cycles. This support is also used for the production of siRNA oligos.

Q-SUPPORTS

Oligonucleotides are routinely prepared on supports to which the first nucleoside is attached via a succinate linkage. Over the years, the succinate linkage has demonstrated stability during the synthesis process but has sufficient lability to be cleaved quickly in the deprotection step. However, if the cleavage step is carried out with ammonium hydroxide manually or on the synthesizer, it consumes one hour of precious time while releasing only about 80% of the oligonucleotide. This step is, therefore, a bottleneck in the productivity of many synthesis groups.

Is it possible to find a replacement to the succinate group which offers good stability to the synthesis reagents while offering a much faster cleavage step? The oxalate group has been shown to be very labile during cleavage but its stability to the normal synthesis reagents is not good, requiring changes for successful use. In a practical but elegant study¹ of various bifunctional carboxylic acids, Richard Pon’s group identified hydroquinone-O,O’-diacetic acid as the most satisfactory alternative to the succinate group. Nucleosides with this linker arm (Q-linker) are attached to supports with the same ease as the succinyl linker arm.

The cleavage time in ammonium hydroxide at room temperature was found to be 2 minutes, but what about the stability during synthesis? How significant was premature cleavage of oligonucleotide on the synthesizer because of the basic reagents in the capping mixes and oxidizer? Pon showed that the Q-linker is stable to the capping reagents but very slightly labile to the oxidizer (8% cleavage in overnight exposure which would correspond to about 2,000 normal synthesis cycles).

We tested the significance of premature cleavage by preparing sixteen 20mer oligonucleotides on a 0.2 µmole scale, eight with succinate and eight with Q-linkers. The succinate supported oligos were cleaved for 1 hour at room temperature, while those on the Q-support were cleaved for 2 minutes. Both sets were then deprotected normally with ammonium hydroxide. The Q-supports actually gave 5% better yields of product than the succinate supports. Oligo purities were equivalent in both sets.

The Q-linker is absolutely compatible with all hydrolytic cleavage procedures, but especially mild procedures like potassium carbonate in methanol. Pon also showed that it is preferable for RNA supports, improving the cleavage time for 2’-silyl protected nucleoside supports from 2 hours (60-65% cleavage) to 5 minutes (95% cleavage).

We are offering Q-linkers on 500Å CPG in 0.2 and 1µmole scales. The four regular nucleosides are offered, as well as a Universal Support.

Catalog No.	Catalog No.	Catalog No.	Catalog No.	Catalog No.	Catalog No.	Pack	Price ($)
dA	dC	Ac-dC	dmf-dG	dT	Universal
500Å Bulk Support
21-2000-01	21-2010-01	21-2013-01	21-2029-01	21-2030-01		0.1g	11.00
21-2000-02	21-2010-02	21-2013-02	21-2029-02	21-2030-02		0.25g	25.00
21-2000-10	21-2010-10	21-2013-10	21-2029-10	21-2030-10		1.0g	95.00
ABI Format (not LV)
21-2100-41	21-2110-41	21-2113-41	21-2129-41	21-2130-41	21-5100-41	4X1µm	60.00
21-2100-42	21-2110-42	21-2113-42	21-2129-42	21-2130-42	21-5100-42	4X0.2µm	40.00
Expedite Format
21-2200-41	21-2210-41	21-2213-41	21-2229-41	21-2230-41	21-5200-41	4X1µm	60.00
21-2200-42	21-2210-42	21-2213-42	21-2229-42	21-2230-42	21-5200-42	4X0.2µm	40.00
500Å Bulk Universal-Q Support
					21-5000-01	0.1g	15.00
					21-5000-02	0.25g	35.00
					21-5000-10	1.0g	135.00

HIGH LOAD CPG

Our high loading support is based on controlled pore silica and it retains the usual 500Å pores. The spacer is also conventional. The only significant difference is the loading which is in the range 80 - 130µmoles/g or about 2.5 times the loading of normal 500Å CPG. Typical loadings for our high load CPG are in the 100 - 120µmoles/g range. As a consequence of the high loading, this support should not be used for sequences longer than 40mers. This high loading support is available in columns for most synthesizers. The 2.5µmole column is identical to our standard 1µmole column (with the exception of the loading). It should be used on occasions when greater than 1µmole is desired but when a 10 or 15µmole synthesis is too high. It should be run using the 1µmole cycle. The 25µmole column is identical to the 10µmole column used on Applied Biosystems synthesizers. It is run using the 10µmole cycle. The 35µmole column is used as an alternative to the 15µmole Expedite column. Again no changes to the standard cycle are recommended. The support is of course available in bulk for use on large-scale synthesizers. A word of caution is in order. When using a column with a higher load than recommended by the instrument manufacturer, there is a much smaller margin for error. All reagents must be fresh and anhydrous diluent and activator must be used. Should you decide to prepare higher-loading columns, ensure that the molar excess of monomer to support nucleoside is at least 5X and preferably 10X.

	Catalog No.	Catalog No.	Catalog No.	Catalog No.	Catalog No.	Pack	Price ($)
	dA	dC	dG	dT	Universal
Columns
(ABI)	25-2100-46	25-2110-46	25-2120-46	25-2130-46		4X2.5µm	75.00
	25-2100-17	25-2110-17	25-2120-17	25-2130-17		1X25µm	125.00
(Expedite)	25-2200-46	25-2210-46	25-2220-46	25-2230-46		4X2.5µm	75.00
	25-2200-18	25-2210-18	25-2220-18	25-2230-18		1X35µm	185.00
Bulk
	25-2000-02	25-2010-02	25-2020-02	25-2030-02		0.25g	25.00
	25-2000-10	25-2010-10	25-2020-10	25-2030-10		1.0g	90.00
High Load Universal Support					25-5000-01	0.1g	15.00
					25-5000-02	0.25g	30.00
					25-5000-10	1.0g	115.00