Oligo Synthesis : CEPs

Catalog Number: 10-1936-xx

Description: Thiol-Modifier C6 S-S

TERMINUS MODIFIERS

Glen Research 5’-Modifiers are designed for use in DNA synthesizers to functionalize the 5’-terminus of the target oligonucleotide. The 5’-Amino-Modifiers are available with a variety of chain lengths to fit exactly the desired application.

The DMS(O)MT-protected amino group is easier to deprotect compared to the MMT-protected one. The sulfoxy derivative survives conditions of oligonucleotide synthesis and can either be cleaved with standard deblock solution, or left intact for HPLC purification. At the same time, the DMS(O)MT group is fully compatible with cartridge purification. When detritylation on a cartridge is carried out, the DMS(O)MT , which is more stable than MMT , does not reattach itself to an amine. We now offer 5’-DMS(O)MT-Amino-Modifier C6 utilizing this new trityl based protecting group.

5’-Amino-Modifier TEG, a hydrophilic triethylene glycol ethylamine derivative, is 12 atoms in length and fully soluble in aqueous media.

The disulfide thiol modifier may be used for introducing 3’- or 5’-thiol linkages. Dithiol Phosphoramidite (DTPA) is a disulfide-containing modifier designed to functionalize synthetic DNA or RNA with multiple thiol groups and can be incorporated at any position of the oligonucleotide. Each DTPA addition leads to two thiol groups. This modifier was designed for optimal tethering of oligonucleotides to a gold surface but it can also be used for multiple reactions with maleimides and other thiol-specific derivatives. 5’-Carboxy-Modifier C10 is a unique linker designed to be added at the terminus of an oligonucleotide synthesis. It generates an activated carboxylic acid N-hydroxysuccinimide (NHS) ester suitable for immediate conjugation on the synthesis column with molecules containing a primary amine, resulting in a stable amide linkage. PC Amino-Modifier is a photocleavable C6 amino-modifier, part of our line of photocleavable (PC) modifiers.

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Material Safety Data Sheet

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FREQUENTLY ASKED TECHNICAL QUESTION

QUESTION: Do you have a biotin phosphoramidite containing a disulfide linker which can be cleaved later with DTT to release the DNA from a streptavidin support?

RESPONSE:No. However, this can be produced on the synthesizer by adding to the 5"- terminus first 5"-thiol-modifier C6 S-S (10-1936) followed by BioTEG phosphoramidite (10-1955). This should generate a biotinylated primer with a long spacer arm containing the disulfide linkage which can be cleaved later with DTT.

QUESTION: What is the best method to make peptide-oligonucleotide conjugates?

RESPONSE:It would seem that the best method to make peptide-oligo conjugates would be to use Fmoc chemistry and synthesize the peptide off an oligo synthesized on amino-CPG. However, deprotection of peptides synthesized using Fmoc chemistry requires 50% TFA and t-boc synthesized peptides require HF both of which would severely damage if not completely hydrolyze the oligo.

The best and most straight foward method is to use a heterobifunctional crosslinking reagent to link a synthetic peptide, containing an N-terminal lysine, to a 5"-Thiol modified oligo or conversely a 5"-amino modified oligo to a cysteine containing peptide . A good crosslinking reagent is N-Maleimido-6-aminocaproyl- (2"-nitro,4"-sulfonic acid)-phenyl ester . Na (mal-sac-HNSA) from Bachem Bioscience (cat. # Q-1615). Reaction of this crosslinker with an amino group releases the dianion phenolate, 1-hydroxy-2-nitro -4-benzene sulfonic acid a yellow chromophore. The chromophore allows both quantitation of the coupling reaction as well as act as an aid in monitoring the seperation of "activated peptide" from free crosslinking reagent using gel filtration.

Method A: Couple Peptide Amine To Oligo Thiol (Note peptide MW must be > 5,000 to be excluded from desalting column). This method best for oligo-enzyme conjugation.

Step 1: Synthesize a peptide with an N-terminal, or internal, lysine (The epsilon amino group is more reactive than an alpha amino group).

Step 2: Synthesize an oligonucleotide with a 5" Thiol group.

Step 3: React peptide with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 4: Seperation of peptide-mal-sac conjugate from free crosslinker and buffer exchange (pH 6.0 Sodium phosphate) using a gel filtration column (Glen Gel-Pak™ or eq.). Note peptide must be large enough to seperate from the free linker which can be visualized as a yellow band. Do not collect yellow band with peptide.

Step 5: Activate thiol modified oligo, desalt and buffer exchange (pH 6 Sodium phosphate) on Glen Gel-Pak™ column.

Step 6: React acitvated peptide with Thiol modified oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

Method B: Couple Oligo Amine To Peptide Cysteine (Note oligos > 15mers are excluded from desalting column). Use above procedure switching oligo for peptide.

Step 1: Synthesize a peptide with an N-terminal, or internal, cysteine

Step 2: Synthesize an oligonucleotide with a 5" amino modifier.

Step 3: Purify oligo Trityl-on by RP HPLC or cartridge.

Step 4: React oligo with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 5: Seperation of oligo-mal-sac conjugate from free crosslinker and buffer exchange (pH 6 Sodium phosphate) using a gel filtration column (Glen Gel-Pak™ or eq.). Note oligo must be large enough to seperate from the free linker which can be visualized as a yellow band.Do not collect yellow band with oligo.

Step 6: Dissolve peptide in pH 6.0 Sodium phosphate buffer and react with activated oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

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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 formore detailed usage informationwith the various synthesizers.

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