Over the past decade, the level of sophistication regarding oligonucleotide synthesis has grown tremendously. Because the applicability of unmodified oligonucleotides is fairly limited, a great deal of time and money is spent on developing modified oligonucleotides. Most researchers find that some degree of oligonucleotide "tweeking" is necessary to get the desired properties for diagnostic and therapeutic applications.
Although the diagnostic and therapeutic industries have different needs, there is considerable overlap in the chemistries being developed. The diagnostic industry is confronted with two primary challenges: stringency and sensitivity (and consequently background noise). Unfortunately, these two requirements are almost mutually exclusive. In order to have high sensitivity, one must have a robust system with probes that will bind available targets with high affinity. However, in driving the equilibrium towards duplex formation, there is an increased binding to non-targets, leading to false positives. In order to solve these problems, researchers have turned to a number of oligonucleotide modifications, including base, sugar and backbone modifications to control hybridization properties. Conjugating reporter groups, such as fluorophores, chemiluminescent reagents and microchips can enhance sensitivity.
The therapeutic industry has a slightly broader range of problems to solve. Although there is disagreement about the severity of the various issues, and the solutions, most would agree that the following are the primary considerations:
It was obvious from the outset that unmodified oligonucleotides would not become a viable drug candidate. A series of modifications enhancing the ability of the oligonucleotide to overcome these obstacles was introduced; phosphorothioates being the most popular of the second generation drugs. These modifications led to the discovery of new modes of action, such as the aptamers of Gilead and ISIS.
As our understanding of biological processes becomes more sophisticated, the demands on chemists increase to produce more radical departures from nature in order to control them. Now we are entering the third generation of drugs. Most of the major players have introduced oligonucleotides with more sophisticated chemistries: Gilead's propynyls, Lynx's phosphoramidates, ISIS's MMIs and MOEs, and Genta's chiral methylphosphonates.
With each new twist, the problems multiply for the synthetic chemists responsible for the preparation of these compounds. Compatibility issues between the new generations of oligonucleotides and the classic methods of synthesis abound. In some cases, such as with peptide nucleic acids, the entire synthesis scheme had to be redeveloped.
A brief review of several case histories demonstrating how to solve some synthesis problems are presented.
In developing the use of chirally pure methylphosphonate dimer synthons to prepare oligonucleotides, it was found that the phosphoramidite synthons rapidly lost their ability to couple, even while stored dry under anhydrous conditions.
Repurify on a silica dimer that coupled at 25% efficiency (95% originally).
Purpose: Try to remove undetectable impurity (salts, etc.)
Result: No improvement. Conclusion: Contaminant co-migrates with product. Not from original synthesis of synthon, or would have been present from beginning.
Activate synthon with tetrazole in NMR tube. Compare to DMT-T amidite standard.Purpose: Determine if synthon converts cleanly to tetrazolide intermediate.
Result: Very little active material observed with dimer synthon (>10%). Most of the amidite converted to hydrolyzed sideproduct.
Conclusion: It's water.
Treat dimer synthon with high quality molecular sieves. Test by NMR and coupling.
Purpose: Try to remove water, and thereby increase coupling efficiency.
Result: After 2 days over sieves, NMR showed over 50% active reagent. Coupling efficiency >95%.
Conclusion: Molecular sieves are a viable solution.
All the dimer synthons are now treated with 3 Å molecular sieves for two days prior to use. In fact, this is done with any new or modified reagent. It is better to be safe, than to lose the results of many hard weeks in the lab. We now prepare our own molecular sieves.
2'-O-silyl RNA monomers have become the most popular reagents for the synthesis of RNA, mainly because of the "first in" principle and cost savings. However, there are a number of complaints about biologically inactive products being produced with these synthons. At the time of this work, tetrabutylammonium fluoride (TBAF) was the reagent of choice for removal of the silyl group.
Synthesize alternating AG and CU RNA oligos, deprotect as usual, analyze on gel.
Purpose: Repeat Miller's observation, and to begin exploring purine vs. pyrimidine issue.
Result: Significant banding of CU oligo with no major band, AG oligo was a clean single band
Conclusion: Definitely a difference between AG and CU oligos.
Retreat CU oligo with fresh TBAF.
Purpose: Determine if incomplete deprotection was problem.
Result: Multiple bands collapsed into single band.
Conclusion: Incomplete removal of silyl. Water content of both TBAF reagents were determined by Karl Fisher titration. The first contained over 10% water, the fresh bottle contained ~ 6%.
Prepare dimers CT, UT, AT and GT and isolate with silyl groups intact. Treat with TBAF containing variable amounts of water. Follow removal of silyl group by HPLC.
Purpose: Quantify affect of water in TBAF on rate of deprotection of individual bases in a model system in a realistic environment with a neighboring phosphate.
Result: Pyrimidines very sensitive to water content of TBAF, with the rate of desilylation rapidly declining with more than 5% water in the TBAF. Purines appear completely impervious, handling up to 20% water with no observable reduction in rate of desilylation.
Conclusion: It's water again.
Treat TBAF with molecular sieves to reduce water. Treat aliquot of reagent containing 20% water with sieves, compare ability to deprotect.
Purpose: Determine if we can not only protect TBAF against water, but actually recover wet reagent. Is water affecting the reagent itself, or the reaction?
Result: The TBAF treated with molecular sieves dried to only 2% water after 3 days. It completely deprotected the CT and UT dimers within 6 hours. The original untreated TBAF with 20% water deprotected about ½ of the dimer by that time.
Conclusion: Molecular sieves come to the rescue again.
We treat the TBAF upon arrival with sieves to ensure proper dryness, and remove the need to test each bottle by Karl Fisher prior to use. (We found that most brand new bottles contain more than 5% water, some contain as much as 8%, which will not deprotect pyrimidines under normal conditions.) We use the small TBAF (5 mL) bottles from Aldrich so that they are not in use for a long period of time. The combination of fresh reagent well dried with sieves has led to a track record of no failed deprotections over several hundred syntheses.
Needless to say, we now treat every water sensitive reagent with high quality molecular sieves prior to use. This work was published by Hogrefe et al. in the paper: The Effect of Excess Water on the Rate of Desilylation of Tetrabutylammonium Fluoride.
The synthesis of methylphosphonate oligonucleotides is no trivial matter. One problem is that the backbone is very sensitive to base and will completely degrade under normal deprotection conditions using ammonium hydroxide. The use of ethylenediamine (EDA) as an alternative was established by Paul Miller. The problem was that EDA transaminated N4-benzoyl cytidine, leading to undesired EDA adducts. We could see these compounds as later eluting species on gel, but not on RP-HPLC. This problem was exacerbated during scale up. Miller used a primary treatment with hydrazine to remove the benzoyl group prior to treatment with EDA. This method was not amenable to scale up. Use of a brief treatment with ammonium hydroxide was not very effective at scale and led to loss of product due to insolubility of the oligo in aqueous ammonium hydroxide. Somehow the NH4OH permanently bound the oligo to the support. Not only that, but degradation of methylphosphonate oligos to single nucleosides showed other modifications as well.
We had two problems here. One was to fix a known problem, EDA adduct formation with benzoyl-C, the second was to first determine what caused the unknown modification, then fix it. To top it off, we also had a solubility problem that limited our options.
Synthesize 9mers consisting of dA, dC, dG or T surrounded by T's using C-bz, C-ibu, G-ibu, and A-bz.
Purpose: Determine extent of problem, compare methods.
Result: dA, dC-ibu and T oligomers deprotect cleanly in all systems. dC-Bz yielded EDA adduct in every case. dG yielded unidentified modification in methods a and c, but not with b. Unfortunately, b yielded half the product the other methods did.
Conclusion: The first problem was neatly solved with a simple change, ibu instead of bz protection on dC. However, dG is also modified, something not reported in the literature. We tried O6 protected dG-ibu (diphenylcarbamoyl, DPC), but that exacerbated the problem.
Now that we knew the culprit, and how to fix it, we merely had to apply our knowledge. We couldn't use method b, which called for a pre-treatment of NH4OH before EDA, because it yielded very low yields on scale-up. EDA is the best solvent for that step, but to use it as the principle deprotecting reagent a new capping routine would have to be discovered to avoid NMI or DMAP. Fortunately, one last experiment gave us the needed clue. It was found that a very short treatment of 10 min with 30% NH4OH, or a 30 min treatment with 2% NH4OH in ACN/EtOH was sufficient to revert the DMAP-dG adduct. Furthermore, it was found that the 2% NH4OH solution degraded the methylphosphonate backbone at a very acceptable low rate of 1% per hour.
We solved the problem by first treating the support bound oligo with the mild NH4OH solution to revert dG adducts, followed with one volume of EDA after 30 min to complete the deprotection. Our deprotections went cleanly and our yields were acceptable. And we had a one pot method that was faster, cleaner and more simple than before. Also, the use of dC-ibu instead of dC-bz and this deprotection method eliminated the transamination side product. This work was also published by Hogrefe et al. in the paper: Deprotection of Methylphosphonate Oligonucleotides Using a Novel One-Pot Procedure.
In the search for an uptake enhancer, a certain peptide with biological activity of its own was deemed a viable candidate for conjugation to the terminus of an oligo. It was synthesized and purified using RP-HPLC with 0.1% Trifluoro acetic acid (TFA)/ACN as the eluent. The oligo was found to be too active. It had the same activity profile as the free peptide.
Try electrophoresis.Purpose: Try to rip the complex apart using fact that peptide is positively charged.
Result: Removed peptide.
Conclusion: Electrophoresis is the route of choice.
After successfully removing the free peptide, the compound was found to be completely inactive. Although not happy about the result, we were not entirely displeased. Have you ever contemplated pilot plant scale electrophoresis gels? Not a thought for the timid.
The lesson from the last case was that analytical methods are needed along with purification methods. The lesson here is to believe what you see. Prior to the development of mass spectroscopy (MS) for the analysis of Methylphosphonate (MP) oligonucleotides, you based purity on the integrated area of the putative product peak as determined by HPLC (MP oligos, having no charge, do not run on gel or CE well). What more can you do? First off, you keep looking for something new, and secondly, you believe what you see.
After much trial and tribulation, Genta developed a new purification scheme using normal phase chromatography. It worked very, very well for a number of compounds early on and was used exclusive of other techniques. However, one particular compound was going to be conjugated and therefore subjected to RP-HPLC analysis.
The chromatographic properties of MP oligos on normal phase HPLC was so well defined by base composition that our consultant, LCResources, was able to develop a formula predicting the retention times of various oligomers. However, that did not solve our problem. The simple solution to this problem was to design our oligomers such that they don't end with a T.