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Marc L. Rothstein and Dianne M. Rothstein Prime Synthesis, Inc. Aston, PA 19014 The prices for phosphoramidite monomers and other synthesis chemicals have.

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Presentation on theme: "Marc L. Rothstein and Dianne M. Rothstein Prime Synthesis, Inc. Aston, PA 19014 The prices for phosphoramidite monomers and other synthesis chemicals have."— Presentation transcript:

1 Marc L. Rothstein and Dianne M. Rothstein Prime Synthesis, Inc. Aston, PA The prices for phosphoramidite monomers and other synthesis chemicals have dropped dramatically, and consequently the solid support has become one of the most expensive materials in a large scale oligo synthesis. At Prime Synthesis, Inc., we have studied a variety of ways to reduce the cost of nucleoside Controlled Porosity Glass (CPG). The use of a proprietary long chain alkyl amine linker (CNA linker) has been demonstrated to significantly increase oligonucleotide yields and purities over other types of linkers. (1) However, the performance of this linker was found to be very dependant on a number of silane reaction parameters such as trace water concentration, reaction temperature and the number of repetitive silane applications. Additionally, the level of “n-1” oligo synthesis impurity was found to be proportional to the level of unreacted silanol groups on the CPG after linker and nucleoside immobilization. Cost effective ways to reduce these groups were also studied. The attributes of CPG as a solid support include high specific surface area, dimensional stability in organic solvents, large and stable pore spaces and high rigidity. Most reaction schemes used to link nucleoside to the CPG surface, begin by coupling a bi-functional organosilane to the native silanol groups of the glass surface. (Fig. 1) With proper immobilization techniques, nucleoside loadings of over 100 (µm/g) can be obtained on a 500 Å pore size support. Under proper deposition conditions, a uniform distribution of reactive groups can be achieved, which will result in higher levels of ligand accessibility. Together with the preferred pore structure, this accounts for the continued popularity of CPG in such difficult syntheses as RNAi’s. Since the silanol groups of the CPG are capable of reacting with nucleoside phosphoramidites, it is important to eliminate unreacted surface silanols on the finished nucleoside support, thus minimizing the occurrence of an oligo impurity lacking only the intended 3’ base, (“n-1”). In one study, NMR Spectroscopy was used to detect and characterize residual silanols on the surface of chemically modified silica (2). Silanol groups were classified as “free” or “associated”. The associated forms (Fig. 2) consisted of two adjacent silanols with a hydrogen bound to each other (vicinal) or a silanol with two hydroxyls sitting on the same atom (geminal). Another study (3) concluded that associated silanols were reactive to trimethylchlorosilane (TCMS), while free silanols were more reactive towards hexamethyldisilazane (HMDS). Preliminary work with aminopropyl linkers on CPG showed two silanization approaches could provide complete coverage of all silanol groups: 1) repetitious silanization (monolayer conditions) and 2) single silanization, followed by a dual silanol capping treatment with TMCS and HMDS. In a similar study CPG was functionalized with a proprietary long chain alkykamine linker (CNA) developed at Ribozyme Pharmaceuticals*. CNA silane was found to be highly reactive towards silanol groups. From this, a high performance LCAA-linked nucleoside CPG process was developed, that utilized both the novel linker AND capping silanes. This product gave the lowest “n-1” impurities of any CPG tested to date, but was costly to produce. With this performance as a benchmark, a more cost-effective process was developed, utilizing the CNA precursor silane to its BEST advantage. Acknowledgements Figure 2: Different Types of Silanol groups Figure 1: “Idealized” Silane Coupling Reaction In order to quickly evaluate the various silanol reduction schemes, a rapid screening test was developed. Once screened, the best performing supports were evaluated in actual oligonucloetide synthesis tests. The screening test (Residual Silanol Test) was based on the fact that nucleoside phosphoramidites can couple to the hydroxyl group of a silanol with low efficiency, but a nucleoside succinate cannot. The test scheme is shown below: Amino CPG is saturated with the nucleoside succinate base #1 in an exhaustive reaction Loading Assay nucleoside base #1 Mono-nucleoside loaded CPG is saturated with the nucleoside phosphoramidite base #2 in an exhaustive reaction Loading Assay nucleoside base #2 (Loading # 2 – Loading #1) = Incremental Loading i.e Amount of nucleoside phosphoramidite base #2 that reacted with RESIDUAL SILANOL groups on CPG % INCREASE in Incremental Loading to actual “n-1” impurity (Figure 3) = Original CPG = CPG with Improved Silanol Capping Process Figure 3: Correlation between Residual Silanol Test and Actual “n-1” Impurities Residual Silanol Test Confirmation:  Used two CPG samples of different silanol reduction schemes  Synthesized 18-mer deoxy oligos with AKTA (GE Healthcare) synthesizer  1mM scale  Analysis of “n-1” related impurities with CE and MALDI  Two “extraneous” impurities explained: -“n-3” phosphorothiolated impurity seen by CE was actually “n-1” with extra charged species -Impurity with MW corresponding to (n-1)+94 = phosphorothiolated impurity of n-1 oligo Product ID % Coupling Oligo Purity % n-1 % yield (full length) N16 (Benchmark) N12 (Low Cost) N/A (Competitor) Table 1: Comparison of “Benchmark” and “Low Cost” CPG Performance  An independent check on residual amine groups showed complete amine capping Once the best low-cost CNA linker was identified by the screening test, it was compared to the more expensive benchmark process in a synthesis test. For each support, a number of 21-mer deoxy-oligo nucleotides were synthesized at Integrated DNA technologies, using their proprietary synthesis instrumentation, developed to operate at maximum reagent efficiency. The oligos were analyzed by CE and MALDI for full length product yield, oligo purity, “n-1” purity, and average coupling efficiency We would like to thank Dr. Nanda Sinha of Avecia Biotechnology for his helpful suggestions and evaluations in this study. We would also like to thank Trey Martin, Mike Marvin and Dr. Yakov Leutchy for their evaluations in this work. Figure 4: Representative CE Test Results References 1. Wincott, F.E., “Further Improvement to the Large Scale Production of Ribozymes”, Tides 2000 Presentation 2. Holik, M. and Matejkova, B. (1981) J. Chromatography, Vol. 213: p Snyder, I.R., Principles of Adsorption Chromatography, M. Decker, New York, 1968.


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