Refractive Index to Monitor Solid-Phase Oligonucleotide Synthesis

Abstract

Automated solid-phase oligonucleotide synthesis (SPOS) is a highly developed and efficient process, which has been used in academia and industry since decades. Still, some chemical transformations as well as all washing steps are run in a black box modus as only a few process analytical technology tools are available, namely, UV–vis spectroscopy and conductivity. To ensure high yields and efficiencies in each of the numerous steps, the process is usually performed using an excess of building blocks, reagents, and solvents. The reaction conditions are defined according to standard procedures and not necessarily to the real or optimal need of the reaction. As a result, SPOS suffers from generating large amounts of toxic waste, and its reduction is a must from the perspective of sustainability. One approach to make the process more sustainable is to add more complementary monitoring tools that provide online information about the progress of the process. Herein, we disclose our promising preliminary results in the use of refractive index to monitor SPOS.

Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates

“…In contrast, the S-Bz group is completely removed during ammonia treatment and the free thiol is susceptible to degradation and alkylation under these circumstances. To minimize the risks, the 2-cyanoethyl protection groups must be removed first by treating the oligonucleotide with 20% ( v / v ) diethylamine (DEA) in ACN [ 39 ], then cleaving with ammonium hydroxide/40% aqueous methylamine 1:1 ( v / v ) (AMA, instead of ammonia). Following this protocol, we detected the free thiol as the major product ( Figure 2 d) and as a minor component corresponding to sulfinic acid, formed during the oxidation step ( Figure 2 e and Figure S3 ) [ 31 ].…”

“…The oligonucleotides with S-Bz-Thiol-Modifier C6-dT (Glen Research, Sterling, VA, USA) were prepared using Ac-C and dmf g using DCI as the coupling reagent. Before cleavage, the synthesis column was treated with 20% diethylamine in acetonitrile to cleave the cyanoethyl protecting groups [ 39 ] to avoid alkylation of the thiol by acrylonitrile [ 40 , 41 , 42 , 43 ]. This was followed by cleavage and deprotection using AMA for 24 h at room temperature, yielding the oligonucleotide with the free thiol group.…”

Coaxial Dielectric Spectroscopy as an In-Line Process Analytical Technique for Reaction Monitoring

“…Because DS often provides insight only into the composition of the whole mixture, its use in reaction monitoring will likely be sensitive to the presence of impurities and parallel processes (e.g., side-reactions, crystallization events, etc.). In this way, it is similar to established ‘integral’ methods including reaction calorimetry and measurements of physico-chemical properties (e.g., density, conductivity, and refractive index , ). Furthermore, as noted above, dielectric property measurements can also be very sensitive to temperature .…”