Derivatization of Solid Supports Using a Carbamate‐Linked Tether Containing a Disulfide Functional Group

Chemical modifications of oligonucleotides are widely used to improve their functional properties. Amino modifiers provide versatile chemical handles for post-synthetic (bio)conjugation, nucleic acid immobilization on solid supports, and studies of nonenzymatic genome replication related to the origins of life. This protocol describes an economical disulfide-containing solid-support linker that enables on-column synthesis of nucleic acids bearing 3'-amino or 3'-phosphate modifications. The orthogonal nature of this linker allows for an on-column protecting group strategy, facilitating the synthesis of DNA and RNA containing 3'-amino-2',3'-dideoxyribosides from commercially available unprotected mononucleosides. This protocol further extends the on-column construction of oligomers containing 3'-amino-2',3'-dideoxyribosides to those containing a 2'-amino-2'-deoxynucleoside at the 3'-end, using an orthogonal protecting group strategy. An improved on-column deprotection procedure for DNA and RNA is also presented, eliminating the precipitation step typically utilized in conventional RNA workflows, resulting in an enhanced strand recovery for certain sequences. Finally, the disulfide-containing solid support is applied to the preparation of amino acid-oligonucleotide conjugates, wherein the identity of the final product was contingent upon the specific deprotection conditions employed. All oligonucleotide conjugates and derivatives were purified using standard methods [(e.g., strong anion exchange high-performance liquid chromatography (SAX-HPLC)] and characterized by mass spectrometry (MS). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-dimethoxytrityl (DMTr) disulfide succinimide and solid-support functionalization reaction Basic Protocol 2: Solid-phase synthesis of DNA containing a 3'-amino-2',3'-dideoxyguanosine using 3'-Disulfide CPG Alternate Protocol 1: Synthesis of DNA containing terminal 2'-amino-2'-deoxyadenosine Alternate Protocol 2: Alternate RNA (terminated with 3'-amino modifiers or phosphate) workflow using D-TentaGel Basic Protocol 3: Amino acid-oligonucleotide conjugate synthesis using Lys and D-TentaGel.