oligonucleotide synthesis


  • In a more recent, more convenient, and more widely used method, the synthesis starts with the universal support where a non-nucleosidic linker is attached to the solid support
    material (compounds 1 and 2).

  • The method, initially developed for the solution-phase synthesis, was also implemented on low-cross-linked “popcorn” polystyrene,[26] and later on controlled pore glass (CPG,
    see “Solid support material” below), which initiated a massive research effort in solid-phase synthesis of oligonucleotides and eventually led to the automation of the oligonucleotide chain assembly.

  • By the use of additional steps in the synthetic cycle[38][39] or alternative coupling agents and solvent systems,[37] the oligonucleotide chain assembly may be carried out
    using dA and dC phosphoramidites with unprotected amino groups.

  • [29] The use of 2-cyanoethyl phosphite-protecting group[30] in place of a less user-friendly methyl group[31][32] led to the nucleoside phosphoramidites currently used in
    oligonucleotide synthesis (see Phosphoramidite building blocks below).

  • More recently, high-throughput oligonucleotide synthesis where the solid support is contained in the wells of multi-well plates (most often, 96 or 384 wells per plate) became
    a method of choice for parallel synthesis of oligonucleotides on small scale.

  • A phosphoramidite respective to the 3′-terminal nucleoside residue is coupled to the universal solid support in the first synthetic cycle of oligonucleotide chain assembly
    using the standard protocols.

  • • After the completion of the coupling reaction, a small percentage of the solid support-bound 5′-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked
    from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) shortmers.

  • Solid supports[edit] In solid-phase synthesis, an oligonucleotide being assembled is covalently bound, via its 3′-terminal hydroxy group, to a solid support material and remains
    attached to it over the entire course of the chain assembly.

  • To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product
    (see Synthetic cycle below).

  • The activated phosphoramidite in 1.5 – 20-fold excess over the support-bound material is then brought in contact with the starting solid support (first coupling) or a support-bound
    oligonucleotide precursor (following couplings) whose 5′-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage.

  • • Special solid supports are used for the attachment of desired functional or reporter groups at the 3’-terminus of synthetic oligonucleotides.

  • The simplest to implement, and hence the most widely used, strategy is to install a base-labile protection group on the exocyclic amino groups.

  • Solid support material[edit] In contrast to organic solid-phase synthesis and peptide synthesis, the synthesis of oligonucleotides proceeds best on non-swellable or low-swellable
    solid supports.

  • • The absence of physical dividers between the sites occupied by individual oligonucleotides, a very limited space on the surface of the microarray (one oligonucleotide sequence
    occupies a square 25×25 μm)[107] and the requirement of high fidelity of oligonucleotide synthesis dictate the use of site-selective 5′-deprotection techniques.

  • [30] Once a phosphoramidite has been coupled to the solid support-bound oligonucleotide and the phosphite moieties have been converted to the P(V) species, the presence of
    the phosphate protection is not mandatory for the successful conducting of further coupling reactions.

  • Step 3: Capping[edit] The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and 1-methylimidazole or, less often, DMAP
    as catalysts and, in the phosphoramidite method, serves two purposes.

  • In one approach, the removal of the 5′-O-DMT group is effected by electrochemical generation of the acid at the required site(s).

  • Synthesis of oligonucleotides by the H-Phosphonate Method Thirty years later, this work inspired, independently, two research groups to adopt the H-phosphonate chemistry to
    the solid-phase synthesis using nucleoside H-phosphonate monoesters 7 as building blocks and pivaloyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and other compounds as activators.

  • The apurinic sites thus formed are readily cleaved in the course of the final deprotection of the oligonucleotide under the basic conditions (see below) to give two shorter
    oligonucleotides thus reducing the yield of the full-length product.

  • Non-nucleosidic phosphoramidites are used to introduce desired groups that are not available in natural nucleosides or that can be introduced more readily using simpler chemical

  • Oligonucleotide synthesis involved the use of CPG (controlled pore glass) which is a rigid support and is more suited for column reactors as described above.

  • The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence.

  • [7][8] The practical implementation of H-phosphonate method resulted in a very short and simple synthetic cycle consisting of only two steps, detritylation and coupling (Scheme

  • The critical advantage of this approach is that the same solid support is used irrespectively of the sequence of the oligonucleotide to be synthesized.

  • It is worth remembering that conducting detritylation for an extended time or with stronger than recommended solutions of acids leads to depurination of solid support-bound
    oligonucleotide and thus reduces the yield of the desired full-length product.

  • • The synthesis of oligonucleotide phosphorothioates (OPS, see below) does not involve the oxidation with I2/water, and, respectively, does not suffer from the side reaction
    described above.

  • [73] Step 4: Oxidation[edit] The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide

  • In a historically first and still popular approach, the 3′-hydroxy group of the 3′-terminal nucleoside residue is attached to the solid support via, most often, 3’-O-succinyl
    arm as in compound 3.

  • The higher selectivity of the method allowed the use of more efficient coupling agents and catalysts,[24][25] which dramatically reduced the length of the synthesis.

  • [68] The mixing is usually very brief and occurs in fluid lines of oligonucleotide synthesizers (see below) while the components are being delivered to the reactors containing
    solid support.

  • [101] Currently, solid-phase oligonucleotide synthesis is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers) and is technically
    implemented in column, multi-well plate, and array formats.

  • However, the fact that a nucleosidic solid support has to be selected in a sequence-specific manner reduces the throughput of the entire synthetic process and increases the
    likelihood of human error.

  • Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2′-deoxynucleosides
    (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g.

  • Typically, three conceptually different groups of solid supports are used.

  • The amino group is then used as an anchoring point for linkers suitable for oligonucleotide synthesis (see below).

  • For example, the commercial[89] solid support 4[90] allows the preparation of oligonucleotides bearing 3’-terminal 3-aminopropyl linker.

  • Of many reported reagents capable of the efficient sulfur transfer, only three are commercially available: Commercial sulfur transfer agents for oligonucleotide synthesis.

  • Similarly to non-nucleosidic phosphoramidites, many other special solid supports designed for the attachment of reactive functional groups, non-radioactive reporter groups,
    and terminal modifiers (e.c.

  • The solid support is contained in columns whose dimensions depend on the scale of synthesis and may vary between 0.05 mL and several liters.

  • [102] Multi-well plate format is designed specifically for high-throughput synthesis on small scale to satisfy the growing demand of industry and academia for synthetic oligonucleotides.

  • The current practices of synthesis of chemically modified oligonucleotides on large scale have been recently reviewed.

  • Below, the protecting groups currently used in commercially available[33][34][35][36] and most common nucleoside phosphoramidite building blocks are briefly reviewed: • The
    5′-hydroxyl group is protected by an acid-labile DMT (4,4′-dimethoxytrityl) group.

  • [107] Post-synthetic processing After the completion of the chain assembly, the solid support-bound oligonucleotide is fully protected: • The 5′-terminal 5′-hydroxy group
    is protected with DMT group; • The internucleosidic phosphate or phosphorothioate moieties are protected with

  • Many later improvements to the manufacturing of building blocks, oligonucleotide synthesizers, and synthetic protocols made the phosphoramidite chemistry a very reliable and
    expedient method of choice for the preparation of synthetic oligonucleotides.

  • [47][69][70][71] The reaction is also highly sensitive to the presence of water, particularly when dilute solutions of phosphoramidites are used, and is commonly carried out
    in anhydrous acetonitrile.

  • The initial platform called the VLSS for very large scale synthesizer utilized large Pharmacia liquid chromatograph columns as reactors and could synthesize up to 75 mmol
    of material.

  • The method seems to be a step back from the more selective chemistry described earlier; however, at that time, most phosphate-protecting groups available now had not yet been

  • A very short selection of commercial phosphoramidite reagents is shown in Scheme for the demonstration of the available structural and functional diversity.

  • A more extensive information on the use of various coupling agents in oligonucleotide synthesis can be found in a recent review.

  • This platform was originally designed as a peptide synthesizer and made use of a fluidized bed reactor essential for accommodating the swelling characteristics of polystyrene
    supports used in the Merrifield methodology.

  • In order to be introduced inside the sequence, a non-nucleosidic modifier has to possess at least two hydroxy groups, one of which is often protected with the DMT group while
    the other bears the reactive phosphoramidite moiety.

  • Only the phosphorothioates having sulfur at a non-bridging position as shown in figure are widely used and are available commercially.

  • Step 2: Coupling[edit] A 0.02–0.2 M solution of nucleoside phosphoramidite (or a mixture of several phosphoramidites) in acetonitrile is activated by a 0.2–0.7 M solution
    of an acidic azole catalyst, 1H-tetrazole, 5-ethylthio-1H-tetrazole,[64] 2-benzylthiotetrazole,[65][66] 4,5-dicyanoimidazole,[67] or a number of similar compounds.

  • Generally, the larger the scale of the synthesis, the lower the excess and the higher the concentration of the phosphoramidites is used.

  • The defining difference from the phosphodiester approach was the protection of the phosphate moiety in the building block 1 (Scheme 4) and in the product 3 with 2-cyanoethyl

  • The 3’-terminal hydroxy group in oligonucleotides synthesized on nucleosidic solid supports is deprotected under the conditions somewhat milder than those applicable for universal
    solid supports.

  • The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5′-terminal hydroxyl group.

  • [15][16] The method is very convenient in that various types of phosphate modifications (phosphate/phosphorothioate/phosphoramidate) may be introduced to the same oligonucleotide
    for modulation of its properties.

  • The selectivity and the rate of the formation of internucleosidic linkages is dramatically improved by using 3′-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides
    (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology.

  • The protection of the exocyclic amino groups has to be orthogonal to that of the 5′-hydroxy group because the latter is removed at the end of each synthetic cycle.

  • The two most often used solid-phase materials are controlled pore glass (CPG) and macroporous polystyrene (MPPS).

  • [37] In contrast, the N2-protected versions of the same compound dissolve in acetonitrile well and hence are widely used.


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