• 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
  designs.

• 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
  2).

• 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
  synthesis.

• 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
  introduced.

• 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
  group.

• 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.

 

Works Cited

[‘o Beaucage, S. L.; Iyer, R. P. (1992). “Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach”. Tetrahedron. 48 (12): 2223. doi:10.1016/S0040-4020(01)88752-4.
o ^ Jump up to:a b Brown, D. M. A brief history of oligonucleotide
synthesis. Methods in Molecular Biology (Totowa, NJ, United States) (1993), 20 (Protocols for Oligonucleotides and Analogs), 1–17.
o ^ Reese, Colin B. (2005). “Oligo- and poly-nucleotides: 50 years of chemical synthesis”. Organic & Biomolecular
Chemistry. 3 (21): 3851–68. doi:10.1039/b510458k. PMID 16312051.
o ^ Iyer, R. P.; Beaucage, S. L. 7.05. Oligonucleotide synthesis. In: Comprehensive Natural Products Chemistry, Vol. 7: DNA and Aspects of Molecular Biology. Kool, Eric T.; Editor.
Neth. (1999), Elsevier, Amsterdam, pp. 105–152.
o ^ Michelson, A. M.; Todd, A. R. (1955). “Nucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3′: 5′-internucleotidic linkage”. J. Chem. Soc.: 2632. doi:10.1039/JR9550002632.
o ^
Hall, R. H.; Todd, A.; Webb, R. F. (1957). “644. Nucleotides. Part XLI. Mixed anhydrides as intermediates in the synthesis of dinucleoside phosphates”. J. Chem. Soc.: 3291. doi:10.1039/JR9570003291.
o ^ Froehler, B. C.; Ng, P. G.; Matteucci, M.
D. (1986). “Synthesis of DNA via deoxynucleoside H-phosphonate intermediates”. Nucleic Acids Res. 14 (13): 5399–5407. doi:10.1093/nar/14.13.5399. PMC 311548. PMID 3737406.
o ^ Garegg, P. J.; Lindh, I.; Regberg, T.; Stawinski, J.; Strömberg, R. (1986).
“Nucleoside H-phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach”. Tetrahedron Lett. 27 (34): 4051. doi:10.1016/S0040-4039(00)84908-4.
o ^ Jump up to:a b Wada, T.; Sato, Y.; Honda, F.; Kawahara,
S.; Sekine, M. (1997). “Chemical Synthesis of Oligodeoxyribonucleotides Using N-Unprotected H-Phosphonate Monomers and Carbonium and Phosphonium Condensing Reagents: O-Selective Phosphonylation and Condensation”. J. Am. Chem. Soc. 119 (52): 12710–12721.
doi:10.1021/JA9726015.
o ^ Agrawal, S.; Goodchild, J.; Civeira, M. P.; Thornton, A. H.; Sarin, P. S.; Zamecnik, P. C. (1988). “Oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus”. Proc. Natl.
Acad. Sci. U.S.A. 85 (19): 7079–7083. Bibcode:1988PNAS…85.7079A. doi:10.1073/pnas.85.19.7079. PMC 282127. PMID 3174622.
o ^ Kamer, P. C. J.; Roelen, H. C. P. F.; Van den Elst, H.; Van der Marel, G. A. & Van Boom, J. H. (1989). “An efficient approach
toward the synthesis of phosphorothioate diesters via the Schoenberg reaction”. Tetrahedron Lett. 30 (48): 6757–6760. doi:10.1016/S0040-4039(00)70669-1.
o ^ Agrawal, S.; Tang, J. Y. (1990). “Efficient synthesis of oligoribonucleotide and its phosphorothioate
analog using H-phosphonate approach”. Tetrahedron Lett. 31 (52): 7541–7544. doi:10.1016/S0040-4039(00)97293-9.
o ^ Jump up to:a b Almer, H.; Stawinski, J.; Strӧmberg, R. (1996). “Solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s”.
Nucleic Acids Res. 24 (19): 3811–3820. doi:10.1093/nar/24.19.3811. PMC 146170. PMID 8871563.
o ^ Tram, K.; Wang, X.; Yan, H. (2007). “Facile Synthesis of Oligonucleotide Phosphoroselenoates”. Org. Lett. 9 (24): 5103–5106. doi:10.1021/ol702305v.
PMID 17973486.
o ^ Froehler, B. C. (1986). “Deoxynucleoside H-phosphonate diester intermediates in the synthesis of internucleotide phosphate analogs”. Tetrahedron Lett. 27 (46): 5575–5578. doi:10.1016/S0040-4039(00)85269-7.
o ^ Froehler, B. C.;
Ng, P. G.; Matteucci, M. D. (1988). “Phosphoramidate analogs of DNA: synthesis and thermal stability of heteroduplexes”. Nucleic Acids Res. 16 (11): 4831–4839. doi:10.1093/nar/16.11.4831. PMC 336699. PMID 3387210.
o ^ Dagle, J. M.; Andracki, M.
E.; DeVine, R. J.; Walder, J. (1991). “Physical properties of oligonucleotides containing phosphoramidate-modified internucleoside linkages”. Nucleic Acids Res. 19 (8): 1805–1810. doi:10.1093/nar/19.8.1805. PMC 328108. PMID 2030962.
o ^ Maier, M.
A.; Guzaev, A. P.; Manoharan, M. (2000). “Synthesis of Chimeric Oligonucleotides Containing Phosphodiester, Phosphorothioate, and Phosphoramidate Linkages”. Org. Lett. 2 (13): 1819–1822. doi:10.1021/ol005842h. PMID 10891166.
o ^ Mohe, N. U.; Padiya,
K. J.; Salunkhe, M. M. (2003). “An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the H-Phosphonate approach”. Bioorg. Med. Chem. 11 (7): 1419–1431. doi:10.1016/S0968-0896(02)00615-6. PMID 12628668.
o ^ Kung,
P. P. & Jones, R. A. (1992). “H-phosphonate DNA synthesis without amino protection”. Tetrahedron Lett. 33 (40): 5869–5872. doi:10.1016/S0040-4039(00)61075-4.
o ^ Gilham, P. T.; Khorana, H. G. (1958). “Studies on Polynucleotides. I. A New and General
Method for the Chemical Synthesis of the C5′-C3′ Internucleotidic Linkage. Syntheses of Deoxyribo-dinucleotides”. J. Am. Chem. Soc. 80 (23): 6212. doi:10.1021/ja01556a016.
o ^ Letsinger, R. L.; Ogilvie, K. K. (1969). “Nucleotide chemistry. XIII.
Synthesis of oligothymidylates via phosphotriester intermediates”. J. Am. Chem. Soc. 91 (12): 3350. doi:10.1021/ja01040a042.
o ^ Reese, C. B. (1978). “The chemical synthesis of oligo- and poly-nucleotides by the phosphotriester approach”. Tetrahedron.
34 (21): 3143. doi:10.1016/0040-4020(78)87013-6.
o ^ Efimov, V. A.; Buryakova, A. A.; Reverdatto, S. V.; Chakhmakhcheva, O. G.; Ovchinnikov, Yu. A. (1983). “Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester
method”. Nucleic Acids Res. 11 (23): 8369–8387. doi:10.1093/nar/11.23.8369. PMC 326588. PMID 6324083.
o ^ Efimov, V. A; Molchanova, N. S.; Chakhmakhcheva, O. G. (2007). “Approach to the synthesis of natural and modified oligonucleotides by the phosphotriester
method using O-nucleophilic intramolecular catalysis”. Nucleosides, Nucleotides and Nucleic Acids. 26 (8–9): 1087–93. doi:10.1080/15257770701516268. PMID 18058542. S2CID 34548367.
o ^ Letsinger, R. L.; Mahadevan, V. (1966). “Stepwise synthesis of
oligodeoxyribonucleotides on an insoluble polymer support”. J. Am. Chem. Soc. 88 (22): 5319–24. doi:10.1021/ja00974a053. PMID 5979268.
o ^ Letsinger, R. L.; Finnan, J. L.; Lunsford, N. B. (1975). “Nucleotide chemistry. XX. Phosphite coupling procedure
for generating internucleotide links”. J. Am. Chem. Soc. 97 (11): 3278–9. doi:10.1021/ja00844a090. PMID 1133350.
o ^ Matteucci, M. D.; Caruthers, M. H. (1981). “Synthesis of deoxyoligonucleotides on a polymer support”. J. Am. Chem. Soc. 103 (11):
3185. doi:10.1021/ja00401a041.
o ^ Beaucage, S. L.; Caruthers M. H. (1981). “Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis”. Tetrahedron Lett. 22 (20): 1859. doi:10.1016/S0040-4039(01)90461-7.
o ^
Jump up to:a b Sinha, N. D.; Biernat, J.; McManus, J.; Kӧster, H. (1984). “Polymer support oligonucleotide synthesis. XVIII: use of β-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments
simplifying deprotection and isolation of the final product”. Nucleic Acids Res. 12 (11): 4539–4557. doi:10.1093/nar/12.11.4539. PMC 318857. PMID 6547529.
o ^ McBride, L. J.; Caruthers, M. H. (1983). “Nucleotide chemistry. X. An investigation of
several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides”. Tetrahedron Lett. 24 (3): 245–248. doi:10.1016/S0040-4039(00)81376-3.
o ^ Adams, S. P.; Kavka, K. S.; Wykes, E. J.; Holder, S. B.; Galluppi, G. R. (1983). “Hindered
dialkylamino nucleoside phosphite reagents in the synthesis of two DNA 51-mers”. J. Am. Chem. Soc. 105 (3): 661–663. doi:10.1021/ja00341a078.
o ^ “Beta-Cyanoethyl Phosphoramidites”. Products.appliedbiosystems.com. Retrieved 2009-05-12.
o ^ “Biosearch
Technologies”. Biosearchtech.com. Retrieved 2009-05-12.
o ^ “ChemGenes Corporation, a Biotechnology company”. Chemgenes.com. Retrieved 2009-05-12.
o ^ Powell, M. (2008-01-17). “Applied Biosystems Instruments”. Glenresearch.com. Retrieved 2009-05-12.
o ^
Jump up to:a b Sekine, M. DNA synthesis without base protection. In: Current protocols in nucleic acid chemistry. Beaucage, S. L., Editor (John Wiley & Sons, Inc.) (2004), Chapter 3, Unit 3.10., pp. 3.10.1-3.10.15. PubMed ID:18428925
o ^ Gryaznov,
S. M.; Letsinger, R. L. (1991). “Synthesis of oligonucleotides via monomers with unprotected bases”. J. Am. Chem. Soc. 113 (15): 5876–5877. doi:10.1021/ja00015a059.
o ^ Sekine, M., Ohkubo, A., and Seio, K. (2003). “Protonblock strategy for the synthesis
of oligodeoxynucleotides without base protection, capping reaction, and P-N bond cleavage reaction”. J. Org. Chem. 68 (14): 5478–5492. doi:10.1021/jo034204k. PMID 12839438.
o ^ Jump up to:a b c Reddy, M. P.; Hanna, N. B.; Farooqui, F (1997). “Ultrafast
Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives”. Nucleosides & Nucleotides. 16 (7): 1589–1598. doi:10.1080/07328319708006236.
o ^ McMinn, D. L.; Greenberg, M. M. (1997). “Synthesis of oligonucleotides containing
3′-alkyl amines using N-isobutyryl protected deoxyadenosine phosphoramidite”. Tetrahedron Lett. 38 (18): 3123. doi:10.1016/S0040-4039(97)00568-6.
o ^ Schulhof, J. C.; Molko, D.; Teoule, R. (1987). “The final deprotection step in oligonucleotide
synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups”. Nucleic Acids Res. 15 (2): 397–416. doi:10.1093/nar/15.2.397. PMC 340442. PMID 3822812.
o ^ Zhu, Q.; Delaney, M. O.; Greenberg, M. M. (2001). “Observation
and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection”. Bioorg. Med. Chem. Lett. 11 (9): 1105–7. doi:10.1016/S0960-894X(01)00161-5. PMID 11354354.
o ^ McBride, L. J.; Kierzek,
R.; Beaucage, S. L.; Caruthers, M. H. (1986). “Nucleotide chemistry. 16. Amidine protecting groups for oligonucleotide synthesis”. J. Am. Chem. Soc. 108 (8): 2040–2048. doi:10.1021/ja00268a052.
o ^ Guzaev, A. P.; Manoharan, M. (2001). “Phosphoramidite
Coupling to Oligonucleotides Bearing Unprotected Internucleosidic Phosphate Moieties”. J. Org. Chem. 66 (5): 1798–1804. doi:10.1021/jo001591e. PMID 11262130.
o ^ Ogilvie, K. K.; Theriault, N.; Sadana, K. L. (1977). “Synthesis of oligoribonucleotides”.
J. Am. Chem. Soc. 99 (23): 7741–7743. doi:10.1021/ja00465a073. PMID 915168.
o ^ Jump up to:a b Usman, N.; Ogilvie, K. K.; Jiang, M. Y.; Cedergren, R. J. (1987). “The automated chemical synthesis of long oligoribuncleotides using 2′-O-silylated
ribonucleoside 3′-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3′-half molecule of an Escherichia coli formylmethionine tRNA”. J. Am. Chem. Soc. 109 (25): 7845–7854. doi:10.1021/ja00259a037.
o ^
Usman, N.; Pon, R. T.; Ogilvie, K. K. (1985). “Preparation of ribonucleoside 3′-O-phosphoramidites and their application to the automated solid phase synthesis of oligonucleotides”. Tetrahedron Lett. 26 (38): 4567–4570. doi:10.1016/S0040-4039(00)98753-7.
o ^
Scaringe, S. A.; Francklyn, C.; Usman, N. (1990). “Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites”. Nucleic Acids Res. 18 (18): 5433–5441. doi:10.1093/nar/18.18.5433. PMC
332221. PMID 2216717.
o ^ Pitsch, S.; Weiss, P. A.; Wu, X.; Ackermann, D.; Honegger, T. (1999). “Fast and reliable automated synthesis of RNA and partially 2′-O-protected precursors (“caged RNA”) based on two novel, orthogonal 2′-O-protecting
groups”. Helv. Chim. Acta. 82 (10): 1753–1761. doi:10.1002/(SICI)1522-2675(19991006)82:10
<1753::AID-HLCA1753>3.0.CO;2-Y.
o ^ Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. (2001). “Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O-tom)-protected phosphoramidites”. Helv. Chim. Acta. 84 (12):
3773–3795. doi:10.1002/1522-2675(20011219)84:12
<3773::AID-HLCA3773>3.0.CO;2-E.
o ^ Guzaev, A.; Salo, H.; Azhayev, A.; Lӧnnberg, H. (1995). “A new approach for chemical phosphorylation of oligodeoxyribonucleotides at the 5′-terminus”. Tetrahedron. 51 (34): 9375–9384. doi:10.1016/0040-4020(95)00544-I.
o ^
Sinha, N. D.; Cook, R. M. (1988). “The preparation and application of functionalized synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or-hexanol”. Nucleic Acids Res. 16 (6):
2659–2669. doi:10.1093/nar/16.6.2659. PMC 336396. PMID 3362678.
o ^ Jones, D. S.; Hachmann, J. P.; Conrad, M. J.; Coutts, S.; Livingston, D. A. Intermediates for providing functional groups on the 5′ end of oligonucleotides, (1995) U.S.
Patent 5,391,785.
o ^ Podyminogin, M. A.; Lukhtanov, E. A.; Reed, M. W. (2001). “Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass”. Nucleic Acids Res. 29 (24): 5090–5098. doi:10.1093/nar/29.24.5090.
PMC 97543. PMID 11812841.
o ^ Lebedev, A. V.; Combs, D.; Hogrefe, R. I. (2007). “Preactivated Carboxyl Linker for the Rapid Conjugation of Alkylamines to Oligonucleotides on Solid Support”. Bioconjugate Chem. 18 (5): 1530–1536. doi:10.1021/bc0603891.
PMID 17877414.
o ^ Alvira, M.; Eritja, R. (2007). “Synthesis of oligonucleotides carrying 5′-5′ linkages using copper-catalyzed cycloaddition reactions” (PDF). Chemistry & Biodiversity. 4 (12): 2798–2809. doi:10.1002/cbdv.200790229. hdl:10261/124969.
PMID 18081090. S2CID 25051865.
o ^ Brush, C. K. “Fluorescein Labelled Phosphoramidites”. (1996) U.S. Patent 5,583,236.
o ^ Pitner, J. B.; Linn, C. P. “Synthesis and use of labelled phosphoramidite compositions”. (2000) U.S. Patent 6,114,518.
o ^
Levenson; C.; Chang; C.-A; Oakes; F. T. “Oligonucleotide functionalizing reagents”. (1990) U.S. Patent 4,914,210.
o ^ Durand, M.; Chevrie, K.; Chassignol, M.; Thuong, N. T.; Maurizot, J. C. (1990). “Circular dichroism studies of an oligodeoxyribonucleotide
containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability”. Nucleic Acids Res. 18 (21): 6353–6359. doi:10.1093/nar/18.21.6353. PMC 332506. PMID 2243780.
o ^ Christiano, A.; McSwiggen, J. “RNA interference-mediated
inhibition of retinoblastoma (RB1) gene expression using short interfering nucleic acid”. PCT Int. Appl. (2006), WO 2006078798 A2.
o ^ Pon, R. T. (1991). “A long chain biotin phosphoramidite reagent for the automated synthesis of 5′-biotinylated
oligonucleotides”. Tetrahedron Lett. 32 (14): 1715–1718. doi:10.1016/S0040-4039(00)74311-5.
o ^ Sproat, B.; Colonna, F.; Mullah, B.; Tsou, D.; Andrus, A.; Hampel, A.; Vinayak, R. (1995). “An efficient method for the isolation and purification
of oligoribonucleotides”. Nucleosides & Nucleotides. 14 (1&2): 255–273. doi:10.1080/15257779508014668.
o ^ Stutz, A.; Hobartner, C.; Pitsch, S. (2000). “Novel fluoride-labile nucleobase-protecting groups for the synthesis of 3′(2′)-O-amino-acylated
RNA sequences”. Helv. Chim. Acta. 83 (9): 2477–2503. doi:10.1002/1522-2675(20000906)83:9
<2477::aid-hlca2477>3.0.co;2-9.
o ^ Welz, R.; Muller, S. (2002). “5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis”. Tetrahedron Lett. 43 (5): 795–797. doi:10.1016/S0040-4039(01)02274-2.
o ^ Vargeese,
C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.; Kropp, E.; Peterson, K.; Pieken, W. (1998). “Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis”. Nucleic Acids Res. 26
(4): 1046–1050. doi:10.1093/nar/26.4.1046. PMC 147346. PMID 9461466.
o ^ Wei, Xia (2013). “Coupling activators for the oligonucleotide synthesis via phosphoramidite approach”. Tetrahedron. 69 (18): 3615–3637. doi:10.1016/j.tet.2013.03.001.
o ^
Ogilvie, K. K.; Usman, N.; Nicoghosian, K.; Cedergren, R. J. (1988). “Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity”. Proc. Natl. Acad. Sci. USA. 85 (16): 5764–5768. Bibcode:1988PNAS…85.5764O.
doi:10.1073/pnas.85.16.5764. PMC 281845. PMID 3413059.
o ^ Wu, T.; Ogilvie, K. K.; Perreault, J. Pierre; Cedergren, R. J. (1989). “Convenient procedure for the preparation of specific mixed DNA-RNA polymers”. J. Am. Chem. Soc. 111 (22):
8531–8533. doi:10.1021/ja00204a043.
o ^ Pon, R. T. (1987). “Enhanced coupling efficiency using 4-dimethylaminopyridine (DMAP) and either tetrazole, 5-(o-nitrophenyl)tetrazole, or 5-(p-nitrophenyl)tetrazole in the solid phase synthesis
of oligoribonucleotides by the phosphoramidite procedure”. Tetrahedron Lett. 28 (32): 3643–3646. doi:10.1016/S0040-4039(00)96344-5.
o ^ Pon, R. T.; Usman, N.; Damha, M. J.; Ogilvie, K. K. (1986). “Prevention of guanine modification and
chain cleavage during the solid phase synthesis of oligonucleotides using phosphoramidite derivatives”. Nucleic Acids Res. 14 (16): 6453–6470. doi:10.1093/nar/14.16.6453. PMC 311657. PMID 3748816.
o ^ Jump up to:a b Guzaev, A. P. (2011).
“Reactivity of 3H-1,2,4-dithiazole-3-thiones and 3H-1,2-dithiole-3-thiones as sulfurizing agents for oligonucleotide synthesis”. Tetrahedron Lett. 52 (3): 434–437. doi:10.1016/j.tetlet.2010.11.086.
o ^ Alul, R. H.; Singman, C. N.; Zhang,
G.; Letsinger, R. L. (1991). “Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives”. Nucleic Acids Res. 19 (7): 1527–1532. doi:10.1093/nar/19.7.1527. PMC 333911. PMID 2027761.
o ^ “New Product: 0.5M CSO
for non-aqueous oxidation in DNA synthesis”. Glenres.com. Retrieved 2013-01-28.
o ^ Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. (2000). “Allyl Group as a Protecting Group for Internucleotide Phosphate and Thiophosphate Linkages in
Oligonucleotide Synthesis: Facile Oxidation and Deprotection Conditions”. Org. Lett. 2 (3): 243–246. doi:10.1021/ol9910518. PMID 10814292.
o ^ Prakash, T. P.; Johnston, J. F.; Graham, M. J.; Condon, T. P.; Manoharan, M. (2004). “2′-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified
oligonucleotides inhibit expression of mRNA in vitro and in vivo”. Nucleic Acids Res. 32 (2): 828–833. doi:10.1093/nar/gkh220. PMC 373344. PMID 14762210.
o ^ Jump up to:a b Guzaev, A. P. Solid-phase supports for oligonucleotide synthesis.
In: Current protocols in nucleic acid chemistry. (John Wiley & Sons, Inc.) (2013), Chapter 3, Unit 3.1., pp. 3.1.1-3.1.60. doi:10.1002/0471142700.nc0301s53
o ^ Pon, R. T. Solid-phase supports for oligonucleotide synthesis. Methods in
Molecular Biology (Totowa, NJ, United States) (1993), 20 (Protocols for Oligonucleotides and Analogs), 465–496 doi:10.1385/0-89603-281-7:465.
o ^ Guzaev, A. P.; Manoharan, M. (2003). “A conformationally preorganized universal solid support
for efficient oligonucleotide synthesis”. J. Am. Chem. Soc. 125 (9): 2380–1. doi:10.1021/ja0284613. PMID 12603111.
o ^ Jensen, M. A.; Anderson, K. M.; Davis, R. W. (2010). “Gas-Phase Cleavage and Dephosphorylation of Universal Linker-Bound
Oligodeoxynucleotides”. Nucleosides, Nucleotides and Nucl. Acids. 29 (11): 867–878. doi:10.1080/15257770.2010.534757. PMC 6815660. PMID 21128173.
o ^ “Glen Research Report of Products for RNA and DNA Oligonucelotide Synthesis, Modification
and Labelling”. Glenresearch.com. 2008-01-17. Retrieved 2009-05-12.
o ^ “AM Chemicals, LLC, a supplier of solid supports and reagents for oligonucleotide and organic synthesis on solid phase”. Amchemicals.com. Archived from the original
on 2011-07-07. Retrieved 2009-05-12.
o ^ “AM Chemicals, LLC, a supplier of solid supports and reagents for oligonucleotide and organic synthesis on solid phase”. Amchemicals.com. Archived from the original on 2011-07-07. Retrieved 2009-05-12.
o ^
Powell, M. (2008-01-17). “Supports”. Glenresearch.com. Retrieved 2009-05-12.
o ^ Azhayev, A. V.; Antopolsky, M. L. (2001). “Amide group assisted 3′-dephosphorylation of oligonucleotides synthesized on universal A-supports”. Tetrahedron.
57 (23): 4977–4986. doi:10.1016/S0040-4020(01)00409-4.
o ^ “Metkinen Universal Solid Support III”. Metkinenchemistry.com. Retrieved 2012-04-04.
o ^ “Glen Research Corporation products for DNA and RNA oligo synthesis – Support – 27-5010,
Universal Support III PS”. Glenresearch.com. 2008-11-14. Retrieved 2009-05-12.
o ^ “Glen Research Report of Products for RNA and DNA Oligonucelotide Synthesis, Modification and Labelling”. Glenres.com. 2008-01-17. Retrieved 2009-05-12.
o ^
Petrie, C. R.; Reed, M. W.; Adams, A. D.; Meyer Jr, R. B. (1992). “An improved CPG support for the synthesis of 3′-amine-tailed oligonucleotides”. Bioconjugate Chem. 3 (1): 85–87. doi:10.1021/bc00013a014. PMID 1616954.
o ^ Lebedev,
A. V.; Wickstrom, E. (1996). “The chirality problem in P-substituted oligonucleotides”. Perspectives in Drug Discovery and Design. 4 (1): 17–40. doi:10.1007/BF02172106.
o ^ Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. (2000).
“Deoxyribonucleoside Cyclic N-Acylphosphoramidites as a New Class of Monomers for the Stereocontrolled Synthesis of Oligothymidylyl- and Oligodeoxycytidylyl- Phosphorothioates”. J. Am. Chem. Soc. 122 (10): 2149–2156. doi:10.1021/ja991773u.
o ^
Jump up to:a b “Glen Research Report of Products for RNA and DNA Oligonucelotide Synthesis, Modification and Labelling”. Glenresearch.com. 2008-01-17. Retrieved 2009-05-12.
o ^ Jump up to:a b “Sulfurizing reagent ii and its use in synthesizing
oligonucleotide phosphorothioates” (PDF). Glen Research. 18 (1). 2006. Retrieved 2009-08-01.
o ^ “AM Chemicals, LLC, a supplier of solid supports and reagents for oligonucleotide and organic synthesis on solid phase”. Amchemicals.com.
Archived from the original on 2009-02-18. Retrieved 2009-05-12.
o ^ “Glen Research Corporation products for DNA and RNA oligo synthesis – Minor Base – 40-4037, Sulfurizing Reagent II”. Glenresearch.com. 2008-11-14. Retrieved 2009-05-12.
o ^
Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. (1990). “3H-1,2-Benzodithiole-3-one 1,1-dioxide as an improved sulfurizing reagent in the solid-phase synthesis of oligodeoxyribonucleoside phosphorothioates”. J. Am. Chem. Soc. 112
(3): 1253–1254. doi:10.1021/ja00159a059.
o ^ Beaucage, S. L. (2001). “3H-1,2-benzodithiol-3-one 1,1-dioxide”. E-EROS Encyclopedia of Reagents for Organic Synthesis. doi:10.1002/047084289X.rn00167. ISBN 978-0471936237.
o ^ “3400/394/392/391
DNA Synthesizer Reagents”. Products.appliedbiosystems.com. Retrieved 2009-05-12.
o ^ Vu, H.; Hirschbein, B. L. (1991). “Internucleotide phosphite sulfurization with tetraethylthiuram disulfide. Phosphorothioate oligonucleotide synthesis
via phosphoramidite chemistry”. Tetrahedron Lett. 32 (26): 3005–3008. doi:10.1016/0040-4039(91)80672-S.
o ^ Tanaka, Toshiki; Letsinger, R. L. (1982). “Syringe method for stepwise chemical synthesis of oligonucleotides”. Nucleic Acids
Res. 10 (10): 3249–3259. doi:10.1093/nar/10.10.3249. PMC 320704. PMID 7099961.
o ^ “OligoMaster LS2”. Azcobiotech.com. Archived from the original on November 10, 2011. Retrieved 2011-10-18.
o ^ “DNA / RNA Oligonucleotide Synthesizer:
MerMade 384”. Bioautomation.com. Archived from the original on September 30, 2011. Retrieved 2011-10-18.
o ^ “QMaster DNA/RNA Synthesizer”. Genomictechnologies.com.
o ^ “QMaster DNA/RNA Synthesizer”. www.genomictechnologies.com/QmasterII.shtml.
Archived from the original on 2016-03-04. Retrieved 2014-04-02.
o ^ Sanghvi, Y. S. (2011). “A status update of modified oligonucleotides for chemoterapeutics applications”. Curr. Protoc. Nucleic Acid Chem. 46 (16): 4.1.1–4.1.22. doi:10.1002/0471142700.nc0401s46.
ISBN 978-0471142706. PMID 21901670. S2CID 41903519.
o ^ Jump up to:a b Pease A. C.; Solas D.; Sullivan E. J.; Cronin M. T.; Holmes C.P.; Fodor S. P. (1994). “Light-generated oligonucleotide arrays for rapid DNA sequence analysis”. Proc.
Natl. Acad. Sci. U.S.A. 91 (11): 5022–5026. Bibcode:1994PNAS…91.5022P. doi:10.1073/pnas.91.11.5022. PMC 43922. PMID 8197176.
o ^ Egeland, R. D; Southern, E. M. (2005). “Electrochemically directed synthesis of oligonucleotides for DNA
microarray fabrication” (Free full text). Nucleic Acids Res. 33 (14): e125. doi:10.1093/nar/gni117. PMC 1183109. PMID 16085751.
o ^ Capaldi, D. C.; Gaus, H.; Krotz, A. H.; et al. (2003). “Synthesis of High-Quality Antisense Drugs. Addition
of Acrylonitrile to Phosphorothioate Oligonucleotides: Adduct Characterization and Avoidance”. Organic Process Research & Development. 7 (6): 832–838. doi:10.1021/op020090n.
o ^ Volume 5: Deprotect to completion in organic solvents.
Glen Report 22 (2)
o ^ Boal, J. H.; Wilk, A.; Harindranath, N.; Max, E. E.; Kempel, T.; Beaucage, S. L. (1996). “Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines”.
Nucleic Acids Res. 24 (15): 3115–7. doi:10.1093/nar/24.15.3115. PMC 146024. PMID 8760903.
o ^ Westman, E.; Stroemberg, R. (1994). “Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF)
is a more reliable alternative to tetrabutylammonium fluoride (TBAF)”. Nucleic Acids Res. 22 (12): 2430–1. doi:10.1093/nar/22.12.2430. PMC 523709. PMID 7518583.
o ^ Krotz, A. H; Gaus, H.; Hardee, G. E. (2005). “Formation of oligonucleotide
adducts in pharmaceutical formulations”. Pharmaceutical Development and Technology. 10 (2): 283–90. doi:10.1081/PDT-54464. PMID 15926677. S2CID 34432071.
o ^ Willems, A.; Deforce, D. L.; Van Bocxlaer, J. (2008). “Analysis of oligonucleotides
using capillary zone electrophoresis and electrospray mass spectrometry, in Methods in Molecular Biology”. Capillary Electrophoresis. Totowa, NJ. 384: 401–414. doi:10.1007/978-1-59745-376-9_14. PMID 18392576.
Photo credit: https://www.flickr.com/photos/renaud-camus/12434194695/’]