Protocols for oligonucleotide conjugates synthesis and analytical techniques
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Protocols for oligonucleotide conjugates synthesis and analytical techniques
Chapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques ic blocks. Each block features at least a nucleophilic and an electrophilic function, i.e., the a-amino and the carboxylic functions for peptides, the 5'-OH and the 3’-function (phosphate, phosphoramidite, or phosphonate), for nucleotides. The nucleophilic and electrophilic sites are linked together Protocols for oligonucleotide conjugates synthesis and analytical techniques at the coupling step. Protection is a necessity. It guarantees the chemoselectivity of coupling and the solubility of synthons in organic solvents.ThProtocols for oligonucleotide conjugates synthesis and analytical techniques
ere are two classes of protecting groups: persistent and transient. The persistent protections remain on the biopolymer during all the synthesis. TheyChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques . They also cap the phosphate oxygen of oligonucleotides. The transient protections block the functions to be coupled at a given time of the synthesis. They are specifically cleaved before each coupling.When the synthesis is performed on a solid support, the first monomer of a hundred-mer has to sur Protocols for oligonucleotide conjugates synthesis and analytical techniques vive to a hundred cleavages of a transient protecting group. The yield of successful removal is thus as limiting as the coupling yield. This is also tProtocols for oligonucleotide conjugates synthesis and analytical techniques
rue for the final deprotection. If each monomeric unit is only 90% deprotected, the yield of a dimer of correct structure is 92%, of a trimer 93/10%, Chapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques mana Press Inc., Totowa, NJ12SonveauxYields drop dramatically with length. Paradoxically, the crucial problem of protection is thus deprotection. Good results obtained with a protection strategy on small sequences do not guarantee that the method is viable. The discriminating test is the success in Protocols for oligonucleotide conjugates synthesis and analytical techniques obtaining high yields of long oligomers.In oligonucleotide synthesis, the academic research is nowadays moving from DNA synthesis to the synthesis ofProtocols for oligonucleotide conjugates synthesis and analytical techniques
RNA and modified DNA/RNA structures. As functions and types of aglycone residues entering oligonucleotide synthesis diversify, protection strategies hChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques f ribonucleotides are considered first. Both are usually introduced at the beginning of the synthesis. The transient protection of the 5'-0H is then discussed. This function is indeed capped before the phosphorylation or phosphitylation of the synthons. The last paragraph describes the protections o Protocols for oligonucleotide conjugates synthesis and analytical techniques f phosphorus. This last topic is much related to coupling strategies. The reader is thus invited to consult the other parts of this book to embrace thProtocols for oligonucleotide conjugates synthesis and analytical techniques
e whole field.2. Protection of the Heterocyclic Bases and Protocols for Oligonucleotides and AnalogsThe protecting groups that have been proposed for Chapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques dineIt is possible to synthesize oligo DNA or RNA by one of the three classical methods (phosphotriester, phosphoramidite, phosphonate stategies) without protecting these residues. However, the N-3 nitrogen being acidic (T, pKa = 9.79) (1), a certain amount of the anionic form is present in basic me Protocols for oligonucleotide conjugates synthesis and analytical techniques dia. In these conditions, the thymine and uracil residues react with electrophiles like alkylating agents (2,3) (inter alia, the methyl phosphate of tProtocols for oligonucleotide conjugates synthesis and analytical techniques
he internucleotidic bond in one of the phosphoramidite strategies (3-5), carbodiimides (6), activated phosphates (7-14) and sulfonates (15,16), bis(diChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques products undergo a nucleophilic attack on C-4 by nucleophiles usually present in the medium, like 1,2,4-triazole, 3-nitro-1,2,4-triazole, 1-hydroxybenzotriazole, N-methylim-idazole or pyridine. In the case of acylation, the N-3 acylated derivative is usually obtained (19). It is the thermodynamic p Protocols for oligonucleotide conjugates synthesis and analytical techniques roduct. The 0-4 acylated derivative, accessible by PTC, spontaneously rearranges to the N-3 acylated isomer (16).In the conditions of a normal oligonuProtocols for oligonucleotide conjugates synthesis and analytical techniques
cleotide synthesis, the contact times with electrophiles are short and these side reactions are limited (20). Thymidine is less sensitive than uridineChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques (e.g., oximate). The side reactions of uracil in phosphotriester synthesis may be a serious nuisance (22). They have been carefully studied by Reese (7,15,23,24).Two protecting groups are well established: the N-3 anisoyl and the 4-(2,4-dimethylphenyloxy) derivatives 1 and 4 (Table 1).The N-3 aniso Protocols for oligonucleotide conjugates synthesis and analytical techniques yl compound is a little more resistant to nucleophilic attack than the N-3 benzoyl (25,26). It is imroduced, by PTC (16) or in homogeneous conditionsProtocols for oligonucleotide conjugates synthesis and analytical techniques
(27) (See also ref. 28), by selective N-3 acylation of 3',5'-O-( 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine, followed by the protection of the Chapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques (29). The selective N-3 acylation is possible by the so called Jones’ method (in situ protection of the 5’, 3', and 2’-0 by trimethylsilyl chloride during acylation, followed by the hydrolysis of the silyl ethers in the workup) (26,28,30).Protection 4 is introduced by reaction of 2’-ớ-(4-methoxytetr Protocols for oligonucleotide conjugates synthesis and analytical techniques ahy-dropyran-4-yl)-3',5'-O-dimethoxyacetyluridine with diphenylphos-phochloridate and 3-nitro-l,2,4-triazole in pyridine, followed by a treatment withProtocols for oligonucleotide conjugates synthesis and analytical techniques
a phenol and triethylamine. The labile 3' and 5'-ớ acyl groups are cleaved by 8M ammonia in methanol (23). Care is needed because this type of uracilChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques on N-4. Cytosine is the most basic of the aglycone residues (dC, pKa = 4.25). It is also the most nucleophilic. The rate of acylation decreases in the order: C(AM) > OH > A(2V-6) > GỢV-2) (52).4SonveauxTable 1Protections of Thymine and Uracil Residues*1Properties of the tabulated protecting groups ( Protocols for oligonucleotide conjugates synthesis and analytical techniques solvent mixtures are expressed in volumic proportions)1.Resists to K2CO3 Q.2M (short time); TBAF 1A//THF; chromatography on silica (CHCI3/CH3OH); NEt3Protocols for oligonucleotide conjugates synthesis and analytical techniques
/pyridine. It IS cleaved by NH3 cc in CH3OH/H2O; n-butylamine 0 04Af/CH3OH; TBAF/pyridine/H2O (81:1). See refs. 25,26,28,29,33-35.2.Resists to CH3C00HChapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeri Protocols for oligonucleotide conjugates synthesis and analytical techniques ts to i-butylamine 0.14A//CH3OH (20 min). It is cleaved by Zn/pyndine. See refs 16,38,39.4.Resists to K2CO3 0.2M; morpholine 0 05Af, NH3 8A//CH3OH (<15 min). It is cleaved by oximate. See refs. 23,24,40. Protocols for oligonucleotide conjugates synthesis and analytical techniques Chapter 1Protecting Groups in Oligonucleotide SynthesisEtienne Sonveaux1. IntroductionA biopolymer is synthesized by assembling monomeric or oligomeriGọi ngay
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