Cholesterol → HCID 66607346, compound

HRID 1053619Notes

Reaction equation

HRID 1053619 reaction equation
[CH3:1][CH:2]([CH2:4][CH2:5][CH2:6][C@H:7]([C@@H:9]1[C@:27]2([CH3:28])[C@H:12]([C@H:13]3[C@H:24]([CH2:25][CH2:26]2)[C@:22]2([CH3:23])[C:16]([CH2:17][C@H:18]([CH2:20][CH2:21]2)[OH:19])=[CH:15][CH2:14]3)[CH2:11][CH2:10]1)[CH3:8])[CH3:3].[NH:29]1[CH2:36][C@H:34]([OH:35])[CH2:33][C@H:30]1[CH2:31][OH:32].P(O[C@H]1CC[C@@]2(C)C(=CC[C@@H]3[C@@H]2CC[C@@]2(C)[C@H]3CC[C@@H]2[C@H](C)CCCC(C)C)C1)(N)O.O[C@H]1CN[C@H](CO)C1.NCCCCCC([O-])=O>>[NH:29]1[CH2:36][C@H:34]([OH:35])[CH2:33][C@H:30]1[CH2:31][OH:32].[CH3:3][CH:2]([CH2:4][CH2:5][CH2:6][C@H:7]([C@@H:9]1[C@:27]2([CH3:28])[C@H:12]([C@H:13]3[C@H:24]([CH2:25][CH2:26]2)[C@:22]2([CH3:23])[C:16]([CH2:17][C@H:18]([CH2:20][CH2:21]2)[OH:19])=[CH:15][CH2:14]3)[CH2:11][CH2:10]1)[CH3:8])[CH3:1]

Participants

5

Reactant 4

HCID 5997Cholesterol2×HCID 5460263HCID 5460263, compoundHCID 15817396(3R,5S)-5-(Hydroxymethyl)pyrrolidin-3-OLHCID 86761550HCID 86761550, compound

Product 1

HCID 66607346HCID 66607346, compound

Procedure

3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled siRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with ten-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes. Deprotection I (Nucleobase Deprotection) After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac. Deprotection II (Removal of 2′-TBDMS Group)

Workup

  1. customare synthesized
  2. additioncontaining the corresponding ligand

Sources & evidence

ORD record
ord-1f43b79fbd424685b7496f464ed63a61
Source dataset
uspto-grants-2016_07
Patent number
US09394234B2

Reaction notes

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