Artificial bioconjugates with naturally occurring linkages: the use of phosphodiester

Artificial orthogonal bond formations such as the alkyne–azide cycloaddition have enabled selective bioconjugations under mild conditions, yet naturally occurring linkages between native functional groups would be more straightforward to elaborate bioconjugates. Herein, we describe the use of a phosphodiester bond as a versatile option to access various bioconjugates. An opposite activation strategy, involving 5’-phosphitylation of the supported oligonucleotides, has allowed several biomolecules that possess an unactivated alcohol to be directly conjugated. It should be noted that there is no need to pre-install artificial functional groups and undesired and unpredictable perturbations possibly caused by bioconjugation can be minimized.


Supported dT (S3)
The titled compound was synthesized according to the reported procedure.

General procedure for elongation of oligonucleotides
Carried out according to the reported procedure. 1 To a solution of supported oligonucleotide (1.0 mmol) in dichloromethane (88 mL) stirred at rt was added amidite monomer and 0.25 M BMT/MeCN (8.8 mL). The resulting reaction mixture was stirred at rt for 20 min, followed by the addition of 1% Ac 2 O, 1% pyridine, and 1% NMI. The resulting reaction mixture was stirred at rt for 10 min, followed by the addition of 0.67% BPO/DMP/CH 2 Cl 2 . The resulting reaction mixture was stirred at rt for 10 min, diluted with MeOH (100 mL), and collected by vacuum filtration. The residue was repeatedly washed with MeOH and dried in vacuo to give the elongated product as a white solid.

General procedure for deprotection of DMTr
To a solution of supported oligonucleotide (1.0 mmol) in CH 2 Cl 2 (20 mL) stirred at rt was added 5% DCA/ CH 2 Cl 2 (20 mL). The resulting reaction mixture was stirred at rt for 1 min, diluted with MeOH (100 mL), neutralized with TEA and collected by vacuum filtration. The residue was repeatedly washed with MeOH and dried in vacuo to give the deprotected product as a white solid.

General procedure for coupling of peptide
To a solution of supported peptide (1.0 mmol) and Fmoc-AA-OH in THF (20 mL) stirred at rt was added COMU (1.4 mmol) and DIPEA (1.4 mmol). The resulting reaction mixture was stirred at rt for 10 min, diluted with MeCN (60 mL), and collected by vacuum filtration. The residue was repeatedly washed with MeCN and dried in vacuo to give the coupled product as a white solid.
The residue was repeatedly washed with MeOH and dried in vacuo to give the desired bioconjugate as a white solid. B) Bioconjugation of supported pentapeptide (7) to 5'-activated supported trinucleotide 2 To a solution of 5'-activated supported trinucleotide 2 (0.025 mmol) in CH 2 Cl 2 (10:1, v/v, 6.4 mL) stirred at rt was add supported pentapeptide 7 and 0.75 M BMT/MeCN (200 µL). The resulting reaction mixture was stirred at rt for 30 min, followed by the addition of 0.67% BPO/DMP/CH 2 Cl 2 . The resulting reaction mixture was stirred at rt for 30 min, diluted with MeOH (30 mL), and collected by vacuum filtration. The residue was repeatedly washed with MeOH and dried in vacuo to give the desired bioconjugate as a white solid. C) Bioconjugation of lipid 9 and suger 10 to 5'-activated suppored trinucleotide 2 To a solution of 5'-activated supported trinucleotide 2 (0.025 mmol) in CH 2 Cl 2 (10:1, v/v, 2 mL) stirred at rt was add lipid 9 or sugar 10 and 0.25 M BMT/MeCN (200 µL

Bioconjugations and deprotection, see Scheme 4
All protocol was carried out according to the procedure of pentapeptide conjugate (8).

Protected pentapeptide conjugate 15
Crude product as white solid. 31