Preparation of oligo conjugates

A method of preparing a compound of the formula

• comprising
• (i) attaching a conjugation component of the formula
• wherein R2 is R2a or R2ap,
• to a solid support
• to form a compound of the formula
• (ii) when R2 is R2ap, converting
• to
• (iii) contacting
• with a conjugation component of the formula
• wherein R3 is R3a, or R3ap;
• to form
• (iv) when R3 is R3ap, converting
• to
• and
• (v) contacting
• with a conjugation component of the formula
• to provide the compound of the formula
• wherein;
• is a solid support material;
• are independently an oligo, where each oligo is independently a polymer, having from 2 to about 150 covalently linked monomer units, that is characterized by a sequence of 2′-deoxyribosenucleotide residues (DNA), ribonucleotide residues (RNA), and peptide nucleic acids (PNAs), or a combination thereof;
• R1a and R1b are complementary conjugation functionalities and L1 is conjugate linker, and R1a, R1b, and L1 are
• (a) R1a is azido, R1b is —C≡C—R23, and L1 is
• or
• (b) R1a is —NHR23, R1b is carboxy, and L1 is —NR23C(═O)—, or
• (c) R1a is carboxy, R1b is —NHR23, and L1 is —C(═O)NR23—, or
• (d) R1a is —NHR23, R1b is halo, and L1 is —NR23—, or
• (e) R1a is —O—P(═O)(OH)(X), R1b is hydroxy, and L1 is —O—P(═O)(OH)—O—, or
• (f) R1a is —O—P(═O)(OH)(X), R1b is —NHR23, and L1 is —O—P(═O)(OH)— NR23—, or
• (g) R1a is —O—P(═O)(OH)(X), R1b is thio, and L1 is —O—P(═O)(OH)—S—, or
• (h) R1a is halo, R1b is thio, and L1 is —S—, or
• (i) R1a is
• R1b is thio, and L1
• or
• (j) R1a is —C≡C—R23, R1b is azido, and L1 is
• or
• (k) R1a is halo, R1b is —NHR23, and L1 is —NR23—, or
• (l) R1a is hydroxy, R1b is —O—P(═O)(OH)(X), and L1 is —O—P(═O)(OH)—O—, or
• (m) R1a is —NHR23, R1b is —O—P(═O)(OH)(X), and L1 is —NR23—P(═O)(OH)—O—, or
• (n) R1a is thio, R1b is —O—P(═O)(OH)(X), and L1 is —S—P(═O)(OH)—O—, or
• (o) R1a is —O—P(═O)(OH)SH, R1b is —X, and L1 is —O—P(═O)(OH)—S—, or
• (p) R1a is —X, R1b is —O—P(═O)(OH)SH, and L1 is —S—P(═O)(OH)—O—, or
• (q) R1a is —(CR25R25)SC(═O)OR23, R1b is —NHR23, L1 is —(CR25R25)sC(═O)NR23—, or
• (r) R1a is —NHR23, R1b is —(CR25R25)sC(═O)OR23, L1 is —NR23C(═O)—(CR25R25)s—, or
• (s) R1a is thio, R1b is halo, and L1 is —S—, or
• (t) R1a is thio, R1b is
• and L1 is
• (u) R1a is —SH, R1b is —O—P(═O)(OH)(O—(CH2)n—SH) and L1 is —S—S—(CH2)n—O—P(OH)(═O)—O—;
• R2a and R2b are complementary conjugation functionalities and L2 is conjugate linker, and R2ap, R2a, R2b, and L2 are
• (a′) R2ap is halo, R2a is azido, R2b is —C≡C—R23, and L2 is
• or
• (b′) R2ap is —NR23Pr, R2a is —NHR23, R2b is carboxy, and L2 is —NR23C(═O)—, or
• (c′) R2ap is carboxy ester, R2a is carboxy, R2b is —NHR23, and L2 is —C(═O)NR23—, or
• (d′) R2ap is —NR23Pr, R2a is —NHR23, R2b is halo, and L2 is —NR23—, or
• (e′) R2ap is —OH, phosphate or phosphate ester, R2a is —O—P(═O)(OH)(X), R2b is hydroxy, and L2 is —O—P(═O)(OH)—O—, or
• (f′) R2ap is —OH, phosphate or phosphate ester, R2a is —O—P(═O)(OH)(X), R2b is —NHR23, and L2 is —O—P(═O)(OH)—NR23—, or
• (g′) R2ap is —OH, phosphate or phosphate ester, R2a is —O—P(═O)(OH)(X), R2b is thio, and L2 is —O—P(═O)(OH)—S—, or
• (h′) R2a is —X, R2b is thio, and L2 is —S—, or
• (i′) R2a is
• R2b is thio, and L2 is
• or
• (j′) R2a is —C≡C—R23, R2b is azido, and L2 is
• or
• (k′) R2a is halo, R2b is —NHR23, and L2 is —NR23—, or
• (l′) R2a is hydroxy, R2b is —O—P(═O)(OH)(X), and L2 is —O—P(═O)(OH)—O—, or
• (m′) R2a is —NHR23, R2b is —O—P(═O)(OH)(X), and L2 is —NR23—P(═O)(OH)—O—, or
• (n′) R2a is thio, R2b is —O—P(═O)(OH)(X), and L2 is —S—P(═O)(OH)—O—, or
• (o′) R2ap is —O—P(═O)(OH)SR24, R2a is —O—P(═O)(OH)SH, R2b is —X, and L2 is —O—P(═O)(OH)—S—, or
• (p′) R2a is —X, R2b is —O—P(═O)(OH)SH, and L2 is —S—P(═O)(OH)—O—, or
• (q′) R2a is —(CR25R25)sC(═O)OR23, R2b is —NHR23, L2 is —(CR25R25)sC(═O)NR23—, or
• (r′) R2a is —NHR23, R2b is —(CR25R25)sC(═O)OR23, L2 is —NR23C(═O)—(CR25R25)s—, or
• (s′) R2a is thio, R2b is halo, and L2 is —S—, or
• (t′) R2a is thio, R2b is
• and L2 is
• or
• (u′) R2a is —SH, R2b is —O—P(═O)(OH)(O—(CH2)n—SH) and L2 is —S—S—(CH2)n—O—P(OH)(═O)—O—;
• R3a and R3b are complementary conjugation functionalities and L3 is conjugate linker, and R3ap, R3a, R3b, and L3 are
• (a″) R3ap is halo, R3a is azido, R3b is —C≡C—R23, and L3 is
• or
• (b″) R3ap is —NR23Pr, R3a is —NHR23, R3b is carboxy, and L3 is —NR23C(═O)—, or
• (c″) R3ap is carboxy ester, R3a is carboxy, R3b is —NHR23, and L3 is —C(═O)NR23—, or
• (d″) R3ap is —NR23Pr, R3a is —NHR23, R3b is halo, and L3 is —NR23—, or
• (e″) R3ap is —OH, phosphate or phosphate ester, R3a is —O—P(═O)(OH)(X), R3b is hydroxy, and L3 is —O—P(═O)(OH)—O—, or
• (f″) R3ap is —OH, phosphate or phosphate ester, R3a is —O—P(═O)(OH)(X), R3b is —NHR23, and L3 is —O—P(═O)(OH)—NR23—, or
• (g″) R3ap is —OH, phosphate or phosphate ester, R3a is —O—P(═O)(OH)(X), R3b is thio, and L3 is —O—P(═O)(OH)—S—, or
• (h″) R3a is —X, R3b is thio, and L3 is —S—, or
• (i″) R3a is
• R3b is thio, and L3 is
• or
• (j″) R3a is —C≡C—R23, R3b is azido, and L3 is
• or
• (k″) R3a is halo, R3b is —NHR23, and L3 is —NR23—, or
• (l″) R3a is hydroxy, R2b is —O—P(═O)(OH)(X), and L3 is —O—P(═O)(OH)—O—, or
• (m′) R3a is —NHR23, R3b is —O—P(═O)(OH)(X), and L3 is —NR23—P(═O)(OH)—O—, or
• (n′) R3a is thio, R3b is —O—P(═O)(OH)(X), and L3 is —S—P(═O)(OH)—(O), or
• (o″) R3ap is —O—P(═O(OH)SR24, R3a is —O—P(═O)(OH)SH, R3b is —X, and L3 is —O—P(═O)(OH)—S—, or
• (p″) R3a is —X, R3b is —O—P(═O)(OH)SH, and L3 is —S—P(═O)(OH)—O—, or
• (q″) R3a is —(CR25R25)sC(═O)OR23, R3b is —NHR23, L3 is —(CR25R25)sC(═O)NR23—, or
• (r″) R3a is —NHR23, R3b is —(CR25R25)sC(═C)OR23, L3 is —NR23C(═O)—(CR25R25)s—, or
• (s″) R3a is thio, R3b is —X, and L3 is —S—, or
• (t″) R3a is thio, R3b is
• and L3 is
• or
• (u″) R3a is —SH, R3b is —O—P(═O)(OH)(O—(CH2)n—SH) and L3 is —S—S—(CH2)n—O—P(OH)(═O)—O—;
• R4 is selected from the group consisting of R3a, R3ap, halo, —NR23Pr, —OH, —O—P(═O)(OH)SR24, carboxy ester, phosphate, phosphate ester, azido, —C≡C—R23, —NHR23, carboxy, hydroxy, —(CR25R25)sC(═O)OR23, —O—P(═O)(OH)SH, or —O—P(═O)(OH)Br, or an oligo;
• X is chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflate,
• n is 1, 2, 3, 4, 5, or 6;
• R23 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
• Pr is an amino protecting group;
• R24 is trityl or benzyl;
• R25 is hydrogen or C1-6 alkyl;
• n is 1, 2, 3, 4, 5, or 6;
• s is an integer of greater than 1;
• provided that R2 does not react with R1a or R1b, R3 does not react with R2a or R2b and R4 does not react with R3a or R3b;
• ----- represents the point of connection to the part of the solid support-bound conjugated molecule that is closer to
• and represents the point of connection to the part of the solid support-bound conjugated molecule that is further away from