Preparation of Oligo Conjugates
Conjugated molecules are prepared that comprise a predetermined number of oligo conjugation components. The conjugated molecules also may comprise one or more detectable labels. Preparation of these molecules can be implemented according to an asymmetric or a symmetric conjugation strategy.
1 . A method of preparing a compound of the formula
comprising
(i) attaching a conjugation component of the formula
wherein R 2 is R 2a or R 2ap ,
to a solid support
to form a compound of the formula
(ii) when R 2 is R 2ap , converting
to
and
(iii) contacting
with a conjugation component of the formula
wherein R 3 is R 3a , R 3ap , hydrogen, a detectable label or an oligo;
to form
wherein:
is a solid support material;
are independently an oligo;
R 1a and R 1b are complementary conjugation functionalities and L 1 is conjugate linker, and R 1a , R 1b , and L 1 are
(a) R 1a is azido, R 1b is —C≡C—R 23 , and L 1 is
or
(b) R 1a is —NHR 23 , R 1b is carboxy, and L 1 is —NR 23 C(═O)—, or
(c) R 1a is carboxy, R 1b is —NHR 23 , and L 1 is —C(═O)NR 23 —, or
(d) R 1a is —NHR 23 , R 1b is halo, and L 1 is —NR 23 —, or
(e) R 1a is —O—P(═O)(OH)(X), R 1b is hydroxy, and L 1 is —O—P(═O)(OH)—O—, or
(f) R 1a is —O—P(═O)(OH)(X), R 1b is —NHR 23 , and L 1 is —O—P(═O)(OH)— NR 23 —, or
(g) R 1a is —O—P(═O)(OH)(X), R 1b is thio, and L 1 is —O—P(═O)(OH)—S—, or
(h) R 1a is halo, R 1b is thio, and L 1 is —S—, or
(i) R 1a is
R 1b is thio, and L 1 is
or
(j) R 1a is —C≡C—R 23 , R 1b is azido, and L 1 is
or
(k) R 1a is halo, R 1b is —NHR 23 , and L 1 is —NR 23 —, or
(l) R 1a is hydroxy, R 1b is —O—P(═O)(OH)(X), and L 1 is —O—P(═O)(OH)—O—, or
(m) R 1a is —NHR 23 , R 1b is —O—P(═O)(OH)(X), and L 1 is —NR 23 —P(═O)(OH)—O—, or
(n) R 1a is thio, R 1b is —O—P(═O)(OH)(X), and L 1 is —S—P(═O)(OH)—O—, or
(o) R 1a is —O—P(═O)(OH)SH, R 1b is —X, and L 1 is —O—P(═O)(OH)—S—, or
(p) R 1a is —X, R 1b is —O—P(═O)(OH)SH, and L 1 is —S—P(═O)(OH)—O—, or
(q) R 1a is —(CR 25 R 25 ) s C(═O)OR 23 , R 1b is —NHR 23 , L 1 is —(CR 25 R 25 ) s C(═O)NR 23 —, or
(r) R 1a is —NHR 23 , R 1b is —(CR 25 R 25 ) s C(═O)OR 23 , L 1 is —NR 23 C(═O)—(CR 25 R 25 ) s —, or
(s) R 1a is thio, R 1b is halo, and L 1 is —S—, or
(t) R 1a is thio, R 1b is
and L 1 is
(u) R 1a is —SH, R 1b is —O—P(═O)(OH)(O—(CH 2 ) n —SH) and L 1 is —S—S—(CH 2 ) n —O—P(OH)(═O)—O—;
R 2a and R 2b are complementary conjugation functionalities and L 2 is conjugate linker, and R 2ap , R 2a , R 2b , and L 2 are
(a′) R 2ap is halo, R 2a is azido, R 2b is —C≡C—R 23 , and L 2 is
or
(b′) R 2ap is —NR 23 Pr, R 2a is —NHR 23 , R 2b is carboxy, and L 2 is —NR 23 C(═O)—, or
(c′) R 2ap is carboxy ester, R 2a is carboxy, R 2b is —NHR 23 , and L 2 is —C(═O)NR 23 —, or
(d′) R 2ap is —NR 23 Pr, R 2a is —NHR 23 , R 2b is halo, and L 2 is —NR 23 —, or
(e′) R 2ap is —OH, phosphate or phosphate ester, R 2a is —O—P(═O)(OH)(X), R 2b is hydroxy, and L 2 is —O—P(═O)(OH)—O—, or
(f′) R 2ap is —OH, phosphate or phosphate ester, R 2a is —O—P(═O)(OH)(X), R 2b is —NHR 23 , and L 2 is —O—P(═O)(OH)—NR 23 —, or
(g′) R 2ap is —OH, phosphate or phosphate ester, R 2a is —O—P(═O)(OH)(X), R 2b is thio, and L 2 is —O—P(═O)(OH)—S—, or
(h′) R 2a is —X, R 2b is thio, and L 2 is —S—, or
(i′) R 2a is
R 2b is thio, and L 2 is
or
(j′) R 2a is —C≡C—R 23 , R 2b is azido, and L 2 is
or
(k′) R 2a is halo, R 2b is —NHR 23 , and L 2 is —NR 23 —, or
(l′) R 2a is hydroxy, R 2b is —O—P(═O)(OH)(X), and L 2 is —O—P(═O)(OH)—O—, or
(m′) R 2a is —NHR 23 , R 2b is —O—P(═O)(OH)(X), and L 2 is —NR 23 —P(═O)(OH)—O—, or
(n′) R 2a is thio, R 2b is —O—P(═O)(OH)(X), and L 2 is —S—P(═O)(OH)—O—, or
(o′) R 2ap is —O—P(═O)(OH)SR 24 , R 2a is —O—P(═O)(OH)SH, R 2b is —X, and L 2 is —O—P(═O)(OH)—S—, or
(p′) R 2a is —X, R 2b is —O—P(═O)(OH)SH, and L 2 is —S—P(═O)(OH)—O—, or
(q′) R 2a is —(CR 25 R 25 ) s C(═O)OR 23 , R 2b is —NHR 23 , L 2 is —(CR 25 R 25 ) s C(═O)NR 23 —, or
(r′) R 2a is —NHR 23 , R 2b is —(CR 25 R 25 ) s C(═O)OR 23 , L 2 is —NR 23 C(═O)—(CR 25 R 25 ) 5 —, or
(s′) R 2a is thio, R 2b is halo, and L 2 is —S—, or
(t′) R 2a is thio, R 2b is
and L 2 is
or
(u′) R 2a is —SH, R 2b is —O—P(═O)(OH)(O—(CH 2 ) n —SH) and L 2 is —S—S—(CH 2 ) n —O—P(OH)(═O)—O—;
X is selected from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflate,
n is 1, 2, 3, 4, 5, or 6;
R 23 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;
R 24 is trityl or benzyl;
R 25 is hydrogen or C 1-6 alkyl;
n is 1, 2, 3, 4, 5, or 6;
s is an integer of greater than 1;
R 3ap is selected from the group consisting of halo, —OH, —NR 23 Pr, —O—P(═O)(OH)SR 24 , carboxy ester, phosphate and phosphate ester; and
R 3a is selected from the group consisting of azido, —C≡C—R 23 , —NHR 23 , carboxy, halo, hydroxy, —C(═O)OR 23 , —O—P(═O)(OH)SH and —O—P(═O)(OH)Br;
provided that R 2 does not react with R 1a or R 1b , and R 3 does not react with R 2a or R 2b ;
----- 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
2 . The method of claim 1 for preparing a compound of the formula
comprising
(i) attaching a compound of the formula
to a solid support
to form a compound of the formula
and
(iii) reacting
with a compound of the formula
to form
wherein:
is a solid support material;
are independently an oligo;
R 1a and R 1b are complementary conjugation functionalities and L 1 is conjugate linker, R 2a and R 2b are complementary conjugation functionalities and L 2 is conjugate linker, and R 1a , R 1b , L 1 , R 2a , R 2b , L 2 , and R 3a are selected from
R 1a , R 2a , or R 3a
R 1b or R 2b
L 1 or L 2
—C≡C—R 23
azido
azido
—C≡C—R 23
carboxy
—NHR 23
—C(═O)NR 23 —
—NHR 23
carboxy
—NR 23 C(═O)—
halo
—NHR 23
—NR 23 —
—NR 23
halo
—NR 23 —
hydroxy
—O—P(═O)(OH)(X)
—O—P(═O)(OH)—O—
—O—P(═O)(OH)(X)
hydroxy
—O—P(═O)(OH)—O—
—NHR 23
—O—P(═O)(OH)(X)
—NR 23 —P(═O)(OH)—O—
—O—P(═O)(OH)(X)
—NHR 23
—O—P(═O)(OH)—NR 23 —
thio
—O—P(═O)(OH)(X)
—S—P(═O)(OH)—O—
—O—P(═O)(OH)(X)
thio
—O—P(═O)(OH)—S—
thio
—X
—S—
—X
thio
—S—
—O—P(═O)(OH)SH
—X
—O—P(═O)(OH)—S—
—X
—O—P(═O)(OH)SH
—S—P(═O)(OH)—O—
—(CR 25 R 25 ) s C(═O)OR 23
—NHR 23
—(CR 25 R 25 ) s C(═O)NR 23 —
—NHR 23
—(CR 25 R 25 ) s C(═O)OR 23
—NR 23 C(═O)—(CR 25 R 25 ) s —
thio
thio
—SH
—O—P(═O)(OH)(O—(CH 2 ) n —SH)
—S—S—(CH 2 ) n —O—P(OH)(═O)—O—
wherein the selection of R 1a , R 2a , or R 3a is independent of each other provided that L 1 and L 2 are different, R 2a does not react with R 1a or R 1b , and R 3a does not react with R 2a or R 2b ;
X is selected from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflate;
R 23 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; and
R 25 is hydrogen or C 1-6 alkyl;
s is an integer of greater than 1;
----- 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
3 . A method of preparing a compound of the formula
comprising
(i) attaching a compound of the formula
to a solid support
to form a compound of the formula
(ii) converting
to
and
(iii) contacting
with a compound of the formula
to form
wherein:
is a solid support material;
are independently an oligo;
R ap , R a , R b , and L are selected from
(a) R ap is halo, R a is azido, R b is —C≡C—R 23 , and L is
or
(b) R ap is —NR 23 Pr, R a is —NHR 23 , R b is carboxy, and L is —NR 23 C(═O)—, or
(c) R ap is carboxy ester, R a is carboxy, R b is —NHR 23 , and L is —C(═O)NR 23 —, or
(d) R ap is —NR 23 Pr, R a is —NHR 23 , R b is halo, and L is —NR 23 —, or
(e) R ap is —OH, phosphate or phosphate ester; R a is —O—P(═O)(OH)(X), R b is hydroxy, and L is —O—P(═O)(OH)—O—, or
(f) R ap is —OH, phosphate or phosphate ester; R a is —O—P(═O)(OH)(X), R b is —NHR 23 , and L is —O—P(═O)(OH)—NR 23 —, or
(g) R ap is —O—P(═O)(OH)SR 24 , R a is —O—P(═O)(OH)SH, R b is —X, and L is —O—P(═O)(OH)—S—, or
(h) R ap is —OH, phosphate or phosphate ester; R a is —O—P(═O)(OH)Br, R b is thio, and L is —O—P(═O)(OH)—S—, or
(i) (i) R ap is —SR 24 , R a is —SH, R b is —O—P(═O)(OH)SH and L is —O—P(═O)(OH)S—S—;
wherein R 23 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;
X is selected from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflate;
R 24 is trityl or benzyl;
----- 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
4 . A method of preparing a compound of the formula
comprising
cleaving the bond between
and L 1 of the compound of the formula
thereby obtaining
wherein:
the compound of
is prepared according to the method of claim 1 ; and
L 1 , L 2 , R 3 ,
are as defined in claim 1 ; and
Z is selected from —OH, OH—(C 1 -C 10 )alkylene-, —COOH, NH 2 C(O)—, NH 2 NH—C(O)—, COOH—(C 1 -C 10 )alkylene-, NH 2 C(O)—(C 1 -C 10 )alkylene-, NH 2 NH—C(O)—(C 1 -C 10 )alkylene-, CH 2 ═CH—(C 1 -C 10 )alkylene-, C≡C—(C 1 -C 10 )alkylene- or HS—(C 1 -C 10 )alkylene- and any alkylene is optionally substituted by one or more groups selected from —OH, halogen, —NHR″, —NHC(O)—(C 1 -C 10 )alkylene-C≡CH, or —NHC(O)—(C 1 -C 10 )alkylene-CH═CH 2 ;
R″ is selected from (C 1 -C 10 )alkyl, (C 3 -C 10 )cycloalkyl, or (C 3 -C 10 )aryl.
5 . A method of preparing a compound of the formula
comprising converting a compound of the formula
to a compound of the formula
wherein:
R is an oligo or a detectable label;
the compound of the formula
is prepared according to the method of claim 1 ; and
L 1 , L 2 , R 3 ,
are as defined in claim 1 .
6 . A method of preparing a compound of the formula
comprising
cleaving the bond between
and L 1 of the compound of the formula
thereby obtaining
wherein:
the compound of the formula
is prepared according to the method of claim 5 ;
Z is selected from —OH, OH—(C 1 -C 10 )alkylene-, —COOH, NH 2 C(O)—, NH 2 NH—C(O)—, COOH—(C 1 -C 10 )alkylene-, NH 2 C(O)—(C 1 -C 10 )alkylene-, NH 2 NH—C(O)—(C 1 -C 10 )alkylene-, CH 2 ═CH—(C 1 -C 10 )alkylene-, C≡C—(C 1 -C 10 )alkylene- or HS—(C 1 -C 10 )alkylene- and the (C 1 -C 10 )alkylene can be optionally substituted by one or more groups selected from —OH, halogen, —NHR″, —NHC(O)—(C 1 -C 10 )alkylene-C≡CH, or —NHC(O) (C 1 -C 10 )alkylene-CH═CH 2 ;
R″ is selected from (C 1 -C 10 )alkyl, (C 3 -C 10 )cycloalkyl, or (C 3 -C 10 )aryl, -and
L 1 , L 2 , R,
are as defined in claim 5 .
7 . A solid support-bound conjugated molecule represented by the formula:
wherein:
is a solid support material;
is an oligo;
R 2 is R 2a , R 2ap , an oligo or a detectable label;
R 2ap is selected from the group consisting of halo, —OH, NR 23 Pr, carboxy ester, phosphate, phosphate ester, and —O—P(═O)(OH)SR 24 ,
R 2a is selected from the group consisting of halo, azido, hydroxy, thio, —NHR 23 , carboxy, —NHR 23 , —O—P(═O)(OH)SH, —O—P(═O)(OH)(X),
—C≡C—R 23 , —(CR 25 R 25 ) s C(═O)OR 23 ;
L 1 is selected from the group consisting of —C(═O)NR 23 —, —NR 23 C(═O)—, —(CR 25 R 25 ) s C(═O)NR 23 —, —NR 23 C(═O)—(CR 25 R 25 ) s —, —NR 23 —, —O—P(═O)(OH)—O—, —NR 23 —P(═O)(OH)—O—, —O—P(═O)(OH)—NR 23 —, —S—, —S—P(═O)(OH)—O—, and —O—P(═O)(OH)—S—,
R 23 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;
X is selected from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflate;
R 24 is trityl or benzyl;
R 25 is hydrogen or C 1-6 alkyl;
s is an integer of greater than 1; and
wherein ----- 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
8 . A compound of the formula
wherein:
R 2 is R 2a , R 2ap , or an oligo or a detectable label;
is an oligo;
R 1b is selected from the group consisting of —C≡C—R 23 , carboxy, —NHR 23 , halo, hydroxy, thio, azido, —O—P(═O)(OH)(X), —O—P(═O)(OH)SH, —(CR 25 R 25 ) s C(═O)OR 23 , and
R 2ap is selected from the group consisting of halo, NR 23 Pr, carboxy ester, phosphate, phosphate ester and —O—P(═O)(OH)SR 24 ;
R 2a is selected from the group consisting of halo, azido, hydroxy, thio, —NHR 23 , carboxy, —NHR 23 , —O—P(═O)(OH)(X), —O—P(═O)(OH)SH,
—C≡C—R 23 , —(CR 25 R 25 ) s C(═O)OR 23 ;
R 23 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;
X is selected from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy triflat;
R 24 is trityl or benzyl; and
and represents the point of connection to