Insulin polypeptide-oligomer conjugates, proinsulin polypeptide-oligomer conjugates and methods of synthesizing same

A method of synthesizing an insulin polypeptide-oligomer conjugate comprising:

• (a) contacting a proinsulin polypeptide comprising an insulin polypeptide coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer comprising the structure of Formula I:
• A-Lj-Gk-R-G′m-R′-G″n-T (I)
• wherein:
• A is an activatable moiety;
• L is an optional linker moiety;
• G, G′ and G″ are each optional spacer moieties;
• R is a lipophilic moiety and R′ is a polyalkylene glycol moiety, or R′ is the lipophilic moiety and R is the polyalkylene glycol moiety, and wherein, (i) R and R′ are both present, or (ii) R and G are absent and L is coupled to G′ if present or to R′ if G′ is not present, or (iii) R′ and G″ are absent and T is coupled to G′ if present or to R if G′ is not present;
• T is a terminating moiety;
• and
• j, k, m and n are individually 0 or 1;
• under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate;
• and
• (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate.

A method of synthesizing an insulin polypeptide-oligomer conjugate comprising:

• (a) contacting a proinsulin polypeptide comprising an insulin polypeptide coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer comprising the structure of Formula II:
• A-X(CH2)mY(C2H4O)nR (II)
• wherein:
• A is —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH2;
• X is an oxygen atom or a covalent bond, with the proviso that X is not an oxygen atom when A is —OH;
• Y is an ester, an ether, a carbamate, a carbonate, or an amide bonding moiety;
• m is between 0 and 30;
• n is between 0 and 50;
• in and n are not both 0;
• and
• R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety, or a fatty acid moiety;
• under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate;
• and
• (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate.

A method of synthesizing an insulin polypeptide-oligomer conjugate comprising:

• (a) contacting a proinsulin polypeptide comprising an insulin polypeptide coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer comprising the structure of Formula Ill:
• A-(CH2)m(OC2H4)nOR (III)
• wherein:
• A is —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH2;
• m is between 0 and 25;
• n is between 0 and 25;
• m and n are not both 0;
• and
• R is alkyl;
• under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate;
• and
• (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate.

A method of synthesizing an insulin polypeptide-oligomer conjugate comprising:

• (a) contacting a proinsulin polypeptide comprising an insulin polypeptide having an A-chain polypeptide and a B-chain polypeptide, which comprises a lysine residue;
• a connecting peptide coupled at a first end to the C-terminus of the B-chain polypeptide and coupled at a second end to the N-terminus of the A-chain polypeptide;
• and a leader peptide coupled to the N-terminus of the B-chain polypeptide with an oligomer comprising the structure of Formula IV:
• 20
• wherein:
• m is between 0 and 30;
• n is between 0 and 50;
• m and n are not both 0;
• and
• R is alkyl;
• under conditions sufficient to couple the oligomer to the lysine residue of the B-chain polypeptide of the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate;
• and
• (b) enzymatically cleaving the connecting peptide and the leader peptide from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate.

A method of synthesizing an insulin-oligomer conjugate comprising:

• (a) contacting a proinsulin polypeptide, which comprises proinsulin coupled at its N-terminus to a leader peptide, with an oligomer comprising the structure of Formula V:
• 21
• under conditions sufficient to couple the oligomer to the B29 lysine residue of the proinsulin and provide a proinsulin polypeptide-oligomer conjugate;
• and
• (b) enzymatically cleaving the C-peptide and the leader peptide from the proinsulin polypeptide-oligomer conjugate to provide the insulin-oligomer conjugate.

A method of synthesizing an X-oligomer conjugate, wherein X is a target polypeptide, the method comprising:

• (a) contacting a pro-X polypeptide comprising one or more X polypeptides with an oligomer comprising a hydrophilic moiety and/or a lipophilic moiety under conditions sufficient to couple the oligomer to the proinsulin polypeptide and provide a pro-X polypeptide-oligomer conjugate, wherein the pro-X polypeptide comprises:
• (i) one or more X polypeptides;
• and
• (ii) one or more non-X polypeptides coupled to the X polypeptide(s) by peptide bond(s) capable of being cleaved to yield the X polypeptide(s);
• and
• (b) cleaving the one or more non-X polypeptides from the pro-X polypeptide-oligomer conjugate to provide the X polypeptide-oligomer conjugate.