A COMPOUND CONTAINING A LABILE DISULFIDE BOND

Abstract: A labile disulfide-containing compound under physiological conditions, comprising: the disulfide-containing compound having a labile disulfide bond that is either a disulfide bond that is cleaved more rapidly than oxidized glutathione or a disulfide bond constructed from thiols in which one of the constituent thiols has a lower pKa than glutathione or a disulfide bond that is activated by intramolecular attack from a free thiol.

Description: A Compound Containing a Labile Disulfide Bond Background Bifunctional molecules, commonly referred to as crosslinkers. are used to connect two molecules together Bifunctional molecules can contain homo or heterobifunctionalitv The disulfide linkage (RSSR') may be used within bifunctional molecules The reversibility of disulfide bond formation makes them useful tools for the transient attachment of two molecules Disulfides have been used to attach a bioactive compound and another compound (Thorpe, P E J Natl Cancer Inst 1987. 79, 1101) The disulfide bond is reduced thereby releasing the bioacftve compound Disulfide bonds may also be used in the formation of polymers (Kishore. K ~ , Ganesh. K. in Advances in Polvmer Science, Vol 21, Saegusa, T Ed , 1993) There are many commercially available reagents for the linkage of two molecules by a disulfide bond Additionally there are bifunctional reagents that have a disulfide bond present Typically, these reagents are based on 3-mercaptopropιonιc acid, l e dithiobispropionate However, the rate at which these bonds are broken under physiological conditions is slow For example, the half life of a disulfide denved from dithiobispropionimidate. an analog of 3-mercaptopropιonιc acid, is 27 hours in vivo (Arpicco. S . Dosio. F . Brusa, P . Crosasso, P . Cattel, L Btoconjugate Chem 1997. 8. 327 ) A stable disulfide bond is often desirable, for example when punfication of linked molecules or long circulation in vivo is needed For this reason. attempts have been made to make the disulfide less susceptible to cleavage It has been demonstrated that both stability, measured as reduction potential, and rate, measured as rate constants, of disulfide reduction are both related to the acidity of the thiols which constitute the disulfide Additional factors that may affect the rate of reduction are stenc interactions, and intramolecular disulfide cleavage Looking at the difference in the rates for the reactions RSH + R'SSR' → RSSR' + R'SH and RSH + R"SSR" → RSSR" + R"SH. it has been demonstrated that log k" k' = β(pKa R - pK_a R " ). where k " and k" are the rate constant for the reactions with R'SSR' and R"SSR" respectively, pKa R and pKa R" are the acidities of the thiol groups R'SH and R"SH. and β is a constant determined empirically to be 0 72 From this equation, one would predict that the reduction of a disulfide composed from relatively acidic thiols would be reduced more quickly than one composed of less acidic thiols In support of this observation, it has been demonstrated that the disulfides cystine (pK * 8 3) and cystamine (pKa 8 2) are reduced 3-15 times faster than oxidized glutathione (pKa 8 9) (Bulaj, G , Kortemme, T . Goldenberg, D P Biochemistry 1998. 37, 8965) It has been demonstrated that both stability (thermodynamics), measured as reduction potential (Keire D A J Org Chem 1992, 57, 123), and rate (kinetics), measured as rate constants, of disulfide reduction are both related to the acidity of the thiols which constitute the disulfide (Szajewski, R.P , Whitesides, G M J Am Chem Soc 1980, 102, 2011) The increase m acidity of a thiol is dependent upon one or more of the following structural factors the presence of electron withdrawing groups which stabilize the thiolate through sigma and pi bonds (inductive effect), the presence of electron withdrawing groups that stabilize the thiolate through space or solvent (field effects), pi bonds which allow the negative charge to be placed on other atoms (resonance stabilization), and hydrogen bond donating groups within the molecule that can interact internally with the thiolate For example, cysteine has an ammo group two atoms from the thiol, which is more electron withdrawing than the amide nitrogen that is two atoms from the thiol in glutathione As a consequence of this difference in electron withdrawing groups, the thiol of cysteine is 0 6 pK units more acidic than glutathione. and as mentioned previously, cystine is reduced 3-15 times faster than oxidized glutathione Another example of a relatively acidic thiol is 5- thιo-2-nιtrobenzoιc acid. pKa 5 Its acidity is due to resonance stabilization and inductive effects Its disulfide is rapidly reduced by all standard alkyl thiols and its colored thiolate makes it a convenient assay for thiol concentration Summary Descnbed in a preferred embodiment is a process for the delivery of a compound to a cell, compnsing associating a compound, containing a disulfide bond that can be cleaved under physiological conditions, with a polymer, then delivering the polymer to the cell The polymer may compnse a first polvmer and a second polymer The first polymer and the second polymer mav compnse nucleic acids, proteins, genes, antisense polymers. DNNRNA hybπds. or synthetic polymers In another preferred embodiment, a biologically active compound is associated with a disulfide-containing compound, compnsing the disulfide-containing compound having a labile disulfide bond that is selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols m which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated by intramolecular attack from a free thiol In another preferred embodiment, a compound is provided for inserting into an organism, compnsing the compound having a disulfide bond that is labile under physiologic conditions selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols in which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated by intramolecular attack from a free thiol In another preferred embodiment, a process is provided for forming a compound having a labile disulfide bond for use with an organism, comprising forming the compound having a disulfide bond selected from the group consisting of (I) a disulfide bond that is cleaved more rapidly than oxidized glutathione, and (n) a disulfide bond constmcted from thiols in which one of the constituent thiols has a lower pKa than glutathione. and (in) a disulfide bond that is activated by intramolecular attack from a free thiol. inserting the compound into the organism In another preferred embodiment, a process is descπbed for compacting a nucleic acid for delivery to a cell, compnsing associating a polymer containing a disulfide bond with a nucleic acid and delivenng the nucleic acid to the cell In another preferred embodiment, a process is descnbed for compacting a nucleic acid for delivery to a cell compnsing associating a polymer with the nucleic acid, then associating a compound containing a disulfide bond that can be cleaved under physiological conditions with the nucleic acid polymer complex, then delivenng the complex to a cell In another preferred embodiment, a process is descπbed for compacting a nucleic acid for delivery to a cell, compnsing associating a polymer containing a disulfide bond with a nucleic acid, then associating another polymer with the disulfide containing polymer - nucleic acid complex, then delivenng the complex to the cell In another preferred embodiment, a process is descnbed for compacting a nucleic acid for delivery to a cell compnsing associating a polymer with the nucleic acid, then associating a compound containing a disulfide bond that can be cleaved under physiological conditions with the nucleic acid polymer complex, then associating another polymer with the complex, then delivenng the complex to a cell In another prefeπed embodiment, a compound is descπbed which contains a disulfide bond that can be cleaved under physiological conditions and possesses heterobifunctional or homobifunctional groups Such a compound can be descnbed as a disulfide containing bifunctional molecule A — S— S— A 2 More particularly, a compound that contains an aliphatic disulfide bond with one or more electronegative (electron withdrawing groups) substituted alpha or beta to one or both of the sulfur atoms These groups serve to lower the pK a of the constituent thiols Where Ri, R , R 3 , R4, R5, Rό, R7, Rs - at least one of which is an electronegative atom or functionality such as OH. OR (an ether). NH 2 (also secondary, tertiary, and quaternary amines), SO0. COOH, COOR (an ester). CONH 2 , CONR 2 (substituted amide), a halogen (F. Cl. Br. I), N0 2 L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups, Ai and A 2 L may or may not be present and may be chosen from a group that includes alkanes, alkenes, alkynes. esters, ethers, glycerol, amide, urea, sacchandes, polysacchandes. heteroatoms such as oxygen, sulfur, or nitrogen The spacer may be charge positive, charge negative, charge neutral, or zwitteπomc Ai and A are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as m a heterobifunctional bifunctional molecule In a preferred embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include (but not limited to) isothiocyanate, isocyanate. acyl azide, acid halide, O-acyl urea, N-hydroxysuccimmide esters, succinimide esters, amide, urea, sulfonyl chlonde, aldehyde, ketone. ether, epoxide, carbonate, alkyl halide, lmidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or anhydndes If functional group A1.A2 is an amine then A1,A2 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate, isocyanate. acyl azide, alkyl halide, acid halide, N-hydroxysuccimmide ester, sulfonyl chlonde, aldehyde, ketone, epoxide, carbonate, lmidoester. amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function A1,A2 is an amine, then an acylating or alkylating agent can react with the amine If functional group A1.A2 is a sulfhydrvl then A1.A2 can react with (but not restncted to) a haloacetyl denvative. activated carboxvhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene deπvatives. or disulfide denvative (such as a pyπdyl disulfide or 5-thio-2-nitrobenzoic acidjTNB} deπvatives) If functional group A1.A2 is carboxylate then A1,A2 can react with (but not restncted to) a diazoacetate, alcohol, thiol or an amine once the acid has been activated If functional group A1.A2 is an hvdroxyl then A1.A2 can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonvldiimidazole is used If functional group A1.A2 is an aldehyde or ketone then A1.A2 can react with (but not restncted to) an hydrazine, hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group A1.A2 is activated carboxyhc acid, isothiocyanate, isocyanate. acyl azide, N-hydroxysuccimmide ester, sulfonyl chlonde, aldehyde, ketone. epoxide. carbonate, lmidoester. alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydπde, alkyl halide. or acid halide, p-mtrophenyl ester, o-nitrophenyl ester. pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyπdinium, or carbonyl dimethylaminopyndinium, then A1,A2 can react with (but not restncted to) an amine. a hvdroxyl, hydrazine. hydrazide, or sulfhydryl group If functional group A1.A2 an activated carboxyhc acid, haloacetyl denvative, maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-mtrobenzoιc acιd{TNB} denvatives) then A1,A2 can react with (but not restncted to) a sulfhydryl If functional group A1.A2 is an aldehyde, ketone, epoxide, oxirane, or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then A1.A2 can react with (but not restncted to) a hvdroxyl If functional group A1.A2 is a hvdrazine, hydrazide denvative. or amine (pπmarv or secondary) then A1.A2 can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Additionally, a compound which contains an aromatic disulfide bond in which the sulfur atom is bonded directlv to the aromatic nng The ring may contain 5 or more atoms Ri-Ri. R6-R9 - The substitution pattern on the nng may be vaned to alter the reduction potential of the disulfide bond The substiuents may be selected from the group that includes but is not limited to OH. OR (an ether), NH 2 ,(also secondary, tertiary, and quaternary amines), S0 3 " , COOH. COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F. Cl. Br, I), N0 , CH^ (or longer branched or straight chain, saturated, or unsaturated aliphatic group) L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes, alkenes. esters, ethers, glycerol, amide, sacchaπdes, polysacchaπdes, heteroatoms such as oxygen, sulfur, or nitrogen. The spacer may be charge positive, charge negative, charge neutral, or zwitteπonic R5, Rio - are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule In a prefened embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include lsothiocynanate, lsocynanate, acyl azide. N-hydroxysuccinimide esters, succinimide esters, sulfonyl chlonde. aldehyde, epoxide. carbonate, imidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or succinic anhydπde If functional group R5. RIO is an amine then R5. RIO can react with (but not restricted to) an activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone. epoxide. carbonate, imidoester. amide, carboxylate. or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function R5, RIO is an amine. then an acvlating or alkvlating agent can react with the amine If functional group R5. RIO is a sulfhydryl then R5, RIO can react with (but not restncted to) a haloacetvl denvative. activated carboxyhc acid, maleimide. azindine denvative, acryloyl denvative. fluorobenzene denvatives. or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-mtrobenzoιc acιd{TNB} deπvatives) If functional group R5. RIO is carboxylate then R5. RIO can react with (but not restncted to) a diazoacetate, alcohol, thiol or an amine once the acid has been activated If functional group R5. RIO is an hvdroxyl then R5, RIO can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane, or an amine m which carbonyldiimidazole is used If functional group R5, RIO is an aldehyde or ketone then R5, RIO can react with (but not restncted to) an hydrazine. hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group R5. RIO is activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydπde, alkyl halide. or acid halide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyπdinium, or carbonyl dimethylaminopyπdinium. then R5, RIO can react with (but not restncted to) an amine. a hydroxv 1. hvdrazme. hydrazide. or sulfhydryl group If functional group R5. RIO an activated carboxyhc acid, haloacetvl denvative. maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} denvatives) then R5. RIO can react with (but not restncted to) a sulfhydryl If functional group R5, R10 is an aldehvde. ketone, epoxide, oxirane. or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then R5. R10 can react with (but not restncted to) a hvdroxy 1 If functional group R5. RIO is a hydrazine. hydrazide denvative, or amine (pπmary or secondary) then R5. RIO can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Additionally, a compound which contains a disulfide bond that is connected directly to a heterocychc πng The heterocychc nng may be aromatic or aliphatic The heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (O, N, S, P), and the rest being carbon atoms H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus Rι-R , R5-R7 are substiuents that may be selected from the group that includes but is not limited to OH, OR (an ether), NH 2 ,(also secondary, tertiary, and quaternary amines), S0 3 " , COOH, COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F, CI, Br, I), N0 2 , CH 3 (or longer branched or straight chain, saturated, or unsaturated aliphatic group) The substitution pattern on the aromatic nng may be vaπed to alter the reduction potential of the disulfide bond L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes. alkenes. esters, ethers, glycerol. amide, sacchandes. polysacchandes. heteroatoms such as oxygen, sulfur, or nitrogen The spacer mav be charge positive, charge negative, charge neutral, or zwittenonic R^ Re are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as m a heterobifunctional bifunctional molecule In a preferred embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include isothiocynanate. lsocynanate. acyl azide, N-hydroxysuccimmide esters, succmimide esters, sulfonyl chlonde. aldehyde, epoxide. carbonate, imidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or succinic anhydπde If functional group R4. R8 is an amine then R4. R8 can react with (but not restocted to) an activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, alkyl halide. acid halide, N-hydroxysuccinimide ester, sulfonyl chlonde. aldehyde, ketone, epoxide. carbonate, imidoester. amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function R4, R8 is an amine, then an acylating or alkylating agent can react with the amine If functional group R4. R8 is a sulfhydryl then R4. R8 can react with (but not restncted to) a haloacetvl denvative, activated carboxyhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene deπvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} denvatives) If functional group R4. R8 is carboxylate then R4, R8 can react with (but not restncted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated If functional group R4. R8 is an hvdroxyl then R4, R8 can react with (but not restncted to) an activated carboxyhc acid, epoxide, oxirane. or an amine m which carbonyldiimidazole is used If functional group R4. R8 is an aldehyde or ketone then R4. R8 can react with (but not restocted to) an hvdrazine. hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group R4. R8 is activated carboxyhc acid, isothiocyanate, isocyanate, acyl azide, N-hydroxysuccimmide ester, sulfonyl chlonde. aldehyde, ketone. epoxide. carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhydπde. alkyl halide. or acid halide, p-mtrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyndinium. or carbonyl dimethylaminopyndinium. then R4. R8 can react with (but not restocted to) an amine. a hydroxvl. hydrazine. hydrazide, or sulfhydryl group If functional group R4. R8 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative. acryloyl denvative. fluorobenzene deπvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} denvatives) then R4. R8 can react with (but not restncted to) a sulfhydryl If functional group R4. R8 is an aldehyde, ketone, epoxide, oxirane, or an amine in which carbonyldiimidazole or N, N'-disuccinimidyl carbonate is used, then R4, R8 can react with (but not restocted to) a hvdroxyl If functional group R4. R8 is a hydrazine. hydrazide denvative. or amine (pπmary or secondary) then R4. R8 can react with (but not restocted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Additionally, a compound which contains a disulfide bond that is connected directly to a nng system(aromatic or non-aromatic) through one of the sulfur atoms and to a aliphatic carbon through the other sulfur atom The cyclic nng may contain 5 or more atoms R1-R4 are substiuents selected from the group that includes but is not limited to H, OH. OR (an ether). NH 2 (also secondary, tertiary, and quaternary amines), S0 3 " , COOH. COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F, Cl, Br, I), N0 2 . CH 3 (or longer branched or straight chain, saturated, or unsaturated aliphatic group) The substitution pattern on the aromatic nng may be vaned to alter the reduction potential of the disulfide bond Rβ-R 9 are substiuents selected from the group that includes but is not limited to H. OH. OR (an ether), NH 2 (also secondary. tertiary, and quaternary amines), S0 3 " . COOH. COOR (an ester), CONH 2 , CONR 2 (substi ted amide), a halogen (F, Cl. Br, I), N0 2 , CH 3 (or longer branched or straight chain, saturated, or unsaturated aliphatic group) L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes. alkenes, esters, ethers, glycerol, amide, sacchaπdes, polysacchandes. heteroatoms such as oxygen, sulfur, or nitrogen The spacer may be charge positive, charge negative, charge neutral, or zwitteπomc R5, and Rio are reactive groups that may be identical as in a homobifunctional bifunctional molecule, or different as m a heterobifunctional bifunctional molecule In a prefened embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include lsothiocynanate. lsocynanate, acyl azide. N-hydroxysuccmimide esters, succinimide esters, sulfonyl chlonde. aldehyde, epoxide, carbonate, imidoester. carboxylate, alkylphosphate, arylhalides (e g difluoro-dinitrobenzene) or succinic anhydπde If functional group R5, RIO is an amine then R5. RIO can react with (but not restocted to) an activated carboxyhc acid, isothiocyanate. isocyanate. acyl aade, alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide. carbonate, imidoester. amide, carboxylate. or alkylphosphate. arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function R5. RIO is an amine. then an acylating or alkylating agent can react with the amine If functional group R5. RIO is a sulfhydryl then R5. RIO can react with (but not restocted to) a haloacetyl denvative. activated carboxyhc acid, maleimide. azindine denvative. acryloyl denvative. fluorobenzene deπvatives. or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} deπvatives) If functional group R5. RIO is carboxvlate then R5. RIO can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated If functional group R5. RIO is an hvdroxyl then R5. RIO can react with (but not restocted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonyldiimidazole is used If functional group R5. RIO is an aldehyde or ketone then R5. RIO can react with (but not restocted to) an hydrazine. hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group R5. RIO is activated carboxyhc acid, isothiocyanate, isocyanate. acyl azide. N-hydroxysuccmimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide, carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhydπde. alkyl halide. or acid halide. p-mtrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole. carbonyl pyndimum, or carbonyl dimethylaminopyndinium, then R5, RIO can react with (but not restncted to) an amine. a hvdroxyl. hydrazine. hydrazide. or sulfhydryl group If functional group R5. RIO an activated carboxyhc acid, haloacetyl denvative, maleimide, azindine denvative. acryloyl denvative, fluorobenzene deπvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thio-2-mtrobenzoic acιd{TNB} deπvatives) then R5. RIO can react with (but not restocted to) a sulfhydryl If functional group R5. RIO is an aldehvde. ketone. epoxide. oxirane. or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then R5. RIO can react with (but not restncted to) a hvdroxyl If functional group R5. RIO is a hydrazine. hydrazide denvative, or amine (pnmary or secondary) then R5. RIO can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Additionally, a compound which contains a disulfide bond that is connected directly to a heterocychc nng system through one of the sulfur atoms and to a aliphatic carbon through the other sulfur atom The heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (0. N. S. P) or combinations of heteroatoms. and the rest bemg carbon atoms H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus Where Ri. R 2 , R 3 , Rt, R 5 , Re, R?, Rs ,Rs>, RIO, Rl l, R12, R14- at least one of which is an electronegative atom or functionality such as OH, OR (an ether), NH 2j (also secondary, tertiary, and quaternary amines), S0 3 " , COOH, COOR (an ester), CONH 2 , CONR (substituted amide), a halogen (F, Cl. Br, I), N0 2 L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups. Ai and R9 L may or may not be present and may be chosen from a group that includes alkanes, alkenes, alkynes. esters, ethers, glycerol. amide, urea, sacchaπdes, polysacchaπdes, heteroatoms such as oxygen, sulfur, or nitrogen The spacer may be charge positive. charge negative, charge neutral, or zwitteπonic Ai and R9 are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule In a prefened embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include (but not limited to) lsothiocynanate. lsocynanate, acyl azide. acid halide. O-acyl urea N-hydroxysuccinimide esters, succinimide esters, amide, urea, sulfonyl chlonde. aldehyde, ketone. ether, epoxide. carbonate, alkyl halide. imidoester. carboxylate. alkylphosphate, arylhalides (e g difluoro- dinitrobenzene) or anhydndes If functional group A1.R9 is an amine then A1.R9 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate. lsocvanate. acyl azide, alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde. aldehyde, ketone, epoxide. carbonate, imidoester, amide, carboxvlate. or alkylphosphate. arylhalides (difluoro-dimtrobenzene) or anhydndes In other terms when function A1.R9 is an amine. then an acylating or alkvlating agent can react with the amine If functional group A1.R9 is a sulfhydryl then A1.R9 can react with (but not restncted to) a haloacetyl denvative. activated carboxyhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene denvatives. or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} denvatives) If functional group A1.R9 is carboxylate then A1.R9 can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated If functional group A1,R9 is an hvdroxyl then A1,R9 can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonvldiimidazole is used If functional group A1.R9 is an aldehyde or ketone then A1,R9 can react with (but not restocted to) an hydrazine, hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group A1.R9 is activated carboxyhc acid, isothiocyanate, isocyanate, acyl azide. N-hydroxvsuccinimide ester, sulfonyl chlonde. aldehyde, ketone, epoxide, carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhydπde. alkyl halide. or acid halide. p-mtrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyπdinium. or carbonyl dimethylaminopyndinium, then A1,R9 can react with (but not restocted to) an amine. a hvdroxyl. hydrazine. hydrazide. or sulfhydryl group If functional group A1.R9 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative. acryloyl denvative. fluorobenzene deπvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} deπvatives) then A1.R9 can react with (but not restncted to) a sulfhydryl If functional group A1.R9 is an aldehyde, ketone. epoxide. oxirane, or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then A1.R9 can react with (but not restncted to) a hvdroxyl If functional group A1.R9 is a hydrazine. hydrazide denvative. or amine (pπmary or secondary) then A1.R9 can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Additionally, a compound which contains a disulfide bond that is connected directly to a heterocychc nng system (aromatic or non-aromatic) through one of the sulfur atoms and to an aromatic nng system through the other sulfur atom The heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (O, N. S, P) or combinations of heteroatoms. and the rest being carbon atoms H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus Where Ri, R 2 , R 3 , R», R5, Re, R 7 , Re , Rio, R11, Rπ, R13- at least one of which is an electronegative atom or functionality such as OH. OR (an ether), NH 2 ,(also secondary, tertiary, and quaternary amines), S0 3 " . COOH. COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F. Cl. Br. I), N0 2 L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups. R9 and Rι 4 L may or may not be present and may be chosen from a group that includes alkanes, alkenes, alkynes. esters, ethers, glycerol, amide, urea, sacchandes, polysacchaπdes, heteroatoms such as oxygen, sulfur, or nitrogen The spacer may be charge positive, charge negative, charge neutral, or zwitteπonic R 9 and Rι are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule In a prefened embodiment, the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include (but not limited to) lsothiocynanate, lsocynanate, acyl azide. acid halide. O-acyl urea, N-hydroxysuccinimide esters, succinimide esters, amide, urea, sulfonyl chlonde, aldehyde, ketone. ether, epoxide, carbonate, alkyl halide. imidoester. carboxylate, alkylphosphate, arylhalides (e g difluoro- dimtrobenzene) or anhydndes If functional group R9.R14 is an amme then R9.R14 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate. isocyanate. acyl azide, alkyl halide, acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide. carbonate, imidoester, amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function R9,R14 is an amine. then an acylating or alkylatmg agent can react with the amine If functional group R9.R14 is a sulfhydryl then R9,R14 can react with (but not restocted to) a haloacetyl denvative, activated carboxyhc acid, maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thio-2-nitrobenzoic acιd{TNB} deπvatives) If functional group R9.R14 is carboxylate then R9,R14 can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amme once the acid has been activated If functional group R9.R14 is an hydroxvl then R9.R14 can react with (but not restocted to) an actπ ated carboxyhc acid, epoxide. oxirane. or an amine in which carbonyldiimidazole is used If functional group R9.R14 is an aldehyde or ketone then R9.R14 can react with (but not restocted to) an hydrazine, hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal If functional group R9.R14 is activated carboxyhc acid, isothiocyanate. isocyanate. acvl azide. N-hydroxvsuccinimide ester, sulfonyl chlonde, aldehyde, ketone. epoxide. carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhydnde. alkyl halide. or acid halide. p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyndimum. or carbonyl dimethylaminopyndinium, then R9,R14 can react with (but not restocted to) an amine. a hydroxvl. hydrazine. hydrazide. or sulfhydryl group If functional group R9.R14 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative, acryloyl denvative, fluorobenzene deπvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thιo-2-nιtrobenzoιc acιd{TNB} denvatives) then R9.R14 can react with (but not restocted to) a sulfhydryl If functional group R9.R14 is an aldehyde, ketone, epoxide, oxirane, or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then R9,R14 can react with (but not restocted to) a hydroxyl If functional group R9.R14 is a hydrazine, hydrazide denvative, or amine (pπmary or secondary) then R9.R14 can react with (but not restocted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) Detailed Description Counteπntuitive to previous efforts to synthesize bifunctional molecules with stabile disulfides. the object of the current invention is to synthesize labile disulfide molecules In vivo, disulfides are pnmanly reduced by the cysteine-based thiol glutathione (γ-glutamylcystylglycine). which is present in millimolar concentrations in the cell To increase the lability of the disulfide bond in a bifunctional molecule and its construct, we have synthesized several disulfide bond-containmg bifunctional molecules that are more rapidly reduced than oxidized glutathione Disulfide Bond Containing Bifunctional molecules Bifunctional molecules, possessing either homo or heterobifunctionality (commonly refeπed to as crosshnkers). are used to connect two molecules together The disulfide linkage (RSSR') may be used within bifunctional molecules The reversibility of disulfide bond formation makes them useful tools for the transient attachment of two molecules Physiologically, disulfides are reduced by glutathione A disulfide bond that is labile under physiological conditions means the disulfide bond is cleaved more rapidly than oxidized glutathione or any disulfide constmcted from thiols in which one of the constituent thiols is more acidic, lower pKa, than glutathione or is activated by intramolecular attack by a free thiol Constituent in this case means the thiols that are bonded together in the disulfide bond Cleavable means that a chemical bond between atoms is broken The present invention descnbes physiologically labile disulfide bond containing bifunctional molecules The present invention is also meant to include constructs prepared from the bifunctional molecules, including polymers, peptides, proteins, nucleic acids, polymer nucleic acid complexes Construct means any compound resulting from the chemical reaction of at least one of the reactive centers of the bifunctional molecule resulting in new chemical bond other that that resulting from hydrolysis of both reactive centers of the bifunctional molecule Further chemical modification may occur after the formation of the construct Cross nkmg refers to the chemical attachment of two or more molecules with a bifunctional reagent A bifunctional reagent is a molecule with two reactive ends The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifunctional molecule Polymers A polymer is a molecule built up b\ repetitive bonding together of smaller units called monomers In this application the term polvmer includes both ohgomers which have two to about 80 monomers and poh mers having more than 80 monomers The polymer can be linear, branched network, star. comb, or ladder types of polymer The polymer can be a homopolymer in which a single monomer is used or can be copolvmer in which two or more monomers are used Types of copolymers include alternating, random, block and graft To those skilled in the art of pohmeπzation. there are several categones of polymenzation processes that can be utilized in the descπbed process The polymenzation can be chain or step This classification descπption is more often used that the previous terminology of addition and condensation polymer "Most step- reaction polymenzations are condensation processes and most chain-reaction polymeπzations are addition processes" (M P Stevens Polymer Chemistry An Introduction New York Oxford University Press 1990) Template polymeπzation can be used to form polymers from daughter polymers Step Polymenzation In step polymenzation. the polymenzation occurs in a stepwise fashion Polvmer growth occurs by reaction between monomers, o gomers and polymers No initiator is needed since there is the same reaction throughout and there is no termination step so that the end groups are still reactive The polymenzation rate decreases as the functional groups are consumed Typically, step polymeπzation is done either of two different ways One way, the monomer has both reactive functional groups (A and B) in the same molecule so that A-B yields -[A-B]-Or the other approach is to have two bifunctional monomers A-A + B-B yields -[A-A-B-B]-Generally. these reactions can involve acylation or alkylation Acylation is defined as the mtroduction of an acyl group (-COR) onto a molecule Alkylation is defined as the introduction of an alkyl group onto a molecule If functional group A is an amine then B can be (but not restocted to) an isothiocyanate. isocyanate. acyl azide. N-hydroxysuccimmide. sulfonyl chlonde, aldehyde (including formaldehyde and glutaraldehyde). ketone, epoxide. carbonate, lmidoester. carboxylate activated with a carbodnmide. alkylphosphate. arylhalides (difluoro-dinitrobenzene). anhvdnde. or acid halide. p-nitrophenyl ester, o- nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole. carbonyl pyndimum. or carbonyl dimethylaminopyndinium In other terms when function A is an amine then function B can be acylating or alkylating agent or amination agent If functional group A is a sulfhydryl then function B can be (but not restncted to) an lodoacetvl denvative. maleimide. azindine denvative. acrylovl denvative. fluorobenzene denvatives. or disulfide denvative (such as a pyndyl disulfide or 5- thιo-2-nιtrobenzoιc acιd{TNB} denvatives) If functional group A is carboxylate then function B can be (but not restocted to) a diazoacetate or an amine in which a carbodiimide is used Other additives may be utilized such as carbonyldiimidazole. dimethylamino pyπdine (DMAP), N- hydroxysuccinimide or alcohol using carbodiimide and DMAP If functional group A is an hydroxyl then function B can be (but not restocted to) an epoxide. oxirane. or an amine in which carbonyldiimidazole or N, N'-disuccinimidyl carbonate, or N-hydroxysuccinimidyl chloroformate or other chloroformates are used If functional group A is an aldehyde or ketone then function B can be (but not restocted to) an hydrazine. hydrazide denvative, amine (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3 ) or hydroxyl compound to form a ketal or acetal Yet another approach is to have one bifunctional monomer so that A-A plus another agent yields -[A-A]- If function A is a sulfhydryl group then it can be converted to disulfide bonds by oxidizing agents such as iodine (12 ) or NaI04 (sodium penodate), or oxygen (02 ) Function A can also be an amine that is converted to a sulfhydryl group by reaction with 2-Imιnothιolate (Traut's reagent) which then undergoes oxidation and disulfide formation Disulfide denvatives (such as a pyndyl disulfide or 5-thio-2-nitiObenzoic acιd{TNB} deπvatives) can also be used to catalyze disulfide bond formation Functional group A or B in any of the above examples could also be a photoreactive group such as aryl azide (including halogenated aryl azide), diazo , benzophenone. alkyne or diazinne deπv ative Reactions of the amme. h\ droxyl. sulfhydryl. carboxylate groups yield chemical bonds that are descnbed as amide, amidine. disulfide. ethers, esters, enamine, lmine. urea, isothiourea. isourea. sulfonamide. carbamate. alkylamine bond (secondary amine), carbon-nitrogen single bonds in which the carbon contains a hydroxyl group, thioether, diol. hydrazone. diazo. or sulfone Cham Polvmenzation In chain-reaction polvmeπzation growth of the polymer occurs bv successive addition of monomer units to limited number of growing chains The initiation and propagation mechanisms are different and there is usually a chain- terminating step The polymenzation rate remains constant until the monomer is depleted Monomers containing (but not limited to) vinyl, acrylate. methacrylate, acrylamide, methacrylamide groups can undergo chain reaction which can be radical, anionic , or catiomc Cham polymeπzation can also be accomplished by cycle or nng opening polymeπzation Several different tvpes of free radical initiators could be used that include peroxides, hydroxy peroxides, and azo compounds such as 2,2'-Azobιs(- amidinopropane) dihydrochloπde (AAP) Tvpes of Monomers A wide vanety of monomers can be used in the polymenzation processes These mclude positive charged organic monomers such as amme salts, lmidine, guanidine, imine. hydroxylamine. hydrozyine. heterocycle (salts) like imidazole, pyπdine. morphohne. pyπmidine. or pyrene The amines could be pH-sensitive in that the pKa of the amine is within the physiologic range of 4 to 8 Specific amines include spermine, spermidine, N.N'-bιs(2-amιnoethyl)-l,3-propanedιamιne (AEPD), and 3.3'- Diamino-N.N-dimethyldipropylammonium bromide Monomers can also be hydrophobic. hydrophi c or amphipathic Amphipathic compounds have both hydrophihc (water-soluble) and hydrophobic (water-insoluble) parts Hydrophihc groups indicate in qualitative terms that the chemical moiety is water-prefernng Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water Examples of hydrophihc groups include compounds with the following chemical moieties, carbohydrates, polyoxyethylene. peptides. oligonucleotides and groups containing amines, amides, alkoxy amides, carboxyhc acids, sulfurs. or hydroxyls Hydrophobic groups indicate m qualitative terms that the chemical moiety is water-avoiding Typically, such chemical groups are not water soluble, and tend not to hvdrogen bonds Hydrocarbons are hydrophobic groups Monomers can also be intercalating agents such as acndine. thiazole organge, or ethidium bromide Monomers can also contain chemical moieties that can be modified before or after the polymeπzation including (but not limited to) amines (pπmary. secondary, and tertiary), amides, carboxv c acid, ester, hydroxyl, hydrazine, alkyl halide. aldehyde . and ketone Other Components of the Monomers and Polymers The polymers have other groups that increase their utility These groups can be incorporated into monomers pnor to polymer formation or attached to the polymer after its formation These groups include targeting groups, signals, reporter or marker molecules, spacers, steπc stabilizers, chelators. polycations, polyanions, and polymers Targeting groups are used for targeting the polymer-nucleic acid complexes to specific cells or tissues Examples of targeting agents include agents that target to the asialoglycoprotein receptor bv using asiologlycoproteins or galactose residues Proteins such as insulin. EGF. or transfernn can be used for targeting Protein refers to a molecule made up of 2 or more amino acid residues connected one to another by peptide bonds between the alpha-ammo group and carboxyl group of contiguous ammo acid residues as in a polypeptide The amino acids may be naturally occurπng or synthetic Peptides that include the RGD sequence can be used to target many cells Peptide refers to a linear senes of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group of contiguous ammo acid residues Polypeptide includes proteins and peptides. modified proteins and peptides. and non-natural proteins and peptides Chemical groups that react w ith sulfhydryl or disulfide groups on cells can also be used to target manv types of cells Folate and other vitamins can also be used for targeting Other targeting groups include molecules that interact with membranes such as fattv acids, cholesterol, dansvl compounds, and amphoteπcin denvatives Other targeting groups can be used to increase the delivery of the drug or nucleic acid to certain parts of the cell For example, agents can be used to disrupt endosomes and a nuclear localizing signal (NLS) can be used to target the nucleus A vaπety of hgands have been used to target drugs and genes to cells and to specific cellular receptors The ligand mav seek a target within the cell membrane, on the cell membrane or near a cell Binding of gands to receptors typically initiates endocytosis Ligands could also be used for DNA delivery that bind to receptors that are not endocytosed For example peptides containing RGD peptide sequence that bmd integnn receptor could be used In addition viral proteins could be used to bind the complex to cells Lipids and steroids could be used to directly insert a complex mto cellular membranes The polymers can also contain cleavable groups within themselves When attached to the targeting group, cleavage leads to reduce interaction between the complex and the receptor for the targeting group Cleavable groups include but are not restncted to disulfide bonds, diols. diazo bonds, ester bonds, sulfone bonds, acetals. ketals, enol ethers, enol esters, enamines and lmines, acyl hydrazones, and Schiff bases In a prefened embodiment, a chemical reaction can be used to attach a signal to a nucleic acid complex The signal is defined in this specification as a molecule that modifies the nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism By modifying the cellular or tissue location of the foreign gene, the expression of the foreign gene can be enhanced The signal can be a protein, peptide. lipid. steroid, sugar, carbohydrate, nucleic acid or synthetic compound The signals enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other mtracellular vesicles Nuclear localizing signals enhance the targeting of the gene mto proximity of the nucleus and/or its entrv into the nucleus Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS These nuclear localizing signals interact with a vanetv of nuclear transport factors such as the NLS receptor (karyophenn alpha) which then interacts with karyophenn beta. The nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus Signals that enhance release from mtracellular compartments (releasing signals) can cause DNA release from mtracellular compartments such as endosomes (early and late), lysosomes. phagosomes. vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN). and sarcoplasmic reticulum Release includes movement out of an mtracellular compartment into cytoplasm or into an organelle such as the nucleus Releasing signals include chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutimn subumt HA-2 peptides and other types of amphipathic peptides Cellular receptor signals are any signal that enhances the association of the gene or particle with a cell This can be accomplished by either increasing the binding of the gene to the cell surface and/or its association with an lntracellular compartment, for example hgands that enhance endocytosis by enhancing binding the cell surface This includes agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues Other proteins such as insulin, EGF, or transfernn can be used for targeting Peptides that include the RGD sequence can be used to target many cells Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells Folate and other vitamins can also be used for targeting Other targeting groups include molecules that interact with membranes such as lipids fatty acids, cholesterol, dansyl compounds, and amphotencin deπvatives In addition viral proteins could be used to bind cells Reporter or marker molecules are compounds that can be easily detected Typically they are fluorescent compounds such as fluorescein, rhodamme, Texas red, cy 5, cy 3 or dansyl compounds They can be molecules that can be detected by UV or visible spectroscopy or by antibody interactions or by electron spin resonance Biotin is another reporter molecule that can be detected by labeled avidin Biotin could also be used to attach targeting groups A spacer is any linker known to those skilled in the art to enable one to join one moiety to another moiety The moieties can be hydrophihc or hydrophobic Prefened spacer groups include, but are not limited to C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl. C6-C18 aralkyl. C6-C18 aralkenyl. C6-C18 aralkynyl. ester, ether, ketone. alcohol, polyol. amide, amine. polyglycol. polvamine. thiol. thio ether, thioester. phosphorous containing, and heterocvchc A Steπc stabilizer is a long chain hvdrophi c group that prevents aggregation of final polymer by stencallv hindeπng particle to particle electrostatic interactions Examples include alkyl groups. PEG chains, polysacchandes, hydrogen molecules, alkyl amines Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges A polvcation is a polymer containing a net positive charge, for example poly-L-lysme hydrobromide The polvcation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive A polvcation also can mean a non-polymeπc molecule that contains two or more positive charges A polyamon is a polymer containing a net negative charge, for example polyglutamic acid The polyamon can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative A polyamon can also mean a non-polymeπc molecule that contains two or more negative charges The term polyion includes polvcation, polyamon. zwittenonic polymers, and neutral polymers The term zwittenonic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule Salts are ionic compounds that dissociate into cations and anions when dissolved in solution Salts increase the lomc strength of a solution, and consequently decrease interactions between nucleic acids with other cations A chelator is a poly dentate ligand. a molecule that can occupy more than one site in the coordination sphere of an ion. particularly a metal ion, pπmary amine, or single proton Examples of chelators include crown ethers, cryptates. and non-cyclic polydentate molecules A crown ether is a cyclic poly ether containing (-X-(CR1- 2)n)m umts. where n = 1-3 and m = 3-8 The X and CR1-2 moieties can be substituted, or at a different oxidation states X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any combination thereof R can be H, C, O, S, N. P A subset of crown ethers descπbed as a cryptate contain a second (-X-(CRl-2)n)z strand where z=3-8 The beginning X atom of the strand is an X atom in the (-X-(CRl-2)n)m unit, and the terminal CH2 of the new strand is bonded to a second X atom in the (-X- (CRl-2)n)m umt Non-cv c c polydentate molecules containing (-X-(CRl-2)n)m umt(s), where n = 1-4 and m = 1-8 The X and CR1-2 moieties can be substituted, or at a different oxidation states X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or am combination thereof A polychelator is a polymer associated with a plurality of chelators by an ionic or co\ alent bond and can include a spacer The polymer can be catiomc. anionic. zwittenonic. neutral, or contain any combination of catiomc, anionic. zwittenonic. or neutral groups with a net charge bemg catiomc, anionic or neutral, and may contain steπc stabilizers, peptides, proteins, signals, or amphipathic compound for the formation of micellar. reverse rmcellar, or umlamellar structures Preferably the amphipathic compound can ha e a hydrophihc segment that is catiomc. anionic. or zwittenonic. and can contain polymeπzable groups, and a hydrophobic segment that can contain a polvmenzable group The present invention provides for the transfer of polynucleotides, and biologically active compounds into parenchymal cells within tissues in situ and in vivo, utilizing disulfide bonds that can be cleaved under physialogicval condidtions, and delivered lntravasculary (U S patent application senal number 08/571,536), lntrarteπally, intravenous, oralh intraduodenaly. v la the jejunum (or lleum or colon), rectally, transdermall . subcutaneously, lntramuscularlv . lntrapentoneally, lntraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including lntraventncular), spinal cord, ganglion, lymph nodes, lymphatic system, adipose tissues, thrvoid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the sali arv or other exocπne glands "Delivered" means that the polynucleotide becomes associated with the cell The polynucleotide can be on the membrane of the cell or inside the cytoplasm, nucleus. or other organelle of the cell The process of deliv enng a polynucleotide to a cell has been commonly termed "transfection" or the process of "transfecting" and also it has been termed "transformation" The poh nucleotide could be used to produce a change m a cell that can be therapeutic The deliv en of polynucleotides or genetic mateπal for therapeutic and research purposes is commonh called "gene therapy" The polynucleotides or genetic matenal being deliv ered are generally mixed with transfection reagents prior to deliverv A biologically activ e compound is a compound having the potential to react with biological components More particularh biologically active compounds utilized in this specification are designed to change the natural processes associated with a living cell For purposes of this specification, a cellular natural process is a process that is associated with a cell before delivery of a biologically active compound In this specification, the cellular production of. or inhibition of a mateπal. such as a protein, caused bv a human assisting a molecule to an in vivo cell is an example of a delivered biologically active compound Pharmaceuticals, proteins, peptides. polypeptides. hormones, cytokines. antigens, viruses, oligonucleotides, and nucleic acids are examples of biologicallv active compounds Bioactive compounds may be used interchangeably with biologically active compound for purposes of this application The term "nucleic acid" is a term of art that refers to a polymer containing at least two nucleotides "Nucleotides" contain a sugar deoxvπbose (DNA) or πbose (RNA), a base, and a phosphate group Nucleotides are linked together through the phosphate groups "Bases" include puπnes and pynmidines. which further mclude natural compounds ademne. thvmine. guanine. cytosine. uracil, inosine, and synthetic denvatives of punnes and pynmidines, or natural analogs Nucleotides are the monomeπc units of nucleic acid polymers A "polynucleotide" is distinguished here from an "oligonucleotide" bv containing more than 80 monomeπc umts, oligonucleotides contain from 2 to 80 nucleotides The term nuclei acid includes deoxynbonucleic acid (DNA) and nbonucleic acid (RNA) DNA may be in the form of anti-sense, plasmid DNA. parts of a plasmid DNA. vectors (PI. PAC, BAC, YAC. artificial chromosomes), expression cassettes, chimeπc sequences, chromosomal DNN or denvatives of these groups RΝA mav be in the form of oligonucleotide RΝN tRΝA (transfer RΝA). snRΝA (small nuclear RΝA), rRΝA (nbosomal RΝA). rnRΝA (messenger RΝA) anti-sense RΝA. πbozvmes. chimenc sequences, or deπvatives of these groups "Anti-sense" is a polv nucleotide that interferes with the function of DΝA and/or RΝA This may result in suppression of expression Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones and bases These include PNAs (peptide nucleic acids), phosphothionates. and other v ariants of the phosphate backbone of native nucleic acids In addition. DNA and RNA may be single double, tπple, or quadruple stranded "Expression cassette" refers to a natural or recombinantly produced polynucleotide molecule which is capable of expressing protem(s) A DNA expression cassette tv picallv includes a promoter (allowing transcnption initiation), and a sequence encoding one or more proteins Optionally, the expression cassette may include trancnptional enhancers, non-coding sequences, splicing signals, transcnption termination signals, and polyadenylation signals An RNA expression cassette typically includes a translation imtiation codon (allowing translation imtiation), and a sequence encoding one or more proteins Optionally, the expression cassette may include translation termination signals, a polyadenosine sequence, internal nbosome entrv sites (IRES), and non-coding sequences The term "naked polynucleotides" indicates that the polynucleotides are not associated with a transfection reagent or other delivery vehicle that is required for the polynucleotide to be deliv ered to the cardiac muscle cell A "transfection reagent" or "delivery vehicle" is a compound or compounds used in the pnor art that bιnd(s) to or complex(es) with oligonucleotides or polynucleotides. and mediates their entry into cells The transfection reagent also mediates the binding and lntema zation of polynucleotides into cells Examples of transfection reagents include catiomc liposomes and lipids. polyamines. calcium phosphate precipitates, histone proteins, polyethylemmine, and polylysine complexes (polyethylenimine and polylysine are both toxic) Typically, the transfection reagent has a net positive charge that binds to the polynucleotide's negative charge The transfection reagent mediates binding of polynucleotides to cell via its positive charge (that binds to the cell membrane's negative charge) or via hgands that bind to receptors in the cell For example, catiomc liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA Other deliverv vehicles are also used, in the pnor art, to transfer genes into cells These include complexmg the polynucleotides on particles that are then accelerated into the cell This is termed "bio stic" or "gun" techniques Ionic (electrostatic) interactions are the non-covalent association of two or more substances due to attractiv e forces between positive and negative charges, or partial positive and partial negativ e charges Condensed Nucleic Acids A method of condensing a polymer is defined as decreasing its linear length, also called compacting Condensing a polymer also means decreasing the v olume that the polymer occupies An example of condensing nucleic acid is the condensation of DNA that occurs in cells The DNA from a human cell is approximately one meter in length but is condensed to fit in a cell nucleus that has a diameter of approximately 10 microns The cells condense (or compacts) DNA by a senes of packaging mechanisms involving the histones and other chromosomal proteins to form nucleosomes and chromatin The DNA within these structures is rendered partially resistant to nuclease DNase) action The process of condensing polymers can be used for delivenng them into cells of an organism A delivered polymer can stay within the cytoplasm or nucleus apart from the endogenous genetic matenal Alternatively, the polymer could recombine (become a part of) the endogenous genetic matenal For example. DNA can insert into chromosomal DNA by either homologous or non-homologous recombination Intravascular An mtrav ascular route of administration enables a polymer or polynucleotide to be delivered to cells more evenly distπbuted and more efficiently expressed than direct injections Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body Within the cavity of the tubular structure, a bodily fluid flows to or from the body part Examples of bodily fluid include blood, lymphatic fluid, or bile Examples of vessels include artenes, arteπoles. capillaπes, venules, sinusoids, veins, lymphatics, and bile ducts The mtrav ascular route includes delivery through the blood vessels such as an artery An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes Buffers are made from a weak acid or weak base and their salts Buffer solutions resist changes in pH hen additional acid or base is added to the solution Biological, chemical, or biochemical reactions involve the formation or cleavage of ionic and/or covalent bonds Biomolecule refers to peptides. polypeptides. proteins, enzymes, polynucleotides. oligonucleotides. viruses, antigens, carbohydrates (and conjugates), lipids. and sacchaπdes Enzvmes are proteins ev ol ed by the cells of living organisms for the specific function of catalyzing chemical reactions A chemical reaction is defined as the formation or cleavage of covalent or ionic bonds As a result of the chemical reaction a polymer can be formed A polymer is defined as a compound containing more than two monomers A monomer is a compound that can be attached to itself or another monomer and thus a form a polymer Transdermal refers to application to mammal skin in which drug delivery occurs by crossing the dermal layer Hydrocarbon means containing carbon and hydrogen atoms, and halohydrocarbon means containing carbon, halogen (F. Cl. Br. I), and hydrogen atoms Alkyl means containing sp 3 hybπdized carbon atoms, alkenyl means containing two or more sp 2 hybridized carbon atoms, aklkynyl means containing two or more sp hybπdized carbon atoms, aralkyl means containing one or more aromatic πng(s) in addition containing sp 3 hybndized carbon atoms, aralkenyl means containing one or more aromatic πng(s) in addition to containing two or more sp 2 hybπdized carbon atoms, aralkvnyl means containing one or more aromatic πng(s) in addition to containing two or more sp hybndized carbon atoms, steroid includes natural and unnatural steroids and steroid deπvatives A steroid denvative means a sterol. a sterol in which the hydroxyl moity has been modified (for example, acylated) or a steroid hormone, or an analog thereof Carbohydrates include natural and unnatural sugars (for example glucose), and sugar denvatives (a sugar denvative means a system in which one or more of the hydroxyl groups on the sugar moiety has been modified (for example acylated), or a system in which one or more of the hv drow 1 groups is not present) Polvoxvethv lene means a polvmer having two to six (n=2-3000) ethylene oxide units (-(CH 2 CH 2 0) n -) or a derivative thereof R is meant to be anv compatible group, for example hydrogen, alkyl, alkenyl, alkynyl. aralkyl. aral env 1. or aralkvnvl. and can include heteroatoms (N. O. S), and carbon l groups A compound is a material made up of t o or more elements Electron withdrawing group is anv chemical group or atom composed of electronegative atom(s), that is atoms that tend to attract electrons Resonance stabilization is the ability to distnbute charge on multiple atoms through pi bonds The inductive effective, in a molecule, is a shift of electron density due to the polanzation of a bond by a nearbv electronegative or electropositive atom Stenc hindrance, or stencs. is the prevention or retardation of a chemical reaction because of neighbonng groups on the same molecule An activated carboxylate is a carboxyhc acid denvative that reacts with nucleophiles to form a new covalent bond Nucleophiles include nitrogen, oxygen and sulfur- containing compounds to produce ureas, amides, carbonates, esters, and thioesters The carboxvhc acid may be activated bv vaπous agents including carbodiimides, carbonates, phosphoniums. uroniums to produce activated carboxylates acyl ureas, acylphosphonates. and carbonates Activation of carboxyhc acid may be used in conjunction with hydroxy and amine-containing compounds to produce activated carboxylates N-hydroxysuccinimide esters, hydroxybenzotnazole esters, N-hydroxy- 5-norbornene-endo-2.3-dicarboximide esters, p-nitrophenyl esters, pentafluorophenyl esters. 4-dιmethvlamιnopyndιnιum amides, and acyl lmidazoles A nucleophile is a species possessing one or more electron-nch sites, such as an unshared pair of electrons, the negative end of a polar bond, or pi electrons Examples Example 1 Sv nthesis of 5.5 " -Dιthιobιs(2-nιtrobenzoate)propιomtole 5,5'-dithiobis(2-nitrobenzoic acid) (500 mg,l 26 mmol. Aldnch Chemical Company) was taken up in 4 0 mL dioxane Dicylohexvlcarbodiimide (540 mg, 2 6 mmol, Aldnch Chemical Compan ) and 3-hvdroxvpropιonιtπle (240 μL. 188 mg, 2 60 mmol. .Aldnch Chemical Companv) ere added The reaction mixture was stirred overnight at room temperature The precipitate was removed by centπfugation, and the solvent concentrated under reduced pressure The residue was washed with saturated sodium bicarbonate water and brine and dried over magnesium sulfate Solvent removal (aspirator) yielded 696 mg yellow/orange foam The residue was puπfied using normal phase HPLC (Alltech econosil. 250 x 22 nm), flow rate = 9 0 mL/min. mobile phase = 1 % ethanol in chloroform, retention time = 13 mm Removal of solvent (aspirator) afforded 233 mg (36 8 %) of 5.5'-dιthιobιs(2- mtrobenzoate)propιonιtnle as a yellow oil TLC (silica 5 % methanol in chloroform. Rf = 0 51) H 1 NMR C8 05 (d. 4 H), 7 75 (m, 4H). 4 55 (t. 4H). 2 85 (t, 4H) Example 2 Synthesis of Dimethyl 5,5'-dιthιobιs(2-nιtrobenzoate)propιonιmιdate-2 HCl 5,5'-Dιthιobιs(2-mtrobenzoate)propιonιtπle ( 113 mg, 0 226 mmol) was taken up in 500 μL anhvdrous chloroform Anhydrous methanol (20 0 μL, 0494 mmol. Aldnch Chemical Company) was added The resulting solution was cooled to 0 °C on an ice bath, and HCl gas was bubbled through the solution for a penod of 10 mmutes The resulting solution was placed in a -20 °C freezer for a penod of 48 hours During this time a yellow oil formed The oil was washed thoroughly with chloroform and dned under vacuum to afford 137 mg (95 8 %) of dimethyl 5.5'-dιthιobιs(2- rutrobenzoate)propιonιmιdate-2 HCl as a yellow foam Example 3 Polv menzation of N-(2-Amιnoethyl)-1.3-propanedιamιne and Dimethyl 5.5'-dιthιobιs(2-nιtrobenzoate)propιonιmιdate-2 HCl on a DNA Template Procedure Template polvmenzation was earned out in 25 mM HEPES buffer, pH 8 0 N-(2-Amιnoethv l)-1.3-propanedιamιne (48 μg, 0 3 mM. Aldnch Chemical Company) was added to a 0 5 mL solution of pCIluc DNA (25 mg. 075 mM in phosphate. 2 6 ug/uL pCIluc. prepared according to Danko. I . Williams. P . Herweijer, H et al Hum Mol Genetics (1997) in press) Dimethyl 5 5"-dιthιobιs(2- nιtrobenzoate)propιonιmιdate -2 HCl (500 μg. 0 78 mM) was added, and the solution was vortexed The reaction w as incubated at room temperature for one hour A fine yellow precipitate was observ ed to form dunng the incubation penod The reaction was centnfuged to remove the precipitate. A portion of the reaction (10 μL) was reduced with 10 mM dithiothreitol (10 μL) to break the disulfide bonds forming the polymer. Portions (0.5 μg) of the intact polymer and the reduced polymer were analyzed on a 1 % agarose gel. Example 4 Formation of DNNPoly-L-Lysine/Dimethyl 5.5'-Dithiobis(2- nitrobenzoate) propionimidate -2 HCl Complexes pDNA/ Poly-L-lysine hydrobromide complexes were prepared by combining plasmid DNA (25 μg) with Poly-L-lysine hydrobromide (95 μg, MW 35 kDa, Aldrich Chemical Company ) in 0.5 mL 25 mM Hepes buffer pH 8.0. and the solution was vortexed to mix. The resulting solution was divided into 3 portions. One portion was incubated at room temperature for 2 hrs. To the second portion was added dimethyl 5,5'-dithiobis(2-nitrobenzoate)propiommidate-2 HCl (472 mg, 1.5 mmol), the solution was mixed, and incubated at room temperature for 2 hrs. To the third sample was added dimethyl 3.3'-dithiobispropionimidate (1 1 mg, 1.5 mmol), the solution was mixed, and incubated at room temperature for 2 hrs. After 2 hrs. the samples were then centnfuged at 12000 rpm for five minutes. Ninety degree light scattering measurements were performed (Shimadzo RF-1501 Fluorescence Spectrophotometer). The wavelength setting was 700 nm for both the incident beam and detection of scattenng light. The slits for both beams were fixed at 10 nm. The particle size of the resulting complex was determined by light scattering (Brookhav en ZetaPlus Particle Sizer) After determining the initial intensity of scattered light. 15 μL 5 M NaCl solution was added to the complexes while the intensity of scattered light was monitored. The addition of salt to the non-caged particles led to an immediate increase in the turbidity of the solution indicating aggregation. The non-caged sample also became visibly cloudy The addition of salt to the particles caged using dimethyl 3,3'- dithiobispropionimidate led to an increase in turbidity of approximately 33%. The addition of salt to the dimethyl 5.5'-dithιobιs(2-mtrobenzoate)propionimidate-2 HCl caged complexes lead to no visible nse in turbidity The particle size of the dimethyl 5,5'-dιthiobιs(2-nιtrobenzoate) propιonιmιdate-2 HCl caged particles was determined (Brookhaven Zeta Plus Particle Sizer) in 150 mM NaCl (physiological concentration). The mean particle diameter was found to be 89.7 nm. 67% of the total number of particles w ere under 100 nm in size The example indicates that dimethy 1 5.5 ' -dιthιobis(2-nιtrobenzoate)propionimidate-2 HCl caged DNA The particles formed are stable in physiological salt, and are under lOO nm in size. Example 5 Demonstration of Reducibi ty of Disulfide Bond in vitro. pDNA (pCI Luc)/poly ethyleneimine (25 kDa. Aldrich Chemical Company)/ dimethyl 3.3 '-dithiobispropionimi date and pDNA/polyethyleneimine/dimethyl 5,5'-dithiobis(2- nitrobenzoate)propιonιmιdate-2 HCl complexes were prepared in 25 mM HEPES buffer pH 8 0 All complexes were prepared at pDNA/polyethyleneimine ratios of 1/3. Dimethyl 3.3'-dithiobιspropιonιmιdate and dimethyl 5,5'-dithiobis(2- nitrobenzoate) propionιmidate-2 HCl w ere added at the following ratios: 0,3,6,12, and 25 Complexes were incubated 0 5 hour at room temperature, and centnfuged 5 minutes at 12.000 rpm pnor to transfection Transfections were carried out in 35 mm wells. At the time of transfection. HepG2 monolayers. at approximately 50% confluency. were washed once with PBS (phosphate buffered saline), and subsequently stored in serum-free media (Opti-MEM. Gibco BRL). The complexes were diluted in Opti-MEM and added by drops. 5 0 μg DNA/well. to the cells. After a 4 hour incubation period at 37 °C. the media containing the complexes was aspirated from the cells, and replaced ith complete growth media. DMEM with 10% fetal bovine serum (Sigma) After an additional incubation of 42 hours, the cells were harvested and the lysate was assayed for luciferase expression (Wolff, J.A.. Malone, R.W.. Williams. P . Chong. W . Acsadi. G . Jani. A and Feigner. P.L. Direct gene transfer into mouse muscle in ivo Science. 1465-1468. 1990.). A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Germany) luminometer was used. pDNA poly ethyleneimine/ dimethy l 3.3 " -dιthιobιspropιonιmιdate and pDNA/polv ethyleneimine /dimethyl 5.5 " -dιthιobιs(2-nιtrobenzoate)propionimidate-2 HCl particles were transfected into Hep G2 cells pDNA/polyethyleneimine complexes were also transfected as a control The cell lysates were then analyzed for the expression of lucifeπn The results show that while the dimethyl 3.3'- dithiobispropionimidate complexes gav e expression results below baseline (< 200 RLU). the dimethyl 5.5 " -dithiobis(2-nitrobenzoate)propiommidate-2 HCl/pDNApolyethyleneimine complexes gave levels of expression that were as high as 120.000 RLU The physiologically labile disulfide bonds present in the dimethyl 5,5'-dιthιobιs(2- mtrobenzoate)propιonιmιdate-2 HCl complexes can be reduced by cultured cells, while the disulfide bonds present in the dimethyl 3.3'-dιthιobιspropιommιdate complexes cannot Example 6 Synthesis of 5.5'-dithiobis[(3"-bromopropyl)-2-nitrobenzoate] 5,5'-dιthιobιs-(2-nιtrobenzoιc acid) (500 mg. 1 26 mmol. Aldnch Chemical Compan ) and 3-bromopropanol (368 mg. 2 65 mmol. Aldnch Chemical Company) were taken up in 7 0 mL THF Dicyclohexylcarbodπmide (545 mg, 2 65 mmol, Aldnch Chemical Company) was added, and the reaction mixture was stirred overnight at ambient temperature The precipitate was removed by filtration, and the solution w as concentrated under reduced pressure to afford 430 mg (54%) of 5,5'- dιthιobιs[(3 "" -bromopropyl)-2-nιtrobenzoate] as a yellow oil Example 7 Sv nthesis of 5.5 " -dιthιobιs[(3 " -ammonιo-{N.N-dιmethyl-N- propionitπle} propyl bromιde)2-nιtrobenzoate] 5.5'-dιthιobιs[(3"-bromopropy l)-2-nιtrobenzoate] was taken up in 2 0 mL THF. and 3-dιmeth\ laminopropiomtnle (193 mg 1 96 mmol. Aldnch Chemical Company) was added Alter 3 days at ambient temperature, the salt was precipitated from solution with Et 2 0 and purified by reverse phase HPLC (C-1 Aquasil 200x20 mm) using a gradient irom 20 to 80 % methanol ov er 20 minutes (elution at 15 minutes). The solvent w as remov ed under reduced pressure to afford 15 2 mg (3%) 5,5'- dιthιobιs[(3 -ammonιo-{N.N-dιmethv l- -propιonιtπle}propyl bromιde)2- mtrobenzoatel H'-NMR (CD,OD) 18 4-8 6 (m. 6H). 5 0 (t. 4 H), 4 35 (t, 4H), 4 1 (m. 4H). 2 85 ( m . 4H). 3 75 (m. 16H) Synthesis of Dimethyl 5.5'-dithiobis[(3"-ammonio-(N.N-dimethyl-N- propιoιmιdate)propy chlonde) 2-nιtrobenzoate] - hydrochloπde Example 8 S nthesis of 5.5'-dithiobisl(3 " -ammonio-(N.N-dimethyl-N- propιoιmιdate)propy 1 chlonde) 2-nιtrobenzoate] 5,5'-dιthιobιs[(3 ' -ammomo-{N.N-dιmethvl-N-propιomtnle}propyl bromιde)2- nitrobenzoate] (15 2 mg. 0 018 mmol) was taken up in 1 mL of methanol The solution was saturated with HCl at 0 °C The resulting solution was held at -20 °C for 1 week Et 2 0 was added and the precipitate collected by filtration to afford 8 3 mg (41%) of dimethyl 5.5'-dιthιobιs[(3"-ammonιo-(N.N-dιmethyl-N- propιoιmιdate)propyl chlonde) 2-nιtrobenzoate] - hvdrochloπde Example 9 Sv nthesis of N.N'-Bιs(t-BOC)-L-cystine To a solution of L-cystine (1 gm,4 2 mmol. Aldnch Chemical Company) in acetone (10 mL) and ter (10 mL) was added 2-(tert-butoxycarbonyloxyimino)-2- phenylacetonitπle (2 5 gm.10 mmol Aldnch Chemical Company) and toethylarmne (1 4 mL. 10 mmol. Aldnch Chemical Company) The reaction was allowed to stir overnight at room temperature The w ater and acetone was then by rotary evaporation resulting in a v ellovv solid The diBOC compound was then isolated by flash chromatographv on silica gel eluting ith ethvl acetate 0 1% acetic acid Example 10 S nthesis of L-cvstine - 1 4-bιs(3-amιnopropyl)pιperazιne copolymer To a solution of N N'-Bιs(t-BOC)-L-cv stine (85 mg. 0 15 mmol) in ethyl acetate (20 mL) was added N N'-dicy clohexylcarbodiimide (108 mg. 0 5 mmol) and N- hydroxv succinimide (60 mg. 0 5 mmol) After 2 hr the solution was filtered through a cotton plug and 1.4-bιs(3-ammopropy piperazine ( 54 μL, 0 25 mmol) was added The reaction w as allowed to stir at room temperature for 16 h The ethyl acetate was then removed bv rotaπ ev aporation and the resulting solid was dissolved in tnfluoroacetic acid (9 5 mL). water (0 5 mL) and tπisopropylsilane (0 5 mL) After 2 h. the tnfluoroacetic acid as remo\ ed bv rotarv e\ aporation and the aqueous solution was dialyzed in a 15.000 MW cutoff tubing against water (2 X 2 1) for 24 h. The solution as then removed from dialysis tubing, filtered through 5 μM nylon syringe filter and then dned by lyophi zation to yield 30 mg of polymer. Example 11. Synthesis of guanidino-L-cystine To a solution of cystine (lgm. 4.2 mmol) in ammonium hydroxide (10 mL) in a screw-capped v lal was added O-methy sourea hydrogen sulfate (1.8 gm, 10 mmol). The vial was sealed and heated to 60° C for 16 h The solution was then cooled and the ammonium hydroxide was remo\ ed by rotary evaporation. The solid was then dissolved in water (20 mL). filtered through a cotton plug. The product was then isolated by ion exchange chromatography using Bio-Rex 70 resin and eluting with hydrochloric acid (100 mM) Example 12: Synthesis of guanidino-L-cystinel.4-bis(3-aminopropyl)piperazine copolymer To a solution of guanidino-L-cystine (64 mg, 0 2 mmol) in water (10 mL) was slowly added N.N'-dicyclohexylcarbodiimide (82 mg, 0.4 mmol) and N-hyroxysuccinimide (46 mg. 0 4 mmol) in dioxane (5 mL) After 16 hr. the solution was filtered through a cotton plug and 1.4-bιs(3-amιnopropyl)pιperazιne ( 40 μL. 0.2 mmol) was added. The reaction w as allowed to stir at room temperature for 16 h and then the aqueous solution was dialyzed in a 1 .000 MW cutoff tubing against water (2 X 2 1) for 24 h. The solution w as then removed from dialysis tubing, filtered through 5 μM nylon syringe filter and then dned by lyophihzation to yield 5 mg of polymer. Example 13 The particle size of pD A- L-cystine - 1.4-bis(3- amιnopropyl)pιperazιne copolymer and DNA-guanιdino-L-cystιnel.4-bis(3- aminopropyl)pιperazιne copolymer complexes To a solution of pDNA (1 μg/mL) in o 5 mL 25 mM HEPES buffer pH 7.5 was added 10 μg/mL L-cystine - 1.4-bιs(3-amιnopropyl)pιperazme copolymer or guanidino-L-cv stιnel.4-bιs(3-amιnopropy ljpiperazine copolymer. The size of the complexes between DNA and the poly mers were measured. For both polymers, the size of the particles were approximately 60 nm. Example 14: Condensation of DNA ith L-cystine - 1.4-bis(3- aminopropy piperazine copolymer and decondensation of DNA upon addition of glutathione Fluorescein labeled DNA w as used for the determination of DNA condensation in complexes with L-cystine - 1.4-bιs( 3 -aminopropyOpiperazine copolymer. pDNA was modified to a level of 1 fluorescein per 100 bases using Minis' Labellt™ Fluorescein kit. The fluorescence was determined using a fluorescence spectrophotometer (Shimadzu RF-1501 spectrofluorometer) at an excitation wavelength of 495 nm and an emission wavelength of 530 nm (Trubetskoy. V S.. Slattum. P. M., Hagstrom, J. E., Wolff. J. A . Budker. V G.. "Quantitative Assessment of DNA Condensation," Anal. Biochem (1999) incorporated by reference). The intensity of the fluorescence of the fluorescein-labeled DNA ( 10 μg/mL) in 0.5 mL of 25 mM HEPES buffer pH 7 5 w as 300 units. Upon addition of 10 μg/mL of L- cystine - 1.4-bιs(3-amιnopropyl)pιperazιne copolymer. the intensity decreased to 100 units. To this DNA-polycation sample was added 1 mM glutathione and the intensity of the fluorescence was measured An increase in intensity was measured to the level observed for the DNA sample alone The half life of this increase in fluorescence was 8 minutes The expenment indicates that DNA complexes with physiologically -labile disulfide- containing polymers are cleavable in trie presence of the biological reductant glutathione. Example 15' Mouse Tail \ ein Injection of DNA-L-cystine - 1.4-bis(3- aminopropyOpiperazine copolymer and DNA-guanιdmo-L-cystmel.4-bis(3- aminopropyOpiperazine copolymer Complexes Plasmid deliv ery in the tail ein of ICR mice was performed as described. To PCILuc DNA (50 μg) in 2 5 mL H:0 was added either L-cystine - 1.4-bis(3- aminopropyOpiperazme copolymer. guanιdιno-L-cystιnel.4-bιs(3- aminopropyOpiperazine copolymer. or poh -L-lysine (34.000 MW, Sigma Chemical Company) (50 μg) The samples w ere then injected into the tail vein of mice using a 30 gauge. 0.5 inch needle One dav after injection, the animal was sacrificed, and a luciferase assay was conducted Polvcation ng/liver poly-L-lysine 6.2 L-cystine- 1.4-bιs(3-amιnopropyl)pιperazιne copolymer 439 guanidino-L-cystine l,4-bιs(3-amιnopropy0pιperazine copolymer 487 The expeπment indicates that DNA complexes with the physiologically-labile disulfide-containing poly mers are capable of being broken, thereby allowing the luciferase gene to be expressed Example 16 Rat Intramuscle injection of DNA-L-cystine - l,4-bis(3- aminopropyOpiperazine copolymer and DNA-guanidino-L-cystιnel,4-bis(3- aminopropyOpiperazine copolymer complexes Plasmid delivery mtro rat leg was performed as descnbed (Wolff, J.A., Malone. R.W . Williams. P . Chong. W . Acsadi. G . Jam. A and Feigner. P L. Direct gene transfer into mouse muscle in viv o Science. 1465-1468. 1990 ) To pCILuc DNA (100 μg/mL. 2.5 mL) as added L-cystine - 1.4-bιs(3-amιnopropyl)pιperazιne copolymer or guamdιno-L-cvstιnel.4-bιs(3-amιnopropyl)pιperazιne copolymer (100 μg/mL) and then injected into the leg muscles of a lat After 7 days, the animal was sacrificed and a luciferase assay was conducted DNA complex amount luciferase (ng) per leg no polvcation 3.3 L-cystine - 1 4-bιs(3-amιnopropyl)pιperazιne copolymer 4 5 guamdιno-L-cystinel.4-bιs(3-amιnopropy 0pιperazιne 6.5 copolymer The expenment indicates that DNA complexes with the physiologically-labile disulfide-comaining poly mers are capable of being broken, thereby allowing the luciferase gene to be expressed Example 17 Injection of DNA-L-cy stine - 1.4-bιs(3-amιnopropyl)pιperazιne copolymer complex and pDNA (pCI Luc)/5.5'-Dιthιobιs(2-mtrobenzoιc acid) - 1,4- Bιs(3-amιnopropy Opiperazine Copolymer Complex and pDNA (pCI Luc)/5,5'- dιthιobιs(2-mtrobenzoιc acid) - L4-bis(3-aminopropyl)piperazine -Folate Copolymer Complexes into the intestinal lumen of mice Intestinal cells were transfected by injecting pDNA solutions into the mesentenc vasculature A 3-cm section of the small intestines was clamped, blocking both vascular inflow and outflow A volume of 250 μl containing 50 μg pCILuc and 50 μg poly(ethylenιmιne) (Aldnch Chemical Co MW 25.000 MW), L-cystine - l,4-bιs(3- aminopropy Opiperazine copolymer. pDNA (pCI Luc)/5,5'-dιthιobιs(2- trobenzoιc acid) - 1.4-bιs(3-amιnopropy Opiperazine copolymer, and pDNA (pCI Luc)/5.5'- dιthιobιs(2-nιtrobenzoιc acid) - 1.4-bιs(3-amιnopropyl)pιperazιne -folate copolymer complexes ere injected into the intestinal lumen of mice After 3 minutes, the clamps were removed One dav aftei DNA deliv ery. the mice were sacπficed. the injected section of the intestines was excised, cut in 3 cm sections and assayed for luciferase expression Different areas of the intestines were targeted (duodenum, jejunum, lleum) Amount luciferase (pg) Complex Duodenum leiunum lleum DNA-poly(ethvlenimine) 0 5 3 0 1 7 DNA-L-cystιne - 1.4-bιs 6.2 3.7 2.8 (3-amιnopropyl)pιperazιne copolymer pDNA (pCI Luc)/5,5'-dithiobis(2-mtrobenzoic 42 20 226 acid) - 1.4-bιs(3-amιnopropy0pιperazιne copolymer pDNA (pCI Luc)/5.5'-dιthιobιs(2-mtrobenzoιc 36 1.9 51 acid) - 1.4-bιs(3-amιnopropy Opiperazine -folate copolymer The expenment indicates that DNA complexes with labile disulfide-contaimng polymers are capable of being broken, thereby allowing the luciferase gene to be expressed Example 18 Synthesis of 5.5 " -Dιthιobιs[succιmmidyl(2-nιtrobenzoate)] 5,5'-dιthιobιs(2-nιtrobenzoιc acid) (50 0 mg. 0 126 mmol. Aldrich Chemical Company) and N-hyroxysuccinimide (29 0 mg. 0.252 mmol. Aldrich Chemical Company) were taken up in 1 0 mL dichloromethane. Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was added and the reaction mixture was stined overnight at room temperature After 16 hr. the reaction mixture was partitioned in EtOAc/H 2 0. The organic layer was washed 2 \ H 2 0. 1 x brine, dried (MgS0 4 ) and concentrated under reduced pressure The residue was taken up in CH 2 C1 2 , filtered, and purified by flash column chromatography on silica gel (130 \ 30 mm, EtOAc:CH 2 Cι 2 1 :9 eluent) to afford 42 mg (56%) 5.5 " -dιthιobιs[succιnιmιdyl(2-nιtrobenzoate)] as a white solid. H' NMR (DMSO) C7 81-7 77 (d. 2H). 7 57-7 26 (m, 4H), 3.69 (s, 8 H). Example 19 Synthesis of 5.5 " -Dιthιobιs(2-nιtrobenzoιc acid) - 1.4-Bis(3- aminopropyOpiperazine Copolymer l,4-Bιs(3-amιnopropyl)pφerazιne ( 10 μL. 0 050 mmol. Aldnch Chemical Company) was taken up in 1 0 mL methanol and HCl (2 mL. 1 M in Et 2 0, Aldrich Chemical Company) was added Et 2 0 w s added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5,5'- dithiobιs[succιnιmιdy l(2-nιtrobenzoate)] (30 mg. 0.050 mmol) was added. The resulting solution was heated to 80 TJ and diisopropylethylamine (35 μL, 0.20 mmol. Aldnch Chemical Company) was added by drops After 16 hr, the solution was cooled, diluted with 3 mL H 2 0. and dialyzed in 12.000 - 14,000 MW cutoff tubing against water (2 X 2 L) for 24 h. The solution was then removed from dialysis tubing and dned by ly ophili/.ation to yield 23 mg (82%) of 5.5'-dithiobis(2- mtrobenzoic acid) - 1.4-bιs(3-amιnopropy Opiperazine copolymer. Example 20 Particle Sizing of pDNA (pCI Luc)/5.5'-Dιthiobis(2-nitrobenzoic acid) - 1.4-Bis(3-amιnopropyl)pιperazιne Copoly mer Complexes To 50 μg pDNA in 3 mL Ringers (0 85% sodium chlonde. 0.03 % potassium chloride. 0 03 % calcium chloride) was added 170 μg 5.5'-Dithiobis(2-nitrobenzoic acid) - 1.4-Bis(3-aminopropy0piperazme Copolymer. Particle sizing (Brookhaven Instruments Coporation. ZetaPlus Particle Sizer. 190, 532 nm) indicated an effective diameter of 92 nm for the complex A 50 μg pDNA m 3 mL Ringers sample indicated no particle formation 5,5'-Dithιobιs(2-nιtrobenzoιc acid) - 1.4-Bιs(3-amιnopropyl)piperazine Copolymer condenses pDNA. forming small particles Example 21 Mouse Tail \ ein Injections of pDNA (pCI Luc)/5,5'-Dithiobis(2- mtrobenzoic acid) -1.4-Bιs(3-amιnopropy Opiperazine Copolymer Complexes Four complexes were prepared as follows Complex I pDNA (pCI Luc. 200 μg) in 1 mL H 2 0 and diluted with 9 mL Ringers pnor to injection Complex II pDNA (pCI Luc. 200 μg) w as mixed with poly-L-lysine (378 μg, MW 3400. Sigma Chemical Compan ) in 1 mL H 2 0 and diluted with 9 mL Ringers pnor to injection Complex 111 pDNA (pCI Luc. 200 μg) w as mixed with 5.5'-Dιthιobis(2- nitrobenzoic acid) - 1.4-Bis(3-amιnopropy Opiperazine Copolymer (400 μg) m 1 mL H 2 0 and diluted ith 9 mL Ringers prior to injection Complex IV pDNA (pCI Luc. 200 μg) was mixed with Histone HI (1.2 mg, Sigma Chemical Company) in 1 mL H 2 0 and diluted with 9 mL Ringers pnor to injection. 2.5 mL and 250 μL tail vein injections of the complex were performed (Zhang, G, Budker. V . Wolff. J. High Levels of Foreign Gene Expression in Hepatocvtes from Tail Vein Injections of Naked Plasmid DNA. Human Gene Therapy. July, 1999, incorporated by reference). Results reported are for liver expression. Luciferase expression was determined as previously reported (Wolff, J.A.. Malone, R.W., Williams. P.. Chong. W.. Acsadi. G. Jam. A. and Feigner. P.L. Direct gene transfer into mouse muscle in vivo Science. 1465-1468. 1990.). A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Germany) luminometer was used. Results from 2.5 mL injections Complex I- 1.976.000 Complex II 128.000 Complex III. 5.025.000 Complex IV 1,960 Results from 250 μL injections Complex I 985 Complex III. 1,140 Results indicate an increased level of luciferase expression in pDNA / 5.5'- Dithiobιs(2-nιtrobenzoιc acid) - 1.4-Bιs(3 -amino propy Opiperazine Copolymer complexes over pCI Luc DNA itself . pCI Luc DNA/poly-L-lysine complexes, and pCI Luc DNA/ Histone HI complexes These results also indicate that the pDNA is being released from the pDNA / 5.5 " -Dithιobis(2-mtrobenzoic acid) - 1,4-Bis(3- aminopropyOpiperazine Copolymer complexes, and is accessible for transcription. 250 μL injection results were similar for both pDNA / 5.5'-Dithiobis(2-nitiObenzoic acid) - 1.4-Bιs(3-amιnopropy Opiperazine Copolymer complexes and pCI Luc DNA. Example 22 Synthesis of 5.5 " -Dιthιobιs(2-nιtrobenzoιc acid) - 1.4-Bis(3- aminopropyOpiperazine - Tπs(2-amιnoethyl)amιne Copolymer l,4-Bιs(3-amιnopropv Opiperazine ( 2 4 μL. 0 012 mmol, Aldnch Chemical Company ) and tπs(2-amιnoethyl)amιne (0 51 μL. 0 0034 mmol, Aldnch Chemical Company ) were taken up in 0 5 mL methanol and HCl (1 mL. 1 M in Et 2 0, Aldnch Chemical Companv) w as added Et 2 0 w as added and the resulting HCl salt was collected bv filtration 5 5 ' -dιthιobιs[succιnιmιd l (2-nιtrobenzoate)] (10 mg, 0 016 mmol) was added and the mixture was taken up in 0 4 mL DMSO and 04 mL THF The resulting solution as stined at room temperature and dusopropylethylamine (5 9 μL, 0 042 mmol. Aldnch Chemical Company) was added by drops After 16 hr, the solution was diluted with 3 mL H 2 0. and dialyzed in 12,000 - 14,000 MW cutoff tubing against w ater (2 X 2 L) for 48 h The solution was then removed from dialysis tubing and dried bv lv ophi zation to yield 2 7 mg (30%) of 5,5'-dιthιobιs(2- mtrobenzoic acid) - 1 4-bιs(3-amιnopropvl)pιperazιne - tns(2-amιnoethyl)amme copolymer Example 23 Sv nthesis of 5 5 * -Dιthιobιs(2-nιtrobenzoιc acid) - Tetraethv lenepentamine Copolymer Tetraethv lenepentamine ( 3 2 μL. 0 017 mmol. Aldnch Chemical Company) was taken up in 1 0 mL dichloromethane and HCl (1 mL. 1 M in Et 2 0, Aldnch Chemical Companv ) was added Et 2 0 as added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5.5'-dιthιobιs[succιnιmιdyl (2- nitrobenzoate) I ( 1 mg 0 017 mmol) w as added The resulting solution was heated to 80°C and dnsopropv lethv lamine (15 μL 0 085 mmol. Aldnch Chemical Company) was added bv drops After 1 (> hr the solution was cooled, diluted with 3 mL H 2 0, and dialv zed in 12.000 - 14 000 MW cutoff tubing against water (2 X 2 L) for 24 h The solution was then remo\ ed from dialv sis tubing and dned by lyophihzation to yield 5 8 mg (62%) of 5 5 -dιthιobιs(2-nιtrobenzoιc acid) - tetraethylenepentamine copolymer Example 24 Mouse Tail \ ein Injections of pDNA (pCI Luc)/5.5'-Dιthιobιs(2- nitrobenzoic acid) - Tetraethv lenepentamine Copolymer Complexes Complexes were prepared as follows Complex I pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 2.5 mL Ringers w as added Complex II pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 5,5'- Dithiobιs(2-nιtrobenzoιc acid) - Tetraethylenepentamine Copolymer (336 μg) was added followed by 2 5 mL Ringers 2.5 mL tail vain injections of the complex were performed as previously described. Results reported are for In er expression, and are the average of two mice. Luciferase expression was determined as previously reported (Wolff, J.A., Malone, R.W., Williams. P , Chong. W . Acsadi. G . Jam. A. and Feigner, P.L. Direct gene transfer into mouse muscle in v IV o Science. 1465-1468. 1990 ). A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Germany) luminometer was used. 250 μL injections Complex I 25.200.000 Complex II 21.000.000 Results indicate that pDNA (pCI Luc) 5.5'-Dιthιobιs(2-nιtrobenzoic acid) - tetraethylenepentamine copolv mer complexes are nearly equivalent to pCI Luc DNA itself in 2 5 mL injections This indicates that the pDNA is being released from the complex and is accessible lor transcnption Example 25 Sv nthesis of 5 5 " -Dιthιobιs(2-mtrobenzoιc acid) - Tetraethv lenepentamine - Tι ιs(2-amιnoethyl)amme Copolymer Tetraethylenepentamine ( 2 3 μL. 0 012 mmol. Aldnch Chemical Company) and 1 μL. 0 0034 mmol. Aldrich Chemical Company) were taken up in 0 5 mL methanol and HCl ( 1 mL. 1 M in Et 2 0, Aldrich Chemical Company) was added EbO w as added and the resulting HCl salt was collected by filtration The salt as taken up in 1 mL DMF and 5.5 " -dithiobis[succinimidyl (2- nitrobenzoate)] ( 10 mg. 0 017 mmol) w as added The resulting solution was heated to 80°C and diisopropylethy lamine (15 μL. 0 085 mmol, Aldrich Chemical Company) was added by drops After 1 hr. the solution was cooled, diluted with 3 mL H 2 0, and dialyzed in 12.000 - 1 .000 MW cutoff tubing against water (2 X 2 L) for 24 h. The solution as then remo\ ed from dialysis tubing and dried by lyophihzation to yield 6 9 mg (77%) of 5.5 " -dιthιobιs(2-nιtrobenzoιc acid) - tetraethylenepentamine - tris(2-aminoethy i)amιne copolymer Example 25 Mouse Tail Vein Injections of pDNA (pCI Luc)/5,5'-Dithiobis(2- mtrobenzoic acid) - Tetraethv lenepentamιne-Tπs(2-amιnoethyl)amine Copolymer Complexes Complexes were prepared as follows Complex I pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 2.5 mL Ringers was added Complex II pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 5,5'- Dithiobιs(2-nιtrobenzoιc acid) - Tetraethv lenepentarnine-Tris(2-aminoethyl)amine Copolymer (324 μg) was added follow ed by 2 5 mL Ringers. 2.5 mL tail vain injections of the complex w ere preformed as previously described. Results reported aie for In er expression, and are the average of two mice. Luciferase expression was determined as prev lously reported (Wolff, J.A.. Malone. R.W., Williams. P . Chong. W . Acsαdi. G . Jam. A and Feigner. P.L. Direct gene transfer mto mouse muscle in o Science. 1465-1468. 1990 ). A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Geimany) luminometer was used. 250 uL injections Complex I 25.200.000 Complex II 37.200.000 pDNA (pCI Luc) 5.5 ' -Dιιhιobιs(2-nιtrobenzoιc acid) - tetraethylenepentamine- Tris(2-amιnoeιhv I lamine C o polvmer Complexes are more effective than pCI Luc DNA m 2 5 mL injections Indicating that the pDNA is released from the complex and is accessible for transcnption Example 26 Sy nthesis of 5 5 " -Dιthιobιs(2-nιtrobenzoιc acid) - N.N'-Bis(2- aminoethyl)-1.3-propanedιamιne Copolymer N,N'-Bis(2-amιnoethyD-1.3-propanedιamιne ( 2 8 μL. 0 017 mmol. Aldrich Chemical Company) was taken up in 1 0 mL dichloromethane and HCl (1 mL, 1 M in Et 2 0, Aldrich Chemical Compan ) as added Et 2 0 was added and the resulting HCl salt was collected bv filtration The salt was taken up in 1 mL DMF and 5,5 " - dithiobisfsuccinimidy l(2-nιtrobenzoate)] (10 mg. 0 017 mmol) was added. The resulting solution w as heated to 80°C and diisopropylethylamine (12 μL, 0.068 mmol. Aldrich Chemical Compan ) w as added by drops After 16 hr, the solution was cooled, diluted with 3 mL H 2 0. and dialyzed in 12.000 - 14,000 MW cutoff tubing against w ater (2 X 2 L) for 24 hr The solution was then removed from dialysis tubing and dned by lvophilization to y leld 5 9 mg (66%) of 5,5'-dithiobis(2- nitrobenzoic acid) -N.N " -bιs(2-amιnoethyl)-1.3-propanediamine Copolymer. Example 27 Mouse Tail \ ein Injections of pDNA (pCI Luc)/5.5'-Dithiobis(2- rutrobenzoic acid) - N.N " -Bιs(2-amιnoethyl)- 1.3-propanediamine Copolymer Complexes Complexes weie prepared α-. follows Complex I pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 2.5 mL Ringers w as added Complex II pDNA (pCI Luc. 200 μg) was added to 300μL DMSO then 5,5'- Dιthiobιs(2-nιtrobenzoιc acid) - N.N'-Bιs(2-amιnoethyl)-l,3-propanediamine Copolymer (474 μg) was added followed by 2 5 mL Ringers Tail vain injections of 2 5 mL of the complex w ere preformed as previously described Results reported are for liver expression, and are the average of two mice. Luciferase expression w as det imined as preuously reported. Results: 2 5 mL miections Complex I 25.200.000 Complex II 341.000 pDNA (pCI Luc).5.5 -Dιthιobιs(2-nitrobenzoιc acid) - tetraethylenepentamine Copolymer Complexes pro\ ides luciferase expression indicating that the pDNA is being released from the complex and is accessible for transcription. Example 28:Synthesιs of 5. ' -Dιthιobιs(2-nιtrobenzoic acid) - N,N'-Bis(2- aminoethy l)-1.3-propanedιamιne - Tπs(2-amιnoethyl)amine Copolymer N,N'-Bis(2-amιnoethyD-1.3-propanedιamιne ( 2.0 μL, 0.012 mmol, Aldrich Chemical Company ) and tris(2-aminoeihyl)amine (0 51 μL. 0.0034 mmol. Aldrich Chemical Company ) were taken up in 0 5 mL methanol and HCl (1 mL. 1 M in Et 2 0, Aldrich Chemical Company ) w as added Et 2 0 w as added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5,5'- dithiobis[succιnιmιdv l(2-nπrobenzoate)] (10 mg. 0.017 mmol) was added. The resulting solution as heated to 80°C and diisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Companv ) w as added by drops After 16 hr, the solution was cooled, diluted with 3 mL H 0. and dialyzed in 12.000 - 14,000 MW cutoff tubing against water (2 X 2 L) for 24 hr The solution as then removed from dialysis tubing and dried bv 1\ ophih/ation to y leld 6 0 mg (70%) of 5.5'-dithiobis(2- nitrobenzoic acid) - VV-bi 2-amιnoethy l)- L3-propanedιamιne - tris(2- aminoethv Damme copolv mei Example 2' ) Mouse Tail \ em injections of pDNA (pCI Luc)/5,5'-Dithiobis(2- nitrobenzoic acid) - W-Bιs(2-amιnoeιhy D- 1.3-propanediamιne- Tris(2- aminoethy 1 (amme C opol mei Complexes Complexes wei e prepared α- follows: Complex 1 pDNA (pC I Luc. 200 μg) w as added to 300μL DMSO then 2.5 mL Ringers w as added Complex II pDNA (pCI Luc. 200 μg) w as added to 300μL DMSO then 5.5"- Dithiobιs(2-nιtroben/oιc acid) - \.N'-Bιs(2-amιnoethyl)-l,3-propanediamme- Tris(2- aminoethy l)amme Copol mer (474 μg) as added followed by 2.5 mL Ringers. Tail vain injections of 2 5 mL of the complex w ere preformed as previously described Results reported aie for liver expression, and are the average of two mice. Luciferase expression w as determined as previously reported. Results: 2 5 mL injections Complex 1 25.200.000 Complex II 1.440.000 Results indicate that pDNA (pCI Luc). 5.5 ' -Dιthιobis(2-nιtrobenzoic acid) - N,N'- Bis(2-amιnoethv l)- 1.3-propanedιamιne- Tns(2-amιnoethyl)amine Copolymer Complexes are less effectn e than pCI Luc DNA in 2.5 mL injections. Although the complex was less effectn e. the luciferase expression indicates that the pDNA is being released from the complex and is accessible for transcnption. Example 30 Intramuscular Injections of Complexes from pDNA (pCI Luc)/Physιologιcally Labile Disulfide Bond Containing Polymers on Mouse. Seven complexes ere prepaied as follows Complex 1 pDN A ( pCI Luc 40 ug ) as added to 586 μL glucose (290 mM)- HEPES (5 mM pH 8) Complex II pDNA ( pCI Luc. 40 μgj w as added to 577 μL glucose (290 mM)- HEPES (5 mM. pH 8 ) To this solution w as added 5.5'-Dithiobιs(2-nitrobenzoic acid) - 1.4-Bιs(3-amιnopι o|)v Opiperazine Copolymer (9 μL. 200 μg). Complex III pDNA ( pCI Luc. 40 μg) as added to 573 μL glucose (290 mM)- HEPES (5 mM. pH 8) To this solution as added 5.5'-Dιthiobis(2-nitrobenzoιc acid) - 1.4-Bιs(3-amιnopropv Dpipeia/ine Copolv mer ( 13 μL. 200 μg). Complex IV pDN A ( pCI Luc 40 μg) w as added to 574 μL glucose (290 mM)- HEPES (5 mM. pH S ) To ιiu.s solution w as added 5.5 " -Dιthiobιs(2-nitrobenzoιc acid) - Tetraethv lenepentamine ( opolv mer ( 12 μL. 70 μg) Complex Y. pDNA ψ I Luc. 40 μg) w as added to 576 μL glucose (290 niM)- HEPES (5 mM. pH 8) To this solution was added 5,5'-Dithiobis(2-nitrobenzoic acid) - Tetraethylenepentamine - Tπs(2-amιnoethyl)amine Copolymer (10 μL, 65 μg). Complex VI: pDNA ( pCI Luc. 40 μg) w as added to 581 μL glucose (290 mM)- HEPES (5 mM. pH S) To mis solution w as added 5,5'-Dithiobis(2-nitrobenzoic acid) - N,N'-Bιs(2-aminoeιhyD- 1.3-propanedιamine Copolymer (5 μL, 94 μg). Complex VII: pDNA ( pCI Luc. 40 μg) w as added to 570 μL glucose (290 mM)- HEPES (5 mM. pH 8 ) To this solution w as added 5,5'-Dithiobis(2-nitrobenzoic acid) - 1.3-propanedιamιne - Tris(2-aminoethyl)amine Copolymer (16 μL, 94 μg). Direct muscle injections of ! 5o μL of the complex were preformed as previously described (See Budker. \ . Zhang. G.. Danko. I.. Williams. P., and Wolff, J., "The Efficient Expression Of Intrav ascularly Delivered DNA In Rat Muscle," Gene Therapy 5. 272-6( 199S). Wolff. J A.. Malone. R.W., Williams, P., Chong, W., Acsadi, G . Jam. A. and Feigner. P L. Direct gene transfer into mouse muscle in vivo. Science. 1465-1468. 1990 w inch are incorporated herein by reference.). Seven days post injection, the animals w ere sacrificed, and the muscle harvested. Samples were homogenized in lux buffer 1 1 mL ). and centnfuged for 15 minutes at 4000 RPM. Luciferase expression as αeiermined as previously reported. Results reported for left quadracep . right quadracep (Complex Iλ ; - only injected into left quadracep). Results: Complex 1 RLL = ■ l.'Hr 4. 16 Complex II: RLL = 13.453 20.640 Complex III: RLL = 10. 1 5 M 30.491 Complex IV RLL = 9.88N Complex V: RLL - 19.5- 5 5.806 Complex VI: RLL = 27o 427 Complex VII: RLL = 973 U HJΠ The complexes prepai ed 10 pC I Luc D\.V Physiologically Labile Disulfide Bond Containing Poly mers ore ei i ctn e in direct muscle injections. The luciferase expression indicates that the pDNA is being released from the complex and is accessible for transcnption Complexes prepared with 5.5'-Dithiobis(2-nitrobenzoic acid) - 1.4-Bιs(3-amιnopropyl)pιperazιne Copolymer were the most effective, giving luciferase expression levels 2 to 10 times as high as pDNA Example 31 Synthesis of 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - Pentaethylenehexamine Copolymer Pentaethylenehexamine ( 4 2 μL. 0 017 mmol. Aldnch Chemical Company) was taken up in 1 0 mL dichloromethane and HCl (1 mL, 1 M in Et 0. Aldnch Chemical Company) was added Et 2 0 was added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5.5'-dιthιobιs[succιmmιdyl(2- mtrobenzoate)] (10 mg, 0 017 mmol) was added The resulting solution was heated to 80°C and dnsopropylethylamine (12 μL. 0 068 mmol, Aldnch Chemical Company) was added by drops After 16 hr, the solution was cooled, diluted with 3 mL H 2 0, and dialyzed m 12,000 - 14,000 MW cutoff tubing against water (2 X 2 L) for 24 hr The solution was then removed from dialysis tubing and dned by lyophihzation to yield 5 9 mg (58%) of 5.5'-dιthιobιs(2-nιtrobenzoιc acid) - pentaethylenehexamine Copolymer Example 32 Synthesis of 5,5'-Dιthιobιs(2-nιtrobenzoιc acid) - Pentaethylenehexamine - Tns(2-amιnoethyl)amme Copolymer Pentaethylenehexamine ( 2 9 μL. 0 012 mmol. Aldnch Chemical Company) and tns(2-amιnoethyl)amιne (0 51 μL, 0 0034 mmol. Aldnch Chemical Company) were taken up in 0 5 mL methanol and HCl (1 mL. 1 M in Et 2 0. Aldnch Chemical Company) was added Et 2 0 was added and the resulting HCl salt was collected by filtration The salt was taken up m 1 mL DMF and 5.5'-dithiobis[succinimidyl(2- nitrobenzoate)] (10 mg, 0 017mmol) was added The resulting solution was heated to 80°C and disopropylethylamme (12 μL. 0 068 mmol. Aldnch Chemical Company) was added by drops After 16 hr. the solution was cooled, diluted with 3 mL H 2 0. and dialvzed in 12.000 - 14.000 MW cutoff tubing against water (2 X 2 L) for 24 h The solution was then removed from dialysis tubing and dned by lyophihzation to yield 6 0 mg (64%) of 5.5'-dithiobis(2-mtrobenzoic acid) - pentaethylenehexamine - tns(2-amιnoethyl)amιne copolymer Example 33 Synthesis of 5.5'-Dιthιobιs(2-nιtrobenzoιc acid) - N-(3-Amιnopropyl)- 1,3-propanediamine Copolymer 5,5'-Dιthιobιs[succιmmιdyl(2-mtrobenzoate)] (2 5 mg, 0 0042 mmol) was taken up in 10 μL of DMF N-(3-aminopropyl)-1.3-propanediamine ( 0 6 μL, 0 004 mmol. Aldnch Chemical Company) was added with 10 μL HEPES 250 mM, pH 7 5 After 1 hr the solution was concentrated under reduced pressure The resulting residue was dissolved in 0 42 mL DMSO Analysis of the solution on SDS-PAGE versus poly-L- lysisne hydrobromide (MW of 1000. 7500, 15000) indicated an approximate molecular weight range of 3500-8000 for the polymer Example 34 Synthesis of 5.5'-dιthιobιs(2-nιtrobenzoιc acid) - 1.4-bιs(3- aminopropyOpiperazme -Folate Copolymer Folate-PEG(3400 MW)-NH2 was prepared according to the known procedure (Lee, R J . Low, P S Biochimica et Biophysica Acta 1233, 1995. 134-144) Folate-PEG- NH2 was acylated with succinylated N-(3-(BOC)aminopropyl)-1.3- propaneamιne(BOC)amιne Removal of the BOC protecting groups afforded the Folate monomer 1.4-bιs(3-amιnopropy Opiperazine ( 5 0 μL. 0 023 mmol. Aldnch Chemical Company) and folate monomer (5 0 mg, 0 0012 mmol) were taken up m 0 4 mL methanol and HCl (1 mL. 1 M in Et 2 0. Aldnch Chemical Company) was added The resulting suspension was concentrated under reduced pressure to afford a white solid The salt was taken up in 0 5 mL DMF and 5.5 " -dιthιobιs[succιnιmιdyl(2- nitrobenzoate)] (14 mg, 0 025 mmol) was added The resulting solution was heated to 80 °C and diisopropylethylamine (18 μL. 0 10 mmol. Aldnch Chemical Company) was added by drops After 16 hr. the solution was cooled, diluted with 3 mL H 0. and dialyzed in 12.000 - 14.000 MW cutoff tubing against water (2 X 2 L) for 24 h The solution was then removed from dialysis tubing and dned by lyophihzation to yield 13 mg (68%) of 5.5 " -dιthιobιs(2-mtrobenzoιc acid) - 1.4-bιs(3-amιnopropyl) piperazine - folate copolymer Example 35 Synthesis of 5.5 '-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer H 2 N-EEEEEEEE-NHCH 2 CH 2 NH 2 (5 0 mg, 0 0052 mmol, Genosis) was taken up m 0 1 mL HEPES (250 mM, pH 7 5) 5.5 ' -dιthιobιs[succιmmιdyl(2-nιtrobenzoate)] (3 1 mg, 0 0052) was added with 0 2 mL DMSO and the mixture was stined ovemight at room temperature After 16 hr the solution was heated to 70°C for 10 mm. cooled to room temperature and diluted to 1 10 mL with DMSO Example 36 Complex Formation with 5.5'-Dιthιobιs(2-nιtrobenzoιc acid) - Poly- Glutamicacid (8mer) Copolymer Fluorescein labeled DNA was used for the determination of DNA condensation m complexes with 5,5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer pDNA was modified to a level of 1 fluorescein per 20 bases using Minis' LabellT™ Fluorescein kit The fluorescence was determined using a fluorescence spectrophotometer (Shimadzo RF-1501 Fluorescence Spectrophotometer), at an excitation wavelength of 497 nm. and an emission wavelength of 520 nm To fluorescein labeled DNA (10 μg) m 1 mL HEPES (25 mM, pH 7 5) was added polyomithine (18 μg, Sigma Chemical Company ) The mixtures were held at room temperature for 5 minutes and the fluorescence was determined (see Trubetskoy, V S . Slattum, P M . Hagstrom. J E . Wolff. J A . Budker. V G . "Quantitative Assessment of DNA Condensation." Anal Biochem (1999) incorporated by reference) Since fluorescence intensity is decreased by DNA condensation, results indicate that polyomithine compacts DNA To the resulting complex was added 5.5'- Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer (60 μg). and the fluorescence was again determined The fluorescence of the sample decreased further Upon the addition of 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer to the sample, the fluorescence decreased, indicating the formation a tople complex No competition of the 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly- Glutamicacid (8mer) Copolymer for the polyomithine was observed (increase in fluorescence) Example 37 Transfection of 3T3 Cells with 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer Three complexes were formed Complex I) To 300 μL Opti-MEM was added LT-1™ (12 μg, Minis Corporation) followed by pDNA (pCI Luc. 4 μg) Complex II) To 300 μL Opti-MEM was added LT-1™ (12 μg, Minis Corporation) followed by pDNA (pCI Luc. 4 μg), and 5.5'-Dιthιobιs(2-nιtiObenzoιc acid) - Poly- Glutamicacid (8mer) Copolymer (4 μg) Complex III) To 300 μL Opti-MEM was added LT-1™ (12 μg. Minis Corporation) followed by pDNA (pCI Luc. 4 μg), and Poly-Glutamicacid (4 μg, MW 49000, Sigma Chemical Company) Transfections were earned out in 35 mm wells At the time of transfection. 3T3 cells. at approximately 50% confluency. were washed once with PBS (phosphate buffered saline), and subsequently stored in serum-free media (2 0 mL, Opti-MEM. Gibco BRL) 150 μL of complex was added to each well After a 3 25 h incubation penod at 37 °C, the media containing the complexes was aspirated from the cells, and replaced with complete growth media. DMEM with 10% fetal bovine serum (Sigma) After an additional incubation of 48 hours, the cells were harvested and the lysate was assayed for luciferase expression as previously reported (Wolff. J A . Malone. R W . Williams. P . Chong, W . Acsadi. G . Jam. A and Feigner, P L Direct gene transfer into mouse muscle in vivo Science. 1465-1468. 1990 ) A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Germany) luminometer was used Results Complex I RLU = 17.000.000 Complex II RLU = 14.000.000 Complex III RLU = 26.000,000 The addition of Poly-Glutamicacid (4 μg, MW 49000. Sigma Chemical Company) in the transfection expenment improved the pDNA expression The addition of 5.5 * - Dιthιobιs(2-nιtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer (4 μg) while not improving the pDNA expression was not detrimental to the expression Example 38 Demonstration of Reduction of by 5.5'-dιthιobιs(2-nιtrobenzoιc acid) - Containing Copolymers by Glutathione To a solution of 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - 1.4-Bιs(3- ammopropyOpiperazine Copolymer (100 μg) in 0 5 mL HEPES (25 mM, pH 8) was added glutathione (final concentration of 2 mM) The absorbance of the sample was measured at λ 412 (The cleaved disulfide has an absorbance maximum at λ 412. See Hermanson, G T Bioconjugate Techniques. Academic Press. New York. New York, 1996. pp 88) versus time (Beckman DU-7 Spectrophotometer) To a solution of 5.5 " -dithiobis(2-mtrobenzoic acid) - tetraethylenepentamine copolymer (50 μg) in 0 5 mL HEPES (25 mM. pH 8) was added glutathione (final concentration of 2 mM) The absorbance of the sample was measured at λ 412 (The cleaved disulfide has an absorbance maximum at λ 412 See Hermanson. G T Bioconjugate Techniques. Academic Press. New York, New York. 1996, pp 88) versus time (Beckman DU-7 Spectrophotometer) To a solution of 5.5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer (50 μg) in 0 5 mL HEPES (25 mM, pH 8) was added glutathione (final concentration of 2 mM) The absorbance of the sample was measured at λ 412 (The cleaved disulfide has an absorbance maximum at λ 412 See Hermanson, G T Bioconjugate Techniques. Academic Press. New York. New York. 1996, pp 88) versus time (Beckman DU-7 Spectrophotometer) Each sample showed a rapid increase in the absorbance at λ 412 upon the addition of glutathione. indicating cleavage of the disulfide bond Half life values were estimated as 5.5'-Dιthιobιs(2-nιtrobenzoιc acid) - 1.4-Bιs(3-ammopropyl)pιperazιne Copolymer ti 2 = 42 sec 5,5'-dithiobis(2-nitrobenzoic acid) - tetraethylenepentamine copolymer tι/ = 75 sec 5,5'-Dιthιobιs(2-mtrobenzoιc acid) - Poly-Glutamicacid (8mer) Copolymer tι/ 2 = 24 sec The expeπment demonstrates rapid cleavage of the disulfide bond of 5.5 ' -dιthιobιs(2- mtrobenzoic acid) - contaimng copolymers with the physiological reducing agent glutathione The foregoing is considered as illustrative only of the pπnciples of the invention Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and descnbed Therefore, all suitable modifications and equivalents fall within the scope of the invention

Claims: We Claim: 1) A biologically active compound associated with a disulfide-contaimng compound, compnsing the disulfide-contaimng compound having a labile disulfide bond that is selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols in which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated bv intramolecular attack from a free thiol 2) The compounds of claim 1 wherein the disulfide-contaimng compound compnses a polymer 3) The compound of claim 2 wherein the polymer is selected from the group consisting of a polycation. a polyamon. a neutral polymer and an amphipathic polymer 4) The compound of claim 1 wherein the biologically active compound is a polynucleotide 5) The compound of claim 1 wherein the biologically active compound is a polypeptide 6) The compound of claim 1 wherein the disulfide-contaimng compound contains a ligand 7) A compound for inserting into an organism compnsing the compound having a disulfide bond that is labile under physiologic conditions selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols m which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated by intramolecular attack from a free thiol 8) The compound of claim 7 wherein the compound compnses an amphipathic compound 9) The compound of claim 7 wherein the compound compnses a polymer 10) The method of claim 7 wherein the polymer is selected from the group consisting of a polycation. a polyamon. a neutral polymer, and an amphipathic polymer 11) The method of claim 7 wherein the compound contains a ligand 12) A process for forming a compound having a labile disulfide bond for use with an organism, compnsing a) forming the compound having a disulfide bond selected from the group consisting of (I) a disulfide bond that is cleaved more rapidly than oxidized glutathione. and (n) a disulfide bond constructed from thiols in which one of the constituent thiols has a lower pKa than glutathione. and (in) a disulfide bond that is activated by intramolecular attack from a free thiol, b) inserting the compound into the organism 13) The process of claim 12 wherein the compound compnses a polymer 14) The process of claim 12 wherem the polymer is selected from the group consisting of a polycation, a polyamon. a neutral polymer, and an amphipathic polymer 15) The process of claim 12 wherein the compound having a labile disulfide bond is associated with a biologically active compound 16) The process of claim 15 wherein the biologically active compound is a polynucleotide 17) The process of claim 15 wherein the biologically active compound is a polypeptide 18) The process of claim 12 wherein the disulfide is a bifunctional molecule

Inventors: WOLFF JON A, WOLFF, JON, A.
Applicants: MIRUS CORP, MIRUS CORPORATION
Application year: 2000
Main classification: C07H21
IPC codes: A61K31, A61K38, A61K47, A61K48, C07H21, C07K7, C12N15