CN103491982B - The polynucleotides delivering in vivo conjugate connected with enzyme sensitiveness - Google Patents

Abstract

The present invention relates to RNA is disturbed to (RNAi) polynucleotides delivering in vivo to the composition of cell.The composition is included with the reversibly modified amphiphilic film activity polyamines of the cleavable dipeptide amide benzyl carbonate screening agent of enzyme.The film activity of the polymer is sheltered in modification, and invertibity provides physiologic response.The reversibly modified polyamines (the poly- conjugate of dynamic or DPC) is also covalently attached RNAi polynucleotide or given jointly with the RNAi polynucleotide targeting molecule conjugate of targeting.

Description

Polynucleotide in vivo delivery conjugates with enzyme sensitive linkages

Background

Delivery of polynucleotides and other compounds that are substantially impermeable to cell membranes to living cells is limited by the complex membrane systems of the cells. Drugs used in antisense, RNAi and gene therapy are relatively highly hydrophilic polymers and are generally highly negatively charged. These physical characteristics all severely limit their direct diffusion across cell membranes. For this reason, the major obstacle to polynucleotide delivery is the delivery of polynucleotides across cell membranes to the cytoplasm or nucleus.

One approach that has been used to deliver small nucleic acids in vivo is to bind the nucleic acid to a small targeting molecule or a lipid or sterol. Although some delivery and activity has been observed in these conjugates, these methods require very large nucleic acid doses.

A number of transfection reagents have also been developed which enable reasonably efficient delivery of polynucleotides to cells in vitro. However, in vivo delivery of polynucleotides with these same transfection reagents is complex and ineffective due to in vivo toxicity, adverse serum interactions, and poor targeting. Transfection reagents, cationic polymers and lipids, which function well in vitro, often form large cationic electrostatic particles and destabilize cell membranes. The positive charge of the in vitro transfection reagent facilitates binding to the nucleic acid by charge-charge (electrostatic) interaction, thus forming a nucleic acid/transfection reagent complex. The positive charge also facilitates non-specific binding of the carrier to the cell and membrane fusion, destabilization or destruction. Destabilization of the membrane facilitates delivery of a polynucleotide that is substantially impermeable to the cell membrane across the cell membrane. While these properties facilitate the transfer of nucleic acids in vitro, they can cause toxicity and targeting failure in vivo. Cationic charges interact with serum components, resulting in instability of polynucleotide transfection reagent interactions, lower bioavailability, and poor targeting. In vitro potent transfection reagent membrane activity often leads to toxicity in vivo.

For in vivo delivery, the carrier (nucleic acid and conjugated delivery agent) should be small, less than 100nm in diameter, preferably less than 50 nm. Even smaller complexes below 20nm or below 10nm may be more useful. Delivery vehicles larger than 100nm rarely pass through cells other than vascular cells in vivo. Complexes formed by electrostatic interactions tend to aggregate or collapse when exposed to physiological salt concentrations or serum components. In addition, cationic charge on the in vivo delivery vehicle leads to poor serum interactions and thus poor bioavailability. Interestingly, the high negative charge also inhibits targeted in vivo delivery, i.e., binding of the targeting ligand to the cellular receptor, by interfering with the interaction required for targeting. Thus, near neutral carriers are required for in vivo distribution and targeting. Without careful regulation, membrane disruption or destabilization activity is toxic when used in vivo. Balancing vehicle toxicity with nucleic acid delivery is easier to achieve in vitro than in vivo.

Rozema et al, in U.S. patent publication 20080152661, provide a method for reversibly modulating the membrane-disrupting activity of membrane-active polyamines using disubstituted maleic anhydride modifications. The maleamic acid linkage formed from the reaction of maleic anhydride and an amine is pH unstable within a suitable pH range for in vivo delivery. The method enables the use of membrane active polymers for in vivo delivery or nucleic acids. We now provide modified membrane active polymers with dipeptide-amide benzyl-carbamate (carbamate) linkages. The dipeptide-amide benzyl-carbamate or ester linkage is reversible and physiologically responsive. Unlike the pH labile maleamic acid linkage formed by the di-substituted maleic anhydride modification, the polymer modifier linkages described herein generate enzymatically cleavable linkages that are more stable in circulation in vivo.

Disclosure of Invention

In a preferred embodiment, we describe a masking agent for reversibly modifying and inhibiting the membrane activity of an amphiphilic membrane active polyamine, the masking agent comprising: sterically hindered stabilizers or targeting ligands that link the dipeptide-amidobenzyl-carbonate (carbonate) are referred to herein as dipeptide masking agents or protease cleavable masking agents. The dipeptide masking agent has the general formula:

R-A¹A²-amidobenzyl-carbonate.

Wherein R is a steric stabilizer or targeting ligand, A¹Is an amino acid, A²Is an amino acid. The masking agent carbonate or ester reacts with the polymeric amine to form a carbamate or ester linkage. The masking agent remains stable until the dipeptide is cleaved in vivo by the endogenous protease and thus the steric stabilizer or targeting ligand is cleaved from the polyamine. In the dipeptide (A)²And amidobenzyl), the amidobenzyl-carbamate or ester spontaneously rearranges, causing polymeric amine regeneration. Preferably, R is neutral. More preferably, R is uncharged. A preferred steric stabilizer is polyethylene glycol (PEG). The targeting ligand may be selected from: haptens such as digoxin, vitamins such as biotin, antibodies, monoclonal antibodies, and cell surface receptor ligands. The targeting ligand may be linked to the dipeptide via a linker (e.g., a PEG linker). Preferred cell surface receptor ligands are asialoglycoprotein receptor (ASGPr) ligands. A preferred ASGPr ligand is N-acetylgalactosamine (NAG). Preferred dipeptides consist of hydrophobic amino acids linked to hydrophobic uncharged amino acids by amide bonds. The preferred amidobenzyl group is a para-amidobenzyl group. Preferred carbonates or esters are activated amine-reactive carbonates or esters.

In a preferred embodiment, the present invention relates to a composition for delivering an RNA interference (RNAi) polynucleotide into a cell in vivo, comprising: masked amphiphilic membrane active polyamines (delivery polymers) and RNAi polynucleotides, wherein the polyamines are masked by reversible modification with dipeptide masking agents as described herein. The delivery polymer can be covalently linked to the RNAi polynucleotide. The preferred linkage for covalently linking the delivery polymer and the RNAi polynucleotide is a physiologically labile linkage. In one embodiment, the linkage and the dipeptide masking agent linkage are orthogonal to each other. Alternatively, the delivery polymer is not covalently linked to the RNAi polynucleotide, which is covalently linked to a targeting molecule.

In a preferred embodiment, we describe a composition comprising: an amphiphilic membrane active polyamine covalently linked to: a) covalently linking a plurality of targeting ligands or steric stabilizers through a dipeptide-amide benzyl-carbamate or ester linkage; and b) covalently linking one or more polynucleotides by one or more reversible linkages. In one embodiment, the dipeptide-amide benzyl-carbamate linkage and the polynucleotide reversible covalent linkage are orthogonal to each other. Administering to the mammal a pharmaceutically acceptable carrier or diluent comprising the polynucleotide-polymer conjugate.

In a preferred embodiment, we describe a composition comprising: a) an amphiphilic membrane active polyamine covalently linked to a plurality of targeting ligands or steric stabilizers by dipeptide-amide benzyl-carbamate or ester linkage; and b) an RNAi polynucleotide covalently linked to a targeting group selected from the group consisting of: a hydrophobic group having 20 or more carbon atoms and a galactose cluster. In this embodiment, the RNAi polynucleotide is not covalently linked to the modified amphiphilic membrane active polyamine. The modified polyamine and RNAi polynucleotide targeting group conjugates are synthesized separately and may be provided in separate containers or in a single container. Administering to the mammal, either in combination or separately, said modified polyamine and RNA polynucleotide targeting group conjugate dissolved in a pharmaceutically acceptable carrier or diluent.

Preferred dipeptide masking agents include protease (peptidase) cleavable dipeptide-p-amidobenzylamine-reactive carbonate derivatives. The protease cleavable masking agents of the present invention have a dipeptide attached to an amidobenzyl activated carbonate or ester moiety. A targeting ligand or steric stabilizer is attached to the amino terminus of the dipeptide. The amidobenzyl activated carbonate or ester moiety is attached to the carboxyl terminus of the dipeptide. Protease cleavable linkers suitable for use in the present invention have the following general structure:

wherein R4 comprises a targeting ligand or steric stabilizer, R3 comprises an amine-reactive carbonate moiety, and R1 and R2 are amino acid side chains. The preferred activated carbonate is p-nitrophenol. However, other amine-reactive carbonates or esters known in the art are readily substituted for the p-nitrophenol. The reaction of the activated carbonate or ester with an amine connects a targeting group or steric stabilizer to the membrane-activated polyamine via a peptidase-cleavable dipeptide-amide benzyl-carbamate or ester linkage. The cleavage between the amino acid and the amidobenzyl group removes R4 from the polymer and triggers an elimination reaction that causes regeneration of the polymeric amine.

The dipeptide masking agents of the invention are useful for reversible modification/inhibition of amphiphilic membrane-activating polyamines. Covalent bonds are formed by reaction of activated carbonate salts or esters of the dipeptide masking agent and polymeric amines, especially primary amine groups. Thus provided herein is a conjugate comprising a dipeptide-amide benzyl-carbonate masking agent as described herein and an amphiphilic membrane-activated polyamine:

the compounds according to the invention can generally be obtained using methods known to the person skilled in the art of organic or pharmaceutical chemistry. Other objects, features and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram showing the structure of a dipeptide masking agent, wherein:

r1 and R2 are the R groups of amino acids,

r4 is a targeting ligand for a steric stabilizer,

x is-NH-, -O-or-CH 2-,

y is-NH-or-O-

R5 is-CH 2-O-C (O) -O-Z in the 2, 4 or 6 position, wherein Z is a carbonate, and

r6 is independently hydrogen, alkyl or halogen each at position 2, 3, 4, 5 or 6 (except the position occupied by R5).

FIG. 2 is a schematic showing the structure of a dipeptide masking agent linked to a polyamine, wherein: r1 and R2 are the R groups of amino acids, R4 is a targeting ligand for a steric stabilizer, X is-NH-, -O-or-CH₂-, Y is-NH-or-O-.

FIG. 3 is a schematic diagram showing the structure of different dipeptide masking agents.

FIG. 4 shows the cycle time of a polymer modified with a dipeptide masking agent compared to two different maleic anhydride-based masking agents.

Detailed Description

Masking agents for reversibly modifying and inhibiting amphiphilic membrane active polyamines, and delivery polymers formed from the dipeptide masking agents modified polyamines, are described. The peptidase cleavable linkage is hydrolytically stable in the absence of enzyme, is electrically neutral, and prolongs the stability of DPC in storage and in vivo circulation. An improved (longer) half-life in circulation helps to increase the potential for ligand-mediated accumulation in tissues such as tumor tissue. The delivery polymers are particularly useful for delivering RNAi polynucleotides in vivo. In vivo delivery of RNAi polynucleotides is used to therapeutically suppress (knock down) gene expression.

The dipeptide masking agent has the general formula:

R-A¹A²-amide benzyl-carbamate.

Wherein R is a steric stabilizer or targeting ligand, A¹Is an amino acid, A²Is an amino acid and the carbonate is an activated amine-reactive carbonate. R is preferably uncharged. The masking agent carbonate or ester reacts with the polymeric amine to form a carbamate linkage. The masking agent remains stable until the dipeptide is cleaved in vivo by an endogenous protease and thus the steric stabilizer or targeting ligand is cleaved from the polyamine. After the dipeptide (A)²And amidobenzyl), the amidobenzyl-carbamate or ester spontaneously rearranges, causing polymeric amine regeneration. A preferred steric stabilizer is polyethylene glycol (PEG). A preferred targeting ligand for hepatic delivery is an ASGPr ligand. A preferred ASGPr ligand is N-acetylgalactosamine (NAG). The preferred amidobenzyl group is a para-amidobenzyl group.

Dipeptide of said dipeptide mask (as A)¹A²Amino acids, including α and β amino acids, are well known in the biological and chemical arts and are molecules containing an amino group, a carboxylic acid group, and side chains that vary between different amino acids₂In a preferred dipeptide, A1 is a hydrophobic amino acid, A2 is an uncharged hydrophilic amino acid¹And additional amino acids can also be inserted between R. Can also be obtained by eliminating amino acid A¹To makeThe dipeptide was replaced with a single amino acid. Any natural amino acid used in the present invention is herein indicated by its common abbreviation. Although charged amino acids can be used, uncharged masking agents are preferably used.

In a preferred embodiment, the amphiphilic membrane active polyamine is reversibly modified by reaction with the dipeptide-amidobenzyl-carbonate masking agent of the present invention to produce a membrane-inactive delivery polymer. The dipeptide masking agent can mask the polymer from non-specific interactions, increase cycle time, enhance specific interactions, inhibit toxicity or alter the charge of the polymer.

The reversibly masked polymers of the present invention include the following structure:

in the formula:

x is-NH-, -O-or-CH 2-

Y is-NH-or-O-

R1 is preferably

-(CH₂)_k-phenyl (k is 1,2, 3, 4, 5, 6; k ═ 1 phenylalanine),

-CH-(CH₃)₂(valine) and (d) in the presence of a pharmaceutically acceptable acid,

-CH-(CH₃)₂(leucine) and (ii) a pharmaceutically acceptable salt thereof,

-CH-(CH₃)₂(isoleucine),

-CH₃(alanine) and (b) a pharmaceutically acceptable salt thereof,

-(CH₂)₂-COOH (glutamic acid),

or

R2 is preferably

Hydrogen (glycine)

-(CH₂)₃-NH-C(O)-NH₂(citrulline) and (C) in the amino acid,

-(CH₂)₄-N-(CH₃)₂(lysine (CH)₃)₂)，

-(CH2)_k-C(O)-NH₂(ii) a (k is 1,2, 3, 4, 5, 6),

-CH₂-C(O)-NH₂(asparagine) in the presence of a base,

-(CH₂)₂-C(O)-NH₂(the amino acid of glutamine),

-CH₂-C(O)-NR¹R²(asparagine-linked amide) or (I),

-(CH₂)₂-C(O)-NR¹R²(glutamic acid amide),

-CH₂-C(O)-OR¹(asparagine ester), or

-(CH₂)₂-C(O)-OR¹(a glutamic acid ester),

R¹and R²Is an alkyl radical

R4 includes polyethylene glycol or a targeting ligand; and

the polyamine is an amphiphilic membrane active polyamine.

The above structure indicates a single dipeptide masking agent attached to the polymer, whereas in the practice of the invention, 50% to 90% or more of the polymer amines are modified by the dipeptide masking agent.

In a preferred embodiment, the reversibly masked polymers of the present invention comprise the following structure:

wherein R1, R2, R4 and the polyamine are as described above.

The reversibly masked polymers of the present invention are formed by reacting the dipeptide masking agents of the present invention with amines on the polymer. The dipeptide masking agents of the invention have the following structure:

in the formula:

x, Y, R1, R2 and R4 are as described above

R5 is-CH 2-O-C (O) -O-Z in position 2, 4 or 6, wherein Z is a-halide,

r6 is independently hydrogen, alkyl, - (CH 5) at each of the 2, 3, 4, 5 or 6 positions (other than the position occupied by R5)₂)_n-CH₃(wherein n is 0 to 4), - (CH)₂)-(CH₃)₂Or a halide.

In a preferred embodiment, as shown below, X is-NH-, Y is-NH-, R4 is uncharged, R5 is at the 4-position, R6 is hydrogen:

in another embodiment, R4 is:

R-(O-CH₂-CH₂)_s-O-Y1-, wherein

R is hydrogen, methyl or ethyl, and s is an integer of 1 to 150,

y1 is a linker selected from the following table:

-O-Y2-NH-C(O)-(CH2)₂-C (O) -, wherein Y2 is- (CH)₂)₃-

-C(O)-N-(CH₂-CH₂-O)_p-CH₂-CH₂- (p is an integer of 1 to 20) and-O-.

The targeting ligand may be selected from: haptens, vitamins, antibodies, monoclonal antibodies and cell surface receptor ligands. The targeting ligand may be linked to the dipeptide via a linker (e.g., a PEG linker).

Non-limiting examples of film active polymers suitable for use in the present invention have been previously described in U.S. patent publication nos. 20080152661, 20090023890, 20080287630 and 20110207799. Suitable amphiphilic membrane active polyamines may also be small peptides, such as melittin.

The polymeric amine is reversibly modified using an enzyme-cleavable linker as described herein. If cleavage of the modifying group regenerates the amine, the amine is a reversible modification. Reaction of the activated masking agent carbonate or ester with the polymer amine allows the targeting ligand or steric stabilizer to be attached to the polymer via a peptidase-cleavable dipeptide-amide benzyl carbamate linkage as shown below:

R¹comprising a targeting ligand (with or without a protecting group) or PEG,

R²is an amphiphilic membrane active polyamine, and the polyamine is a polyamine,

AA is a dipeptide (with or without a protecting group), and

z is an amine reactive carbonate or ester.

The protecting group may be used during the synthesis of the dipeptide masking agent. If present, the protecting group may be removed before or after modification of the amphiphilic membrane active polyamine.

Reversibly modifying a sufficient percentage of the polymeric amine with the dipeptide masking agent can inhibit the membrane activity of the membrane active polyamine. Modification of the polymeric amine with the dipeptide masking agent also preferably neutralizes the charge of the amine. The dipeptide-amide benzyl-carbamate linkage is susceptible to cleavage by proteases (or peptidases). Cleavage of the anilide bond in the presence of a protease produces an intermediate that undergoes an immediate 1,6 elimination reaction to release the free polymer:

in the above reaction scheme, AA is a dipeptide and R¹Comprising a targeting ligand or steric stabilizer, R²Is an amphiphilic membrane active polyamine. Importantly, the free polymer is unmodified and therefore membrane activity is restored.

In the masked state, the reversible, masked, membrane-active polyamine exhibits no membrane-disrupting activity. Inhibiting membrane activity and providing cell targeting functions (i.e., forming a reversibly masked membrane active polymer (delivery polymer)) may require reversible modification of more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% of the amines on the polyamine with a dipeptide masking agent.

The invention also provides methods for delivering a biologically active substance into a cell. More specifically, the invention relates to compounds, compositions and methods for delivering RNAi polynucleotides to mammalian cells in vivo.

In one embodiment, the RNAi polynucleotide is linked to the delivery polymer of the invention through a physiologically labile covalent bond. By using physiologically labile linkages, the polynucleotide can be cleaved from the polymer, releasing the polynucleotide to participate in a functional interaction with cellular components.

The present invention comprises a conjugated delivery system of the general structure:

wherein N is an RNAi polynucleotide, L¹Is a physiologically labile linkage, P is an amphiphilic membrane-active polyamine, M¹Is a targeting ligand linked to P by a dipeptide-amidobenzyl-carbamate linkage, M²Is a steric stabilizer linked to P via a dipeptide-amide benzyl-carbamate linkage. y and z are each integers greater than or equal to 0, provided that the value of y + z is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the primary amines on the polyamine P, as measured by the amine content on P in the absence of any masking agent. In its unmodified state, P is a membrane active polyamine. Delivery of Polymer M¹ _y–P–M² _zThere is no membrane activity. By combining M¹And/or M²Reversible modification of primary amines P reversibly inhibits or inactivates the membrane activity of P. It should be noted that small amphiphilic membrane active polyamines, such as melittin, contain as few as 3-5 primary amines. A certain percentage of amine modification is intended to reflect the percentage of modification of the amine in the polymer population. Cutting M¹And M²Thereafter, the amine of the polyamine is regenerated, thereby restoring P to its unmodified membrane active state.

In another embodiment, the RNAi polynucleotide and the delivery polymer of the invention are co-administered in vivo. Accordingly, the present invention includes compositions having the following general structure:

M¹ _y–P–M² _zadding N-T into the mixture, adding N-T,

wherein N is an RNAi polynucleotide, T is a targeting group, P is an amphiphilic membrane active polyamine, and M¹Is a targeting ligand linked to P by a dipeptide-amidobenzyl-carbamate linkage, M²Is sterically stabilized by a dipeptide-amide benzyl-carbamate linkage to PAnd (3) preparing. y and z are each integers greater than or equal to 0, provided that the value of y + z is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the primary amine on polyamine P, as determined by the amine content on P in the absence of any masking agent. In its unmodified state, P is a membrane active polyamine. Delivery of Polymer M¹ _y–P–M² _zThere is no membrane activity. By combining M¹And/or M²Reversible modification of primary amines P reversibly inhibits or inactivates the membrane activity of P. It should be noted that small amphiphilic membrane active polyamines, such as melittin, contain as few as 3-5 primary amines. Thus, a certain percentage of amine modification is intended to reflect the percentage of modification of the amine in the polymer population. Cutting M¹And M²Thereafter, the amine of the polyamine is regenerated, thereby restoring P to its unmodified membrane active state. The RNAi polynucleotide-targeting group conjugate is formed by covalently linking N to T using standard methods in the art. Preferred covalent bonds are the physiologically labile bonds N-T. The delivery polymer and N-T are synthesized or produced separately. Neither T nor N directly nor indirectly with P, M¹Or M²And (3) covalent linkage. Electrostatic or hydrophobic attachment of the polynucleotide or polynucleotide conjugate to the masked or unmasked polymer is not required for in vivo hepatic delivery of the polynucleotide. The masking polymer and the polynucleotide conjugate may be provided in the same container or in separate containers. They may be combined, co-administered or administered sequentially prior to administration.

For hepatocyte delivery, whether the RNAi polynucleotide is linked to the delivery polymer by a covalent bond or co-administered with the delivery polymer, the value of y is greater than 50% of the primary amine on polymer P and up to 100%. Thus, the value of z is greater than or equal to zero percent (0%) of primary amine on polymer P, but less than 50%.

For hepatic tumor cell delivery, the value of z is greater than up to 100% of the primary amine on polymer P. In a preferred embodiment, for tumor cell delivery, the value of z is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the primary amines on polyamine P, and y is zero.

Membrane active polyamines are capable of disrupting the plasma membrane or the lysosomal/endocytic membrane. Such membrane activity is an essential feature of polynucleotide cell delivery. However, the membrane activity generates toxicity when the polymer is administered in vivo. Polyamines also readily interact with many anionic components in vivo, resulting in undesirable biodistribution. Thus, in vivo use requires reversible masking of the membrane activity of the polyamine.

Masking is achieved by the reversible binding of the dipeptide masking agent to a membrane active polyamine to form a reversible masked membrane active polymer, i.e., a delivery polymer. In addition to inhibiting membrane activity, the masking agents shield the polymers from non-specific interactions, reduce serum interactions, neutralize polyamines to reduce positive charges and form near-neutral charged polymers, increase circulation time and/or provide cell-specific interactions, i.e., targeting.

The essential feature of the masking agent is that the masking agents together inhibit the film activity of the polymer. Masking agents can shield the polymer from non-specific interactions (reduce serum interactions, increase circulation time). The membrane-active polyamine is membrane-activated in the unmodified (unmasked) state and non-membrane-activated (inactivated) in the modified (masked) state. A sufficient amount of masking agent is attached to the polymer to achieve the desired level of inactivation. The desired level of polymer modification by binding of the masking agent is readily determined by a suitable polymer activity assay. For example, if a polymer has membrane activity in a given assay, a sufficient level of masking agent is attached to the polymer to achieve a desired level of inhibition of membrane activity in that assay. Masking requires modification of greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, or greater than or equal to 90% of the primary amine groups on the polymer population, as measured by the amount of primary amine on the polymer in the absence of any masking agent. Preferred features of the masking agent also include that its binding to the polymer reduces the positive charge of the polymer, thereby forming a more neutral delivery polymer. Ideally the masking polymer remains water soluble.

In the presence of excess masking agent, the membrane-active polyamine may be coupled to the masking agent. Excess masking agent may be removed from the coupled delivery polymer prior to administration of the delivery polymer.

As used herein, a "steric stabilizer" is a nonionic hydrophilic polymer (natural, synthetic, or non-natural), a polymer that is relatively free of steric stabilizers, which prevents or inhibits intramolecular or intermolecular interactions of the polymer to which it is bound. Steric stabilizers hinder the polymers to which they are bound from participating in electrostatic interactions. Electrostatic interactions are non-covalent associations between two or more substances due to attractive forces between positive and negative charges.

Steric stabilizers inhibit interaction with blood components and thus opsonization, phagocytosis, and uptake by the reticuloendothelial system.

Steric stabilizers can therefore increase the cycle time of molecules bound thereto. Steric stabilizers can also inhibit aggregation of the polymer. Preferred steric stabilizers are polyethylene glycol (PEG) or PEG derivatives. As used herein, preferred PEGs may have about 1 to 500 or 2 to 25 ethylene glycol monomers. As used herein, preferred PEGs have an average molecular weight of about 85-20,000 daltons (Da), about 85-1000 Da. As used herein, a steric stabilizer prevents or inhibits intermolecular or intramolecular interactions of a polymer to which it is bound, relative to a polymer in aqueous solution that does not contain the steric stabilizer.

A "targeting ligand" enhances the pharmacokinetic or biodistribution properties of the conjugate to which it binds to improve the cell or tissue specific distribution and cell specific uptake of the conjugate. For clarity, the term "targeting ligand" is used herein to refer to a targeting ligand linked to a dipeptide masking agent, while a "targeting group" is a targeting ligand linked to an RNAi polynucleotide in an RNAi polynucleotide-targeting group conjugate. The targeting ligand enhances the attachment of the molecule to the target cell. Thus, the targeting ligand can enhance the pharmacokinetic or biodistribution properties of the conjugate to which it binds to improve the cellular distribution and cellular uptake of the conjugate. Binding of the targeting ligand to the cell or cellular receptor may trigger endocytosis. The targeting ligand may be monovalent, divalent, trivalent, tetravalent, or have a higher valency. The targeting ligand may be selected from the group consisting of: compounds having affinity for cell surface molecules, cell receptor ligands, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics having affinity for cell surface molecules. Preferred targeting ligands include cellular receptor ligands. Various ligands have been used to target drugs and genes to cells and specific cellular receptors. The cellular receptor ligand may be selected from the group consisting of: carbohydrates, polysaccharides, sugars (including but not limited to galactose, galactose derivatives, mannose and mannose derivatives), vitamins, folic acid, biotin, aptamers, and peptides (including but not limited to RGD-containing peptides, insulin, EGF, and transferrin).

For hepatocyte targeting, the preferred targeting ligand is a carbohydrate with affinity for asialoglycoprotein (ASGPr). Galactose and galactose derivatives were used to target molecules to hepatocytes in vivo by binding to ASGPr expressed on the surface of hepatocytes. As used herein, "ASGPr targeting ligand" includes galactose and galactose derivatives having an affinity for ASGPr equal to or greater than that of galactose. The binding of the galactose targeting ligand to the ASGPr facilitates cell specific targeting of the delivery polymer to the hepatocytes and endocytosis of the delivery polymer into the hepatocytes.

The ASGPr targeting ligand may be selected from the group consisting of: lactose, galactose, N-acetylgalactosamine (NAG), galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-butyrylgalactosamine, and N-isobutyryl-galactosamine (Iobst, s.t. and Drickamer, k.j.b.c.1996,271, 6686). The ASGPr targeting moiety can be monomeric (e.g., having a single galactosamine) or multimeric (e.g., having multiple galactosamines).

In one embodiment, the membrane active polyamine is reversibly masked by binding 50%, > 60%, > 70%, > 80%, or > 90% of the amines on the polyamine with an ASGPr targeting ligand masking agent. In another embodiment, the membrane active polyamine is reversibly masked by binding 50%, ≧ 60%, > 70%, > 80%, or ≧ 90% primary amine on the polymer by an ASGPr targeting ligand masking agent and a PEG masking agent. When both an ASGPr targeting ligand masking agent and a PEG masking agent are employed, the ratio of PEG to ASGPr targeting ligand is about 0 to 4:1, more preferably about 0.5 to 2: 1.

"amphiphilic" or amphiphilic polymers having hydrophilic (polar, water-soluble) and hydrophobic (non-polar, fat-soluble, water-insoluble) groups or moieties are well known and understood in the art.

The term "hydrophilic group" is qualitatively intended to mean that the chemical moiety is water-preferred. Typically, the chemical group is water soluble and is a hydrogen bond donor or acceptor for water. The hydrophilic group may be charged or uncharged. The charged groups may be positively charged (anionic) or negatively charged (cationic) or both (zwitterionic). Examples of hydrophilic groups include the following chemical components: carbohydrates, polyoxyethylene, certain peptides, oligonucleotides, amines, amides, alkoxyamides, carboxylic acids, sulphur and hydroxyl groups.

The "hydrophobic group" is qualitatively expressed as the chemical moiety being kept away from water. Typically such chemical groups are not water soluble and do not tend to form hydrogen bonds. Lipophilic groups are soluble in fats, oils, lipids and non-polar solvents and have little or no ability to form hydrogen bonds. Hydrocarbons containing two (2) or more carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol derivatives are examples of hydrophobic groups and compounds.

The hydrophobic groups are preferably hydrocarbons, comprising only carbon and hydrogen atoms. However, nonpolar substituted or nonpolar heteroatoms which remain hydrophobic and include, for example, fluorine may be permitted. The term includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which may be linear, branched, or cyclic. The term hydrophobic group also includes: sterols, steroids, cholesterol and derivatives of steroids and cholesterol.

As used herein, when referring to an amphoteric polymer, a moiety is defined as a molecule that is derivatized when one covalent bond is broken and replaced with a hydrogen. For example, cleavage and replacement of carbon and nitrogen bonds in butylamine by hydrogen will yield ammonia (hydrophilic) and butane (hydrophobic). If 1, 4-diaminobutane is cleaved at the nitrogen-carbon bond and replaced with hydrogen, the resulting molecules are again ammonia (2 ×) and butane. However, 1, 4-diaminobutane is not considered amphoteric because the formation of a hydrophobic moiety requires the cleavage of two bonds.

As used herein, a surface active polymer reduces the surface tension of water and/or the interfacial tension with other phases, and is thus adsorbing to the liquid/gas interface. Surface-active characteristics are generally due to the amphiphilicity or amphiphilicity of the molecules of the substance.

As used herein, a "membrane active" polymer is a surface activated amphiphilic polymer that is capable of inducing one or more of the following effects on a biological membrane: membrane alteration or disruption allows non-membrane permeable molecules to enter cells or to pass through the membrane, forming pores in the membrane, membrane disruption, or disruption or lysis of the membrane. As used herein, a membrane or cell membrane comprises a lipid bilayer. The change or disruption of the membrane can be functionally defined by polymer activity using at least one of the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell lysis, and endocytotic release. Membrane active polymers that cause lysis of cell membranes are also referred to as membrane lysis polymers. Polymers that cause destruction of endocytosis or lysosomes at the plasma membrane are preferably considered as endocytic cleavage. The effect of the membrane active polymer on the cell membrane may be transient. The membrane activity has affinity for the membrane and causes denaturation or deformation of the bilayer structure. The membrane active polymer may also be a synthetic or non-natural amphiphilic polymer.

As used herein, membrane active polymers are distinct from a class of polymers known as cell penetrating peptides or polymers represented by compounds such as arginine-rich peptides derived from the HIV TAT protein, antennal peptides, VP22 peptides, transporters, arginine-rich artificial peptides, guanidine salt-rich artificial small polymers, and the like. Although cell penetrating compounds appear to be able to transport molecules across the membrane, from one side of the lipid bilayer to the other, apparently without endocytosis and without disrupting membrane integrity, the mechanism is not understood.

Delivery of polynucleotides into cells is mediated by membrane active polymers that disrupt or destabilize the plasma membrane or the inner vesicle membrane (e.g., endocytosis or lysosomes), including forming pores in the membrane, or disrupting endocytosis or lysosomal vesicles thereby allowing release of the contents of the vesicles into the cytoplasm.

The amphiphilic membrane active polyamine copolymers of the present invention are the result of the copolymerization of two or more monomeric species. In one embodiment, the amphiphilic membrane active heteromers of the present invention have the general structure:

-(A)_a-(B)_b-

wherein A comprises a pendant primary or secondary amine functional group and B comprises a pendant hydrophobic group. a and b are integers > 0. The polymer may be a random, block or alternating polymer. Additional monomer incorporation is allowed.

An "endocytolytic polymer" is a polymer that is capable of causing disruption or lysis of endosomes or release of normally cell membrane impermeable compounds (e.g., polynucleotides) from vesicles (e.g., endosomes or lysosomes) encapsulated within the intracellular membrane in response to endosomal specific environmental factors (e.g., the presence of a lytic enzyme). In endosomes, the physicochemical properties of endocytosed lytic polymers change. The change may be a change in the solubility of the polymer or the ability to interact with other compounds or membranes due to a change in charge, hydrophobicity, or hydrophilicity. It is contemplated that the reversible masking membrane active polyamine of the present invention is an endocytosis cleaving polymer.

The melittin is an amphiphilic membrane active small peptide naturally generated in bee venom. Melittin may be isolated from biological sources or may be synthesized. Synthetic polymers are "formulated or produced by chemical processes by humans" and not by naturally occurring biological processes. Melittin as used herein includes a variety of melittin peptides of the naturally occurring melittin family, which can be found in the venom of the following species: western bees (Apis meliera), Chinese bees (Apis cerana), frontal spotted wasps (Vespula maculifrons), hornet (Vespa magnica), black-chest wasps (Vespa velutinitririthorax), hornet HQL-2001(Polistes sp.HQL-2001), small bees (Apis florae), large bees (Apis dordata), Chinese bees (Apis cerana cerana), and Asian hornet (Poles hebraeus). Melittin as used herein also includes synthetic peptides having an amino acid sequence identical or similar to that of naturally occurring melittin. In particular, the melittin amino acid sequences include those shown in table 1. Synthetic melittin may comprise naturally occurring L-form amino acids or enantiomeric D-form amino acids (retro). However, melittin should include substantially all L-or all D-amino acids, but may have amino acids appended to opposite stereocenters at the amino or carboxyl terminus. The melittin amino acid sequence may also be reversed (retro). The retro-melittin may have an L-type amino acid or a D-type amino acid (retro-inverso). Two melittin peptides may also be covalently linked to form a melittin dimer. Melittin may have a non-masking agent modifying group attached to the amino terminus or the carboxy terminus to enhance tissue targeting or promote circulation in vivo.

A linkage or "linker" is a link between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. For example, the linking can be by linking a masking agent or polynucleotide to the polymer. The labile linkage comprises a labile bond. Attachment may optionally include a spacer that increases the distance between the two attached atoms. Spacers may also increase the flexibility and/or length of the connection. Spacers may include, but are not limited to: alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, each of which may include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art and the foregoing list is not intended to limit the scope of the invention.

A "labile bond" is a covalent bond other than a covalent bond with a hydrogen atom that can be selectively broken or cleaved without breaking or cleaving other covalent bonds in the same molecule. More specifically, a labile bond is a covalent bond that is less stable (thermodynamic) or breaks down more rapidly (kinetic) under appropriate conditions than a labile covalent bond in the same molecule. Cleavage of an intramolecular labile bond will result in the formation of two molecules. For those skilled in the art, cleavage or destabilization of a bond is generally termed the half-life of bond cleavage (ii)t_1/2) (time required to cut half of the bond). Thus, labile bonds encompass bonds that selectively cleave faster than other bonds in the molecule.

As used herein, a "physiologically labile bond" is a labile bond that can be cleaved under conditions normally encountered or similar to those encountered in a mammalian body. The physiologically labile bond is selected such that it undergoes a chemical transformation (e.g., cleavage) when present under certain physiological conditions.

As used herein, a physiologically labile bond of a cell is a labile bond cleavable under conditions within a mammalian cell. Conditions within mammalian cells include chemical conditions such as pH, temperature, oxidizing or reducing conditions or reagent and salt concentrations found within or similar to those encountered within mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally occurring in mammals such as proteolytic or hydrolytic enzymes. Physiologically labile bonds in cells can also be cleaved in response to administration of a pharmaceutically acceptable exogenous agent.

RNAi interfering targeting group conjugates: targeting groups can be attached to the 3 'or 5' end of the RNAi polynucleotide. For siRNA polynucleotides, the targeting moiety may be attached to the sense strand or the antisense strand, preferably the sense strand.

In one embodiment, the targeting group consists of a hydrophobic group. More specifically, the targeting group consists of a hydrophobic group having at least 20 carbon atoms. Hydrophobic groups for use as polynucleotide targeting moieties are referred to herein as hydrophobic targeting moieties. Exemplary suitable hydrophobic groups may be selected from the group consisting of: cholesterol, di-cholesterol, tocopherol, di-tocopherol, didecyl, didodecyl, dioctadecyl, isoprenoid and cholenamide (choleamide). Hydrophobic groups having 6 or fewer carbon atoms are less effective than polynucleotide targeting moieties, while hydrophobic groups having 8-18 carbon atoms can deliver more polynucleotides having larger size hydrophobic groups (i.e., an increase in the number of carbon atoms). Without co-administration of a delivery polymer, the binding of a hydrophobic targeting group to the RNAi polynucleotide does not provide effective functional in vivo RNAi polynucleotide delivery. Although siRNA-cholesterol conjugates have been reported for in vivo delivery of siRNA (siRNA-cholesterol) to the liver, high concentrations of siRNA are required and delivery efficiency is poor without any additional delivery vehicle. Delivery of the polynucleotides is significantly improved when combined with the delivery polymers described herein. By providing siRNA-cholesterol with the delivery polymer of the present invention, the efficiency of siRNA-cholesterol is increased by about 100-fold.

Hydrophobic groups for use as polynucleotide targeting moieties may be selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl and aralkynyl (each of which may be linear, branched or cyclic), cholesterol derivatives, sterols, steroids and steroid derivatives. The hydrophobic targeting group is preferably a hydrocarbon, comprising only carbon and hydrogen atoms. However, substitutions or heteroatoms, such as fluorine, which maintain hydrophobicity may be allowed. The hydrophobic targeting group can also be linked to the 3 'or 5' end of the RNAi polynucleotide using methods known in the art. For RNAi polynucleotides having 2 strands, e.g., sirnas, the hydrophobic group can be attached to either strand.

In another embodiment, the targeting group comprises a galactose cluster (galactose cluster targeting moiety). As used herein, a "galactose cluster" comprises molecules having 2-4 terminal galactose derivatives. As used herein, the term galactose derivative includes galactose and galactose derivatives having an affinity for ASGPr equal to or greater than galactose. The terminal galactose derivative is linked to the molecule through its C-1 carbon. Preferred galactose clusters have 3 terminal galactosamines or galactosamine derivatives each having affinity for asialoglycoprotein receptor. More preferred galactose clusters have 3 terminal N-acetylgalactosamine. Other common terms in the art include trigeminal (tri-antennary) galactose, trivalent galactose and galactose trimers. The triantennary galactose derivative cluster is known to bind ASGPr with a stronger affinity than the structure of either the two-touch or one-touch galactose derivatives (Baenziger and Fiete,1980, Cell,22, 611-945; Connolly et al, 1982, J.biol.chem.,257, 939-945). Multivalence is required to obtain neutrality in nM. When co-administered with a delivery polymer, the monogalactose derivative that binds with affinity for the asialoglycoprotein receptor is unable to deliver the RNAi polynucleotide functionally in vivo into the liver cell.

The galactose cluster contains 2 to 4, preferably 3 galactose derivatives each linked to a central branch point. The galactose derivative is bound to a central branch point through the C-1 carbon of the saccharide. The galactose derivative is preferably linked to the branch point by a linker or a spacer. Preferred spacers are flexible hydrophilic spacers (U.S. Pat. No. 5885968; Biessen et al J.Med.chem.1995 Vol.39, page 1538-1546). The preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is PEG₃A spacer. The branch point may be any small molecule that allows the three galactose derivatives to bind and also allows the branch point to bind to the RNAi polynucleotide. An exemplary branch point group is dilysine. The dilysine molecule contains three amine groups through which three galactose derivatives can be bound and a carboxyl reactive group through which the dilysine can bind the RNAi polynucleotide. Binding of the branch point to the RNAi polynucleotide can occur through a linker or spacer. Preferred spacers are flexible hydrophilic spacers. The preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is PEG₃Spacer (three ethylene units). The galactose cluster may also be linked to the 3 'or 5' end of the RNAi polynucleotide using methods known in the art. For RNAi polynucleotides having 2 strands, e.g., sirnas, the galactose cluster may be linked to either strand. U.S. patent publication 20110207799 describes suitable galactose clusters.

The term "polynucleotide", or nucleic acid or polynucleic acid, is a term of art and refers to a polymer comprising at least two nucleotides. Nucleotides are monomeric units of polynucleotide polymers. Polynucleotides having fewer than 120 monomer units are often referred to as oligonucleotides. Natural nucleic acids have a ribose-or ribose-phosphate backbone. A non-natural or synthetic polynucleotide is a polynucleotide that is polymerized in vitro or in a cell-free system and contains the same or similar bases but may contain backbone types other than the natural ribose or deoxyribose phosphate backbone. Polynucleotides can be synthesized by any technique known in the art. Polynucleotide backbones known in the art include: PNA (peptide nucleic acids), phosphorothioates, phosphodiamides (phosphorodiamidites), morpholinos, and other phosphate backbone variants of natural nucleic acids. Bases include purines and pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic analogs of purines and pyrimidines include, but are not limited to, modifications that place a new reactive group on the nucleotide, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. The term base encompasses any known analogs of DNA and RNA bases. Polynucleotides may also include ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination. Polynucleotides may be polymerized in vitro, they may be recombinant, including chimeric sequences or derivatives of these groups. The polynucleotide may also include terminal cap moieties at both the 5 'and 3' ends, or both the 5 'and 3' ends. The headpiece can be, but is not limited to, an inverted deoxyabasic moiety, an inverted deoxythymidine moiety, a thymidine moiety, or a 3' glycerol modification.

An "RNA interference (RNAi) polynucleotide" is a molecule capable of inducing RNA interference that degrades or inhibits translation of a transgenic messenger RNA (mrna) transcript in a sequence-specific manner by interacting with a component of the RNA interference pathway of a mammalian cell. The two major RNAi polynucleotides are small (or short) interfering rnas (sirnas) and micrornas (mirnas). The RNAi polynucleotide may be selected from the group consisting of: siRNA, microrna, double-stranded RNA (dsrna), short hairpin RNA (shrna), and an expression cassette encoding a polypeptide capable of inducing RNA interference. siRNAs comprise double-stranded structures typically comprising 15-50 base pairs, preferably 21-25 base pairs, and having a nucleotide sequence that is identical (perfectly complementary) or nearly identical (partially complementary) to the coding sequence in the target gene or RNA expressed in the cell. siRNA also has 3' dinucleotide protrusions. The siRNA may consist of two annealed polynucleotides or a single nucleotide forming a hairpin structure. The siRNA molecules of the invention comprise a sense region and an antisense region. In one embodiment, the siRNA of the conjugate is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siRNA molecule and the second fragment comprises the nucleotide sequence of the sense strand of the siRNA molecule. In another embodiment, the sense strand is linked to the antisense strand by a linker molecule such as a polynucleotide linker or a non-nucleotide linker. Micro RNA (mirna) is a non-coding small RNA gene product of about 22 nucleotides in length that directs the destruction or translational inhibition of its mRNA target. If there is partial complementarity between the miRNA and the target mRNA, translation of the target mRNA is inhibited. If broadly complementary, the target mRNA is cleaved. For mirnas, the complex binds to a target site that is usually located in the 3' UTR of the mRNA, which is usually only partially homologous to the miRNA. The "seed region" is a stretch of about seven (7) contiguous nucleotides at the 5' end of the miRNA, forming perfect base pairing with its target-playing a key role in miRNA specificity. Binding of the RISC/miRNA complex to mRNA can lead to protein translation inhibition or mRNA cleavage and degradation. Recent data indicate that mRNA cleavage occurs preferentially if there is perfect homology to its target along the full length miRNA rather than showing perfect base pairing only in the seed region (pilai et al, 2007).

The RNAi polynucleotide expression cassette can be transcribed in a cell to produce a small hairpin RNA that can function as a siRNA, an isolated sense and antisense strand linear siRNA, or a miRNA. The DNA transcribed by RNA polymerase III contains a promoter selected from the following list: the U6 promoter, the H1 promoter, and the tRNA promoter. RNA polymerase II promoters include U1, U2, U4 and U5 promoters, snRNA promoters, microrna promoters and mRNA promoters.

Lists of known miRNA sequences can be found in databases maintained by research organizations, such as the welcongkin Sanger Institute (wellcorn Trust Sanger Institute), the Penn Center for bioinformatics, the slogan katerin Memorial Center for cancer (Memorial Center) and the european molecular biology laboratory, among others. Known effective siRNA sequences and associated binding sites are also well described in the relevant literature. RNAi molecules are readily designed and produced using techniques known in the art. In addition, computational tools can be used to increase the likelihood of finding effective and specific sequence motifs (Pei et al 2006, Reynolds et al 2004, Khvorova et al 2003, Schwarz et al 2003, Ui-Tei et al 2004, Heale et al 2005, Chalk et al 2004, Amarzguioui et al 2004).

The polynucleotides of the invention may be chemically modified. Non-limiting examples of such chemical modifications include: phosphorothioate internucleotide linkages, 2 '-O-methyl ribonucleotides, 2' -deoxy-2 '-fluoro ribonucleotides, 2' -deoxyribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when applied to various polynucleotide structures, are shown to maintain polynucleotide activity in the cell while increasing the serum stability of these compounds. Chemically modified sirnas may also minimize the possibility of activation of interferon activity in humans.

In one embodiment, the chemically modified RNAi polynucleotides of the invention comprise duplexes having two strands, one or both of which may be chemically modified, wherein each strand is from about 19 to about 29 nucleotides. In one embodiment, the RNAi polynucleotides of the invention include one or more modified nucleotides while maintaining the ability to mediate RNAi in a cell or to be reconstituted in an in vitro system. RNAi polynucleotides can be modified, wherein a chemical modification includes one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides. The RNAi polynucleotides of the invention can include modified nucleotides that account for a percentage of the total number of nucleotides present in the RNAi polynucleotide. Likewise, the RNAi polynucleotides of the invention can typically include modified nucleotides at about 5 to about 100% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotide positions). The actual percentage of modified nucleotides present in a given RNAi polynucleotide depends on the total number of nucleotides present in the RNAi polynucleotide. If the RNAi polynucleotide is single-stranded, the percentage modification can be based on the total number of nucleotides present in the single-stranded RNAi polynucleotide. Likewise, if the RNAi polynucleotide is double-stranded, the percentage modification can be based on the total number of nucleotides present in the sense strand, the antisense strand, or both the sense and antisense strands. In addition, the actual percentage of modified nucleotides present in a given RNAi polynucleotide can also depend on the total number of purine and pyrimidine nucleotides present in the RNAi polynucleotide. For example, wherein all pyrimidine nucleotides and/or all purine nucleotides present in the RNAi polynucleotide are modified.

The RNAi polynucleotides regulate expression of RNA encoded by the gene. Since multiple genes may share some degree of sequence homology with each other, RNAi polynucleotides can be designed to target a class of genes with sufficient sequence homology. Thus, the RNAi polynucleotide can include sequences that are complementary to sequences that are shared between different gene targets or that are unique to a specific gene target. Thus, RNAi polynucleotides can be designed to target conserved regions of RNA sequences with homology between several genes, thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In another embodiment, the RNAi polynucleotide can be designed to target sequences unique to specific RNA sequences in a single gene.

The term "complementary" refers to the ability of a polynucleotide to form hydrogen bonds with another polynucleotide sequence through conventional watson crick or other unconventional types. In relation to the polynucleotide molecules of the present invention, the free energy of binding of the polynucleotide molecule to its target (effective binding site) or complementary sequence is sufficient to facilitate the relevant function of the polynucleotide, such as mRNA cleavage or translational inhibition of the enzyme. Determination of the amount of free energy associated with a nucleic acid molecule is well known in the art (Frier et al 1986, Turner et al 1987). Percent complementarity refers to the percentage of bases of contiguous strands in a first polynucleotide molecule that can form hydrogen bonds (e.g., watson crick base pairing) with a second polynucleotide sequence (e.g., five tenths, six, seven, eight, nine, ten, i.e., 50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfect complementarity means that all bases in the contiguous strand of a polynucleotide sequence form hydrogen bonds with the same number of consecutive bases in the second polynucleotide sequence.

Inhibiting, down-regulating or knocking down gene expression means that the expression of the gene is lower than that observed without blocking the polynucleotide conjugate of the invention, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the RNA. Gene expression inhibition, down-regulation or knockdown with a polynucleotide delivered with a composition of the invention is preferably below the level observed in the presence of a control inactivating nucleic acid (a sequence scrambled nucleic acid or a passivation mismatched nucleic acid) or when the polynucleotide is not coupled to a masking polymer.

It was found that stabilization of siRNA against degradation by endosomal/lysosomal localization nucleases (e.g., dnase II) greatly facilitates targeted knockdown. The stabilization can directly affect the amount of siRNA released into the cytoplasm in which the cellular RNAi machinery is located. Only the siRNA moiety available in the cytoplasm triggers the RNAi effect.

In addition to poor pharmacokinetic properties, sirnas are also susceptible to nucleases in the biological environment when so administered into the circulation without a protective delivery vehicle. Thus, many sirnas degrade rapidly in extracellular tissues and blood streams or after intracellular uptake (endosomes). Nuclease cleavage can be inhibited by nucleotides lacking a2 '-OH group (e.g., 2' -deoxy, 2 '-O-methyl (2' -OMe) or 2 '-deoxy-2' -fluoro (2 '-F) nucleotides), and by non-nucleotide moieties at the 5' -terminus of the polynucleotide, such as cholesterol, aminoalkyl-linker, or phosphorothioate at the first internucleotide linkage. Preferably, the RNAi polynucleotide strand is devoid of any 2' -OH nucleotide, starting at the 2' -OMe nucleotide and linked at the 5 ' -terminus to a second nucleotide by a Phosphorothioate (PTO) linkage.

siRNA can be significantly stabilized using the following design, wherein an oligonucleotide is provided having the following modification pattern of the antisense strand: 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'and sense strand 5' - (Z3) n of the following modification modes_s-3', wherein

w is independently 5 '-phosphate or 5' -thiophosphate or H,

z1 is an independent 2' -modified nucleoside.

Z2 is an independent 2 '-deoxynucleoside or 2' -fluoro-modified nucleoside,

z3 is an independent 2' -modified nucleoside,

n_ais 8 to 23, n_sIs 8 to 25.

In a preferred embodiment, the provided oligonucleotides have antisense strands with the following modification patterns: 5' - (w) - (Z1) - (Z2) - (Z3) n_a-3' and sense strand of the following modification pattern: 5' - (Z3) n_s-3 ', wherein Z1 is a 2' -fluoro-modified nucleoside or a 2-deoxy-nucleoside, and all remaining substituents as well as the variable n_aAnd n_sHaving the meaning given above.

In a preferred embodiment, the provided oligonucleotides have antisense strands with the following modification patterns: 5' - (w) - (Z1) - (Z2) - (Z3) n_a-3' and sense strand of the following modification pattern: 5' - (Z3) n_s-3 ', wherein Z3 is a 2' -O-methyl modified nucleoside, a 2' -fluoro-modified nucleoside or a 2-deoxy-nucleoside, and all remaining substituents as well as the variable n_aAnd n_sHaving the meaning given above.

In a preferred embodiment, the provided oligonucleotides have antisense strands with the following modification patterns: 5' - (w) - (Z1) - (Z2) - (Z3) n_a-3' and sense strand of the following modification pattern: 5' - (Z3) n_s-3 ', wherein Z1 is a 2' -fluoro-modified nucleoside or 2-deoxy-nucleoside, Z3 is a2 '-O-methyl-modified nucleoside, 2' -fluoro-modified nucleoside or 2-deoxy-nucleoside, and all remaining substituents as well as the variable n_aAnd n_sHaving the meaning given above.

The nucleosides in the nucleic acid sequence of the oligonucleotides with the novel modification patterns can be linked by 5 '-3' phosphodiesters or 5 '-3' phosphorothioates.

As used herein, an "antisense" strand is an siRNA strand complementary to a target mRNA that binds to the mRNA once it is uncoiled. The sense strand of the siRNA comprising a novel modification pattern is complementary to the antisense strand.

In principle, the nuclease cleavage site between the RNAi polynucleotide covalently linked thereto and the targeting moiety or delivery polymer can be introduced by a 3 ' -or 5 ' -overhang containing at least one 2' -OH nucleotide in the sense or antisense strand. The final active siRNA species are generated by intracellular nuclease processing. In addition, a defined cleavage site consisting of a 2' -OH nucleotide within the base-pairing region may be used. This can be done using at least one 2' -OH nucleotide complementary to the opposite strand or by introducing at least one mismatched 2' -OH nucleotide or a hairpin/bulge containing at least one 2' -OH nucleotide.

Ligation of polynucleotides to delivery polymers

In one embodiment, the RNAi polynucleotide delivers the polymer through a physiologically labile bond or linker linkage. The physiologically labile linker is selected such that chemical transformation (e.g., cleavage) occurs in the presence of certain physiological conditions (e.g., cleavage of a disulfide bond in a cytoplasmic reducing environment). Releasing the polynucleotide from the polymer by cleavage of the physiologically labile linkage facilitates interaction of the polynucleotide and the appropriate cellular components in terms of activity.

The polynucleotide polymer conjugate is formed by covalently linking the polynucleotide and the polymer. The polymer is polymerized or modified so that it contains a reactive group a. The polynucleotide is also polymerized or modified such that it comprises a reactive group B. The reactive groups a and B are selected such that they can be linked to each other by reversible covalent linking using methods known in the art.

The coupling of the polynucleotide and the polymer can be carried out in the presence of an excess of polymer. Because the polynucleotide and the polymer may be oppositely charged during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the conjugate. Alternatively, an excess of carrier polymer, such as a polycation, can be used. Excess polymer may be removed from the conjugated polymer prior to administration of the conjugate to an animal or cell culture. Alternatively, the excess polymer can be co-administered with the conjugate to an animal or cell culture.

In vivo administration

In pharmacology and toxicology, the route of administration is the route by which a drug, liquid, toxic agent, or other substance contacts the body. Generally, drugs and nucleic acid administration methods for treating mammals are well known in the art and may be used to administer the compositions of the present invention. The compounds of the invention may be employed in formulations adapted appropriately for the route by any suitable route, most preferably parenterally. Thus, the compounds of the present invention may be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal injection. Accordingly, the present invention also provides pharmaceutical compositions containing a pharmaceutically acceptable carrier or excipient.

Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal (subdermal), subcutaneous (subductous), intratumoral, intraperitoneal, intrathecal, subdural, epidural, and intralymphatic injection, using syringes and needles or catheters. Intravascular as used herein refers to within a tubular structure, referred to as a vessel, that connects tissues or organs within the body. Within the lumen of the tubular structure, bodily fluids flow to or from the body part. Examples of body fluids include blood, cerebrospinal fluid (CSF), lymph, or bile. Examples of vessels include coronary arteries, arterioles, capillaries, venules, sinusoidal tubules, veins, lymphatic vessels, bile ducts, and salivary ducts or other exocrine ducts. Intravascular routes include delivery through a blood vessel such as an artery or vein. The blood circulation system provides for systemic diffusion of the drug.

The composition is injected in a pharmaceutically acceptable carrier solution. Pharmaceutically acceptable refers to those properties and/or substances that are acceptable to a mammal from a pharmacological/toxicological standpoint. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a mammal. The term "pharmaceutically acceptable" as used herein preferably refers to approval by a regulatory agency of the federal or a state government, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

These carriers may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the occurrence of microorganisms can be ensured by sterilization methods (supra) and the incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol and phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like may also be included in the compositions as desired. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the addition of substances which delay absorption, for example, aluminum monostearate and gelatin.

In one embodiment, an RNAi polynucleotide-targeting group conjugate is co-administered with a delivery polymer of the invention. Co-administration means that the RNAi polynucleotide conjugate and the delivery polymer are administered to a mammal such that both are present within the mammal. The RNAi polynucleotide targeting group conjugate and the delivery polymer can be administered simultaneously or they can be delivered sequentially. For simultaneous administration, they may be mixed prior to administration. For sequential administration, the RNAi polynucleotide targeting moiety conjugate or delivery polymer may be administered first.

Therapeutic effects

The RNAi polynucleotides can be delivered for research purposes or to produce changes in the treated cells. In vivo delivery of RNAi polynucleotides is used for research reagents and various therapeutic, diagnostic, target validation, genome development, genetic engineering, and pharmacogenomic applications. Our disclosed RNAi polynucleotide delivery results in the inhibition of endogenous gene expression in hepatocytes. The reporter (marker) gene expression level measured after polynucleotide delivery indicates a reasonable expectation that the gene expression levels are similar after other polynucleotide delivery. The level of treatment that would be considered beneficial by one of ordinary skill in the art varies between diseases. For example, hemophilia a and B are caused by X-linked deficiencies in coagulation factor VIII and IX, respectively. Its clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or IX: < 2%, severe; 2-5%, moderate, and 5-30% mild. Thus, an increase in the normal level of circulating factor from 1% to 2% in severe patients may be considered beneficial. Levels above 6% prevent spontaneous bleeding but do not prevent secondary bleeding of those following surgery or injury. Similarly, inhibition of a gene need not be 100% to provide a therapeutic benefit. One of ordinary skill in the art of gene therapy can reasonably expect a beneficial level of disease-specific gene expression based on a sufficient level of marker gene outcome. In the hemophilia example, if the marker gene is expressed to produce protein at a level equivalent to 2% by volume of the normal level of factor VIII, it is reasonable to expect that the gene encoding factor VIII will also be expressed at a similar level. Thus, reporter or marker genes are often used as useful paradigms for intracellular protein expression.

The liver is the most important target tissue for gene therapy because it plays a key role in metabolism (e.g., lipoprotein metabolism in various hypercholesterolemia) and secretion of circulating proteins (e.g., coagulation factors in hemophilia). Furthermore, commonly acquired diseases such as chronic hepatitis and cirrhosis and may also be treated with polynucleotide-based liver therapies. Many diseases or conditions that affect or are affected by the liver can be treated by knocking down (inhibiting) gene expression in the liver. The liver diseases and conditions may be selected from the list consisting of: liver cancer (including hepatocellular carcinoma, HCC), viral infections (including hepatitis), metabolic disorders (including hyperlipidemia and diabetes), fibrosis, and acute liver injury.

The actual dosage level of the active ingredient in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and which is non-toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, body weight, condition, general health, prior medical history of the patient being treated, and like factors well known in the medical arts.

The amount (dose) of delivery polymer, RNAi polynucleotide-targeting group conjugate, or delivery polymer-RNAi polynucleotide conjugate to be administered can be determined empirically. We show that using siRNA at 0.1-10mg/kg animal body weight and delivery polymer at 1.5-60mg/kg animal body weight can effectively knock down gene expression. Preferred amounts in mice are 0.25-2.5mg/kg siRNA-conjugate and 10-40mg/kg delivery polymer. More preferably, about 1.5-20mg/kg of the delivery composition is administered. The amount of RNAi polynucleotide conjugate is readily increased because it is generally non-toxic at high doses.

As used herein, in vivo refers to processes occurring within an organism and more specifically refers to processes occurring in or on living tissue of an intact, living multicellular organism (animal), such as a mammal, as opposed to a partially or dead individual.

As used herein, a "pharmaceutical composition" comprises a conjugate of the invention, a pharmaceutical carrier or diluent, and any other media or agents necessary for formulation.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

Examples

Example 1 Synthesis of protease (peptidase) cleavable masking agents.

Aqueous NaHCO₃All reactions were carried out under anhydrous conditions using fresh anhydrous solvent, except for the amino acid coupling and silyl group deprotection in (1). Column purification was done on silica gel using the indicated eluent. Mass Spectrometry (MS) was performed using electrospray ionization.

In the preparation of active p-nitrophenyl-p-amidobenzyl carbonate derivatives of NAG and PEG (NAG-L-AA-PABC-PNP and PEG-AA-PABC-PNP), we applied NHS ester or NAG-containing derivatives of each PEG to acylate the amino terminus of the dipeptide-p-amidobenzyl alcohol precursor. In the next step, the benzyl hydroxyl group is converted to p-nitrophenyl carbonate, and then the protecting group is removed from the amino acid and NAG moieties. In some applications, when p-nitrophenyl (PNP) carbonate is used for modification of certain polymers, the protecting group is removed prior to modification of the polymer.

R comprises an ASGPR ligand (protected or unprotected) or PEG, and

A¹and A²Is an amino acid (protected or unprotected)

The synthesis starts from H-A¹A²Preparation of PABA (Table 1) derivatives. These adducts were obtained using the synthetic scheme described by Dubowchik et al (2002) with some modifications. Fmoc-protected amino acid Fmoc-A¹-OH is converted into N-hydroxysuccinimide ester (Fomc-A) by reaction with Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS)¹-NHS). These reactive NHS esters are added with aqueous NaHCO to maintain the reactivity of the amino group₃In the presence of a protected amino acid A²And (3) coupling. For the preparation of 1e and 1f (table 1), commercially available pentafluorophenyl ester (OPfp) was used instead of NHS ester for coupling.

Synthesis of Fmoc dipeptides 1 a-h.

a) The NHS ester of AA was prepared from the corresponding amino acid containing NHS and DCC and used without additional purification.

Conditions are as follows: (i) n-hydroxysuccinimide (NHS) and N-N' -Dicyclohexylcarbodiimide (DCC) at 0-20 ℃.

For Fmoc-Ala-NHS, DCC (286mg,1.38mmol) was added to an ice-cold solution of Fmoc-Ala-OH (412mg,1.32mmol) and NHS (160mg,1.38mmol) in DCM (13mL), stirred for 30 min, then held at 20 ℃ for 16 h. The solid Dicyclohexylurea (DCU) was filtered off and the solvent was removed in vacuo.

For Fmoc-Asn (DMCP) -NHS, DCC (148mg,0.72mmol) was added to an ice-cold solution of Fmoc-Asn (DMCP) -OH (298mg,0.68mmol) and NHS (83mg,0.72mmol) in DCM (13mL), stirred for 30 min, and then at 20 ℃ for 16 hours. The solid DCU was filtered off and the solvent removed in vacuo.

For Fmoc-Gly-NHS, Fmoc-Gly-OH (891mg,3mmol) and NHS (380mg,3.3mmol) were stirred in THF (10mL) at 0 ℃ for 5 min, then treated with DCC (650mg,3.15mmol) in THF (5 mL). The cooling bath was removed within 30 minutes and the reaction mixture was stirred at 20 ℃ for 10 hours. The solid DCU was filtered off, washed with THF and the solvent removed on a rotary evaporator. The product was weighed and dissolved in DME to make a 0.2mM solution.

For Fmoc-Glu (O-2PhiPr) -NHS, DCC (217mg,1.05mmol) was added to an ice-cold solution of Fmoc-Glu (O-2PhiPr) -OH (487mg,1mmol) and NHS (127mg,1.1mmol) in THF (5mL), stirred for 15 minutes, and then continued at 20 ℃ for 10 hours. Treatment was done as described for Fmoc-Gly-NHS.

For Fmoc-Phe-NHS, DCC (1.181g,5.72mmol) was added to an ice-cold solution of Fmoc-Phe-OH (2.11g,5.45mmol) and NHS (664mg,5.77mmol) in DCM (50mL), stirred for 30 min, then at 20 ℃ for 10 h. The solid DCU was filtered off and the solvent removed in vacuo.

For Fmoc-Val-NHS, DCC (227mg,1.1mmol) was added to an ice-cold solution of Fmoc-Val-OH (339mg,1mmol) and NHS (127mg,1.1mmol) in DCM (13mL), stirred for 30 min, then held at 20 ℃ for 16 h. The solid DCU was filtered off and the solvent removed in vacuo.

b) Preparation of the amino acids H-Asn (DMCP) -OH and H-Lys (MMT) -OH from the available Fmoc-protected derivatives

Conditions are as follows: (i) triethylamine (Et) in Dimethylformamide (DMF)₃N)

H-Asn(DMCP)-OH

In DMF (9mL) and Et₃Fmoc-Asn (DMCP) -OH (576mg,1.32mmol) was stirred in N (3.7mL,26.4mmol) for 15 h. All volatiles were removed on a rotary evaporator at 40 deg.c/oil pump vacuum. The residue was triturated 3 times with ether (30mL) and dried in vacuo. Yield 271mg (96%). MS 643.6[3M +1 ]]⁺；451.3[2M+Na]⁺；429.5[2M+1]⁺；236.7[M+Na]⁺；215.3[M+1]⁺；132.8[M-DMCP+1]⁺。

H-Lys (MMT) -OH in DMF (100mL) and Et₃Fmoc-Lys (MMT) -OH (4.902g,7.65mmol) was stirred in N (32mL,30 equivalents 229.4mmol) for 10 h. All volatiles were removed on a rotary evaporator at 40 deg.c/oil pump vacuum. The residue was triturated with ether 2 times and dried in vacuo. Yield 3.1g (97%). MS (negative mode) 455,453.3[ M + Cl]^-；417.8[M-1]^-。

c) Synthesis of Fmoc-A₁A₂-OH。

A¹＝Gly、Glu(2PhiPr)、Asn(DMCP)、Phe、Ala、Val。

A²＝Gly、Lys(MMT)、Cit、Asn(DMCP)、Lys(CH₃)₂。

Conditions are as follows: (i) H-A₂-OH、NaHCO₃Dimethoxyethane (DME), Tetrahydrofuran (THF) and H₂A mixture of O. (ii) H-A₂-OH、NaHCO₃、DME/THF/H₂O。(iii)H-Cit-OH、NaHCO₃Dissolved in H₂THF of O.

For Fmoc-GlyGly-OH 1a, glycine (75mg,1mmol) and NaHCO₃(100mg,1.2mmol) in H₂O (10mL) and Dimethoxyethane (DME) (5 mL). A solution of Fmoc-Gly-NHS in DME (5ml,1mmol) was added. THF (2.5mL) was added and the mixture was sonicated to homogenize and stir for 20 hours. All volatiles were removed on a rotary evaporator using EtOAc and 5% KHCO₃H of (A) to (B)₂The residue was treated with O solution. The product was extracted four times with EtOAc, washed with pH 3 brine, and dried (Na)₂SO₄) Concentrated and dried in vacuo. Yield 321mg (90%). MS 775.0[2M +2Na ]]⁺；377.4[M+Na]⁺；355.1[M+1]⁺。

For Fmoc-Glu (O-2PhiPr) Gly-OH 1b, glycine (75mg,1mmol) and NaHCO₃(84mg,1mmol) in H₂A mixture of O (2mL), THF (4mL) and DME (5 mL). A solution of Fmoc-Glu (O-2PhiPr) -NHS in DME (5mL,1mmol) was added and stirred for 10 hours. All volatiles were removed on a rotary evaporator and 20mL 0.1M MES buffer (pH 5) was added followed by EtOAc (25 mL). The reaction mixture was stirred on ice and washed with 5% KHSO₄The solution was acidified to pH 5. The product was extracted four times with EtOAc, washed with pH 5 brine, and dried (Na)₂SO₄) Concentrated and dried in vacuo. Yield 528mg (96%). MS 567[ M + Na]⁺；562[M+NH₄]⁺；545.0[M+1]⁺；427.1[M-2PhiPr]⁺。

Fmoc-Asn (DMCP) Gly-OH1c was prepared from Fmoc-Asn (DMCP) -NHS and H-Gly-OH as described above for 1 b. The yield was 96%. MS 987.4[2M +1 ]]⁺；516.3[M+Na]⁺；494.4[M+1]⁺；412.2[M-DMCP+1]⁺。

Fmoc-Phe Lys (MMT) -OH1d was prepared from Fmoc-Phe-NHS and H-Lys (MMT) -OH as described above for 1 b. The yield was 94%. MS 788.5[ M +1 ]]⁺，273.1[M-MMT+1]⁺。

For Fmoc-PheCit-OH 1 e:

i) to DME (40mL) containing Fmoc-Phe-NHS (4.96g,10.26mmol) was added L-citrulline (1).80g,10.26mmol) and NaHCO₃(0.86g,10.26mmol) of H₂A mixture solution of O (40mL) and THF (20 mL). The reaction was stirred for 15 hours. The activated residual DCC was filtered off and the organic solvent was removed on a rotary evaporator. Adding H to the residue₂O (100mL) and iPrOH (10 mL). With 5% KHSO₄The suspension was acidified to pH 3, the product was extracted with EtOAc/iPrOH 9:1 solution (3 ×,500mL), washed with a mixture of brine/iPrOH 9:1(2 ×,50mL), dried (Na)₂SO₄) Filtered and concentrated, and dried with an oil pump. Trituration with ether afforded the pure product 1 e. Yield 3.84g (68%). MS 545.6[ M + Na ]]⁺；528.5[M-H₂O]⁺；306.3[M-Fmoc+H₂O]⁺。

ii) to a solution of H-Cit-OH (184mg,1.05mmol) and NaHCO₃(88.2mg,1.05mmol) of H₂O (2.6mL) solution was added Fmoc-Phe-OPfp (553mg,1mmol) in THF (5 mL). THF (2mL) was added to homogenize the solution and stirred for 10 hours. THF is removed on a rotary evaporator and the residue is freed from H₂O (10mL) and iPrOH (1mL) were diluted and acidified to pH 1 with 3% HCl. The product was extracted 5 times with a solution of EtOAc iPrOH 9:1, washed with a mixture of brine iPrOH 9:1, and dried (Na)₂SO₄) And concentrated in vacuo. Trituration with ether afforded 313mg of pure product 1e (57%).

Fmoc-AlaCit-OH 1f was prepared from Fmoc-Ala-NHS and H-Cit-OH as described above for 1e- (a). The yield was 77%. MS 959.8[2M + Na]⁺；938.1[2M+1]⁺；491.4[M+Na]⁺；469.9[M+1]⁺。

Crude Fmoc-ValCit-OH 1g was prepared from Fmoc-Val-NHS and H-Cit-OH as described above for 1 b. Final purification was done by trituration with ether. The total yield is 76%. MS 1060.3[2M +3Na]⁺；1015.7[2M+Na]⁺；519.7[M+Na]⁺；497.9[M+1]⁺。

Fmoc-Ala-Asn (DMCP) -OH1H was prepared from Fmoc-Ala-NHS and H-Asn (DMCP) -OH as described above for 1 b. The yield was 95%. MS 530.2[ M + Na ]]⁺；508.2[M+1]⁺；426.0[M-DMCP+1]⁺。

Coupling with p-aminobenzyl alcohol to prepare Fmoc-AA-PABA and Fmoc-A-PABA 2 a-m.

Coupling the product 1a-h with p-aminobenzyl alcohol (PABA) in the presence of 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) to form 2 a-h. Four representatives 3j-l with only one amino acid attached to the PABA moiety were also prepared.

Conditions are as follows: (i) PABA, EEDQ, THF

For Fmoc-GlyGly-PABA 2a, a solution of 1a (318mg,0.9mmol) and PABA (220mg,1.8mmol) in DCM (17mL) and MeOH (6mL) was stirred with EEDQ (444mg,1.8mmol) for 10 h. All volatiles were removed on a rotary evaporator and the residue was Et₂Triturate with O, filter the product and dry in vacuo. Yield 348mg (84%).

For Fmoc-Glu (O-2PhiPr) Gly-PABA 2b, a solution of 1b (524mg,0.96mmol) and PABA (142mg,1.55mmol) in DCM (10mL) was stirred with EEDQ (357mg,1.44mmol) for 10 hours. The process is completed as described above with respect to 2 a. Yield 462mg (74%).

Fmoc-Asn (DMCP) Gly-PABA 2c was prepared as described above for 2 a. The yield was 64%. MS 621.5[ M +22 ]]⁺；599.3[M+1]⁺。

Fmoc-PheLys (MMT) -PABA 2d was prepared as described above for 2 b. The yield was 70%.

For Fmoc-PheCit-PABA 2e, a solution of 1e (5.98g,10.97mmol) and PABA (2.70g,21.95mmol) in DCM (150mL) and MeOH (50mL) was treated with EEDQ (5.43g,21.95mmol) and stirred for 15 h. The process is completed as described above with respect to 2 a. Yield 6.14g (86%). MS 650.7[ M +1 ]]⁺；527.3[M-PABA+1]⁺。

For Fmoc-AlaCit-PABA 2f, a solution of 1f (2.89g,6.17mmol) and PABA (1.52g,12.34mmol) in DCM (45mL) and MeOH (15mL) was treated with EEDQ (3)05g,12.34mmol) and stirred for 15 hours. The process is completed as described above with respect to 2 a. Yield 4.56g (74%). MS (ES, negative mode) 307.4[ M-263.6-1]^-；349.9[M-Fmoc-1]^-；610,608.4[M+HCl-1]^-。

Fmoc-ValCit-PABA 2g was prepared as described above for 2 b. (98%).

Fmoc-AlaAsn (DMCP) -PABA 2h was prepared as described above for 2 a. The yield was 59%. MS 613.2[ M +1 ]]⁺；531.4[M-DMCP+1]⁺；408.2[M-205+1]⁺。

For Fmoc-Lys (CH)₃)₂-PABA 2i，Fmoc-Lys(CH₃)₂HCl salt (433mg,1mmol) and PABA (246mg,2mmol) were dissolved in DCM (10mL) and MeOH (1.5mL), cooled to 5 deg.C, then EEDQ (495mg,2mmol) was added. The cooling bath was removed and the mixture was stirred at room temperature for 10 hours. All volatiles were removed on a rotary evaporator and the residue was taken up in Et₂O grinding, and filtering out a crude product. The crude product was redissolved in a mixture of DCM (2mL) and MeOH (1mL) and added dropwise to Et₂O (40mL) was reprecipitated. The product was filtered and dried in vacuo. Yield 448mg (83%).

For Fmoc-Leu-PABA 2j, a solution of Fmoc-Leu-OH (353mg,1mmol), EEDQ (495mg,2mmol) and PABA (222mg,1.8mmol) in DCM (10mL) was stirred for 10 h. All volatiles were removed on a rotary evaporator and the residue was dissolved in Et₂O (40mL), frozen on dry ice for 2 hours, and the solids were separated by centrifugation. The resulting crude material was purified on a column eluting with a gradient of MeOH (1-2%) in CHCl₃. Yield 444mg (97%). MS 459.4[ M +1 ]]⁺。

Fmoc-Asn (DMCP) -PABA 2k was prepared as described above for 2 j. In the work-up after removal of DCM, the residue was taken up in Et₂Triturate, freeze to 0 ℃ and filter the crude product without column purification. This treatment was repeated once and then dried under vacuum. The yield was 77%. MS 542.5[ M +1 ]]⁺。

For Fmoc-Cit-PABA 2l, the Fmoc-Cit-OH-containing solution (345.7mg, 0) was treated with EEDQ (430mg,1.74mmol)87mmol) and PABA (214mg,1.74mmol) in DCM (10mL) and MeOH (4mL) and stirred for 15 h. The solid product was triturated with ether three times, the product was filtered off and dried. Yield 288mg (67%). MS 502.3[ M +1 ]]⁺；485.5[M-H₂O+1]⁺；263[M-Fmoc-H₂O+1]⁺；179.0[M-306+1]⁺；120.2[M-365.3+1]⁺。

The product 2m was prepared using a different protocol: H-Lys (CH)₃)₂PABA derivative 3 coupled Fmoc-Phe-NHS.

Conditions are as follows: (i) containing triethylamine (Et)₃N) in DMF for 10 hours. (ii) Fmoc-Phe-NHS, Diisopropylethylamine (DIEA), DMF.

For Fmoc-PheLys (CH)₃)-PABA 2m，Fmoc-Lys(CH₃)₂-PABA (2i) (448mg,0.83mmol) by treatment with Et-containing solution₃The Fmoc protection was removed by stirring N (3.5mL) in DMF (11mL) for 10 h. All volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum to obtain crude product 3 i. The product was dissolved in DMF (7mL), Fmoc-Phe-NHS (482mg,0.996mmol) followed by DIEA (0.42mL,2.2mmol) was added and the mixture was stirred for 10 h. The solvent with DIEA was removed in oil pump vacuum at 40 ℃/on a rotary evaporator to obtain crude 2m, which was used without additional purification. MS 549.4[ M +1 ]]⁺。

Preparation of H-AA-PABA 3a-H, m and H-A-PABA 3 j-l.

Conditions are as follows: (i) containing Et₃DMF of N, 10 hours.

Et-containing solutions as described above for 3i₃DMF treatment of Fmoc-derivative 2a-h, j-l with N followed by concentration and drying in vacuo. The crude product is dissolvedIn DMF to give a 0.1M solution, which was used without additional purification.

TABLE 1 intermediates of H-AA-PABA (1-3)

	A1	A2
			1,2,3a	Gly	Gly
1,2,3b	Glu(2PhiPr)	Gly
			1,2,3c	Asn(DMCP)	Gly
1,2,3d	Phe	Lys(MMT)
			1,2,3e	Phe	Cit
1,2,3f	Ala	Cit
			1,2,3g	Val	Cit
1,2,3h	Ala	Asn(DMCP)
			1,2,3i	Lys(CH3)2
1,2,3j	Leu	-
			1,2,3k	Asn(DMCP)	-
1,2,3l	Cit	-
			2,3m	Phe	Lys(CH3)2

Preparation of protease cleavable NAG-masking reagent.

NAG(R¹,R²,R³) Preparation of (E) -L-AA-PABC-PNP (tables 2, 3)

Preparation of NAG (R)¹,R²,R³) -L-AA-PABC-PNP, wherein R¹、R²And R³Is a protecting group, L is the linkage between the galactosamine moiety (NAG) and the dipeptide (AA), said preparation starting from NAG-L-CO₂Preparation of H acids 6,10a, b,13 and 17, the NAG-L-CO₂The H acid was used to acylate H-AA-PABA 2 after conversion to NHS ester. In the carbonates 21a-f designed for base-sensitive polymers, the protecting groups must be removed before the polymer can be modified. For this reason, in the preparation of 10a, b,13 and 17, the Ac-protecting group of the GAL moiety was replaced by a labile Triethylsilyl (TES) and tert-butyldimethylsilyl (TBDMS) group. Those groups can be removed at 0 ℃ using a 70% trifluoroacetic acid solution in water without damaging the base sensitive PNP-carbonate moiety.

a) In NAG-L¹-CO₂H6 (wherein R¹＝R²＝R³Z protected OAc) by reacting NAG-tetraacetic acid 4[3-5 ]]Carrying out Z-deprotection (H)₂、Pd/C(10％)、MeOH、CHCl₃(20%)) to obtain NAG-amine 5, which is then acylated with succinic anhydride. (succinic anhydride, Et₃N, DCM, 1 hour).

Conditions are as follows: (i) h₂、Pd/C(10％)、MeOH、CHCl₃(20%), (ii) succinic anhydride, Et₃N, CDM, 1 hour.

NAG-amine 5: for the preparation of 5, NAG 4(6.74g,11.85mmol) in MeOH (144mL) and CHCl containing NAG 4(6.74g,11.85mmol) were hydrogenated in the presence of 10% Pd/C (674mg) at 1 atmosphere₃(36mL) of the solution was 10 hours. The catalyst was filtered off through celite, the product was concentrated and dried in vacuo. Yield 5.04g (98%).

NAG-L¹-OH 6: for preparation of 6, to DCM (50mL) containing NAG-amine 5(4g,9.15mmol) was added a solution of succinic anhydride (966mg,9.65mmol) in DCM (30mL) followed by Et₃N (1.964mL,14 mmol). After 1 hour, the reaction mixture was concentrated and dried in vacuo. The product was purified on a column with a gradient of MeOH in CHCl (5-7%) eluent₃. Yield 3.1g (63%). MS 535.3[ M +1 ]]⁺(ii) a 330.3[ deglycosylated product]⁺。

b) NAG derivatives with easily removable silyl ether protecting groups were prepared by O-deacetylation of 4 in an aqueous methanol mixture containing triethylamine followed by treatment with a trialkylchlorosilane.

i)NAG-L¹-OH 10a,b.10a：R¹OTES and OTBDMS, R²＝OH，R³＝OTES；10b：R¹＝R³＝OTBDMS，R²＝OH。

Preparation of NAG 8a, b.

Conditions are as follows: (i) et (Et)₃N、MeOH、H₂O (5:7:6) for 10 hours. (ii) TBDMSCl (1 eq), imidazole, 1 hour, followed by TESCl (3 eq) in DMF for 10 hours. (iii) TBDMSCl (3 equivalents), imidazole, in DMF for 10 hours.

NAG derivative 7.

10a, b.

Conditions are as follows: (i) h₂Pd/C (10%), THF. (ii) Succinic anhydride, Et₃N, DCM, 1 hour.

For preparation of 7, NAG 4(2g,3.52mmol) was purified by washing in MeOH (10mL), H₂O (32mL) and Et₃The solution was stirred in N (25mL) for 10h for O-deacetylation. All volatiles were removed on a rotary evaporator at 40 ℃ and the residue was dried by evaporating toluene twice from the reaction mixture. Product 7 was used directly in the subsequent step. MS 544.3[ M + Et₃N+1]⁺；443.7[M+1]⁺(ii) a 204[ deglycosylation product]⁺。

For the preparation of 8a, DMF (15mL) containing product 7(1.76mmol) was treated with imidazole (718mg,10.54mmol) and TBDMSCl (265mg,1.76mmol), stirred for 2h, then the reaction mixture was cooled to 0 ℃. TESCl (531mg,3.52mmol) was added, stirred for 10h, concentrated and dried in vacuo. The residue was taken up in EtOAc (110mL) and H₂O (30 mL). The organic layer was separated, cooled to 5 ℃ and washed with citric acid (5%), H₂O、NaHCO₃Washed and dried (Na)₂SO₄). The crude product was passed through a column (eluent 2% MeOH in CHCl)₃) To obtain a mixture of TBDMS and TES bis-Si protected NAG derivative 8 a. Yield 575mg (49%). MS 672.0[ M +1 ]]⁺(ii) a 432.5[ deglycosylation product]⁺。

For preparation of 8b, a solution of 7(1.76mmol) in DMF (15mL) was stirred with imidazole (718mg,10.56mmol) and TBDMSCl (1.061g,7mmol) for 10 hours. The reaction mixture was worked up as described above for preparation 8 a. The yield after column purification was 767mg (65%). 672.0[ M +1 ]]⁺(ii) a 432.7[ deglycosylation product]⁺.

Product 10a was prepared as a mixture of TBDMS and TES bissi protected NAG derivatives according to the method described below for 10 b.

For the preparation of 10b, compound 8b (920mg,1.37mmol) was hydrogenated in THF (20mL) in the presence of Pc/C10% (150mg) at 1 atmosphere for 10 h. The catalyst was filtered off through celite, and the product 9b was concentrated and dried in vacuo.

The NAG-amine 9b was dissolved in DCM (12mL) without additional purification, DCM (7mL) containing succinic anhydride solution (140mg,1.40mmol) was added, followed by Et₃N (0.236mL,1.676mmol) and stirred for 2 h.

The solvent was removed on a rotary evaporator and the product was purified on a column eluting with 1% AcOH, 10% MeOH in CHCl₃. Yield 614mg (72%).

ii)NAG-L²-OH 13。R¹＝R³＝OTBDMS，R²OH. NAG derivatives contain a longer PEG spacer.

For analogs containing longer PEG spacers, precursor 5 was first deprotected to the acetyl group to give 11 (Et)₃N、MeOH、H₂O (5:7:6) for 10 hours). Then, with bis-dPEG₅Acylation of the half benzyl hemiNHS ester (Quanta product #10237) 11 to yield benzyl ester 12 (NHS-PEG)₅-CO₂Bn、Et₃N, DCM). The 12 bis-silylation was then carried out with TBDMSCl (TBDMSCl (3 eq.), imidazole, 10H in DMF) and by hydrogenation (H)₂Pd/C (10%), THF) to give the acid 13.

Preparation of NAG-derivative 12.

Conditions are as follows: (i) et (Et)₃N、MeOH、H₂O (5:7:6) for 10 hours. (ii) NHS-PEG₅-CO₂Bn、Et₃N、DCM。

NAG-L²Preparation of-OH 13.

Conditions are as follows: TBDMSCl (3 equivalents), imidazole, DMF for 10 hours. (vi) H₂，Pd/C(10％)，THF。

NAG-PEG₈-SA benzyl ester 12. For the preparation of NAG-amine 11, O-deacetylation of NAG-amine 5(0.381mmol) was carried out as described for precursor 7 (method of 8a, b). Product 11 is subjected to rotary evaporationDried on the instrument by evaporating toluene twice and dissolving in DMF (25 mL). Adding bis-dPEG to the reaction mixture₅Half benzyl half NHS ester (200mg,0.381mmol) then DIEA (0.079mL,0.457mmol) was added, stirred for 8 hours and concentrated on a rotary evaporator at 40 deg.C/oil pump vacuum pump. The crude product 12 was used in the next step without additional purification. MS 719.4[ M +1 ]]⁺(ii) a 516.4[ deglycosylation product]⁺。

For NAG-L²Preparation of OBn, dried product 12 dissolved in DMF (5mL) and treated with TBDMSCl (230mg,1.524mmol) followed by imidazole (156mg,2.29 mmol). The reaction mixture was stirred for 10 hours, all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum, then the residue was taken up in EtOAc (85mL) and washed with HCl (1%), H₂And O, cleaning. The water was combined and back extracted with EtOAc. The combined organic solutions were dried (Na)₂SO₄) Concentrated and purified on a column eluting with a gradient of MeOH (3-6%) in CHCl₃. Benzyl ester yield 291mg (80%). MS 965.3[ M + NH ]₄]⁺；948.0[M+1]⁺(ii) a 516.4[ deglycosylation product]⁺。

For NAG-L²Preparation of-OH 13, ester NAG-L²The hydrogenation of OBn is carried out as described for 9b (method 10 b). The yield was 98%. MS 858.0[ M +1 ]]⁺(ii) a 426.1[ deglycosylated product]⁺. The product was used without additional purification.

iii)NAG-L³-OH 17。R¹＝R³＝OTBDMS，R²OH. NAG derivatives contain a longer PEG spacer.

Preparation 17 by using PEG₄mono-tBu ester (TMSOTf, DCE/HO-PEG)₄-CO₂tBu、SnCl₄DCM) gave pentaacetate 14[ 3-5%]Glycosylation to produce 15. Hydrolysis 15 (HCO)₂H,10 hours) to obtain the acid 16, followed by O-deacetylation (Et) of the acid 16₃N、MeOH、H₂O (5:7:6)10h) and then treated with TBDMSCl (TBDMSCl (3 equiv.), imidazole, 10h in DMF) to obtain the bis-silylated NAG-acid 17.

Ester NAG (OAc)₃-L³Preparation of-O-tBu 15.

Conditions are as follows: (i) trimethylsilyl trifluoromethanesulfonate (TMSOTf), Dichloroethane (DCE). (ii) 12-hydroxy-4, 7, 10-Trioxadodecanoic acid tert-butyl ester (HO-PEG)₄-CO₂tBu)，SnCl₄Dichloromethane (DCM).

Conditions are as follows: (i) HCO₂H,10 hours. (ii) Et (Et)₃N、MeOH、H₂O (5:7:6) for 10 hours. (iii) TBDMSCl (3 equivalents), imidazole, DMF for 10 hours.

For ester NAG (OAc)₃-L³O-tBu 15, drying the pentaacetyl derivative of galactosamine 14 (10g,25.64mmol) by two toluene evaporations. The resulting white glass was treated with DCE (223mL) containing TMSTf (5.18mL,28.6mmol) and stirred at 60 ℃ for 16 h. The reaction mixture was cooled to 0 ℃, quenched with TEA (2.6mL), quenched with CHCl₃Diluted (300mL) and NaHCO₃The solution and brine were washed twice. The separated organic solution was MgSO₄Treated, concentrated and dried in vacuo. The crude oxazoline derivative is used without additional purification. Yield 8.14g (96%). MS 368.1[ M + K ]]⁺；352.2[M+Na]⁺；330.2[M+1]⁺。

To a mixture of an oxazoline-containing derivative (5.28g,16mmol), tert-butyl 12-hydroxy-4, 7, 10-trioxadecanoate (5.12g,18.4mmol) and CaSO₄(20g) DCM (270mL) stirred the mixture and SnCl was added dropwise₄(0.84mL,0.84 mmol). The solution was stirred for 16 hours, filtered and washed with CHCl₃Diluted (250mL) with NaHCO₃The solution and brine were washed twice. The product was MgSO₄Dried and concentrated. The crude product is purified on a columnEluent gradient of ethyl acetate with MeOH (0-7%). Yield 4.83g (50%). MS 630.8[ M + Na ]]⁺；625.5[M+NH₄]⁺；608.4[M+1]⁺；552.6[M-t-Bu+1]⁺(ii) a 330.2[ deglycosylated product]⁺。

For NAG (OAc)₃-L³-OH 16, tert-butyl ester 15(1.99g,3.27mmol) was stirred in pure formic acid (54mL) for 16 h, all volatiles were removed in vacuo, and toluene was evaporated three times. The product was dried with a vacuum oil pump for 2 hours and used without additional purification. Yield 1.77g (98%). MS 330.2[ deglycosylated product]⁺；590.4[M+K]⁺；574.6[M+Na]⁺；569.6[M+NH₄]⁺；552.6[M+1]⁺。

For NAG (R)¹,R²,R³)-L³-OH 17(R¹＝R³＝OTBDMS,R²= OH), the product 16O-was deacetylated, treated with TBDMSCl (preparation method 8 b), and purified on a column, eluent CHCl with 3% MeOH, 0.5% AcOH₃. The yield was 18%. MS 1228.7[ M +1 ]]⁺796.7[ deglycosylation products]⁺。

All five obtained acids 6,10a, b,13,17 were converted to NHS esters 18a-e in reaction with NHS and DCC (NHS, DCC, DCM, 10 hours).

Conditions are as follows: (i) NHS, DCC, DXCM,10 hours.

For the preparation of NAG-L-NHS 18a-e, the procedure described below for 18c was used. For product 18c, an ice-cold solution of 10b (614mg,0.964mmol) and NHS (122mg,1.061mmol) in DCM (15mL) was treated with DCC (219mg,1.061mmol), stirred on ice for 30 min at 20 ℃ for 8 h. The reaction mixture was cooled to 0 ℃, the DCU was filtered off, the residue was concentrated and dried in vacuo. The crude product was dissolved in DMF to make a 0.05M solution, which was used without additional purification.

Products 18b-e were prepared as described for 18 a.

c) Formation of 20a-l, acylation of 3a-h with NHS ester of hydroxy protected NAG-derivative 18a-e (DIEA, DMF, 5-10h) to afford 19 a-l. The product 19a-l ((PNP) s) was then treated with 5 equivalents of bis (p-nitrophenyl) carbonate₂CO)((PNP)₂CO, dioxane or DCM, 40-50 ℃, 15-24h) to yield O-acetyl protected PNP carbonate derivative 20 a-l. The products 20a-e were used directly for peptide modification. After modification with TFA and Et₃Removing acetyl groups and protecting groups 2PhiPr, DMCP and MMT from amino acid in the continuous treatment process of the DPC by N.

Conditions are as follows: (i) DIEA, DMF, 5-10 hours. (ii) (PNP)₂CO, dioxane or DCM, 25-60 deg.C, 16-48 hr.

NAG(R¹R²R³)-L-AA-PABA 19a-l。

For product 19a (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ GlyGly), a DMF solution (0.05M) containing NAG-NHS ester 18a (0.282mmol) was treated with a 0.1M 3a (0.282mmol) solution of DMF and DIEA (59 μ L,0.338 mmol). All volatiles were removed in a rotary evaporator at 40 deg.C/oil pump vacuum over 3 hours and Et₂Triturate and purify on column, eluent gradient: EtOAc CHCl₃MeOH is 8:7:5 to 8:7: 6. Yield 114mg (53%). MS 754.4[ M +1 ]]⁺。

Product 19b (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ Glu (2PhiPr) Gly) was prepared as described for 19a and purified on a column, eluent EtOAc: CHCl₃MeOH: 8:7: 3. The yield was 64%. MS 944.5[ M +1 ]]⁺。

For product 19c (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ asn (dmcp) Gly), to a solution of 3c (0.43mmol) and DIEA (83 μ L,0.476mmol) in DMF (2.15mL) was added a solution of 18a (0.43mmol) in DMF (2.15 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum. The crude product was treated with Et₂O triturated and purified on column eluting with CHCl₃Acetone to MeOH (5:5: 1). Yield 242mg (62%). MS 915.3[ M + Na ]]⁺；910.6[M+NH₄]⁺；893.6[M+1]⁺。

Product 19d (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ phelys (mmt)) was prepared as described for 19a, purified on column, eluent CHCl with MeOH (5-6%)₃. The yield was 56%. MS 1187.9[ M +1 ]]⁺。

For product 19e (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ phemit), to a solution of 3e (0.57mmol) and DIEA (119 μ L,0.684mmol) in DMF (3mL) was added a solution of 18a (0.57mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum. Crude product from CHCl₃MeOH (5mL) precipitated into Et₂O (45mL), used without additional purification. Yield 392mg (73%). MS 966.8[ M + Na ]]⁺；944.7[M+1]⁺；926.8[M-H₂O]⁺；821.5[M-PABA+1]⁺；615.6[M-NAcGal+1]⁺；492.3[M-PABA-NAcGal+1]⁺。

Product 19f (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ AlaCit) was prepared as described in 19e and used without additional purification. The yield was 50%. MS 993.2[ M + Na ]]⁺；971.0[M+1]⁺(ii) a 539.6[ deglycosylation product]⁺。

Product 19g (R)¹＝R³＝OTBDMS，R²＝OH L＝L¹AA ═ ValCit) was prepared as described for 19f and used in the next step without additional purification. Yield 67%: 998.9[ M +1 ]]⁺。

Product 19h (R)¹＝R³＝OTBDMS，R²＝OH L＝L¹AA ═ Glu (2PhiPr) Gly) was prepared as described for 19a and purified on a column, eluent NH-containing₄OH 3% and MeOH 7.5% in DCM. The yield was 15%. MS 1047.2[ M +1 ]]⁺615.7, 432.6[ deglycosylated product]⁺。

Product 19i (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ phemit) was prepared as described in preparation 19e and used without additional purification. The yield was 50%. MS 1068.7[ M + Na ]]⁺；1047.3[M+1]⁺(ii) a 615.4[ deglycosylation product]⁺(ii) a 432.5[ deglycosylation product]⁺。

Product 19j (R)¹OTBDMS and OTES, R₂＝OH，R³＝OTES，L＝L¹AA ═ phemit) was prepared as described for preparation 19e from 3e and 18b as a mixture of C-3 and C-6O-TBDMS and O-TES protected NAG derivatives and used in the next step without additional purification. The yield was 76%. MS 1047.4[ M +1 ]]⁺615.8[ deglycosylation products]⁺。

Product 19k (R)¹＝R³＝OTBDMS，R²＝OH，L＝L²AA ═ phemit) was prepared as described for 19e and used in the next step without additional purification. The yield was 67%. 1268.2[ M +1 ]]⁺(ii) a 835.9[ deglycosylation product]⁺。

Product 19l (R)¹＝R³＝OTBDMS，R²＝OH，L＝L³AA ═ phemit) was prepared as described for 19e, purified on column, eluent CHCl with 5% MeOH₃And (3) solution. The yield was 60%. MS 1064.0[ M +1 ]]⁺(ii) a 632.7[ deglycosylation product]⁺。

NAG-AA-PABC-PNP 20a-l

For product 20a (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ GlyGly), containing 19a (100mg,0.132 mmol))、(PNP)₂A suspension of CO (202mg,0.663mmol) and DIEA (0.07mL,0.396mmol) in dioxane (5mL) was stirred at 40 ℃ for 8 hours in the dark. Adding another part (PNP)₂CO (121mg,0.397mmol) and DIEA (0.04mL,0.226mmol) and stirring was continued for a further 8 hours at 40 ℃. All volatiles were removed on a rotary evaporator and the product was purified on a column, eluent: CHCl₃EtOAc: MeOH: 7:8: 3. Yield 84mg (69%).

For product 20b (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ Glu (2PhiPr) Gly), containing 19b (160mg,0.169mmol), (PNP)₂A solution of CO (258mg,0.847mmol) and DIEA (0.09mL,0.507mmol) in DCM (10mL) was stirred for 10h in the dark, concentrated on a rotary evaporator and the product purified on a column, eluent: 5-6% MeOH in CHCl₃And (3) solution. Yield 174mg (92%).

For product 20c (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ asn (dmcp) Gly), 19c (127mg, 0.142mmol), (PNP)₂A solution of CO (216mg,0.710mmol) and DIEA (74 μ L,0.426mmol) in DCM (5mL) was stirred for 16 h protected from light, concentrated on a rotary evaporator and the product purified on a column, eluent: CHCl₃EtOAc: MeOH (7:2.2: 0.8). Yield 110.6mg (74%). MS 1080.9[ M + Na ]]⁺；1058.7[M+1]⁺。

Product 20d (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ phelys (mmt)) was prepared as described for 20b and purified on the column, eluent: CHCl₃EtOAc: MeOH: 9:7: 1. Yield 76mg (47%).

For product 20e (R)¹＝R²＝R³＝OAc，L＝L¹AA ═ phemit) containing 19e (164mg,0.173mmol), (PNP)₂A solution of CO (528mg,1.73mmol) and DIEA (182 μ L,1.04mmol) in dioxane (17mL) was stirred at 60 ℃ for 16 h in the dark and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two successive DMF evaporations on a rotary evaporator at 40 ℃/oil pump vacuum, the product was purified on a column, elutedPreparation: CHCl₃EtOAc: MeOH (8:1.5:0.5) followed by CHCl₃MeOH (7: 1). Yield 85mg (44%). MS 1132.0[ M + Na ]]⁺；1110.1[M+1]⁺(ii) a 780.8[ deglycosylation product]⁺。

Product 20f (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ AlaCit) was prepared as described for 20e and purified on a column, eluent: CHCl₃EtOAc: MeOH: 9:10: 1. Yield 153mg (36%).

Product 20g (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ ValCit) was prepared as described for 20e and purified on a column, eluent: CHCl₃EtOAc: MeOH: 16:3: 1. The yield was 44%. MS 1164.5[ M +1 ]]⁺。

Product 20h (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ Glu (2PhiPr) Gly) was prepared as described for 20b and purified on a column, eluent: CHCl₃EtOAc: MeOH: 8:7: 1. The yield was 77%.

For product 20i (R)¹＝R³＝OTBDMS，R²＝OH，L＝L¹AA ═ phemit) containing 19i (316mg,0.297mmol), (PNP)₂A solution of CO (913mg,2.97mmol) and DIEA (310 μ L,1.78mmol) in dioxane (7mL) was stirred at 65 ℃ for 40 hours in the dark and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two successive DMF evaporations on a rotary evaporator at 40 ℃/oil pump vacuum, the product was purified on a column, eluent: CHCl₃EtOAc: MeOH (8:1.5:0.5) followed by CHCl₃MeOH (92: 08). Yield 297mg (81%). MS 1246.7[ M + NH ]₄]⁺；1228.7[M+1]⁺(ii) a 797.6[ deglycosylation product]⁺(ii) a 432.7[ deglycosylation product]⁺。

Product 20j (R)¹OTBDMS and OTES, R₂＝OH，R³＝OTES，L＝L¹AA ═ phemit) was prepared as described for 20e as a mixture of C-3 and C-6O-TBDMS and O-TES protected NAG derivativesForm, and purified on a column, eluent: CHCl₃EtOAc-MeOH-16: 3:1 followed by 10% MeOH in CHCl₃. The yield was 50%. MS 1212.0[ M +1 ]]⁺480.0[ deglycosylated product]⁺。

Product 20k (R)¹＝R³＝OTBDMS，R²＝OH，L＝L²AA ═ phemit) was prepared as described for 20e, purified on a column, eluent: MeOH (8-10%) in CHCl₃. The yield was 85%. The product was used directly in the next step.

For product 20l (R)¹＝R³＝OTBDMS，R²＝OH，L＝L³AA ═ phemit) containing 19l (316mg,297mmol), (PNP)₂A solution of CO (912mg,3mmol) and DIEA (0.31mL,1.78mmol) in dioxane (8mL) was stirred under argon at 60 ℃ for 48 hours in the dark. All volatiles were removed on a rotary evaporator and the product was purified on a column, eluent: CHCl₃EtOAc: MeOH: 16:3: 1. Yield 297mg (81%).

NAG-L-AA-PABC-PNP 21a-f(R¹＝R²＝R³OH), deprotection of 20f-l

21 a-f. For modification of base-sensitive polyacrylates by addition of TFA to H prior to attachment of the polymer₂Treatment of 20f-l (TFA/H) with a 3:1 mixture₂O ═ 3:1, 5 ℃, 2-3 hours) to remove the protecting groups on NAG and dipeptide AA to provide NAG-dipeptide masking reagents 21 a-f.

Conditions are as follows: TFA/H₂O3: 1, 5 deg.C for 2-3 hours

For product 21a (AA ═ AlaCit, L ═ L)¹) Compound 20f (150mg,0.132mmol) in ice-cold TFA: H₂O-3: 1 solution (2mL) was stirred for 4 hours and added dropwise to stirring Et₂O (20 mL). Separating the precipitate by evaporation on a rotary evaporator at 30 deg.CThe mixture was evaporated to dryness and then dried in vacuo. Yield 112mg (94%). MS 907.2[ M +1 ]]⁺(ii) a 704.4[ deglycosylated product]⁺。

For product 21b (AA ═ ValCit, L ═ L¹) Compound 20g (305mg,0.26mmol) in ice-cold TFA: H₂O-3: 1 solution (5mL) was stirred for 1 hour and then added dropwise to stirring Et₂O (45 mL). The solid product was isolated, dried by evaporation of toluene on a rotary evaporator/30 ℃ and then dried in vacuo. Yield 193mg (79%). MS 935.8[ M +1 ]]⁺732.7[ deglycosylation products]⁺。

Product 21c (AA ═ GluGly, L ═ L¹) Prepared as described for 21 b. Yield 55mg (98%). MS 865.5[ M +1 ]]⁺662.3[ deglycosylation products]⁺。

Product 21d (AA ═ pheci, L ═ L)¹) Prepared as described for 21 b. MS 983.7[ M +1 ]]⁺780.9[ deglycosylation products]⁺。

For product 21e (AA ═ phemit, L ═ L)²) Compound 20k TFA H in ice-cold conditions as described for 21b₂The solution (5mL) was stirred for 1.5 hours. The yield was 25% calculated from 19 k. MS 1203.9[ M +1 ]]⁺1001.0[ deglycosylation products]⁺。

Product 21f (AA ═ pheci L ═ L)³) Prepared from 20l with 3 hours deprotection under the conditions described for 21 b. The yield was 75%. MS 1203.9[ M +1 ]]⁺1001.0[ deglycosylation products]⁺。

TABLE 2 intermediates NAG-L-AA-PABA (19) and NAG-L-AA _ PABC (20)

TABLE 3 Final NAG-L-A for DPC preparation₁A₂-PABC(20,21)

Compound (I)	A1	A2	L
				20a	Gly	Gly	L1
20b	Glu	Gly	L1
				20c	Asn	Gly	L1
20d	Phe	Lys	L1
				20e	Phe	Cit	L1
21a	Ala	Cit	L1
				21b	Val	Cit	L1
21c	Glu	Gly	L1
				21d	Phe	Cit	L1
21e	Phe	Cit	L2
				21f	Phe	Cit	L3

Preparation of protease cleavable PEG-masking reagents.

Acylation of any amino group in H-AA-PABA 3b, e, g, H, j, k-m with NHS ester of PEG-acid (DIEA, DMF, 5-10H) to give 22 a-k. The hydroxyl groups in the product 22a-k are then converted to p-nitrophenyl carbonate ((PNP)₂CO, dioxane or THF, 40-60 ℃,10 h) to yield 23 a-k. For 23a, d, g, protecting groups (TFA/H) were removed from Asn and Glu by treatment with aqueous TFA₂O ═ 3:1, 5 ℃, 2-3 hours) to obtain the desired product 24 a-c. (can identity be maintained by converting 23a, d, g to 24a, d, g?

PEG_nPreparation of-AA-PABA 22 a-k.

Conditions are as follows: (i) DIEA, DMF, 5-10 hours.

Product 22a (n ═ 11, AA ═ GluGly). 0.1M 3b in DMF (3.5mL,0.35mmol) with PEG₁₁-NHS ester (240mg,0.35mmol) and DIEA (0.061mL,0.35mmol) were stirred for 10 hours. All volatiles were removed on a rotary evaporator at 40 ℃/oil pump and the product was purified on a column, eluent: CHCl₃MeOH: AcOH: 38:2: 1. Yield 274mg (78%). MS 1015.6[ M + NH ]₄]⁺，998.7[M+1]⁺。

Product 22b (n 11, AA phemit). To a solution of 3e (0.88mmol) and DIEA (167. mu.L, 0.96mmol) in DMF (3mL) was added PEG₁₁-NHS ester (0.80mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum. Crude product from CHCl₃MeOH (5mL) precipitated into Et₂O (45mL) and then purified on a column with a gradient of MeOH in CHCl (10-16%)₃. Yield 420mg (53%). MS 1015.9[ M + H ]₂O]⁺；998.8[M+1]⁺；981.1[M-H₂O]⁺。

Product 22c (n 11, AA ValCit). Product 22f from crude product 3g (obtained from 300mg,0.5mmol of 2g), PEG as described for 22a₁₁-NHS ester (298mg,0.435mmol) and DIEA (0.09mL,0.522 mmol). After concentration on a rotary evaporator at 40 ℃/oil pump, the product was suspended in a mixture of MeOH: DCM ═ 1:1 (6mL), sonicated, filtered and concentrated in Et₂Precipitate in O (50 mL). Separating the solids andand repeating the steps. The residual solvent was removed in vacuo. Yield 283mg (60%). MS 951.5[ M +1 ]]⁺。

Product 22d (n ═ 11, AA ═ alaasn (dmcp)). To a solution of 3h (0.56mmol) and DIEA (116. mu.L, 0.67mmol) in DMF (3mL) was added PEG₁₁-NHS ester (0.56mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum. The residue was dissolved in CHCl₃MeOH-1: 1 mixture (5mL) and Et at cold (0 ℃ C.)₂Precipitate in O (45 mL). The solid was purified on a column, eluting with a gradient of MeOH (3-14%) in DCM. Yield 261mg (49%). MS 983.7[ M + Na ]]⁺；979.1[M+NH₄]⁺；961.8[M+1]⁺；943.9[M-H₂O+1]⁺。

Product 22e (n-11, AA-PheLys (Me)₂) ). product 22e was prepared as described for 22a purification was done using HPLC column Nucleodur C-18, 250 × 4.6.6, eluent ACN-H₂O (0.1% TFA), gradient 15-30%. MS 998.1[ M +1 ]]⁺The isolated product was desalted on Dowex 1 × 8 resin, eluent H₂And O. The yield was 40%.

The product 22f (n 11, AA Leu). Product 22f was prepared as described for 22a and purified on the column, eluent: CHCl₃EtOAc: MeOH: AcOH: 9:7:2: 0.04. The yield was 48%. MS 824.9[ M + NH ]₄]⁺。

Product 22g (n ═ 11, AA ═ asn (dmcp), crude product 3k (obtained from 419mg,0.77mmol of 2k), Peg₁₁NHS ester (200mg,0.292mmol) and DIEA (0.06mL,0.35mmol) were stirred in DCM (5mL) for 10 h. The solvent was removed on a rotary evaporator and the product was purified on a column, eluent: CHCl₃EtOAc: MeOH AcOH: 4.5:3.5:1: 0.02. Yield 254mg (37%). MS 891.1[ M +1 ]]⁺。

The product 22h (n 11AA Cit). To a solution of 3L (0.50mmol) and DIEA (104. mu.L, 0.60mmol) in DMF (2.5mL) was added PEG₁₁-NHS ester (0.50mmol) in DMF (2.5 mL). The mixture was stirred for 16 hours and filteredAnd all volatiles were removed on a rotary evaporator at 40 deg.c/oil pump vacuum. The residue was dissolved in CHCl₃MeOH-1: 1 mixture (5mL) in Et₂Precipitate in O (45 mL). Precipitation was repeated two more times and the product was used without additional purification. Yield 340mg (80%). MS 869.4[ M + NH ]₄]⁺；851.9[M+1]⁺。

The product 22i (n 23, AA phemit). To a solution of 3e (0.72mmol) and DIEA (130. mu.L, 0.74mmol) in DMF (3mL) was added PEG₁₁-NHS ester (0.60mmol) in DMF (3 mL). The mixture was stirred for 16 hours, filtered and all volatiles were removed on a rotary evaporator at 40 ℃/oil pump vacuum. The residue was dissolved in CHCl₃MeOH-1: 1 mixture (5mL) in Et₂Precipitate in O (45 mL). The solid product was purified on a column with a gradient of eluent CHCl with MeOH (7-12%)₃. Yield 487mg (53%). MS 1555.2[ M + Na ]]⁺；1544.7[M+NH₄]⁺；1527.7[M+1]⁺。

A mixture of product 22j (PEG average MW 1000.AA ═ phemit), mPEG-1000-ol (Fluka) (0.173g,0.173mmol), N-disuccinimidyl carbonate (62mg,0.242mmol) and TEA (0.101mL,0.726mmol) was stirred in MeCN (1mL) for 16 h. All volatiles were removed on a rotary evaporator and the crude residue was dissolved in CHCl₃(10 mL). H for organic layer₂O (1mL, pH 5) was washed with brine, and Na was added₂SO₄Dried and concentrated to obtain PEG-1000-NHS carbonate. The product was stirred with 3e (0.121mmol) and DIEA (30. mu.L, 0.173mmol) in DMF (1mL) for 16 h, filtered, and all volatiles were removed on a rotary evaporator under vacuum at 40 ℃ under an oil pump. The residue was dissolved in CHCl₃MeOH-1: 1 mixture (5mL) in Et₂Precipitate in O (45 mL). The precipitation was repeated two more times and the product obtained was used without additional purification. Yield 134mg (79%).

The product 22k (n 23, AA ValCit). To a solution of 3g (1.0mmol) and DIEA (183. mu.L, 1.04mmol) in DMF (4mL) was added PEG₂₃-NHS ester (0.87mmol) in DMF (4 mL). The mixture was stirred for 16 hours and,filter and remove all volatiles on a rotary evaporator at 40 ℃/oil pump vacuum. The residue was dissolved in CHCl₃MeOH-1: 1 mixture (5mL) in Et₂Precipitate in O (45 mL). Precipitation was repeated two more times and the product was used without additional purification. Yield 1.0g (77%). MS 1496.1[ M + NH ]₄]⁺；1479.3[M+1]⁺。

PEG-AA-PABC-PNP 23a-k

Conditions are as follows: (i) (PNP)₂CO, dioxane or THF, 40-60 deg.C, 10 hr.

For product 23a (n 11, AA Glu (2PhiPr) Gly), DCM (15mL) and (PNP) containing product 22a (274mg,0.274mmol)₂CO (418mg,1.372mmol) and DIEA (0.143mL,0.823mmol) were stirred for 15 h protected from light. The solvent was removed on a rotary evaporator and the product was purified on a column eluting with 4% MeOH, 0.2% AcOH in CHCl₃. Yield 260mg (81%). MS 1180.7[ M + NH ]₄]⁺。

For product 23b (n 11, AA phemit), 22b (419mg,0.42mmol), (PNP)₂A solution of CO (766mg,2.52mmol) and DIEA (263 μ L,1.52mmol) in dioxane (4mL) was stirred at 50 ℃ for 15 hours in the dark and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two successive DMF evaporations on a rotary evaporator at 40 ℃/oil pump vacuum, the product was purified on a column, eluent: CHCl₃EtOAc: MeOH (4.5:5:0.5) followed by CHCl₃MeOH (9: 1). Yield 390mg (80%). MS 1181.2[ M + NH ]₄]⁺,1164.2[M+1]⁺。

For the product 23c (n 11, AA ValCit), 22c (273mg,0.287mmol), (PNP)₂A solution of CO (874mg,2.88mmol) and DIEA (0.3mL,1.72mmol) in 1, 4-dioxane (22mL) was stirred at 50 deg.C in the dark for 24 h. Remove solvent on a rotary evaporator at 40 deg.C/oil pump and columnPurify the product, eluent: CHCl₃EtOAc-MeOH-16: 3:1 followed by CHCl containing 12-15% MeOH₃. Yield 163mg (51%). MS 1116.0[ M +1 ]]⁺。

Product 23d was prepared as described for preparation 23 b. The product was purified on a column, eluent CHCl₃EtOAc: MeOH (9:2: 1). The yield was 77%. MS 1144.0[ M + NH ]₄]⁺；1127.3[M+1]⁺。

Product 23e (n-11, AA-phelys (me))₂) Prepared as described for 23a and purified on column, eluent: 10% MeOH, CHCl with 0.2% AcOH₃. The yield was 63%. MS 1163.1[ M +1 ]]⁺。

The product 23f (n ═ 11, AA ═ Leu) was prepared as described for 23c, using only 5 equivalents of (PNP)₂CO and 3 equivalents DIEA, heated for 24 hours. The product was purified on a column with a gradient of MeOH in CHCl (7-12%) eluent₃. The yield was 75%. MS:972[ M +1 ]]⁺。

The product 23g (n-11, AA-asn (dmcp)) was prepared as described for 23f, and the crude product was used in the subsequent steps without additional purification. MS 1073.4[ M +18 ]]⁺。

For the product 23h (n 11, AA Cit), 22h (340mg,0.40mmol), (PNP)₂A solution of CO (608mg,2.00mmol) and DIEA (208. mu.L, 1.20mmol) in DCM (4mL) was stirred at 30 ℃ for 15 h in the dark and all volatiles were removed on a rotary evaporator. Residual DIEA was removed by two successive DMF evaporations on a rotary evaporator at 40 ℃/oil pump vacuum, the product was purified on a column, eluent: CHCl₃EtOAc: MeOH (7:2.5:0.5) followed by CHCl with MeOH (8-14%)₃And (4) gradient. Yield 390mg (80%). MS 1034.3[ M + NH ]₄]⁺；1016.9[M+1]⁺。

The product 23i (n-23, AA-phemit) was prepared as described in preparation 23b and purified on the column, eluent CHCl₃EtOAc: MeOH (4.5:5:0.5) followed by CHCl with MeOH (6-12%)₃And (4) gradient. The yield was 86%. MS:1711.4[M+NH₄]⁺；1694.4[M+1]⁺。

Product 23j (PEG 1000K AA ═ phemit) was prepared as described for preparation 23b and purified on the column, eluent CHCl₃EtOAc: MeOH (4.5:5:0.5) followed by CHCl with MeOH (6-12%)₃And (4) gradient. The yield was 72%.

The product 23k (n 23, AA ValCit) was prepared as described for preparation 23b and the product was purified by HPLC. Column: luna (Phenomenex) 5u, C-8, 100A. Mobile phase: ACN-H₂O(F₃CO₂H0.01%), ACN gradient 30-37%, 31 min. Yield: 530mg (48%). MS 1666.4[ M + Na ]]⁺；1644.2[M+1]⁺。

PEG-AA-PABC-PNP 24a-c, AA deprotection.

Conditions are as follows: (i) TFA/H₂O ═ 3:1, 5 ℃, 2-3 hours.

The product 24a (n ═ 11, AA ═ GluGly). Product 23a (250mg,0.215mmol) in 3% TFA in CHCl₃(16mL) the solution was stirred for 35 min, concentrated on a rotary evaporator and dried in vacuo. Yield 224mg (100%). MS 1062.6[ M + NH ]₄]⁺.；1045.9[M+1]⁺。

Product 24b (n 11, AA AlaAsn-PABC-PNP). Compound 23d was stirred in a mixture of TFA: DCM (3:1) for 1.5 h, and all volatiles were removed on a rotary evaporator at 20 ℃. The product was purified on a column with a gradient of eluent CHCl containing MeOH (6-12%)₃. The yield was 30%. MS 1066.7[ M + Na ]]⁺，1062.0[M+NH₄]⁺；1045.2[M+1]⁺。

Product 24c (n 11, AA Asn). A reaction flask containing 23g (160mg,0.143mmol) was chilled to 0 deg.C and then cold TFA H was added₂O (9:1) (12.5mL) mixture. The mixture is stirred1.5 hours, combined with cold H₂Dilution with O (50 mL). Stirring was continued for 20 minutes at 20 ℃. The precipitate is filtered off and washed with H₂And (4) flushing. All volatiles were removed on a rotary evaporator at 40 ℃ and the product was purified on a column, eluent: CHCl₃EtOAc, MeOH: AcOH 4.5:3.5:1.2: 0.02. Yield 43mg (30%). MS 974.0[ M +1 ]]⁺。

TABLE 4 Final PEG-L-A for DPC preparation₁A₂-PABC

Example 2. attachment of protease cleavable masking agent to amine containing polymer-formation of para-amidobenzyl carbamate linkage.

A. Melittin is modified with a protease-cleavable masking agent. 1 × mg melittin and 10 × mg HEPES base (1-10mg/mL peptide) were masked by the addition of 2-6 × mg amine-reactive p-nitrophenyl carbonate or N-hydroxysuccinimide carbonate derivative of a protease cleavable substrate containing NAG. The solution was then incubated at Room Temperature (RT) for at least 1 hour prior to injection into the animals.

B. Polyamines are modified with protease cleavable masking agents. Reacting the carbonate of the activated (amine-reactive) para-amidobenzyl alcohol derivative with the amino group of the amphiphilic membrane-active polyamine in H₂In (pH)>8) To form p-amidobenzylcarbamate.

R¹Including ASGPr ligands (protected or unprotected) or PEG,

R²is an amphiphilic membrane active polyamine, and the polyamine is a polyamine,

AA is a dipeptide (protected or unprotected), and

z is an amine reactive carbonate or ester.

To Xmg of polymer was added isotonic glucose containing 12 Xmg HEPES free base. DMF containing 2X-16X mg 200mg/ml dipeptide mask was added to the buffered polymer solution. In some applications, the polymer is modified with 2 × mg dipeptide masking agent, followed by attachment of siRNA. The polymer-siRNA conjugate was then further modified with 6X-8 Xmg dipeptide masking agent.

Example 3.sirna has the following sequence:

factor VII siRNA

Sense (Chol) -5 'GfcAFaFgGfcGfccGfcCfaAfcUfcAf (invdT) 3' (Seq ID 1)

Antisense 5 'pdTsgfaGfuUfgGfcaFcGfccCfuUfuGfccdTdT 3' (Seq ID 2)

Or

Sense 5 'GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT 3' (Seq ID 3)

Antisense 5 'GUfAAGACfUfUfGAUfGAUfGAUfCfCfdTdT 3' (Seq ID 4)

Or

Sense (NH)₂C₆)GfuUfgGfuGfaAfuGfgAfgCfuCfaGf(invdT)3′(Seq ID 5)

Antisense pCfsUfgAfgCfuCfcAUfcAfcCfaAfcdTdT 3' (Seq ID 6)

Or

Sense: 5' (NH)₂C₆)uGuGfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)3′(Seq ID23)

Antisense 5 'pdTsgfaGfuUfgGfcaFcGfccCfuUfuGfccdTdT 3' (Seq ID 24)

Factor VII siRNA (Primate)

Sense (chol) -5 'uuAGGfuUfGfuGfaAffGfAfGfcufafaGf (invdT) 3' (Seq ID7)

Antisense 5 'pCfsUfgAfgCfuCfcAfufcAfcAfcCfaAfcdTsdT 3' (Seq ID 8)

ApoB siRNA:

Sense (cholC6SSC6) -5 'GGAAUCuAuuAuuGAUCcAsA 3' (Seq ID 9)

Antisense 5 'uuGGAUcAAAuAuAAGAuUCcscu 3' (Seq ID 10)

AhaI siRNA:

Sense (NH)₂C₆)GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)3′(Seq ID 11)

Antisense pdAsCfuAfufUfcCfaCfufcAfCcdTsdT 3' (Seq ID 12)

Luc siRNA

Sense (chol)5 '-uAuCfuFafCgfuGfaGfuAfcUfuCfgAf (invdT) -3' (Seq ID13)

Antisense 5 '-UfcgaafUfaCfuCfaGfcgcGfcgCfuaGfaGdTdT-3' (Seq ID 14)

Or

Sense (NH)₂C₆)cuuAcGcuGAGuAcuucGAdTsdT 3′(Seq ID 15)

Antisense UCGAAGuACUcAGCGuAAGdTdT 3' (Seq ID 16)

Eg5-KSP

Sense (NH)₂C₆)UfcGfaGfaAfuCfuAfaAfcUfaAfcUf(invdT)3′(Seq ID 17)

Antisense pAfGfufaGfuUfuAfgAfuUfcUfUfcUfcGfadTdT 3' (Seq ID 18)

Or

Sense AGUuAGUuAGAUUCGAdTdT 3' (Seq ID 19)

Antisense (NH)₂C₆)ucGAGAAucuAAAcuAAcudTsdT 3′(Seq ID 20)

EGFP

Sense: 5' (NH)₂C₆)AuAucAuGGccGAcAAGcAdTsdT 3′(Seq ID 21)

Antisense 5 'UGCUUGUCGGCcAAUAUdTdT 3' (Seq ID 22)

2' -O-CH₃Substitution

s ═ phosphorothioate linkages

Post-nucleotide F-2' -F substitution

Pre-nucleotide d-2' -deoxy

RNA synthesis on solid phase by conventional phosphoramidite chemistry, useOligopilot 100 (general electro-medical group, Frisburg, Germany) and Controlled Pore Glass (CPG) as solid supports.

Example 4 RNAi polynucleotides in vivo administration and hepatocyte delivery. DPC was prepared as described above.

6-8 week old mice (C57BL/6 or ICR strain, about 18-20 g each) were obtained from HSD corporation (Harlan Sprague Dawley, Indianapolis, Ind). Mice were housed for at least 2 days prior to injection. Free feeding was performed with haan (Harlan) Teklad rodent chow (Harlan, madison, wisconsin). DPC was synthesized as described herein. Conjugate solution (0.4mL) was injected by infusion into the tail vein. The composition is soluble and non-aggregating under physiological conditions. Injection into other vessels (e.g., retrobulbar injection) is contemplated to be equivalent. 175-200g Wistar Han rats were obtained from Charles River laboratories (Wilmington, Mass.). Rats were housed for at least 1 week prior to injection. The volume injected into rats is typically 1 mL. Unless otherwise indicated, serum samples were taken 48 hours after injection and/or liver samples were harvested.

Serum ApoB levels were determined. Mice were fasted for 4 hours (rats 16 hours) and then serum was collected by submandibular bleeding. Rat blood was collected from the jugular vein. Serum ApoB protein levels were determined by standard sandwich ELISA methods. Briefly, polyclonal goat anti-mouse ApoB antibodies and rabbit anti-mouse ApoB antibodies (biosignal International) were used as capture and detection antibodies, respectively. HRP conjugated goat anti-rabbit IgG antibody (Sigma) was then applied to bind the ApoB/antibody complex. The absorbance of the tetramethylbenzidine (TMB, Sigma) colorimetric development at 450nm was then measured by a Tecan Safire2 (Austria, Europe) microplate reader.

Plasma factor VII (F7) activity measurement. Plasma samples of animals were prepared by collecting blood (9 volumes) either from mice via mandibular bleeding or rats via jugular vein and filled into microcentrifuge tubes containing 0.109mol/L sodium citrate anticoagulant (1 volume) according to standard procedures. Plasma F7 activity was measured using the BIOPHEN VII kit (Hyphen BioMed/anira) using a chromogenic method according to the manufacturer's recommendations. The absorbance of the colorimetric development was measured at 405nm by a Tecan Safire2 microplate reader.

Example 5 in vivo delivery of siRNA to hepatocytes using amphiphilic membrane active polyacrylic polyamines reversibly modified with a dipeptide cleavable masking agent.

100mM HEPES buffer pH7.5 containing polyacrylate Ant-41658-111 was modified with 0.5 wt% of activated disulfide reagent succinimidyloxycarbonyl- α -methyl- α (2-pyridyl-dithio) toluene (SMPT) (Pierce) to obtain thiol reactive groups for subsequent attachment of siRNA. The thiol-reactive polymer was then diluted to 5mg/mL in 60mg/mL HEPES base. To this solution was added 10mg/mL of each of the enzyme-cleavable masking agents. The amounts represent the molar ratio of 1 polymer amine to 2 masking agent. The polymer modification reaction is preferably carried out in a molar ratio of polymer amine/masking agent of 1:1 to 1: 5. More preferably, the ratio is 1:2 to 1: 4. A more preferred ratio is 1: 2. One hour later, acetate protected thiol endogenous rodent factor VII siRNA (0.1-0.2 weight equivalents relative to polymer) was added to the polymer solution. After overnight incubation, the conjugate was further modified by addition of an N-acetylgalactosamine derivative of maleic anhydride (NAG-CDM; Table 5). NAG-CDM was added at 25mg/mL and incubated for 30 minutes to 4 hours.

For NAG-CDM polymer modification, NAG-CDM was lyophilized from 0.1% glacial acetic acid in water. Adding a polymer solution to the dried NAG-CDM. After the anhydride is completely dissolved, the solution is incubated at room temperature for at least 30 minutes before administration to the animal. Reacting dNAG-CDM with the polymer to form:

wherein R is the polymer and R1 includes an ASGPR ligand (e.g., N-acetylgalactosamine).

As shown in table 5, factor VII expression was reduced by 49-85% in animals treated with DPC masked with dipeptide reagent.

TABLE 5 knock-down of factor VII in vivo in mice treated with PEG-AA-p-nitrophenylcarbamate + NAG-CDM-DPC.

^amg Polymer or siRNA/kg animal body weight

^bRelative to the control tested for the first time

Example 6 in vivo siRNA delivery using NAG/PEG-AA-p-nitrophenyl carbamate poly (acrylate) DPC.

A) PEG + NAG modification. 100mM HEPES buffer pH7.5 containing polyacrylate Ant-41658 111 was modified with 0.5 wt% of succinimide oxycarbonyl- α -methyl- α (2-pyridyl-dithio) toluene (SMPT) from Pierce, an activated disulfide reagent. The thiol-reactive polymer was diluted to 5mg/mL in 60mg/mL HEPES base. To this solution was added 10mg/mL of various PEG-AA-p-nitrophenyl carbonate masking agents. After one hour, acetate protected thiol factor VII siRNA (polymer to siRNA ratio range 5-10 to 1) was added to the polymer solution. After overnight incubation, NAG-AA-p-nitrophenyl carbonate masking agent was added to 40 mg/mL. After at least 30 minutes but not more than 4 hours of incubation, the DPC was injected into the tail vein of 20g ICR mice. Serum samples were harvested 48 hours after injection and factor VII levels were measured.

B) NAG was modified individually. 100mM HEPES buffer pH7.5 containing polyacrylate Ant-41658 111 was modified with 0.5 wt% of succinimide oxycarbonyl- α -methyl- α (2-pyridyl-dithio) toluene (SMPT) from Pierce, an activated disulfide reagent. The thiol-reactive polymer was diluted to 5mg/mL in 60mg/mL HEPES base. Acetate protected thiol siRNA factor VII (ratio of polymer to siRNA ranging from 5-10 to 1) was added to the polymer solution. After overnight incubation, NAG-AA-p-nitrophenyl carbonate masking agent was added to 50 mg/mL. After at least 30 minutes but not more than 4 hours of incubation, the polymer-conjugated siRNA was injected into the tail vein of a 20gm ICR mouse. Serum samples were harvested 48 hours after injection and factor VII levels were measured.

TABLE 6 in vivo factor VII knockdown in mice treated with PEG/NAG-AA-p-nitrophenyl carbamate DPC

^amg Polymer or siRNA/kg animal body weight

^bRelative to the control tested for the first time

^cBody weight equivalent

Example 7 in vivo siRNA delivery using NAG/PEG-AA-p-nitrophenyl carbamate poly (vinyl ether) DPC.

Amphiphilic membrane active poly (vinyl ether) polyamine DW1360 was modified as described above for polyacrylate Ant-41658-. After polymer modification, the amino acid protecting groups were removed by incubating to remove TFA and NAG acetate protecting groups in the presence of 30 vol.% triethylamine, 50% methanol and 20% water. The acetate deprotection solution was removed by rotary evaporation. The masked polymer was co-injected with cholesterol-ApoB siRNA conjugate into mice.

TABLE 7 in vivo ApoB knockdown in mice co-injected with NAG-AA-p-nitrophenyl carbamate poly (vinyl ether) and cholesterol-ApoB siRNA conjugate.

^amg Polymer or siRNA/kg animal body weight

^bRelative to the control tested for the first time

Example 8 in vivo knockdown of endogenous ApoB levels following ApoB siRNA delivery using melittin delivery peptide, enzyme cleavable masking agent in mice.

Melittin was reversibly modified with the indicated amount of enzyme cleavable masking agent as described above. Then, 200-300. mu.g of the masked melittin was co-injected with 50-100. mu.g of ApoB siRNA-cholesterol conjugate. The effect on ApoB levels was determined as described above. Peptidase-cleavable dipeptide-amide benzyl carbamate modified melittin is an effective siRNA delivery peptide. Preferably, melittin in D form is used in combination with an enzyme-cleavable masking agent. The same level of target gene knockdown requires more polypeptide because the peptide masking is more stable and the therapeutic index is not altered or improved (compared to masking with the same peptide using CDM-NAG).

Table 8 inhibition of factor VII activity in normal hepatocytes in mice treated with factor VII-siRNA cholesterol conjugate and G1L-melittin (form D) (Seq ID 25), which G1L-melittin was reversibly inhibited with the enzyme-cleavable masking agent shown.

^aAmount of masking agent/melittin amine for masking reaction

Example 9 tumor targeting using protease cleavable DPC.

A) Target gene knockdown detection. In all studies described below, siRNA was specific for Aha1 gene transcript (target gene). siRNA against Enhanced Green Fluorescent Protein (EGFP) was used as off-target control. The Aha1siRNA is complementary to sequence motifs in Aha1 that are 100% homologous in human and mouse genes. Thus, delivery of Aha1siRNA into host cells or tumor cells of human xenografts results in nRNA cleavage and degradation. Using different sequence motifs within the mouse and human Aha1 genes, PCR primers were designed to enable quantitative detection of human Aha1 and mouse Aha1mRNA levels in tissue samples containing mixed populations of different cell types. Tumors that were attached to liver tissue of some healthy mice were harvested 24, 48, or 72 hours after siRNA delivery and processed in Tri-Reagent (Invitrogen) for total RNA isolation. Human Cyc-A and mouse beta-actin were then used as reference genes to detect human and mouse Aha1mRNA levels by qPCR experiments. The Aha1mRNA levels in mock-injected animals or mice receiving off-target control GFP siRNA were considered to be 100%. The results are expressed as a percentage of Aha1mRNA levels relative to the control and are shown in the table below.

B) Mice tumor model hepatocellular carcinoma (HCC) were transplanted in situ. HegG2, Hep3B or HuH7 hepatocellular carcinoma cells were co-transfected with two expression vectors, human secreted placental alkaline phosphatase (SEAP) vector pMIR85 and neomycin/kanamycin resistance gene vector pMIR3, to develop cell lines with stable SEAP expression. Cells were grown in DMEM supplemented with 10% FBS and 300ug/ml g418, collected, counted, and then mixed (50% volume) with matrigel (BD Biosciences). Athymic nude mice or Scid beige mice were anesthetized with approximately 3% isoflurane and placed in sternal horizontal position. A small incision of 1-2 cm is cut in the middle of the abdomen and is close to the lower part of the sword-shaped protrusion. The left liver lobe was gently removed using a wet cotton swab. The left lobe of the liver was gently pulled and an injection needle was inserted into the middle of the left lobe. The needle was inserted approximately 0.5cm obliquely below the liver capsule. 10 μ l of cell/matrigel mixture containing 100,000 cells was injected into the liver using a syringe pump. The needle is allowed to remain in the liver for a period of time (15-20 seconds) to ensure that the injection is complete. SEAP-HepG2 cells were injected into athymic nude mice. SEAP-Hep3B and SEAP-HuH7 cells were injected into Scid beige mice. The syringe was then removed from the liver by needle and a cotton swab was placed on the injection site to prevent cell leakage or bleeding. The matrigel/cell mixture formed visible aggregates and did not disappear after removal of the needle. The lobe was then gently returned to the abdominal cavity, closing the abdominal wall. Sera were collected weekly after tumor transplantation and SEAP experiments were performed to monitor tumor growth. For most studies, tumor bearing mice 4-5 weeks after transplantation are used when tumor measurements of about 4-8 mm (based on SEAP values) are expected.

C) Colorectal metastatic tumor model. HT29 cells were grown in McCoy's 5a medium supplemented with 10% FBS, harvested, counted and then mixed (50% by volume) with matrigel (BD Biosciences). Athymic nude mice were anesthetized with approximately 3% isoflurane and placed in a sternal horizontal position. A small incision of 1-2 cm is cut in the middle of the abdomen and is close to the lower part of the sword-shaped protrusion. The left liver lobe was gently removed using a wet cotton swab. The left lobe of the liver was gently pulled and an injection needle was inserted into the middle of the left lobe. The needle was inserted approximately 0.5cm obliquely below the liver capsule. Mu.l of cell/matrigel mixture containing 40,000 cells was injected into the liver using a syringe pump. The needle is allowed to remain in the liver for a period of time (15-20 seconds) to ensure that the injection is complete. The syringe was then removed from the liver and a cotton swab was placed on the injection site to prevent cell leakage or bleeding. The matrigel/cell mixture formed visible aggregates and did not disappear after removal of the needle. The lobe was then gently returned to the abdominal cavity, closing the abdominal wall. Tumor-bearing mice 4-5 weeks after transplantation were used.

Example 10 PEG₂₄In vivo knockdown of target gene expression in HepG2-SEAP in situ transplantation hepatocellular carcinoma (HCC) model following Val-Cit DPC administration.

Overweight PEG with 18 × as described above₂₄Phe-Cit masking Agents (or PEG)₂₄Val-Cit masking agent) or with 7 × PEG₅₅₀-CDM modification (masking) (2011062805) Ant-129-1 polymer DPC. The polymers were linked (4:1 wt ratio) using Aha1-siRNA (RD-09070) or GFP-siRNA (RD-05814; off-target control) as described above. Before delivery, DPC was purified without gel filtration and without addition of targeting ligand. Mu.l of an aliquot of glucose/animal containing 320. mu.g (polymer weight) of DPC conjugate was administered by tail vein injection (n-3/group). After 24 hours, the animals received a second 200 μ l isotonic glucose injection containing 320 μ g (polymer weight) of DPC conjugate. 48 hours after the second injection, serum samples were collected to assess toxicity by measuring liver enzyme (ALT and AST) and Blood Urine Nitrogen (BUN) levels, followed by tissue harvest and qPCR analysis.

Using PEG₂₄Val-Cit-Ant-129-1-siRNA DPC delivered Aha1siRNA, causing 46% knockdown of the Aha1 gene in human tumor cells (Table 9). Mouse Aha1 response to PEG in contrast to human Aha1 knockdown levels₂₄Val-Cit-Ant-129-1-siRNA DPC administration resulted in a 70% knockdown (Table 1). Endogenous hepatocyte Aha1 knockdown was reduced compared to similar DPC generated with disubstituted maleic anhydride masking agent (PEG 550-CDM). PEG as indicated by ALT, AST and BUN levels₂₄Val-Cit DPC resistanceWell received and showed no toxicity (see table).

Table 9 Aha1 knockdown in HepG2 liver tumor model by maleic anhydride modification versus peptide cleavable modified Aha1siRNA DPC.

TABLE 10 blood chemotoxicity markers following administration of maleic anhydride-modified or peptide cleavable modified Aha1siRNA DPC.

		PEG550-CDM DPC	PEG24-Val-Cit DPC
					Control siRNA	Aha1siRNA	Aha1siRNA
ALT	44.3±5.1	58.0±37.5	35.0±11.3
				AST	81.7±4.0	102.3±57.5	67.7±14.4
BUN	25.3±4.2	23.0±3.5	21.3±1.2

Example 11 Targeted Gene expression knockdown with bispecific antibody (bsAb) -targeted DPC (2011090701).

Modification of Ant-129-1 Polymer with 5 × Dig-PheCit (Dig-FCit) masking agent as described above, followed by siRNA ligation of the conjugate, finally, PEG 8 × (weight) _ PEG₁₂-FCit further modifies Dig-FCit-Ant-129-1-siRNA conjugates. Aha1-siRNA (RD-09070) or GFP siRNA (RD-05814) were ligated at a polymer to siRNA weight ratio of 4: 1. Purification of PEG on Sephadex G50 spin columns₁₂-FCit DPC to remove unbound reagents.

Cell-targeting bispecific antibodies (bsAb) specific for heparan sulfate proteoglycan phosphatidyl ethanolprotein-3 (GPC3) and digoxin (Dig) were prepared, GPC3 is a cell surface heparan sulfate proteoglycan known to be highly expressed in HepG2-SEAP cells. As a control, bispecific antibodies specific for the proteins CD33 (myeloid-derived hematopoietic hepatocyte marker) and Dig were prepared. HepG2-SEAP cells do not express CD 33. BsAb and modified DPC were complexed at a weight ratio of 1.25:1 to provide the estimated 1:1 molar ratio. Complexes were formed in PBS for at least 30 minutes prior to delivery.

DPC (with or without bsAb targeting agent) was administered to HepG2-SEAP tumor-bearing mice. Each animal (n-3/group) received a single dose of 250 μ g (polymer weight) of DPC. mu.L of sterile PBS containing DPC was injected into the tail vein of mice. Serum and tissue samples were harvested after 48 hours and analyzed as described above. As shown in Table 11, a single dose of bsAb-targeted DPC (250. mu.g polymer, 62.5. mu.g siRNA) resulted in 21-32% knockdown of the target gene.

Table 11 Aha1 knockdown in HepG2 liver tumor model of Aha1siRNA DPC modified by using bispecific antibody targeted peptide cleavable masking agent.

Example 12 Targeted Gene expression knockdown with bispecific antibody (bsAb) -Targeted DPC.

DPC was prepared as described above except a) PEG was used₂₄-FCit instead of PEG₁₂FCit and b) Using Dig-PEG₁₂-NHS links Dig to the polymer. PEG₂₄-FCit DPC compared to PEG₁₂-FCit DPC aggregates less and is smaller and more homologous. In addition to being a stable linkage, Dig-PEG₁₂-NHS also comprises longer PEG. DPC was complexed with bsAb and injected into animals as described above. Serum and tissue were harvested 24 or 48 hours after injection. As shown in table 12, a single dose of DPC (250 μ g polymer weight) caused a 46-56% knock-down of human Aha 124 hours post-injection.

Table 12 Aha1 knockdown in HepG2 liver tumor model of Aha1siRNA DPC modified by using a peptide cleavable masking agent with increased PEG length.

Example 13 targeting of DPC to human colorectal adenocarcinoma metastatic liver tumor tissue by bsAb-targeted DPC.

Ant-129-1 Polymer Using 5 × molar excess of Dig-PEG₁₂-NHS and 8 × weight excess PEG₂₄-an FCit modification. The modified Aha1siRNA or GFP siRNA are linked at a polymer to siRNA weight ratio of 4:1A polymer. DPC was purified on a sephadex g50 spin column to remove unbound reagents. Before injection, Dig-DPC was complexed with equimolar amounts of IGF1R-DigbsAb or CD33-Dig bsAb or bsAb in sterile PBS for at least 30 minutes. Animals containing HT29 tumor cells (human colorectal adenocarcinoma; ATCC number HTB-38) were injected with the DPC. HT29 cells overexpress insulin-like growth factor 1 receptor protein (IGF1R) and are able to bind and internalize IGF1R-Dig bispecific antibodies. Animals (n-3) received DPC (320 μ g polymer). Injections were repeated after 24 hours. Serum and tissue samples were collected 48 hours after the second dose. The knockdown of human Aha1 in tumor cells was 26-38% (table 13). FCit-DPC showed less knockdown of off-target liver Aha1 (78-83% compared to 24-36%) compared to CDM-DPC. FCit-DPC also showed reduced liver accumulation compared to CDM-DPC.

TABLE 13 Aha1 knockdown in HT29 colorectal adenocarcinoma metastatic liver tumors by dipeptide cleavable Aha1siRNA DPC.

Example 14 in vivo knockdown of endogenous Aha1 in liver tumors.

400 μ g Lau41648-106 PEG 8 × (by weight)₁₂ValCit or 16 × PEG₂₄-a phemit modification. 100 μ g of Aha1siRNA or 100 μ g of Eg5 control siRNA was ligated to the modified polymer as described above. Injecting the DPC into an animal comprising Hep3B-SEAP tumor cells. Serum and tissue samples were collected 48 hours after injection. The knockdown of human Aha1 in tumor cells was 26-38%.

TABLE 14 Aha1 knockdown in Hep3B-SEAP liver tumors by dipeptide cleavable Aha1siRNA DPC.

Example 15 in vivo circulation and tissue targeting of masking polymers.

At room temperature with a solvent containing¹²⁵Lau24AB polyacrylate (100. mu.g) was treated with 50mM HEPES (pH 8.0) buffer, I-pall Hunter (Bolton-Hunter) (BH) reagent (50. mu. Ci) for 1 hour. The labeled polymer was purified in water in a 2mL Sephadex QEA spin column. The solution of the labeled polymer was stored at 4 ℃. Unlabeled polymer is supplemented with¹²⁵I-labeled Polymer to inject about 1mg of polymer, per 200g of rat with 0.2. mu. Ci. With PEG as described above₂₄-FCit or PEG-CDM (2mg/ml polyacrylate, 14mg/ml PEG-CDM reagent, 14mg/ml NAG-PEG-CDM reagent, 16mg/ml PEG₂₄FCit reagent) modifies a mixture of labelled and unlabelled polymers (calculated on about 3.5 animals). Incubate for 1 hour. The reaction mixture was then diluted with isotonic glucose to generate an injected dose of 1mL volume per animal. 3 animals/group were injected. Animals were bled (0.1-0.2ml) at the given time. The amount of polymer present in the test sample was counted by a gamma counter. As shown in fig. 4, polymers modified with protease-cleavable masking agents cleared from serum less rapidly than polymers masked with pH-labile maleic anhydride masking agents. Increasing the circulation time is beneficial for targeting non-liver tissues.

EXAMPLE 16 Synthesis of amphiphilic Membrane active Polymer.

A) Poly (vinyl acrylate)

i) RAFT copolymerization of N-Boc-ethyl ethoxy acrylate and propyl methacrylate:

in the formula: a is boc-protected ethyl-ethoxyaminoacrylate

B is propyl methacrylate

C is RAFT reagent CPCPCPCPAP (4-cyano-4- (thiobenzoyl) pentanoic acid)

n and m are integers.

After synthesis, the boc protecting group is removed to generate the amine monomer.

For other membrane active polymers, a may also be protected ethyl, propyl or butyl amino acrylate. B may be a highly hydrophobic (10-24 carbon atoms, showing C18) acrylate, a less hydrophobic (1-6 carbon atoms, showing C4) acrylate or a combination of a less hydrophobic acrylate and a highly hydrophobic acrylate.

The copolymer consisting of amine acrylate/C3 methacrylate was synthesized as follows. Monomers and RAFT agent were weighed and butyl acetate was added in the proportions indicated. AIBN (azobisisobutyronitrile) was added and nitrogen was bubbled through the reaction for 1 hour at room temperature. The reaction mixture was then placed in an 80 ℃ oil bath for 15 hours. The polymer was then precipitated with hexane and further fractionated with a DCM/hexane solvent system (see below). Then, the polymer was dried under reduced pressure. The polymer was deprotected by treatment with 7ml2M HCl in acetic acid for 30 min at room temperature. After 30 minutes, 15mL of water was added to the reaction mixture and the mixture was transferred to a 3.5kda mwco dialysis tube. The polymer was dialyzed against NaCl overnight and then against dH another day₂O dialyses overnight. The water was then removed by lyophilization and the polymer was dissolved in dH₂And (4) in O.

(Ant 41658-111). 2, 2' -azobis (2-methylpropanenitrile (AIBN, radical initiator), 4-cyano-4- (thiobenzoyl) pentanoic acid (CPCPCPAP, RAFT reagent) and butyl acetate were purchased from Sigma Aldrich (Sigma Aldrich.) propyl methacrylate monomer (Alfa Aesar) was filtered to remove inhibitors.

In a 2L round bottom flask equipped with a stir bar 2- (2-aminoethoxy) ethanol (21.1g,202.9mmol (Sigma Aldrich)) was dissolved in 350mL of dichloromethane in a separate 1L flask, BOCAnhydride (36.6g, 169.1mmol) was dissolved in 660mL of dichloromethane. The 2L round bottom flask was fitted with an additional funnel and the BOC anhydride solution was added to the flask over 6 hours. The reaction was stirred overnight. In a 2L separatory funnel, 300ml each of 10% citric acid and 10% K was used₂CO₃Saturated NaHCO₃And saturated NaCl wash product. Product BOC protected 2- (2-aminoethoxy) ethanol over Na₂SO₄Dry, gravity filter, and evaporate DCM with rotary evaporation and high vacuum.

To a 500mL bottom flask equipped with a stir bar and purged with argon was added BOC-protected 2- (2-aminoethoxy) ethanol (27.836g, 135.8mmol) followed by 240mL of anhydrous dichloromethane. Diisopropylethylamine (35.5mL, 203.7mmol) was added and the system placed in a dry ice/acetone bath. Acryloyl chloride (12.1mL, 149.4mmol) was diluted with 10mL of dichloromethane and added dropwise to the argon purge system. The system was kept under argon and allowed to cool to room temperature and stir overnight. Each with 100mL of dH₂O, 10% citric acid, 10% K₂CO₃Saturated NaHCO₃And saturated NaCl wash product. The product BOC-aminoethoxyacrylic acid ethyl ester (BAEEA) is taken over Na₂SO₄Dry, gravity filter, and evaporate DCM with rotary evaporation. The product was purified by column chromatography on 29cm silica using a 7.5cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf: 0.30. the fractions were collected and the solvent was removed by rotary evaporation and high vacuum. The BAEEA yield obtained was 74%. BAEEA is stored in a refrigerator.

Polymer Ant-41658-111: a solution of AIBN (1.00mg/mL) and RAFT agent (4-cyano-4- (thiobenzoyl) pentanoic acid (CPCPCPA), 10.0mg/mL) in butyl acetate was prepared. The monomer molar feed ratio was 75BAEEA:25 propyl methacrylate (CAS:2210-28-8) with 0.108CPCPA RAFT reagent and 0.016AIBN catalyst (total molar amount of 0.00562).

20ml was provided with stirringA glass vial of rods was charged with BAEEA (1.09g,4.21mmol) (A), propyl methacrylate (.180g,1.41mmol) (B), CPCPCPAP solution (.170ml,.00609mmol) (C), AIBN solution (.150ml,.000915mmol) and butyl acetate (5.68 ml). The vial was sealed with a rubber cap and nitrogen bubbled through a long syringe needle, the other short syringe needle was used as the outlet, for 1 hour. The syringe needle was removed and the system was heated to 80 ℃ with an oil bath for 15 hours. The solution was allowed to cool to room temperature and transferred to a 50mL centrifuge tube, then hexane (35mL) was added to the solution. The solution was centrifuged at 4,400rpm for 2 minutes. The supernatant layer was carefully decanted and the bottom (solid or gel) layer was washed with hexane. The bottom layer was then redissolved in DCM (7mL), precipitated in hexane (35mL) and centrifuged once more. The supernatant was decanted and the bottom layer was washed with hexane and the polymer was then dried under reduced pressure for several hours. Molecular weight was obtained by MALS 73,000(PDI 1.7); polymer composition using H¹NMR gave 69:31 Amine: Alkyl (Amine: Alkyl).

And (4) fractional precipitation. The dried precipitated product was dissolved in DCM (100 mg/mL). Hexane was added until just past the cloud point (. about.20 mL). The resulting milky white solution was centrifuged. The bottom layer (thick liquid presenting about 60% polymer) was extracted and precipitated well with hexane. The remaining upper solution was also precipitated well by further addition of hexane. Both portions were centrifuged before the polymer was isolated and dried in vacuo. Component 1: mw 87,000(PDI 1.5); and (2) component: mw 52,000(PDI 1.5-1.6).

MALS analysis. About 10mg of polymer was dissolved in 0.5mL of 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and Polydispersity (PDI) were measured with a Wyatt Helos II multi-angle light scattering detector connected to Shimadzu corporation (Shimadzu) science HPLC using a Jordi 5. mu.7.8X 300 mixed bed LS DVB column. Crude polymer: MW:73,000(PDI 1.7), fraction 1: MW 87,000(PDI:1.5), fraction 2: MW 52,000(PDI 1.5-1.6).

The purified BOC-protected polymer was reacted with 2M HCl in acetic acid (7mL) for 0.5 hours to remove the BOC protecting group and generate the amine. 15mL of dH₂O is added for reaction, and the solution is transferred to cellulose with 3500MW cut-off for dialysisIn bags, dialyzed against high salt for 24 hours, then against dH₂O dialysis for 18 hours. The contents were lyophilized and then dissolved in DI H at a concentration of 20mg/mL₂And O. The polymer solution is stored at 2-8 ℃.

ii) Polymer Lau24B was prepared as described above, except that the monomer feed rate was 72.5BAEEA:27.5 propyl methacrylate.

iii) Ant-129-1 was prepared essentially as described above, except that the following monomers were used:

TABLE 15 Ant-129-1 Polymer Synthesis reactions.

For N-Boc-amino-propyl acrylate (BAPA), 3- (BOC-amino) 1-propanol (TCI) (135.8mmol) was added to a 500mL round bottom flask equipped with a stir bar and purged with argon, followed by 240mL dry dichloromethane. Bis-isopropylamine (203.7mmol) was added and the system placed in a dry ice/acetone bath. Acryloyl chloride (149.4mmol) was diluted with 10mL of dichloromethane and added dropwise to the argon purge system. The system was kept under argon and brought to room temperature and stirred overnight. Each with 100mL of dH₂O, 10% citric acid, 10% K₂CO₃Saturated NaHCO₃And saturated NaCl wash product. Product BOC-aminopropyl aminoacrylate (BAPA) with Na₂SO₄Dry, gravity filter, and evaporate DCM with rotary evaporation. The product was purified by column chromatography on 29cm silica using a 7.5cm diameter column. The solvent system used was 30% ethyl acetate in hexane. Rf is 0.30. The fractions were collected and the solvent was removed by rotary evaporation and high vacuum. The BAPA yield obtained was 74%. BAPA is stored in a refrigerator.

iv) N-Boc-ethylethoxyacrylate and methylRandom copolymerization of propyl acrylate. From amine acrylates/C_nThe copolymer consisting of methacrylic acid esters was synthesized as follows. The monomers were weighed and dioxane was added at the ratio indicated. AIBN (azobisisobutyronitrile) was added and nitrogen was bubbled through the reaction for 1 hour at room temperature. The reaction mixture was then placed in a 60 ℃ oil bath for 3 hours. Then, the polymer was dried under reduced pressure. The resulting polymer was purified by GPC. Thereafter, the polymer fraction was treated with 7ml2M HCl in acetic acid for 30 minutes at room temperature for deprotection. After 30 minutes, 15mL of water was added to the reaction mixture and the mixture was transferred to a 3.5kDa MWCO dialysis tube. The polymer was dialyzed overnight against NaCl and then with dH another day₂O dialyses overnight. The polymer is then dissolved in dH by removing the water by lyophilization₂And (4) in O.

Polymers Lau 41648-106. The monomer molar feed ratio was 80BAEEA:20 propyl methacrylate (CAS:2210-28-8) and 3% AIBN catalyst (based on total monomer moles). To a 50mL glass tube equipped with a stir bar were added BAEEA (6.53g,25.2mmol) (a), propyl methacrylate (0.808g,6.3mmol) (B), AIBN (0.155g,0.945mmol) and dioxane (34.5 mL). Compounds A and B were prepared as described above in example 16 Ai. The reaction was repeated three times. Nitrogen was bubbled through each solution for 1 hour using a long pipette. The straws were removed and the tubes carefully capped. Each solution was then heated at 60 ℃ for 3 hours using an oil bath. The solutions were allowed to cool to room temperature and combined in a round bottom. The crude polymer was dried under reduced pressure. Molecular weight was obtained by MALS 55,000(PDI 2.1); polymer composition using H¹NMR gave 74:26 Amine: Alkyl (Amine: Alkyl).

And (4) GPC fractionation. The dried crude polymer was added with 75% dichloromethane, 25% tetrahydrofuran and 0.2% triethylamine at 50 mg/mL. Then in Jordi Gel DVB500mm/22mm columnThe polymer was fractionated by injecting 10mL with a flow rate of 5 mL/min. Early fractions were collected from 15-17 minutes and late fractions were collected from 17-19 minutes. Components 15-17 Mw 138,000(PDI 1.1); components 17-19 Mw64,000(PDI 1.2).

MALS analysis. About 10mg of polymer was dissolved in 0.5mL of 89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. Molecular weight and Polydispersity (PDI) were measured with a Wyatt Helos II multi-angle light scattering detector connected to Shimadzu corporation (Shimadzu) science HPLC using a Jordi 5. mu.7.8X 300 mixed bed LS DVB column. Crude polymer: MW:55,000(PDI 2.1), fraction 15-17: MW 138,000(PDI:1.1), fraction 17-19: MW64,000(PDI 1.2).

The purified BOC-protecting polymer was reacted with 2M HCl in acetic acid (7mL) for 0.5 hours to remove the BOC protecting group and generate the amine. 15mL of dH₂O is added for reaction, the solution is transferred into a cellulose dialysis bag with 3500MW cut-off, dialyzed for 24 hours with high salt and then with dH₂O dialysis for 18 hours. The contents were lyophilized and then dissolved in DI H at a concentration of 20mg/mL₂And O. The polymer solution is stored at 2-8 ℃.

v) synthesizing water-soluble, amphiphilic and membrane active poly (vinyl ether) polyamine terpolymer under the protection of nitrogen gas layerXmol% of an amine protected vinyl ether (such as 2-ethyleneoxyethylphthalimide) was added to dry methylene chloride in an oven dried round bottom flask. Adding into the solutionYmol% of a low-hydrophobic group (e.g. propyl, butyl) vinyl ether and optionallyZmol% of highly hydrophobic groups (e.g.dodecyl, octadecyl) vinyl ether (FIG. 1). The solution was placed in a bath at-50- -78 ℃ to precipitate 2-ethyleneoxyethylphthalimide. To this solution was added 10 mol% BF₃·(OCH₂CH₃)₂Then reacting for 2-3 hours at-50 to-78 ℃. The polymerization was terminated by adding a methanol solution containing ammonium hydroxide. The polymer was dried under reduced pressure and then precipitated into 1, 4-dioxane/methanol (2/1). 20mol equivalents of hydrazine were added per phthalimide to remove the protecting groups on the amine. The solution was refluxed for 3 hours and then dried under reduced pressure. The obtained solid solutionAfter refluxing in 0.5mol/L HCl for 15 min to form the hydrochloride salt of the polymer, it was diluted with distilled water and refluxed for another 1 h. The solution was then neutralized with NaOH, cooled to Room Temperature (RT), transferred to a molecular cellulose dialysis bag, dialyzed against distilled water, and then lyophilized. Size exclusion or other chromatography may be used to further purify the polymer. The molecular weight of the polymer was evaluated with columns according to standard procedures, including analytical size exclusion chromatography and multi-angle light scattering (SEC-MALS) size exclusion chromatography.

Polymer DW 1360. An amine/butyl/octadecyl poly (vinyl ether) terpolymer was synthesized from 2-vinyloxyethylphthalimide (5g, 23.02mmol), butyl vinyl ether (0.665g, 6.58mmol), and octadecyl vinyl ether (0.488g, 1.64mmol) monomers. 2-ethyleneoxyethylphthalimide was added under an argon blanket to 36mL of anhydrous dichloromethane in a 200mL oven dried round bottom flask containing a magnetic stir bar. To this solution was added butyl vinyl ether and n-octadecyl vinyl ether. The monomers were completely dissolved at Room Temperature (RT) to give a clear homogeneous solution. The reaction vessel containing the clear solution was then placed in a-50 ℃ bath generated by adding dry ice to a 1:1 solution of ACS grade denatured alcohol and ethylene glycol, enabling the formation of a visible precipitate of phthalimide monomer. After cooling for about 1.5 minutes, BF was added₃·(OCH₂CH₃)₂(0.058g,0.411mmol) to start the polymerization. The phthalimide monomer dissolves after the polymerization starts. The reaction was carried out at-50 ℃ for 3 hours. The polymerization was stopped by adding 5mL of 1% ammonium hydroxide in methanol. The solvent was then removed by rotary evaporation.

The polymer was then dissolved in 30mL of 1, 4-dioxane/methanol (2/1). To this solution was added hydrazine (0.147g,46mmol) and the mixture was heated to reflux for 3 hours. The solvent was then removed by rotary evaporation and the resulting solid precipitated in 20mL of 0.5mol/L HCl and refluxed for 15 minutes, diluted with 20mL of distilled water, and refluxed for another 1 hour. The solution was then neutralized with NaOH, cooled to room temperature, transferred to a 3,500 molecular weight cellulose dialysis bag, dialyzed against distilled water for 24 hours (2 × 20L), and then lyophilized.

B) Melittin. All melittin peptides were prepared using standard peptide synthesis techniques in the art. Suitable melittin may be all L-amino acids, all D-amino acids (trans). The melittin sequence may be reversed (retro) independent of L or D type.

Claims (17)

1. A compound for reversibly modifying an amphiphilic membrane active polyamine, the compound comprising: a targeting ligand covalently linked to a dipeptide-amidobenzyl-carbonate, having the formula:

wherein

R1 is-CH₃，

R2 is- (CH)₂)₃-NH-C(O)-NH₂，

R4 is uncharged and comprises a targeting ligand, an

Z is

2. The compound of claim 1, wherein the targeting ligand comprises an asialoglycoprotein receptor (ASGPr) ligand.

3. The compound of claim 2, wherein the ASGPr ligand is selected from the group consisting of: lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine and N-isobutyrylgalactosamine.

4. The compound of claim 1, wherein Z is

5.A compound for reversibly modifying an amphiphilic membrane active polyamine, comprising the structure shown below:

wherein,

r comprises a steric stabilizer, wherein the steric stabilizer is a compound of formula (I),

r1 is-CH₃、-CH₂-C₆H₅or-CH- (CH)₃)₂；

R2 is- (CH)₂)₃-NH-C(O)-NH₂Or- (CH)₂)-C(O)-NH₂(ii) a And is

Z is

6. The compound of claim 5, wherein R is neutral.

7. The compound of claim 6, wherein the steric stabilizer is polyethylene glycol (PEG).

8. A delivery polymer for delivering a polynucleotide to a cell in vivo, the delivery polymer comprising:

M¹ _y–P–M² _z

wherein:

p is an amphiphilic membrane-active polyamine,

M¹comprises the following structure:

wherein R4 is uncharged and comprises a targeting ligand, R1 is-CH₃、-CH₂-C₆H₅or-CH- (CH)₃)₂And R2 is- (CH)₂)₃-NH-C(O)-NH₂Or- (CH)₂)-C(O)-NH₂Wherein the uncharged hydrophilic amino acid is uncharged at neutral pH,

M²comprises the following structure:

wherein R comprises a steric stabilizer and R1' is-CH₃、-CH₂-C₆H₅or-CH- (CH)₃)₂And R2' is- (CH)₂)₃-NH-C(O)-NH₂Or- (CH)₂)-C(O)-NH₂，

y and z are each integers greater than or equal to zero,

the value of y + z is greater than 50% of the primary amine on the polyamine P, as determined by the amine content on P in the absence of any masking agent, and

the delivery polymer M¹ _y–P–M² _zHas no membrane activity

9. The delivery polymer of claim 8, wherein the steric stabilizer is PEG.

10. The delivery polymer of claim 8, wherein the targeting ligand comprises a cell receptor ligand.

11. The delivery polymer of claim 10, wherein the cell receptor ligand comprises an ASGPr ligand.

12. The delivery polymer of claim 11, wherein the ASGPr ligand is selected from the group consisting of: lactose, galactose, N-acetylgalactosamine, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine and N-isobutyrylgalactosamine.

13. The delivery polymer of claim 8, wherein the amphiphilic membrane active polyamine is selected from the group consisting of: random, block or alternating polymers are synthesized.

14. The delivery polymer of claim 8, wherein the amphiphilic membrane active polyamine is melittin.

15. The delivery polymer of any one of claims 13 to 14, wherein the amphiphilic membrane active polyamine is further covalently linked to the polynucleotide.

16. The delivery polymer of claim 8, wherein the polynucleotide comprises an RNA interference polynucleotide.

17. The delivery polymer of claim 16, wherein the RNA interference polynucleotide is selected from the group consisting of: DNA, RNA, dsRNA, siRNA and miRNA.