WO1993013788A1 - Conjugated peptide derivatives containing an acceptor site for glycosylation and therapeutic uses thereof - Google Patents

Abstract

The present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation by oligosaccharyltransferase, and the use of such peptide derivatives for delivery of active agents to the lumen of the endoplasmic reticulum (ER). Therapeutic methods and compositions based on such delivery are also provided. The conjugates of the invention comprise (a) a tripeptide derivative of the formula R1-Asn-X-Y in which X is any amino acid except Pro, and Y is Ser or Thr or a derivative thereof, and R1 is an N-terminal group that is not one or more amino acids; and (b) an active agent conjugated to the tripeptide derivative. The conjugates are capable of being glycosylated by oligosaccharyl transferase, and are permeable to cell membranes until glycosylated by oligosaccharyltransferase in the lumen of the Er. In a specific embodiment, the active agent in the conjugate of the invention is an oxidizing agent, e.g. a diazene dicarbonyl covalently linked to amiloride. In another embodiment, the invention provides methods of treating cystic fibrosis, by administering a conjugate of the invention in which the active agent is an oxidizing agent.

Description

CONJUGATED PEPTIDE DERIVATIVES CONTAINING AN ACCEPTOR SITE FOR GLYCOSYLATION AND THERAPEUTIC USES THEREOF

1. INTRODUCTION

The present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation, and use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum. Therapeutic methods based on such concentration are also provided.

2. BACKGROUND OF THE INVENTION The synthesis and maturation of secretory, lysosomal, and membrane proteins in vertebrate cells involves the participation of various subcellular organelles. After synthesis in the rough endoplasmic reticulum, the protein is moved to the Golgi complex, and then sorted to lysoso es or the plasma membrane or secretory vesicles.

Ribosomes are complexes that carry out protein synthesis within the cell by reading the three letter genetic code (codon) of each messenger RNA. The endoplasmic reticulum (ER) is an interconnected series of flattened, generally layered, sacs within the cell. Ribosomes that are synthesizing secretory and integral membrane (ER, Golgi, and plasma membrane) proteins are tightly bound to the membrane of the ER (which is termed the rough ER with such bound ribosomes) . Secretory proteins are transported across the ER membrane into the lumen, or cisterna, of the ER during synthesis; membrane proteins become inserted into the ER membrane during synthesis. A signal sequence, characteristically near the N-terminus of the newly synthesized protein and consisting of one or more positively charged amino acids followed by 6-12 continuous hydrophobic residues, directs a protein to the ER, and inserts itself into the ER membrane, with the aid of the signal recognition particle. The signal sequence is cleaved off by signal peptidase, localized in the lumen of the ER. Other topogenic sequences within membrane proteins, e.g., stop- transfer membrane anchor sequences, function to orient the protein within the membrane. The protein traverses the ER membrane in an unfolded state. After insertion into or traversal of the ER membrane, the newly synthesized proteins can undergo additional maturation modifications in the ER lumen, including formation of disulfide bonds and proper folding of the protein, formation into oligomers, and addition and modification of carbohydrates. Disulfide bonding stabilizes the tertiary structure of proteins, and is important for proper maturation and activity of the protein. Formation of multi-chain oligomeric proteins from their subunit constituents also occurs in the ER. Polypeptides that are misfolded are prevented from moving out of the ER and along their normal maturation pathway; such proteins either accumulate or are degraded in the ER via an active degradative pathway (Stafford and Bonifacino, 1991, J. Cell Biol.

115(5) :1225-1236; Klausner and Sitia, 1990, Cell 62:611-614; Bonifacino and Lippincott-Schwartz, 1991, Curr. Opin. Cell Biol. 3:592-600).

In eukaryotes, glycosylation of proteins can be classified as O-linked (linked to the hydroxyl group oxygen of serine, threonine, and in collagen, hydroxylysine) and N-linked (linked to the amide nitrogen of asparagine) . Glycosyltransferases are enzymes that catalyze the transfer of sugar to newly synthesized proteins; a different type of glycosyltransferase catalyzes the addition of different sugars. All known glycosyltransferases are integral membrane proteins with their active sites within the lumen of the ER or Golgi, where sugar transfer thus occurs.

All N-linked oligosaccharides are synthesized from a common precursor in the ER. In the lumen of the ER, the complete branched oligosaccharide, consisting of three glucose, nine mannose, and two N-acetylglucosamine molecules, is transferreα by the enzyme oligosaccharyltransferase from oligosaccharylpyrophosphoryldolichol to an asparagine residue in an -Asn-X-Ser/Thr- acceptor site (where X is any amino acid except proline) on the nascent protein (Czichi et al., 1977, J. Biol. Chem. 252:7901-7904; Hart et al., 1979, J. Biol. Chem. 254:9747-9753). Oligosaccharyltransferase is a luminally oriented integral membrane protein of the ER, and the glycosylated protein formed by transfer of the oligosaccharide is sequestered within the endoplasmic reticulum (Hanover and Lennarz, 1980, J. Biol. Chem. 255:3600-3604). An in vitro study has shown that amino-ter inal derivatives of Asn-Leu-Thr can act as substrates for oligosaccharyltransferase, while asparagine derivatives of the tripeptide were inactive as substrates or inhibitors of the enzyme ( elply et al., 1983, J. Biol. Chem. 258:11856-11863). A study has suggested that transport from the ER to the cell surface is an unselective process, by comparing the rate of transport of exported proteins with that of an intracellular bulk phase marker; the bulk phase marker used was a tripeptide derivative containing the Asn-X-Ser/Thr acceptor site for glycosylation ( ieland et al., 1987, Cell 50:289-300). i

Immediately after transfer of the oligosaccharide to the protein, catalyzed by oligosaccharyltransferase, certain sugar residues are removed by different enzymes. Further processing of the N-linked oligosaccharide, to the high-mannose or complex form, is completed in the Golgi vesicles. The glycoprotein is transported via transport vesicles from the cis Golgi to the trans Golgi to the trans Golgi reticulum, from where it is sorted to lysosomes or to transport vesicles, or secretory vesicles which eventually fuse with the plasma membrane.

For a general discussion of the foregoing, see Darnell et al. , 1990, Molecular Cell Biology. 2d Ed., W.H. Freeman & Co., New York, pp. 639-680; Pfeffer and Rothman, 1987, Ann. Rev. Biochem. 56:829- 852.

3. SUMMARY OF THE INVENTION The present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation by oligosaccharyltransferase, and the use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum (ER) . Therapeutic methods and compositions based on such delivery are also provided. The conjugates of the invention comprise (a) an amino- terminal blocked derivative of the tripeptide Asn-X-Y, in which X is any amino acid except Pro, and Y is Ser or Thr; and (b) an active agent conjugated to the derivative. The conjugates are capable of being glycosylated by oligosaccharyl transferase, and are permeable to cell membranes until glycosylated by oligosaccharyltransferase in the lumen of the ER. In a specific embodiment, the active agent in the conjugate of the invention is a thiol-oxidizing agent, e.g. , a diazene dicarbonyl compound. In another embodiment, the active agent in the conjugate is a sulfhydryl-alkylating agent such as maleimide or a maleimide derivative. In yet another embodiment, the invention provides methods of treating cystic fibrosis, by administering a conjugate of the invention.

4. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation by oligosaccharyltransferase, and the use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum (ER) . Therapeutic methods and compositions based on such delivery are also provided. The conjugates of the invention comprise (a) an amino- terminal blocked derivative of the tripeptide Asn-X-Y, in which X is any amino acid except Pro, and Y is Ser or Thr, which derivative is an acceptor substrate for glycosylation by oligosaccharyltransferase; and (b) an active agent conjugated to the tripeptide derivative. Such conjugation is preferably by direct derivatization of the tripeptide derivative with the active agent. Such an active agent exerts a biological effect by virtue of its interaction with other molecule(s) in the lumen of the ER, by chemical reaction or by noncovalent or covalent binding with such molecule(s). The conjugate is biocompatible (nontoxic and not highly immunogenic) . The conjugate is permeable to cell membranes until glycosylated by oligosaccharyltransferase within the lumen of the ER, at which point it becomes impermeable to cell membranes and thus localized within the ER lumen. Moveover, the active agents present in the conjugates of the invention are compounds which, by performing their activity in the lumen of the ER, exert a therapeutic effect. It also follows that the active agent is not an inhibitor of oligosaccharyltransferase.

The invention is further detailed in the subsections below.

4.1. THE CONJUGATES CONTAINING AN ACCEPTOR SITE FOR GLYCOSYLATION BY OLIGO- SACCHARYLTRANSFERASE

The tripeptide derivatives in the conjugates of the invention are substrates of oligosaccharyltransferase that contain an acceptor site for glycosylation by the enzyme. The conjugates of the invention are preferably such tripeptide derivatives which have incorporated into their structure an active agent. In a specific embodiment, the active agent is a thiol-oxidizing agent.

Alternatively, the active agent is a sulfhydryl- alkylating agent, such as a maleimide or a maleimide derivative. The thiol-oxidizing agents are preferably diazene dicarbonyl compounds, containing the following structural feature:

0 O

-C-N=N-C-

In a specific embodiment, the active agent is an agent that prevents the abnormal misfolding, assembly or increased levels of degradation in the ER lumen of a defective lysosomal or secretory or cell membrane protein associated with a disease or disorder, thus allowing the protein to continue along its normal maturation pathway to secretion or to the plasma membrane or a lysosome. In a particular embodiment, such an agent is an oxidizing agent. Other specific examples of active agents are described below.

The tripeptide derivatives in the conjugates of the invention are N-blocked derivatives of the tripeptide Asn-X-Y (written in the amino- to carboxy- terminal direction) , in which X is any amino acid except Pro, and Y is Ser. or Thr. In the tripeptide derivatives of the conjugates of the invention, the amino terminus is derivatized, preferably to contain a lipophilic group, since unblocked tripeptides are not acceptors of glycosylation by oligosaccharyltransferase (Hart et al. , 1979, J. Biol. Chem. 254:9747-9753). Furthermore, the side chain group of Asn should not be derivatized since such derivatization abolishes oligosaccharide acceptor activity ( elply et al., 1983, J. Biol. Chem. 258:11856-11863). The tripeptide can also be derivatized at its C-terminus by groups including, but not limited to, amino and alkylamino groups. Tripeptide derivatives with N- or C-terminal groups that increase lipophilicity of the* molecule are preferred. It is preferred to derivatize both the N- terminus and C-terminus of the tripeptide, preferably by amidation, thereby eliminating ionic charge, decreasing the hydrophilicity of the molecule, and facilitating diffusion of the tripeptide across the cell membranes.

The conjugates of the invention are of the general formula (I)

(I)

? in which R is H or methyl. One or more of R^l, R², R³, and R⁴ can be an active agent, to form a conjugate of the invention.

In one embodiment, in compound (I) , the active agent is R¹, R¹ being a diazene dicarbonyl compound of structure

0 0 0 0 0

, II II or || || I R⁵-C-N=N-C- R^S-C-N=N-C-NH-(CH₂)_n-C- in which R⁵ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy; and n is an integer of 1 or more. In this embodiment, R² is H or a lower alkyl; R³ is the side chain of any natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; and R⁴ is OH,

NH₂, a mono- or di-substituted amino, or alkoxy or aralkoxy.

In a second embodiment, in which the active agent is formed by R^l and R², R¹ and R² together form a ring so that the conjugate has the following structure:

(ID

°l with R, R³, and R⁴ corresponding to the chemical groups as described above.

In a third embodiment, in which.the active agent in compound (I) is R³, R³ has the following structure:

R⁶ 0 O

in which n is an integer, of 1 or more, preferably in the range of 1-4; R⁶ is H or a lower alkyl; R⁷ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, a mono- or di- substituted amino, or an alkoxy, aryloxy, or aralkoxy; or R⁶ and R⁷ can together form a ring structure. In this embodiment, R¹ is an acyl group, including but not limited to acetyl, alkanoyl (e.g., octanoyl) , benzoyl, benzyloxycarbonyl, tert-butoxycarbonyl, and the like. The acyl group is preferably in the range of C₂ to C₁₀, most preferably C₂ to C₈. In this embodiment, R² is H. Alternatively, R¹ and R² can together form a ring structure, e.g., such that

In this embodiment, R⁴ is OH, NH₂, a mono- or di- substituted amino, or alkoxy or arylkoxy. In a fourth embodiment, in which the active agent in compound (I) is R⁴, R⁴ has the following structure:

R⁸ R⁹ 0 0

-N-(CH₂)₀-N-C-N=N-C-R 10 to in which R⁸ and R⁹ are each independently H, a lower alkyl, aryl, or aralkyl; R¹⁰ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, mono- or di-substituted amino, or an alkoxy, aryloxy, or aralkoxy; or R⁸ and R⁹ can together form a ring structure; or R⁹ and R¹⁰ can together form a ring structure, As one example where R* and R^ιu form a ring:

in this embodiment, R¹ is an acyl group, R² is H, or R¹ and R² can together form a ring structure. R³ is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl.

In a fifth embodiment, in which the active agent in compound (I) is a maleimide derivative formed of R¹ and R², the compound has the structure of (III) or (IV) :

(III)

//

in which Rⁿ and R¹² are each independently H or a lower alkyl; R³ is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, a mono- or di-substituted amino, or alkoxy or aralkoxy; and n is an integer of 1 or more.

In a sixth embodiment, in which R³ in compound (I) is a maleimide derivative, R³ has the following structure:

in which n is an integer of 1 or more, preferably in the range of 1-4 , and R¹¹ and R¹² are each independently H or a lower alkyl. In this embodiment, R^l is an acyl group; R² is H; or R¹ and R² together form a ring structure; R⁴ is OH, NH₂, a mono- or di-substituted amino, or alkoxy, or aralkoxy.

In a seventh embodiment, in which R⁴ in compound (I) is a maleimide derivative, R⁴ has the following structure:

in which n is an integer of 1 or more, preferably in the range of 1-4; R¹³ is H, alkyl, aryl, or aralkyl; R^u and R¹² are each independently H or a lower alkyl; R^l is an acyl group; R² is H; or R¹ and R² together form a ring structure; R³ is the side chain of a natural amino thacid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl.

In an embodiment in which R¹ or R² is not the active agent, in preferred aspects of the embodiments described above for compound (I) , R² is H, and R^l is an acyl group, in the range of C₂ to C,₀, preferably C-_ to C_g, including but not limited to, one of the following:

CH₃-CO- = acetyl

'BuOCO- = tert-butoxycarbonyl c₆H₅-CO- = benzoyl

C₇H₁₅-CO- = octanoyl

In an aspect in which R³ is not the active agent, preferably R³ is the side chain of any natural amino acid, and is most preferably

(the side chain of Leu) , or

_

(the side chain of Tyr) .

In an aspect in which R⁴ is not the active agent, R⁴ is preferably NH₂.

Many other active agents can be used, as will be known to one skilled in the art. The selection of an active agent will depend on the /5 therapeutic effect it is desired to achieve (see Section 4.4, infra) .

4.2. SYNTHESIS OF THE CONJUGATES OF THE INVENTION The conjugates of the invention are synthesized by any methods known in the art.

For example, any peptide portion of the conjugate can be prepared in brief, by solid phase peptide synthesis which consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha-protected amino acids. The protecting groups may be any known in the art or those described infra. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394-1397;

Marki et al., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Patent Nos. 4,305,872 and 4,316,891. In a specific embodiment, tripeptide derivatives can be synthesized as described ( elply et al., 1983, J. Biol. Chem. 258:11856-11863), using various protected amino acids and either the mixed anhydride procedure (Anderson et al., 1967, J. Am. Chem. Soc. 89:5012- 5017) or the p-nitrophenyl ester method (Bodanski, M. , 1979, in The Peptides: Analysis. Synthesis, Bioloσv. Gross and Meienhofer, eds.. Vol. 1, Academic Press, New York, pp. 105-196) . In another embodiment, peptides are synthesized as described in Section 6, infra, prior to derivatization with the active agent.

The active agent is preferably conjugated directly to the tripeptide derivative, i.e., the ⁱf tripeptide derivative is derivatized by the active agent, rather than through a polyfunctional linker molecule. To so derivatize the tripeptide derivative, any available reactive group on the molecule is used, or else one can be incorporated into the tripeptide derivative or active agent, for use in conjugating the active agent and tripeptide derivative. As used herein, the term "reactive group" refers to a functional group that can react with a second functional group so as to form a covalent bond between the active agent and tripeptide derivative. The term "functional group" retains its standard meaning in organic chemistry. Typical functional groups are thiol groups and amino groups. Protecting groups for use in synthesis can be any of the large number of protecting groups known in the art. For example, an acetyl group can be added to a free amino group by treatment with acetic anhydride. Alternatively, a carbobenzoxy group can be added by treatment with carbobenzoxy chloride. Other N-protecting groups that are useful include the formyl, L-butoxycarbonyl, trifluoroacetyl, tosyl, p- nitrocarbobenzoxy, cyclopentyloxycarbonyl, and phenoxycarbonyl groups. n a specific embodiment in which the active agent is a diazene dicarbonyl compound, the following compounds for use in the synthesis of the conjugates of the invention can be obtained or synthesized as described below:

CH₃CH₂0₂C-N=N-C0₂CH₂CH₃ is commercially available, or alternatively, can be synthesized as described by Rabjohn (1955, in Organic Syntheses. Collective Vol. 3, Organic Syntheses, Inc., p. 375) or Kauer (1963, in Organic Syntheses. Collective Vol. 4, Organic Syntheses, Inc., p. 411). Maleimides can be synthesized by methods known in the art (see, e.g., U.S. Patent No. 4,623,734 granted November 18, 1986 by Kita et al.) or purchased from a commercial vendor.

Representative syntheses of conjugates of the invention, presented by way of example, but not limitation, are described in the subsections below.

4.2.1. SYNTHESIS OF A CONJUGATE OF THE PRESENT INVENTION

A conjugate of the present invention having the formula (V) in which the active agent comprises a diazene dicarbonyl oxidizing agent can be prepared by the method described below. The groups R and R³ have the meanings described previously, above.

A tripeptide having a primary amino group, such as compound (VI) , illustrated below, is allowed to react with an

(VI)

ώ excess amount of a diazene dicarboxylic acid ester, such as dimethyl azodicarboxylate or diethyl azodicarboxylate, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature. Preferably, the inert solvent is dioxane. The resulting diazene monoester (VII) is

then isolated using techniques well known in the art including, but not limited to, fractional crystallization, liquid chromatography and the like. The diazene monoester (VII) is then allowed to react with an excess amount of a preselected nucleophile, Nu, such as ammonia, dimethylamine, piperidine, pyrrolidine and the like, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature. Preferably, the inert solvent is dioxane. The product, which is the therapeutic oxidizing agent of the formula (V) , is then isolated using standard methods well known in the art. kf

4.2.2. SYNTHESIS OF A SECOND CONJUGATE OF THE PRESENT INVENTION

The therapeutic conjugate of the formula (VIII) , in which R can be an H or methyl and n can have the values described previously, above (e.g., or 4) , can be prepared by the following method.

A tripeptide having a primary amino group, such as compound (IX) , illustrated below, is allowed to react with an

excess amount of a diazene dicarboxylic acid ester, such as dimethyl azodicarboxylate or diethyl azodicarboxylate, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature. Preferably, the inert solvent is dioxane. The resulting diazene monoester (X) is

then isolated using techniques well known in the art including, but not limited to, fractional crystallization, liquid chromatography and the like.

The diazene monoester (X) is then allowed to react with an excess amount of a preselected nucleophile, such as ammonia, dimethylamine, piperidine, pyrrolidine and the like, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature. Preferably, the inert solvent is dioxane. The product, which is the therapeutic oxidizing agent of the formula (VIII) , is then isolated using standard methods well known in the art.

4.2.3. SYNTHESIS OF A THIRD CONJUGATE OF THE PRESENT INVENTION

The therapeutic conjugate of the formula

(XI) , in which R can be H or methyl, and R² and R³ have the meanings described previously above, and n can be an integer greater than or equal to 2, can be prepared by the following method. '?

An amino-protected serine or threonine derivative, such as a compound of the formula (XII) , in which R can be H or methyl, respectively, illustrated below, is

allowed to react under standard amide bond forming conditions with an excess amount of a diamino compound of the formula (XIII) , in which one of the amino groups is blocked

H

H₂N-(CH₂)_n-N-^tBOC (XIII) 0 by a suitable protecting group, such as a tertiarybutoxycarbony1 group (^lBOC) . The isolated condensation product is then partially deprotected at the amino acid portion of the molecule by hydrogenolysis, in the case of a benzyloxycarbon l (Cbz) protecting group to provide an intermediate product of the formula (XIV) . Additional amino acid residues may then be added to the growing peptide chain, as desired.

For example, a tripeptide having the general formula Asn-Xaa-Ser(Thr) -NH₂, illustrated below as the compound (XV) (in which R³ is the side chain of an amino acid)

is obtained by sequential, addition of the amino acid residues of choice to the intermediate product of the formula (XIV) . The total synthesis of the therapeutic 4/ conjugate (XI) , which bears the diazine dicarbonyl group, is then carried out by removal of the terminal amino-protecting group, such as 'BOC, condensation with a diazene dicarboxylic acid ester, and conversion of the resulting monoester to the diamide of the present invention, as already discussed in detail above.

4.3. IN VITRO ASSAYS After the conjugate is made as set forth in Section 5.3 hereof, it is preferably tested in vitro to ensure that it is permeable to cell membranes and can act as an acceptor for glycosylation by oligosaccharyltransferase. Such assays can be carried out by any method known in the art. In preferred aspects, the assay is carried out by exposing intact cells or rough microsomes to the conjugate, and detecting glycosylation of the conjugate within the lumen of the ER. Such assays can be carried out as described in Section 7, infra (see also Welply et al., 1983, J. Biol. Chem. 258:11856-11863; Wieland et al., 1987, Cell 20:289-300. Rough microsomes are small closed vesicles formed by fragments of the rough ER produced upon homogenization of cells; microsomes have the same orientation (ribosomes on the outside of the vesicles) as that of the ER within the cell (Darnell et al., 1990, Molecular Cell Biology. 2d Ed., .H. Freeman & Co. , New York, p. 646).

4.4. UTILITY OF THE INVENTION The conjugates of the invention can be administered therapeutically, where a therapeutic effect is mediated by the active agent upon concentration in the ER lumen by virtue of the ability of the conjugate to be glycosylated therein by oligosaccharyltransferase. Such glycosylation renders the conjugate impermeable to cell membranes such that the active agent is thereby concentrated in the ER lumen, where it performs its chemical activity(ies) or binding. The therapeutic methods of the invention are carried out by administration to a subject of an effective amount of the conjugates of the invention. The subject is preferably a mammal, including but not limited to animals such as cows, pigs, etc., and is most preferably human. Methods for prevention of disorders, by administering a therapeutic conjugate of the invention, are also provided.

In a preferred embodiment, the active agent is an oxidizing agent, and the conjugate is administered to a patient for treatment of a disorder involving a genetically mutated lysosomal or secretory or plasma, ER or Golgi membrane protein. Although Applicants do not intend to be limited to any specific mechanism, it is believed that delivery of such an oxidizing agent to the ER lumen allows oxidation of cysteine sulfhydryl groups therein, thereby permitting proper folding and cellular targeting of the protein that otherwise would not occur, or preventing its degradation. Diseases or disorders which can be treated in this manner include but are not limited to cystic fibrosis, emphysema, Tay-Sachs, lysosomal storage diseases, insulin receptor deficiency, familial hypercholesterolemia, Hunter's syndrome, and Hurler's syndrome. As discussed infra. cystic fibrosis is associated with a mutation in the transmembrane protein CFTR. The major genetic cause of emphysema and difficulty in breathing is due to a mutation in the secretory protein α,-antiprotease (α,- antitrypsin) (Darnell et al. , 1990, Molecular Ceil Biology. 2d Ed., .H. Freeman & Co., New York, p. OS

660) . Tay-Sachs disease is caused by a defect in the lysosomal enzyme beta-N-hexosaminidase A (id., p. 671) . Other lysosomal storage diseases are caused by defective lysosomal enzymes. Insulin receptor deficiency results from a mutant (plasma membrane) insulin receptor while familial hypercholesterolemia results from a mutant LDL (low density lipoprotein) (plasma membrane) receptor. Hunter's syndrome and Hurler's syndrome are caused by genetic defects in the lysosomal enzymes which catabolize sulfated mucopolysaccharides (Darnell et al., supra at p. 671).

In a specific embodiment, the active agent is a diazene dicarbonyl compound (I) such as diamide, or a thiol-oxidizing agent such as a maleimide derivative, and the conjugate of the invention is administered to treat cystic fibrosis. Cells from cystic fibrosis patients show a defect in the putative protein product of the cystic fibrosis gene (Rommens et al., 1989, Science 245:1059-1065; Riordan et al., 1989, Science 245:1066-1073; Kerem et al., 1989, Science 245:1073-1080) designated the CFTR (cystic fibrosis transmembrane conductance regulator; Riordan et al., 1989, Science 245:1066-1073). CFTR is an integral membrane protein that appears to act as a chloride channel (Anderson et al., 1991, Cell 67:775- 784; Rich et al., 1990, Nature 347:358-363; Drumm et al., 1990, Cell 62:1277-1233). The present invention provides for treatment of cystic fibrosis by exposure of mutant CFTR in the lumen of the ER, to the oxidizing or alkylating agent that is the active agent in the conjugate of the invention. Although Applicants do not intend to be limited to a specific mechanism, it is believed that the oxidizing or alkylating agent inhibits degradation and/or promotes the correct folding/assembly in the ER lumen of the mutant CFTR that otherwise *1would be abnormally processed or degraded and never reach the plasma membrane, thus achieving proper processing of CFTR to the cell membrane. The conjugate is administered so as to allow, or preferably target, delivery to the in vivo cellular location of CFTR, (Crawford et al. , 1991, Proc. Natl. Acad. Sci. USA 88:9262-9266), namely epithelial cells, such as. those lining sweat ducts, small pancreatic ducts, and intestinal crypts, and in the kidney, and in the lung.

Suitable in vitro and n vivo assays can be used to demonstrate therapeutic utility of the conjugates of the invention. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

An animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/1 mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R. , et al., eds. , Elsevier/North-Holland, New York, pp. 207-219) , that develop a spontaneous rheumatoid arthritis-like disease (Hang et al. , 1982, J. Exp. Med. 155:1690-1701).

4.4.1. THERAPEUTIC ADMINISTRATION AND COMPOSITIONS Various delivery systems are known and can be used to administer the conjugates of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, etc. In view of the somewhat hydrophobic character of the conjugate (so as to render it cell membrane- permeable) , encapsulation in liposomes or other type of lipid layer is preferred. Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active compounds.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. In a preferred aspect, the conjugate of the invention is formulated as an inhalant.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The conjugates of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. The amount of the conjugate of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.

5. EXAMPLE: SYNTHESIS OF TRIPEPTIDE DERIVATIVES

5.1. SYNTHESIS OF N-ACYL-ASN-TYR-THR-NH,

Tripeptide derivatives of the form N-acyl- Asn-Tyr-Thr-NH₂ are synthesized as follows (Wieland, et al., 1987, Cell 50:289-300):

Peptide Synthesis and Purification: Solid phase syntheses are performed according to Stewart and Young (1984, Solid Phase Peptide Synthesis. 2d Ed., Pierce Chemical Co. , Rockford, Illinois) . Benzyhydrylamine-Thr-resin is used throughout. Boc- protected amino acids as well as the resin are from Peninsula Laboratories. The extent of the reaction is monitored at each step by assaying with ninhydrin. The peptides are cleaved from the resin by solvolysis with anhydrous hydrogen fluoride. The resultant peptides are purified by washing the resin with ether and subsequent extraction with 50% acetic acid. The extract is dried by lyophilization, and the peptides are further purified by high performance liquid chromatography (HPLC) . The following conditions are used: 5% acetonitrile in 0.1% trifluoroacetic acid (TFA) for 10 min after injection; thereafter, a linear gradient of 5%-65% acetonitrile in a 0.1% is applied at 1 ml/min. The NH₂-Asn-Tyr-Thr-NH₂ eluted at about 21% acetonitrile. A reversed phase RP18 column (Lichrosorb, Merck) is used with a Beckman controller and delivery system, and the effluent is monitored in a flow cell at 280 nm. The purity of the resulting peptides is confirmed by amino acid analysis after acid hydrolysis (8 N HCl, 18 hr, 105°C) .

Acylation of the Peptides: Acetylation is performed with the resin-linked, boc-protected peptides according to Stewart and Young (1984, Solid Peptide Synthesis, 2d Ed., Pierce Chemical Co.,

Rockford, Illinois) , and the N-acetyltripeptide is solvolyzed and purified as above. Acylation with butyric, octanoic, decanoic, dodecanoic, myristic, and palmitic acids is performed essentially according to Bodanski (1979, in The Peptides: Analysis. Synthesis. Biology. Vol. 1, Gross and Meienhoser, eds.. Academic Press, New York, pp. 105-196) using the corresponding fatty acyl p-nitrophenyl esters, p- Dimethylaminopyridine is used as a catalyst. The reactions are performed in anhydrous dimethyl formamide (DMF) for 24 hour at room temperature. After addition of 5 vol of 70% methanol, the samples are treated with an equimolar amount of NaOH for 20 min at 37°C. Thereafter they are purified by chromatography on AG 1 x 8 ion exchange resin (acetate form) in 70% methanol. The eluants are chromatographed on Dowex AG 50 (H⁺ form) in 70% methanol, and the flow-throughs are lyophilized in a

Speed-Vac concentrator centrifuge. Final purification is achieved by HPLC on reversed phase columns as described for the purification of the free peptides.

The N-acetyl derivative elutes at about 23% acetonitrile, the N-octyl derivative at about 35% acetonitrile. fif

5.2. SYNTHESIS OF TRIPEPTIDE

DERIVATIVES OF ASN-LEU-THR

Various tripeptide derivatives of Asn-Leu-

Thr are synthesized as described by Welply et al.

(1983, J. Biol. Chem. 258:11856-11863). Various protected amino acids and either the mixed anhydride procedure (Anderson et al. , 1967, J. Am. Chem. Soc

89:5012-5017) or the p-nitrophenyl ester method

(Bodanski, M. , 1979, in The Peptides: Analysis.

Synthesis. Biology. Gross and Meienhofer, eds.. Vol.

1, Academic Press, New York, pp. 105-196) are used for peptide bond formation. Acyl substrates are made by reaction of a suitable p-nitrophenyl ester with the α- amino group of the tripeptide.

As a representative example, N^β-acyl-Asn-Leu-

Thr-NH₂ is synthesized as follows (Welply et al., 1983,

J. Biol. Chem. 258:11856-11863):

N°-Boc-Leu-Thr-NH₂ — N-methylmorpholine

(NMM) (1.32 ml, 12 mmol) is added to a stirred solution of Boc-Leu (2.77 g, 12 mmol) in tetrahydrofuran (25 ml) at -15°C, followed by the addition of isobutyl chlorofor ate (1.56 ml, 12 mmol).

Stirring continues for 7 min. A precooled (-15°C) solution of Thr-NH₂-HCl (1.85 g; 12 mmol) and NMM (1.32 ml, 12 mmol) in DMF (dimethylformamide) (14 ml) is then added. After stirring for 30 min at -15°C and 2 h at room temperature, the solvent is evaporated in vacuo. A citric acid solution (5%, 25 ml) is added to the residue, which causes Boc-Leu-Thr-NH₂ to separate out as a crystalline solid. The product is filtered, washed with water (30 ml) , and then with ether (30 ml) , and dried.

N^α-Boc-Asn-Leu-Thr-NH-, — Boc-Leu-Thr-NH₂ (0.93 g. 2.8 mmol) is dissolved in CH₂C1₂- ^xj* trifluoroacetic acid (15 ml; 1:1, v/v), and the solution is let stand at room temperature for 30 min. The solvent is then evaporated j-n vacuo. and the residue is precipitated by the addition of ether. The trifluoroacetate salt is isolated and dried. It is then coupled with Boc-Asn-ONp (0.99 g, 2.8 mmol) in the presence of NMM (0.31 ml, 2.8 M) in DMF (5 ml). After a reaction time of 24 h, the solvent is evaporated in vacuo. and the residue is dissolved in aqueous citric acid (5%, 25 ml) . The solution is then extracted with three 25-ml portions of ethyl acetate, which removes unreacted active ester and p- nitrophenol. The aqueous phase is extracted with six 25-ml portions of l-butanol, and the combined extracts are washed once with 20 ml of H₂0. The butanol layer is dried and evaporated n vacuo, and the residue is precipitated by the addition of ether. N^α-Boc-Asn-Leu- Thr-NH₂ is isolated and dried.

N°-Ac-Asn-Leu-Thr-NH₂ — The Boc-group from Boc-Asn-Leu-Thr-NH₂ (178 g, 0.4 mmol) is cleaved as described above. The resulting CF₃COOH-Asn-Leu-Thr-NH₂ peptide is then coupled with p-nitrophenyl acetate (72 mg, 0.44 mmol) in the presence of NMM (0.044 ml, 0.4 mM) in DMF (1 ml) . After a reaction time of 20 h, ether (50 ml) is added to the mixture. The solid obtained is filtered, and washed with ether. After drying, it is dissolved in water (10 ml) and stirred with Dowex 50W resin (-3 g) for 30 min to remove the salts and unreacted nucleophile. The resin is then filtered off, and the aqueous solution of N^β-Ac-Asn- Leu-Thr-NH₂ is evaporated to dryness. The resulting product is dissolved in ethanol (3 ml) and reprecipitated by the addition of ether (30 ml) . The solid obtained is filtered and dried. 6. IN VITRO ASSAYS OF THE ABILITY OF A COMPOUND TO PERMEATE CELL MEMBRANES . AND BE GLYCOSYLATED BY " OLIGOSACCHARYLTRANSFERASE The following assays can be used to demonstrate the ability of a tripeptide derivative or conjugate of the invention to permeate cell membranes and be glycosylated by oligosaccharyltransferase.

6.1. INTACT CELL ASSAYS

The compounds to be assayed for its ability to permeate cell membranes and be glycosylated by an oligosaccharyltransferase is labeled with ¹²⁵I. Where the compound contains a tyrosine, the following procedure can be used (Wieland et al., supra) :

Iodination of the Acceptor Compound: Up to 50 nmol of tyrosine containing compound in 50 μl of acetonitrile is added to 100 μl of 0.5 M NaP* (pH 7.5). Between 0.5 and 10 mCi of [¹²⁵I] Nal (carrier-free, ICN) is added. To this solution, 100 μl of chloramine T (Sigma) (2 mg/ml) in 0.05 M NaP* (pH 7.5) is added. After 2 min at room temperature, the reaction is stopped by addition of 400 μl of a solution of sodium bisulfite (2.4 mg/ml) in 0.05 M NaP, (pH 7.5). An additional 600 μl of water is added, and the solution is loaded onto a Sep Pack C18 cartridge (Waters) and washed first with 20 ml of 0.1% TFA and then with 20 ml of 5% acetonitrile in 0.1% TFA. The radiolabeled compound is eluted with 60% acetonitrile in 0.1% TFA. The purity of the iodinated peptides is confirmed by HPLC and by thin layer chromatography on Silica gel plates (S1250-PA Baker) in butanol/acetic acid/water (5:2.2 [v/v/v]) . After drying in a Speed-Vac concentrator centrifuge, the radiolabeled compound is dissolved in DMSO at about 5 x 10⁸ to 10 x 10⁸ cpm/μl. Cell Growth and Media. CHO Cells or HepG2 cells are used. Wild-type CHO cells are grown in suspension cultures in αMEM (GIBCO) as described (Balch et al. , 1984, Cell 39:405-416). CHO clone 15B cells are grown in monolayer.

HepG2 cells are grown as described (Strous and Lodish, 1980, Cell 22:709-717). Media are usually changed the day after passing, and the cells are used 3 days later. Incubation of Cells With the Compound to be

Assayed: In a typical experiment, CHO cells (2 x 10⁷ total cells) are washed once with buffer B (25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 5 mM KC1, 0.7 mM Na₂HP0₄) and then resuspended in growth medium without serum (that is additionally buffered with 20 mM HEPES [pH 7.4]) at a density of 1 x 10⁷ cells per ml. Then cycloheximide (100 μg/ml) is added. After incubation in suspension for 2-3 hr at 37°C, the ^12SI compound is added from a stock solution in DMSO (not exceeding a final concentration of 1% DMSO) . Typically, between 5 and 50 μCi is added per ml of suspension. Incubation is at 37°C with gentle stirring. Aliquots of 200 μl are removed after one hour and immediately chilled on ice and centrifuged in the cold in a Eppendorf centrifuge for 1 min. The supernatant media are separated from the cell pellets. Each cell pellet is extracted with 200 μl of buffer A (10 mM Tris-HCl [pH 7.4], 0.15 M NaCl, l mM CaCl₂, l mM MnCl₂, 0.5% Triton X-100) , and the supernatant after centrifugation in the microfuge for 1 min can be saved for further analysis. The media is made 0.5% in Triton X-100, and 1 mM MnCl₂ and 1 mM CaCl₂ are added from stock solutions.

For experiments with HepG2 cells, monolayers (35 mm) are washed once with buffer B at 32°C. Then 3$

300 μl of serum-free αMEM containing 100 μg/ml cyclohexi ide and 20 mM HEPES (pH 7.4) is added. After 2-3 hr at 32°C, ^,25I compound (5-50 μci/ml) is added, and the plates are further incubated at 32°C. Each time point consists of duplicate plates. The media are removed and kept on ice. The cells are extracted as follows: Each plate is rinsed with 0.9 ml of ice-cold buffer B containing 10% FCS. The monolayer is extracted with 300 μl of ice-cold buffer B containing 1% Triton X-100. Cell extracts and media are then analyzed by chromatography on Con-A-Sepharose columns as described below.

For treatment with tunicamycin, CHO cells and HepG2 cells are incubated for 1 or 5 hr at 37°c or 32°C, respectively, in the presence of 10 μg/ml tunicamycin (Sigma) . The tunicamycin stock solution used is 1 mg/ml in 250 mM NaOH.

Analysis of Glycocompounds from Cell Pellets and Media: Equivalent aliquots of Triton-extracts of cells and media (typically 100-200 μl volume) are passed through small columns (200-400 μl bed volume) of Con A-Sepharose (Pharmacia) in buffer A. The columns are washed with 5 successive 1 ml portions of buffer A and then eluted with three 500 μl portions of 0.5 M α-methylmannoside in buffer A. ^I25I radioactivity in the combined α-methylmannoside eluates is determined. ¹²⁵I-glycocompounds intended for further analysis (e.g., thin layer chromatography) are prepared similarly, but after the five washes with buffer A, five washes are performed with 1 ml each of buffer A without Triton X-100. Elution with α- methylmannoside is without Triton X-100 as well.

Using the above procedures, observation of binding to Con A (and elution with the hapten sugar α- methylmannoside) and inhibition by tunicamycin si

(decrease in the ^,25I counts) indicates that the ^l25 - labeled compound is taken up by the CHO or HepG2 cells, glycosylated in the lumen of the ER by oligosaccharyltransferase, and then secreted.

6.2. MICROSOMAL ASSAYS

The ability of a compound to act as a substrate of oligosaccharyltransferase is assayed in intact hen oviduct microsomes (as described by Welply et al., 1983, J. Biol. Chem. 258:11856-11863):

Oviduct Microsome Preparation — The magnum portion of freshly killed laying hens is freed from connective tissue, minced, homogenized, and centrifuged as described by Pless and Lennarz (1977, Proc. Natl. Acad. Sci. USA 74:134-138). Prior to use, microsomes are stored at -70°C at a concentration of -30 mg protein/ml.

Oligosaccharyltransferase Assays Using Endogenous Oligosaccharide Lipid — Standard reaction mixtures (50 μl final volume) contain 30 μM tritiated compound to be assayed (~5 X 10³ cpm) , 10 mM MnCl₂, and approximately 300 μg of microsomal protein in 50 mM Tris-HCl, pH 7.4, containing 140 mM sucrose, 25 mM NaCl, and 1 mM EDTA. Reactions are initiated by the addition of microsomes, and allowed to proceed at 30°C for 5 or 10 min. Reactions are terminated by addition of 50 μl of 20% trichloracetic acid. After 5 min on ice, samples are centrifuged in a Beckman microfuge 11 at setting 10 for 5 min. A 75 μl aliquot of the resulting supernatant is backwashed three times with 1 ml of ether, and gassed with N₂ to remove residual ether. The sample is then analyzed by gel filtration, affinity chromatography or paper chromatography; these procedures detect differences in migration between the glycosylated and unglycosylated compound. In addition, concanavalin A-agarose binding, and elution therefrom in the presence of methylmannoside also evidence glycosylation (see Welply et al., supra) . Any additions to the reaction mixture, such as dimethyl sulfoxide or detergent, are made prior to icrosome addition.

Alternatively, the oligosaccharyl¬ transferase assay can be carried out using newly synthesized oligosaccharide-lipid (see Welply et al., supra) or by quantitating the incorporation of labeled (radioactive) sugars into non-labeled acceptor substrates (Hart et al., 1979, J. Biol. Chem. 254:9747-9753; Hanover and Lennarz, 1990, J. Biol. Chem. 255:3600-3604).

The present invention is not to be limited in scope by the specific embodiments described herein since such embodiments are intended as but single illustrations of one aspect of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and describe herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference herei.n in thei.r enti.reties.

Claims

WHAT IS CLAIMED IS:

A compound of formula (I)

in which R is H or methyl; R¹ is an active agent; R² is H or a lower alkyl; R³ is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, a mono- or di-substituted amino, alkoxy or aralkoxy; which compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

2. The compound of claim 1 in which R¹ is

in which R⁵ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, or a mono- or di-substituted amino, or an alkoxy, aryloxy, or aralkoxy.

3. The compound of claim 1 in which R¹ is O O O

, II II II

R⁵-C-N=N-C-NH-(CH₂)_n-C- in which R^s is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, or a mono- or di-substituted amino, or an alkoxy, aryloxy, or aralkoxy; and n is an integer of 1 or more.

4. The compound of claim 1 in which R¹ is O O

(CH₃)₂N-C-N=N-C- and R² is H.

A compound of formula (II)

in which R is H or methyl; R³ is the side chain of any natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, a mono- or di-substituted amino, alkoxy, or aralkoxy; which compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

6. A compound of formula (I)

3Z in which R is H or methyl; R¹ is an acyl group; R² is H; or R^l and R² together form a ring structure; R⁴ is OH, NH₂, a mono- or di-substituted amino, alkoxy, or aralkoxy; R³ is an active agent; which compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

7. The compound of claim 6 in which R³ has the structure

R⁶0 O

in which n is an integer of 1 or more; R⁶ is H or a lower alkyl; R⁷ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, a mono- or di-substituted amino, or an alkoxy, aryloxy, or aralkoxy; or R⁶ and R⁷ together form a ring structure.

8. The compound of claim 7 in which R⁷ is dimethylamino, n is in the range of 1-4; R¹ is an acyl group in the range of two to ten carbon atoms; and R₂ is H.

9. A compound of formula (I)

(I)

3? in which R is H or methyl; R¹ is an acyl group; R² is H; or R¹ and R² together form a ring structure; R³ is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is an active agent; which compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

10. The compound of claim 9 in which R⁴ has the structure

R⁸ R⁹0 O

-N I-(CH₂)_n-N I-C II-N=N-C II-R _I ¹ ₀ ⁰

in which R⁸ and R⁹ are each independently H, a lower alkyl, aryl, or aralkyl; or R⁸ and R⁹ together form a ring structure; R¹⁰ is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, mono- or di-substituted amino, or alkoxy, aryloxy, or aralkoxy, or R⁹ and R¹⁰ together form a ring structure.

11. A compound of formula (III)

in which R is H or methyl; R¹¹ and R¹² are each independently H or a lower alkyl; R³ is the side chain of a natural amino acid, ^"or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, a mono- or di-substituted amino, or alkoxy, or aralkoxy; which compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

12. A compound of formula (IV)

in which R is H or methyl; R^u and R^t2 are each independently H or a lower alkyl; R³ is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, a mono- or di-substituted amino, or alkoxy, or aralkoxy; n is an integer of 1 or more; whiclr-compound is capable of (a) permeating the membranes of cells, and (b) being glycosylated by oligosaccharyltransferase.

13. The compound of claim 6 in which R³ has the structure

y in which n is an integer of 1 or more, and R¹¹ and R¹² are each independently H or a lower alkyl.

14. The compound of claim 8 in which R⁴ has the structure

in which n is an integer of 1 or more; R¹³ is H, an alkyl, aryl, or aralkyl; R¹¹ and R¹² are each independently H or a lower alkyl.

15. The compound of claim 10 in which R² is H, and R¹ is an acyl group in the range of two to eight carbon atoms.

16. The compound of claim 13 in which R² is H, and R¹ is an acyl group in the range of two to eight carbon atoms.

17. The compound of claim 14 in which R² is H, and R¹ is an acyl group in the range of two to eight carbon atoms.

18. The compound of claim 8 in which R¹ is selected from the group consisting of acetyl,'' tert- butoxycarbony1, benzyloxycarbonyl, benzoyl, and octanoyl. 19. The compound of claim 2 in which R³ is the side chain of a natural amino acid.

20. The compound of claim 3 in which R³ is the side chain of a natural amino acid.

21. The compound of claim 4 in which R³ is the side chain of a natural amino acid.

22. The compound of claim 5 in which R³ is the side chain of a natural amino acid.

23. The compound of claim 10 in which R³ is the side chain of a natural amino acid.

24. The compound of claim 13 in which R³ is the side chain of a natural amino acid.

25. The compound of claim 14 in which R³ is the side chain of a natural amino acid.

26. The compound of claim 15 in which R³ is

27. The compound of claim 2 in which R⁴ is

NH. 2"

28. The compound of claim 8 in which R⁴ is ^m-' 29. The compound of claim 18 in which R⁴ is NH₂.

30. A pharmaceutical composition comprising the compound of claim 1; and a pharmaceutically acceptable carrier.

31. A pharmaceutical composition comprising the compound of claim 6; and a pharmaceutically acceptable carrier.

32. A pharmaceutical composition comprising the compound of claim 9; and a pharmaceutically acceptable carrier.

33. A method of treating a mammal with a disease or disorder involving a defective cell membrane, secretory or lysosomal protein comprising administering to the mammal a therapeutically effective amount of the compound of claim 1.

34. A method of treating a mammal with a disease or disorder involving a defective cell membrane, secretory or lysosomal protein comprising administering to the mammal a therapeutically effective amount of the compound of claim 6.

35. A method of treating a mammal with a disease or disorder involving a defective cell membrane, secretory or lysosomal protein comprising administering to the mammal a therapeutically effective amount of the compound of claim 9.

36. The method according to claim 33 in which the disease or disorder is cystic fibrosis. if

37. The method according to claim 34 in which the disease or disorder is cystic fibrosis.

38. The method according to claim 35 in which the disease or disorder is cystic fibrosis.

39. The method according to claim 33 in which the mammal is a human.

40. A method of delivering a compound intracellularly to the lumen of the endoplasmic reticulum, comprising:

(a) conjugating a compound to a substrate of oligosaccharyltransferase, which substrate is capable of permeating the membranes of cells and has the formula

(I):

in which R is H or methyl; R¹ is an acyl group; R² is H; or R¹ and'R² together form a ring structure; R³ is the side chain of a natural amino acid,' or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; R⁴ is OH, NH₂, , a mono- or di- substituted amino, alkoxy or aralkoxy; and in which the conjugation is via a covalent bond to any suitable functional group on R¹, R³ or R⁴; and (b) exposing a cell to the conjugated compound, in which the conjugated compound is capable of (i) permeating the membranes of cells, and (ii) being glycosylated by oligosaccharyltransferase.