Synthesis and characterization of a trigalactosylated bisacridine

Sep 28, 1992 - to cell surface receptors for use in gene-delivery systems. Each of the ... asialoglycoprotein receptor on primary hepatocytes. A triga...
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Bioconlugate Chem. 1093, 4, 85-93

85

Synthesis and Characterization of a Trigalactosylated Bisacridine Compound To Target DNA to Hepatocytes Jean Haensler and Francis C. Szoka, Jr.’ School of Pharmacy, University of California, San Francisco, California 94143-0446. Received September 28, 1992

We have synthesized three bisacridine intercalators containing a galactose residue(s) to target DNA to cell surface receptors for use in gene-delivery systems. Each of the bisacridines could intercalate into DNA with micromolar dissociation constants. Bisacridines containing a single galactose on either a three- or six-carbon spacer from the secondary amine of spermidine-bisacridine could mediate binding of DNA to the soluble galactose receptor Ricinus communis lectin (RCA I), but not to the asialoglycoprotein receptor on primary hepatocytes. A trigalactosyl dilysyl bisacridine [ (Gal-6)sLyszbA] compound could mediate the binding of DNA to both the ricin lectin and to primary hepatocytes. Binding of the (Gal-6)aLys~bA-DNAto the hepatocytes could be blocked by asialoorosomucoid. On the basis of luciferase expression, (Gal-6)aLysz-bAdid not induce transfection of the hepatocytes when attached to the pCLUC4 plasmid encoding the firefly luciferase gene.

INTRODUCTION The increasing knowledge of the molecular basis of disease has opened the way for correction of pathology using gene therapy. Success in this goal depends, to a large degree, on the development of safe and efficient carriers for selective gene delivery to the target cells. Although viral vectors currently are the most efficient systems for gene transfer into eucaryotic cells (11,a concern exists that the use of viruses for gene therapy might lead to deleterious side effects (2). This concem has encouraged the development of gene transfer techniques that do not use viruses. A nonviral gene carrier will have to surmount all the barriers that viruses have evolved to overcome: The carrier will have to persist in the biophase long enough to gain access to the target cell. I t will have to recognize the target cell and mediate entry of the gene into the cell. Finally, the gene will have to find its way into the nucleus, where the genetic information can be transcribed. Our aim is to use biochemical and biophysical principles to design and synthesize nonviral gene-delivery systems that have the characteristics necessary to achieve a high level of gene delivery into the nucleus of cells. Viruses take advantage of specific cell surface molecules, viral receptors, to bind to the host cells. The initial binding of the virus to its receptor is also essential for viral entry and infection. Binding allows close contact with the host cell surface, which is necessary for membrane fusion or triggers receptor-mediated endocytosis (3). To mimic the binding and entry functions of viruses one can exploit the specificity and efficiency afforded by receptor-mediated endocytosis to deliver recombinant DNA (e.g. plasmids) via attached ligands. Methods for the noncovalent attachment of cell-specificligands to DNA using polycationic anchors or an ethidium homodimer anchor have been devised (4-6). Polylysineligand conjugates which tightly bind to DNA by forming a charge complex have been used to carry foreign DNA into selected target cells and/or organs expressing the cognate receptor (4-1 7). As an alternative to these high molecular weight polylysine-ligand carriers, we propose novel bimolecular conjugates that consist of a low molecular weight targeting

* Address correspondence to this author. 1043-1802/93/2904-0085$04.00/0

ligand covalently attached to a chemically well-defined DNA intercalator. We have applied this approach to create anew carrier system for selective gene delivery to the liver, wherein we attach galactose, a targeting ligand for hepatocytes, to a DNA bis-intercalator. We describe here the synthesis and characterization of a series of compounds obtained by attaching thiogalactosyl residues to the central amino group of spermidine-bisacridine via various spacer arms. The binding constant of the bisacridine compounds for DNA, as well as their ability to mediate binding of a 5 kb plasmid to a soluble receptor for galactose (Ricinus communis agglutinin, RCA-I)’ or to the asialoglycoprotein receptor of isolated rat hepatocytes, is described in this paper. EXPERIMENTAL PROCEDURES Radioactive Na1251was obtained from Du Pont-New England Nuclear (North Bilerica, MA). [1251110do-2/deoxycytidine 5’-triphosphate (lZ5I-dCTP)was obtained from Amersham Co. (Arlington Heights, IL). The plasmid pCLUC4 (18) was a generous gift from Dr. E. Wagner (Institute of Molecular Pathology, Vienna, Austria). It was radiolabeled with lz51-dCTPusing a nick translation kit (Bethesda Research Laboratories, Gaithersburg, MD). Asialoorosomucoid (AsOR), prepared from human orosomucoid by neuraminidase treatment, was a generous gift from Dr. S. Oie (UCSF) and was radioiodinated using Enzymobead radioiodination reagent (Bio-Rad Chemical Division,Richmond, CAI. Freshly isolated rat hepatocytes were obtained from Dr. M. Bissel (Liver Center, UCSF). The silica gel for column chromatography (grade 60,230400 mesh, 60 A; Merck) and all chemicals (unless otherwise specified) were from Aldrich Chemical Co. (Milwaukee, WI).Organicsolvents were obtained from Fisher Scientific Co. (Santa Clara, CAI. Thin-layer chromatography (TLC) was carried out on analytical silica gel 60-Aprecoated glass plates (Whatman) supplied by Baxter Scientific Products Abbreviations used: AsOR,human asialoorosomucoid;DCC,

N,”-dicyclohexylcarbodiimide; FCS, fetal calf serum; HEPES, N-(2-hydroxyethy1)piperazine-N’(2-ethanesulfonicacid);MEM, minimal essential Eagle’s medium; PBS, phosphate-buffered saline;RCA-I,Ricinus communis agglutinin;TFA, trifluoroacetic acid; TMS, tetramethylsilane; Tris, tris(hydroxymethy1)ami-

nomethane.

0 1993 American Chemical Society

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Haensler and Szoka

Bb2on)ugate Chem., Vol. 4, No. 1, 1993

B

n=5

.i

1) TFA

I) DEPROTECTION

Gal-S

2) MeOHINEt&20 5:4:1 v/v

II) PHENOXYACRIDINE I PHENOL 90°C

Gal-S Gal-S

'

2

=

(OAC)~G~-S.(CH,),--COO~NO,

3

=

(0A~)~Gal-s (OAC)~G~~-S-(CH~)~-COO~NO~

s

Gal4

I (FH2)4

=

0=?""

= AcO " # G s

(OAC)~G~I-S-(CH,),-CONH

4

\

(OAC)~G~~--S-(CH,)~-CONH -CH-CONH-fjH

-COO-N (CH2)4

I

(OAC)~G~I-S-(CH,)~-CONH

=

HO

OH

0

Figure 1. Synthetic schemes for galactosyl bisacridines.

(McGaw Park, IL). Proton NMR were obtained on General Electric GN 500 and QE 300 spectrometers (Fremont, CAI. Spectra were recorded on samples diluted in deuterated methanol containing TMS. Proton chemical shifts are reported in ppm downfield from internal TMS (0.00 ppm). Liquid secondary ion mass spectrometry (LSIMS) measurements were made in the positive-ion mode with a Kratos MS-50 mass spectrometer (Ramsey, NJ) at the UCSF mass spectrometry facility. Samples were run in a thioglycerol matrix. Chemical Synthesis. The synthetic scheme for preparation of the bisacridine targeting ligands is shown in

Figure 1. NJP-Bis(tert-butoxycarbony1)spermidinehydrochloride (11, a substrate for selective N4-acylation of spermidine, was prepared according to published methods (19,20). The intermediate N4-benzylspermidine (19) is commercially available (Sigma Chemical Co., St Louis, MO). L-Lysyl-L-lysinebis(trifluoroacetate), for the synthesis of the branched galactosyl ligand, was obtained by standard peptide chemistry by reacting N-(tert-butoxycarbony1)L-lysine (Bachem, Torrance, CA) with 2 equiv of N P bis(tert-butoxycarbony1)-L-lysine p-nitrophenyl ester (Bachem) in N-methylmorpholine containing N,N-diiao-

Intercalator Ligands for DNA Targeting

Bioconjugate Chem., Vol. 4, No. 1, I993

87

propylethylamine (1.5 equiv). The resulting protected from the columns were lyophylized. Spermidine-bisacrilysine dimer was purified on a silica gel column eluted dine and galactosylated derivatives were synthesized with the system chloroform/methanol/water 9O:lO:l and according to a procedure adapted from the general method was further deprotected in TFA. of Barbet and co-workers (24) for the preparation of acridine dimers. 3-(2,3,4,6-Tetra-0-acetyl-l-thio-8-~-galactop~anosyl)propionate was prepared from 2-S-(2,3,4,6-tetra-O-acetylSpermidine-Bisacridine Trihydrochloride (bA-3HCl). 8-~-galactopyranosyl)-2-thiopseudourea hydrobromide (21) 9-Chloroacridine (4.27 g, 20 mmol) and spermidine free and 3-iodopropionic acid as previously described (22). base (1.45 g, 1.57 mL, 10 mmol) were dissolved in 30 mL 642,3,4,6-Tetra-O-acetyl-l-thio-~-~-galactopyranosyl)hexof phenol at 80 "C. The temperature was raised to 130 "C anoate was prepared similarly from 2-S-(2,3,4,6-tetra-Oand the solution stirred under argon. After stirring for acetyl-/3-~-galactopyranosyl)-2-thiopseudourea hydrobro1.5 h, the resulting dark solution was cooled to ca. 40 "C mide and 6-bromohexanoicacid. These acid-functionalized and poured into 60 mL of 1N NaOH. The precipitate was galactose derivatives were transformed into the correcollected by filtration and washed on the filter with 1N NaOH and water. The solid was redissolved in methanol sponding activated esters (respectively 2 and 3) by esterification with p-nitrophenol according to published and purified on a silica gel column eluted with increasing procedures (22, 23). N'-[NuJV-Bis[6-(2,3,4,6-tetra-0- amounts of diethylamine in methanol (0%,0.25 % ,Oh%, acetyl-l-thio-/3-~-galactopyranosyl)hexanoyl] -~-lysyll-N~- 1%,and 2% v/v). The column chromatography separates [(6-(2,3,4,6-tetra-0-acetyl-l-thio-~-~-galactopyranosyl)spermidine-bisacridine from the mono- and trisubstituted byproducts and from the unreacted or degraded starting hexanoyll lysine was synthesized from compound 3 and materials. The fractions containing the acridine dimer, L-lysyl-L-lysine bis(trifluoroacetate) according to the found in the 0 . 5 2 % diethylamine wash, were pooled method of Ponpipom and co-workers (23). This derivative was activated by esterification with N-hydroxysuccinimide together and evaporated to dryness in vacuo. The residue (1.1equiv) in the presence ofDCC (1.1equiv) inanhydrous was redissolved in boiling methanol and crystallized as a trihydrochloride by dropwise addition of concentrated dichloromethane to yield the activated ester 4. HC1. The crystals were collected and recrystallization from Acylation of N'P-Bis(tert-butoxycarbony1)spermimethanol yielded 1.77 g of the title compound (yield 29%1. dine Hydrochloride with the p-Nitrophenyl Esters Zand 3. In a typical reaction, 1mmol of acylating agent (557 Galactosylated Spermidine-Bisacridines Diacetates mg of compound 2 or 599 mg of compound 3) in 10 mL [(Gal-3-bA,Gal-6-bA and (GalB)&ys~bA)].Bisacridine derivatives of the galactosylated spermidines were preof acetonitrile was added dropwise under argon to a stirred pared by a modification of the procedure described for solution of 0.7 mmol of W,P-bis(tert-butoxycarbony1)spermidine-bisacridine. 9-Phenoxyacridine was used as spermidine hydrochloride (1; 270 mg) in 10 mL of reagent instead of 9-chloroacridine. It was prepared from acetonitrile containing 140 pL of triethylamine (1mmol). 9-chloroacridine according to the procedure of Dupr6 and After the addition was completed, the reaction mixture Robinson (25). In a typical reaction, 0.2 mmol of 9-phewas further stirred for 36 h under argon and evaporated noxyacridine (54.2 mg) and 0.1 mmol of a galactosylated in vacuo to a residue. This residue was redissolved in 50 mL of ethyl acetate, washed with 3% hydrochloric acid (3 spermidine derivative were dissolved in 3 mL of phenol at 80 "C. This reaction mixture was further stirred under X 10 mL), water (3 X 10 mL), 5% sodium hydrogen argon at 80-85 "C. After stirring for 2 h, the dark solution carbonate (3 X 10 mL), and water (3 X 10 mL), dried over was cooled to ca. 40 "C and poured into 30 mL of diethyl MgS04, and evaporated to a syrup. This crude product ether. The precipitate was isolated by filtration, washed was further purified on a silica gel column eluted with on the filter with diethyl ether, and redissolved in a mixture ethyl acetate/dichloromethane (1:l v/v) to afford the of n-butanollmethanol (3:l v/v). The resulting solution desired conjugate (5 or 6) as a pale yellow syrup with an was concentrated in vacuo and chromatographed on a average yield from 30 to 40 '3%. column of silica gel eluted with n-butanol/pyridine/acetic Acylation of N'P-Bis(tert-butoxycarbony1)spermiacid/water (6:2:1:2 v/v) in order to separate the desired dine Hydrochloride with the N-Hydroxysuccinimide bisacridine from the monosubstituted byproduct and from Ester 4. A solution containing 400 mg of 4 (0.23 mmol) the unreacted or degraded starting materials. The fracin 5 mL of acetonitrile was added dropwise to a stirred tions containing the bisacridines were pooled together and solution of 80 mg of WP-Bis(tert-butoxycarbony1)evaporated in vacuo to yellow residues. spermidine hydrochloride (1; 0.22 mmol) in 5 mL of This method led to the diacetate salts of the expected acetonitrile containing 42 p L of triethylamine (0.3 mmol). bisacridines with yields averaging from 25 to 35 5%. These The reaction mixture was further stirred for 48 h under compounds, used for the targeting study, were stored at argon and evaporated in vacuo to a crude product which 4 "C as concentrated stock solutions in ethanol. The exact was chromatographed on a silica gel column eluted with concentration of these solutions were determinated from chloroform/methanol/water (90101 v/v) to afford the the absorbance at 412 nm using a spermidine-bisacridine desired conjugate 7 as a syrup in 45% yield. standard. TLC analysis after a 6-month storage period Deprotection of Compounds 5-7. In the first step, the showed a slight degradation (ca. 10%) of the diacridines t ert-butoxycarbonyl protecting groups were quantitatively into the corresponding monoacridines. removed from the N1 and N8 atoms of the spermidine bA.3HCl. lH NMR (CD30D) 6 (ppm): 8.64-8.55 (dd, linker by a 30-min treatment with TFA according to the J = 8.5 Hz, 4 H, acr H-1 and H-8), 7.99-7.95 (m, 4 H, acr procedure described in ref 20. In the second step, the H-4 and H-5), 7.84-7.81 (m, 4 H, acr H-3 and H-6),7.62acetyl protecting groups were removed from the galactosyl 7.56 (m, 4 H, acr H-2 and H-71, 4.32 (t,J = 7.1 Hz, 2 H, headgroups by an overnight treatment with a mixture of spermidine CH2-a), 4.23 (t, J = 6.8 Hz, 2 H, spermidine methanol/water/NEts (5:4:1), according to a procedure CH2-g),3.23 (t, J = 7.4 Hz, 2 H, spermidine CH2-c), 3.15 from reference 23. After the deprotections were completed, (t, J = 7.6 Hz, 2 H, spermidine CH2-d), 2.43 (q, J = 7.1 the dried residues of galactosylated spermidines were Hz, 2 H, spermidine CH2-b), 2.12 (q, J = 7.4 Hz, 2 H, redissolved in water and passed through small Bio-Rad AG 1X 2 (OH-) columns in order to convert the salts into spermidine CH2-0, 1.92 (q, J = 7.2 Hz, 2 H, spermidine CH2-e). LSIMS: m/z = 500 (M + H). Rf (n-butanol/ the corresponding free amines. The flow-through products

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Bloconlugate Chem., Vol. 4, No. 1, 1993

acetic acid/pyridine/water 4:1:2:1) = 0.12 (Rj of the monoacridine homolog = 0.02). Gal-3-bA. lH NMR (CD30D) 6 (ppm): 8.55-8.48 (dd, J = 8.5 Hz, 4 H, acr H-1 and H-8),7.9&7.90 (m, 4 H, acr H-4 and H-5), 7.85-7.78 (m, 4 H, acr H-3 and H-6), 7.567.48 (m, 4 H, acr H-2 and H-7),4.28 (d, J = 10 Hz, 1H, gal. H-1, @-configuration),4.20 (t, J = 7.1 Hz, 2 H, spermidine CHz-a), 4.11 (t,J = 6.5 Hz, 2 H, spermidine CH2-g),3.85 (d, J = 3.2,l H, gal. H-4),3.75-3.60 (m,4 H, spermidine CH2-cand CHz-d), 3.54-3.38 (m, 5 H, gal. H-2, H-3, H-5, H-6a, and H6-b), 3.02-2.93 (m, 2 H, spacer arm 2.24SCH2),2.85-2.70 (m, 2 H, spacer arm CH~CONRZ), 2.10 (m, 2 H, spermidine CH2-b), 2.06-1.94 (m, 2 H, spermidine CH2-f),1.90-1.70 (m, 2 H, spermidine CH2-e). LSIMS: m/z = 750.4 (M + H). Rf (n-butanol/acetic acid/ pyridine/water 4:1:2:1) = 0.22 (Rf of the monoacridine homolog = 0.10). Gal-6-bA. lH NMR (CD30D) 6 (ppm): 8.48-8.45 (dd, J = 8.5 Hz, 4 H, acr H-1 and H-8),7.93-7.88 (m, 4 H, acr H-4 and H-5), 7.85-7.79 (m, 4 H, acr H-3 and H-6), 7.547.48 (m, 4 H, acr H-2 and H-71, 4.26 (d, J = 10 Hz, 1H, gal. H-1, @-configuration),4.18 (t, J = 7.1 Hz, 2 H, spermidine CH2-a), 4.05 (t, J = 6.5 Hz, 2 H, spermidine CHz-g), 3.90 (d, J = 3.2,l H, gal. H-4), 3.76-3.66 (m, 4 H, spermidine CH2-c and CHz-d), 3.58-3.38 (m, 5 H, gal. H-2, H-3, H-5, H-6a, and H-6b), 2.74-2.54 (m, 2 H, spacer arm SCHZ),2.28 (t, J = 7.2, 2 H, spacer arm CH2CONR2), 2.20-2.14 (m, 2 H, spermidine CH2-b), 2.01-1.94 (m, 2 H, spermidine CH2-f),1.93-1.70 (m, 2 H, spermidine CH2-e), 1.6-1.0 (bm, spacer arm CHz). LSIMS: mlz = 792.5 (M + H). Rf (n-butanollacetic acid/pyridine/water 4:1:2:1) = 0.26 (Rf of the monoacridine homolog = 0.14). (Gal-6)aLysz-bA. lH NMR (CD30D) 6 (ppm): 8.558.45 (m, 4 H, acr H-1 and H-8), 7.98-7.90 (m, 4 H, acr H-4 and H-5), 7.85-7.82 (m, 4 H, acr H-3 and H-6),7.60-7.52 (m, 4 H, acr H-2 and H-7), 4.33-4.17 (m, 7 H, gal. H-1, spermidine CH2-a and CHz-g), 4.15-4.07 (m, 1 H, Lys aCH), 4.05-3.95 (m, 1 H, Lys aCH), 3.87 (m, 3 H, gal. H-4), 3.76-3.34 (bm, 19 H, spermidine CH2-c and CH2-d, gal. H-2, H-3, H-5, H-6a, and H-6b), 3.29-2.95 (bm, 4 H, Lys tCH2), 2.78-2.57 (m, 6 H, spacer arm SCHz), 2.272.10 (bm, 8 H, spacer arm CHzCONHR, spermidine CH2b), 2.06-1.80 (bm, spermidine CHz-f, and CHZ-e, acetate), 1.80-1.00 (bm, spacer arm CH2). LSIMS: mlz = 1632.8 (M + H), m/z = 1654.8 (M + Na). R f (n-butanollacetic acid/pyridine/water 4:1:2:1) = 0.18 (Rfof the monoacridine homolog = 0.08). Ethidium Bromide Displacement Assay. Ethidium bromide (19 pM) was mixed with calf thymus DNA (5.7 pM as nucleotide equivalents) in a 10mM Tris-HC1buffer, 0.2 M NaC1, pH 7.4. Then increasing amounts of bAq3HC1, Gal-3-bA, Gal-6-bA, or (Gal-6)aLysz-bA were added in order to displace ethidium bromide from DNA (26,27). This competitive displacement was monitored by recording the decrease of ethidium bromide fluorescence (excitation = 540 nm; emission = 610 nm). To provide values for the decrease of the fluorescenceof the ethidium-DNA complex alone, that of free ethidium was subtracted. Fluorescence values are displayed as a percentage of the maximum (fluorescence of ethidium-DNA complex alone). The intrinsic fluorescence of the bisacridine occurs at 450 nm and does not contribute to the fluorescence signal at 610 nm. Gel Retardation Assay. Samples of pCLUC4 plasmid (200 ng, 300 pmol base pair equivalents) in 5 pL of water were mixed with 21 pmol of Gal-3-bA, Gal-6-bA, or Gal3Lysz-bA in 5 p L of water. The intercalator to plasmid ratio is 300:l. When indicated, 33.3 pmol of RCA-I

Haensler and Szoka

(Boehringer-Mannheim) in 1 pL of a 5 mM phosphate buffer, 0.2 M NaC1, pH 7.2, was added. After 20-min incubation, the samples were electrophoresed through a 0.8% agarose gel using a 40 mM Tris-acetate buffer system (pH 8.0) and stained with ethidium bromide to visualize DNA. Binding and Uptake of AsOR by Isolated Rat Hepatocytes. The assayson rat hepatocytes in suspension were performed within 2 h after cell isolation. The assays on adheregt cells were performed 24 h after cell isolation. AsOR binding was determined using (0.6-0.7) X lo6viable hepstocytes incubated in 1.5-mL polypropylene tubes in 0.5 mL of binding medium [Eagle's minimal essential medium (MEM),20mMHEPES, pH 7.51 containing0.1-3 pg of lz51-AsOR/mL(lo6dpm/pg). The cells were kept in suspension on a rotator for 2 h at 4 "C in the presence of the ligand. At the end of the incubation, the cells were centrifuged and the cell pellets were washed with ice-cold PBS (containing 0.1 g/L CaClz and 0.1 g/L MgC12.6H20). This was repeated three times. The pellets were counted for radioactivity in a y scintillation spectrometer (Beckman Gamma 8000) and lysed in 1 mL of 1 N NaOH prior to protein determination using a Bradford dye-binding protein assay (Bio-Radprotein assay kit). The time course of AsOR uptake was determined similarly using 1251-AsOR (lo6dpm/pg) at a concentration of 1pg/mL in 0.5 mL of binding medium containing (0.6-0.7) X lo6 cells rotated for increasing periods of time (0.5,1, 2, 3, and 4 h) at 37 "C (Figure 5A). AsOR uptake by adherent hepatocytes was determined under the conditions given below for the plasmid uptake assay using 1 pg of 1251-AsOR/mLof medium. Nonspecific binding and uptake were determined in the presence of a 100-fold excess of unlabeled ligand. The amount of cell-associated AsOR is expressed as nanograms of AsOR per milligram of cell protein. Inhibition of AsOR Binding to Hepatocytes. Increasing amounts of (Gal-6)3Lysz-bA,free or complexed to the plasmid pCLUC4 (500 intercalators per plasmid), and 10 ng of 1251-AsOR(3.6 X lo5 dpm) in a total volume of 50 pL of phosphate buffer, pH 7.2, were added in duplicate to 0.5 mL of a suspension of freshly isolated rat hepatocytes (1.25 X lo6 cells) in binding medium in 1.5mL polypropylene tubes. After incubation for 2 h at 4 "C with rotation, 200 pL of the cell suspension was centrifuged over 200 pL of 4:l silicone/light mineral oil at 104gfor 30 s. The radioactivity associated with the cell pellets was measured. The percent cell-associated AsOR was computed as the radioactivity in the presence of inhibitor divided by the radioactivity in the absence of inhibitor times 100. Uptake of Targeted Plasmids by Isolated Rat Hepatocytes. Cells in suspension. Freshly isolated rat hepatocytes (lo6cells) were kept in suspension in 0.5 mL of binding medium in 1.5-mL polypropylene tubes. Ten microliters of water containing 200 ng of 1251-pCLUC4 plasmid (8 X lo4 dpm), complexed or not with (Gd-6)3Lysz-bA (500 bisacridines per plasmid), was added to the tubes in duplicates. The tubes were rotated for different periods at 37 "C (0.5,1,2, and 3 h), and the cell suspensions were centrifuged over 0.5 mL of 4:l silicone/light mineral oil at lO4g for 30 s. The cell pellets were counted for radioactivity and lysed in 0.5 mL of 1 N NaOH for subsequent protein determination as described above. Cellassociated plasmid is expressed as nanograms of plasmid per milligram of cell protein. Adherent cells. Freshly isolated rat hepatocytes (lo6 cells) were plated in 60-mm petri dishes in 3 mL of MEM containing 5% fetal calf serum (FCS) and antibiotics (penicillin, 100 units/mL; streptomycin, 100 pg/mL; gen-

Intercalator Ligands for DNA Targeting

tamycin, 25 pg/mL). After 18 h a t 37 "C, the medium was removed and replaced with 1 mL of serum-free MEM. One microgram of 1251-pCLUC4plasmid complexed to bA-3HC1, Gal-3-bA, Gal-6-bA, or (Gal-6)~Lysz-bAin 100 p L of water was added in duplicate (with 150 pg of AsOR, when indicated). The bisacridine to plasmid ratio was 500:l or 1001. The cells were further incubated for 1 h at 37 "C and then rinsed and digested in 1 mL of 1 N NaOH. The cell lysates were counted for radioactivity and protein was measured as described above. Transfection Assay. The cells were plated and grown as described above. After 18 h at 37 "C, the medium was removed and replaced with 2 mL of serum-free MEM. Then 4 pg of pCLUC4 plasmid complexed to (Ga1-6)3LyszbA at a ratio of 500 intercalators per plasmid in 100 pL of water was added. After 5-h incubation at 37 "C, the medium was removed and replaced with 3 mL of fresh MEM containing 5% FCS and antibiotics. The cells were grown for an additional 48 h at 37 "C and tested for luciferase activity as described previously (28).

Bioconjugate Chem.. Voi. 4, No. 1, 1993 80

U I

112g 80

RESULTS

Synthesis of Bisacridine Intercalators for Use as DNA Targeting Ligands. To create a targeting system for directing DNA to hepatocytes we have synthesized bifunctional molecular conjugates consisting of a targeting ligand for hepatocytes (galactose) that is covalently attached via spacer arms of various lengths to the DNA bis-intercalator spermidine-bisacridine. The synthesis of the delivery system is based upon the trifunctionalization of the N1, N4,and N8 atoms of spermidine (Figure 1)and involves a selective N4-acylation starting with N1,iV-bis(tert-butoxycarbony1)spermidinehydrochloride as proposed by Bergeron and colleagues (20). The acylating agents are the mono (linear) or branched acetyl-protected galactosides 2-4 terminated by an activated carboxylic acid function and prepared as described in refs 22 and 23. The acylation of N,W-bis(tert-butoxycarbonyl)spermidine hydrochloride with these agents gives the protected galactose-spermidine conjugates 5-7. The N4-acylation step is followed by deprotection and subsequent reaction of the primary N1 and N8 atoms with 9-phenoxyacridine. This final reaction leads to the desired galactose-spermidine-bisacridine conjugates [Gal-3-bA, Gal-6-bA, and (Gal-6)3Lysz-bA]. This chemistry is versatile and can easily be used to attach other carbohydrates or any similarly functionalized targeting ligand to a bisacridine anchor. For instance, a similar strategy has been used by Nielson and co-workers to link nitrobenzamido ligands to a l,bbis(9-acridinylamin0)-3-azapentaneanchor (29). Binding of Intercalators to DNA. Intercalation of the bisacridine conjugates into DNA was demonstrated by an ethidium bromide displacement assay (26,27).This assay is based upon the marked fluorescence enhancement seen when ethidium bromide intercalates into DNA. This property is used here for spectrofluorometric quantitation of ethidium displacement from its intercalation sites. As shown in Figure 2, the acridine dimers competitively displace ethidium bromide from calf thymus DNA. The binding constants of the bisacridines are calculated from this assay by using a computation method described by Wolfe and Meehan (30) and an intrinsic dissociation constant of 6.7 X 10+ M for ethidium bromide (31). As a result of the loss of one positive charge, a slight but significant decrease in affinity is observed when spermidine-bisacridine is N4-acylated by the galactosyl derivatives. An additional small decrease in affinity is observed when the size and hydrophilicity of the targeting moiety

0

"

.1

1

10

100

1000

inhibitor concentration (pM)

Figure 2. Displacement of ethidium bromide from calf thymus DNA by the bisacridine derivatives. The decrease of the fluorescence of the ethidium-DNA complex was recorded as described in the Experimental Procedures. Fo, fluorescence of free ethidium bromide (19 pM);Fmax, fluorescence of the ethidium-DNA complex alone (19 pM ethidium, 5.7 pM nucleotide); F, fluorescence of the ethidium-DNA complex in the presence of the bisacridine derivative. ( 0 )bA.3HC1, Kd = 4.31 X lo-@M; (0) Gal-3-bA, Kd = 5.42 X M; (0)Gal-6-bA, Kd = 1.2 X lo4 M; (A)(Gal-6)~Lysn-bA), Kd = 8.64 X lo* M.

are increased [& (bA.3HCl) = 4.31 X M, Kd (Gal3-bA) = 5.42 X M, & (Gal-6-bA) = 1.2 X lo4 M, & [(Gal-6)3Ly~z-bA)]= 8.64 X lo+ MI. Interaction of DNA Ligands with a Soluble Receptor. DNA binding of the galactosylated intercalators and subsequent interaction of the intercalated galactosyl ligands with a soluble receptor, RCA-I, is shown by a gelmobility-shift assay with the 5 kb plasmid pCLUC4 (Figure 3). Binding of intercalating agents extends the double helix, which leads to a relaxation of the supercoiling of circular DNA (32,33). As a result, a shift in the electrophoretic mobility of the plasmid-intercalator complex is observed. Intercalation of Gal-&bA, Gal-6-bA,and (Gal-6)3Lyss-bAinto pCLUC4 is shown by the retardation of the DNA movement observed in Figure 3, lanes 2,4, and 6. Intercalation of the conjugates into the plasmid results in the expected relaxation of the supercoiled form to a relaxed circular form, which migrates slower. Due to their higher affinity for DNA, Gal-3-bA and Gal-6-bA are more potent in producing this shift in mobility of the plasmid than (Gal-6)3Lysz-bAs The capability of Gal-3bA, Gal-6-bA, and (Gal-6)~Lysz-bAto bind pCLUC4 to a soluble receptor for galactose is shown by the complete retardation of the complexes in the presence of RCA-I (Figure 3, lanes 3,5, and 7). The lectin RCA-I is a tetramer selective for terminal P-D-galactoseresidues and is able to cross-link galactosylated particles via four binding sites (34). As a consequence of this property, the ability of Gal-3-bA, Gal-6-bA, and (Gal-613Lys2-bA to promote the binding of pCLUC4 to RCA-I results in the formation of large plasmid aggregates which do not penetrate the gel.

90 Blacanjugate Chem., Vol. 4,

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Figure 3. Effect of Gal-&bA, Gal-&bA, and (Gal-6)sLysz-bAon plasmid DNA electrophoretic migration in the presence and absence of ricin lectin RCA-I. Electrophoresis was performed as described in the Experimental Procedures. Lane 1, pCLUC4; lane 2, pCLUC4 + Gal-3-bA;lane 3, pCLUC4 + Gal-3-bA + RCAI; lane 4, pCLUC4 + Gal-6-bA; lane 5, pCLUC4 + Gal-6-bA + RCA-I; lane 6, pCLUC4 + (Gal-G),Lysz-bA; lane 7,pCLUC4 + (Gal-G)sLys,-bA + RCA-I.

Despite its lower affinity for DNA, (Gal-6)sLyspbAis more efficient (Figure 3, lane 7) in promoting RCA-I-induced plasmid-plasmid aggregation than Gal-3-bA or Gal-6-bA (Figure3, lanes 3 and 5). The efficiency of RCA-I-mediated plasmid aggregation is limited by the accessibility of the attached galactosyl ligand and by the electrostatic repulsion between adjacent plasmids. Thus interaction between RCA-I and the ligands on the DNA should be favored by increasing the spacing between the plasmid and the attached galactosyl ligands; the gel retardation results are consistent with this expectation. As a result of a multivalent interaction, the plasmid aggregates formed by RCA-I are quite stable. No visible dissociation occurred when a 100-fold excess of free galactose was added and incubated with the aggregates (data not shown). Interactionof Galactose-TargetedDNA with Hepatocytes. The interaction of the targeted plasmid with cells expressing a receptor for galactose was studied in vitro with isolated rat hepatocytes. Mammalian hepatocytes express a well-characterized asialoglycoprotein receptor which binds and internalizes glycoproteins exposing terminal galactosyl residues (35). To ensure that the receptor was not damaged during the hepatocyte isolation procedure, the cells were checked for specificAsOR binding and uptake using the conditions of Harris and co-workers (36). The binding of AsOR is saturable with an apparent Kd of about M (Figure 4A). The time course of lZ5I-AsORuptake is shown in Figure 5A; 1251-AsORuptake saturates at about 2 h after addition and is efficiently inhibited in the presence of a 100-fold excess of the unlabeled protein. These results are in agreement with published data (36) and confirm the presence of a functional receptor for galactose on the isolated cells. Interestingly, when the uptake study was performed on adherent cells 24 h after isolation, the 1-h specific uptake decreasedfrom 133.1 ng/mg of cell protein to 52.6 ng/mg of cell protein. Under these conditions the total uptake is about 2 times greater than the nonspecific uptake, indicating a down regulation of the receptor upon culturing of the cells. We examined the interaction of the galactose-targeted plasmids with both adherent (Table I) and suspension cells (Figures 4B and 5B). In adherent cells, (Gal-6)3Lys2bA mediates a saturable uptake that can be inhibited by a 100-fold AsOR excess over the targeting ligand concentration. The other ligands tested do not mediate targeting

1 .o

0.0

[

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1 2 s I]-AsOR

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Added to the cells (pg)

B 1201

Figure 4. Binding of T-AsOR to rat hepatocytes in a suspension in the presence and absence of galactosylated ligands. Panel A: cell-associated 12sI-AsORafter 2-h incubation a t 4 "C with increasingconcentrations of the radiolabeled ligand. Each value is the mean of duplicate determinations that agreed to within 10%;( 0 )total binding, (0) nonspecific binding in the presence of a 100-fold excess of nonlabeled AsOR as described in the experimental procedures. Panel B: cell-associated 1251-AsOR after 2-h incubation with 20 ng/mL of the radiolabeled ligand and in the presence of increasing concentrations of free (Gal6)3Lys2bA(A)or (Gal-6)3Lys2bAcomplexed to pCLUC4 (m, 500 bisacridines per plasmid). The values are the mean of duplicate measurements that agreed to within 10%. AS OR]^ = cellassociated 12sI-AsORin the presence of the inhibitor; [AsORIo = cell-associated 12511-AsOR in the absence of the inhibitor. Table I. Plasmid Uptake by Adherent Rat Hepatocytes

~~~

complexing agent none bASHCl(5oO:l) Gal-3-bA (500:l) Gal-6-bA (500:l) (Gal-G)~Lys2-bA (5oO:l) (Gal-6)sLysz-bA(100:l)

ng 1251-pCLUC4/mgof cell proteina -ASOR +AsOR 10.4 f 0.1 10.5 f 0.1 ND 9.9 f 1.8 10.7 f 1.7 ND 11..1 f 1.8 ND 9.9 i 0.6 17.0 f 1.2 14.5 f 0.6 ND

a The cells were incubated with 1pg of 1251-pCLUC4plasmid in the presence or absenceof the bisacridine derivativesand of an excess of AsOR. The ratio of bis-intercalator to plasmid is shown in parentheses. The amount of plasmid that became cell-associated was determined as described in the Experimental Procedures. The values are the mean of duplicates that agreed within 10%. (ND, not determined).

and do not increase cell-associatedplasmid over that found in the absence of ligand (Table I). The targeting is also sensitive to the density of ligand. At a ratio of 500 (Gal-

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degradation in the lysosomes and/or cross the membrane to enter the cell after its interaction with the asialoglycoprotein receptor. DISCUSSION

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Figure 5. Time course of uptake of lZ5I-AsORand of targeted plasmid DNA by freshly isolated rat hepatocytes in suspension. Panel A the cells were incubated with 1pg/mL of lZ5I-AsORand the amount of ligand that became cell-associatedwas determined as a function of time as described in the Experimental Procedures. Each value is the mean and the range of duplicates agreed within 10%;( 0 )total uptake, (0) nonspecific uptake in presence of 100 pg of nonlabeled AsOR. Panel B: the cells were incubated with 200 ng of 125I-pCLUC4plasmid and the amount of DNA that became cell-associated in presence and absence of the targeting ligand (Gal-6)3Lys2-bAwas determined as a function of time as described in the Experimental Procedures. Each value is the mean and the range of duplicates agreed within 10%;(w) plasmid with (Gal-6)aLysz-bA(500 bisacridines per plasmid), (0) plasmid without (Gal-B)3Lys,-bA.

6)sLysz-bAper plasmid, the specific plasmid-cell association corresponds to 6.5 ng of DNA/mg of cell protein. (Gal-6)sLysz-bAwas found to be less potent at a reduced conjugate to plasmid ratio. At aratio of 100:1, the specific association was reduced to 4 ng/mg of cell protein. In suspension cells, binding of AsOR was inhibited by the targeting ligand (Gal-6)sLysz-bAwith an apparent IC50 of about 5 X lo-' M. Only a slight increase of the IC50 to 10-6 M was observed when (Gal-6)sLysz-bAwas attached to plasmid DNA (Figure 4B). In addition, (Gal-6)sLyszbA mediates a 2-fold increase in plasmid uptake by the cells (Figure 5B). Taken together, these results suggest that the conjugate (GalS)3Lysz-bAis able to target plasmid DNA to the asialoglycoprotein receptor of isolated rat hepatocytes. In spite of AsOR-inhibitable binding of the plasmid to the hepatocytes, no expression of luciferase could be detected from the cells treated with the (Gal-6)sLysz-bApCLUC4 complex and subsequently cultured for 48 h. This is due either to the inability of the plasmid to escape

We have designed, synthesized, and characterized a low molecular weight conjugate useful for targeting of DNA to the galactose receptor of various cells including hepatocytes. The design of this conjugate was motivated by recent reports showing that the AsOR-polylysine conjugates can be used for selective and efficient gene delivery to hepatocytes via a receptor-mediated endocytosis pathway (4, 7-10). Related to previous work, our aim was to separate the binding function from the membrane destabilizing function of the targeting complex. Polylysine has at least two effects when used to attach ligands to DNA. The obvious one is the attachment of the ligand. A less obvious one relates to polylysine's ability to introduce DNA into cells in a nonspecific fashion (37). Our strategy is based upon the attachment of a synthetic galactosyl ligand to a DNA intercalator. A synthetic approach was selected in order to create a low molecular weight and well-defined system. We have evaluated the ability of three synthetic conjugates, Gal-3-bA, Gal-&bA, and (Gal-6)~Lysz-bA,to complex to DNA and subsequently promote binding to a soluble or a cell-surface-localized galactose-binding protein. These conjugates consist of thiogalactosyl head groups that are covalently attached to spermidinebisacridine via variable spacer arms. The selection of spermidine-bisacridine as a DNA anchor for the targeting ligands was motivated by the following criteria: (1) Spermidine can be functionalized stepwise and without ambiguity as to the stoichiometry of the targeting ligands and the acridines. (2) Acridines and related compounds are used for the treatment of malignant and parasitic deseases in humans (38)and thus are a reasonable starting point for a novel human therapeutic. (3) Spermidinebisacridine is a known DNA bis-intercalator (33). Bisintercalation provides tight binding and slow off-rate (39, 40), two important considerations for the formation of stable noncovalent complexes. Intercalation of the targeting conjugates into DNA was demonstrated by competitive displacement of ethidium bromide. The dissociation constants of monoacridines from DNA are in the viscinity of 10-3-10-4 M whereas the dissociation constants of bisacridines, capable of bisintercalation, are about 3-4 orders of magnitude smaller (27). The high binding affinities of the targeting conjugates for DNA strongly support their binding via bis-intercalation. Since the length of the spermidine linker (10.2 A) is sufficient to encompass two successive base pairs in the DNA double helix, a model has been proposed where spermidine-bisacridine spans two base pairs of the helix (33). Thus, and in further agreement with the neighboring site exclusion principle (411,a single spermidine-bisacridine molecule excludes a total of four adjacent base pairs. As a consequence, a maximum of 1225targeting conjugates can be attached to a 5 kb plasmid. The intercalation of the targeting conjugates into DNA results in both helix extension and distortion, which produced the relaxation of supercoiled plasmid DNA seen on the gel (Figure 3). This ability of the intercalators to distort the helical structure of DNA may be a disadvantage in the use of these compounds for gene transfer since condensation of DNA has been observed by a number of groups to promote transfection activity of various cationic facilitators (37)

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and intercalators oppose DNA condensation (32,331.T h e helix extension subsequent t o intercalation may render t h e plasmid too large for efficient inclusion in a 100-200nm endosome. It is also conceivable that intercalation of the bisacridines could interfere with t h e transcription process. However, we have not observed this to be t h e case in experiments where transfection from bisacridinecontaining plasmids was compared to t h a t of unmodified plasmids subsequent to their microinjection into the nucleus of cultured cells (unpublished observation). Modulation of t h e spacer arm between the galactosyl ligands and their bisacridineanchors allowed us t o examine some structural parameters which are important for receptor binding. All three conjugates tested promoted to some extent t h e interaction of plasmid DNA with t h e galactose-binding lectin from RCA-I (gel shift). However, only (Gal-6)3Lysz-bA was able t o efficiently bind t h e plasmid both t o t h e soluble lectin and t o t h e cellular galactose receptor of hepatocytes as shown in t h e competition experiments with AsOR (Figure 4B and Table I). The hepatic asialoglycoprotein receptor is known to have very stringent structural requirements for high-affinity ligand binding (42). Thus, improved accessibility and optimal ligand clustering provided by t h e branched spacer arm in (Gal-6)sLysz-bA may account for its efficient binding. It is not surprising that targeting t h e DNA t o t h e galactose receptor did not lead t o transfection since this bisacridine construct provides no membrane destabilizing functions, such as provided by defective adenovirus particles in the endosome, to help t h e DNA enter t h e cytoplasm (13). Although we have not yet studied t h e fate of t h e cell-associatedDNA, we suspect it is internalized via endocytosis and ultimately degraded in t h e lysosome. Thus the bisacridine-ligands differ from the polycationligands which can bind t o DNA as well as mediate transfection. One can envision a collection of effectors conjugated t o intercalators that could be combined t o create a tailormade gene-delivery system. These effectors might include DNA masking molecules, membrane-destabilizing peptides, targeting ligands, and nuclear localization peptides. The galactosyl bisacridines provide an example of t h e flexibility and robustness of bisacridine chemistry for such a synthesis. ACKNOWLEDGMENT

The authors are grateful t o Dr. Anthony Shaw, Dr. Alan Wolfe, Dr. Jean-Yves Legendre, Gary Green, and KunBee Chang for helpful discussionsand technical assistance. Thanks are also extended to the UCSF mass spectrometry facility supported by the NIH Division of Research Resources, Grant RR01614, a n d to the UCSF Liver Center, Grant POAM26743A-08. This work was supported by NIH Grant G M 26691. LITERATURE CITED (1) Gilboa, E., Eglitis, M. A., Kantoff, P. W., and Anderson, W. F. (1986) Transfer and expression of cloned genes using retroviral vectors. Biotechniques 4, 504-512. (2) Felgner, P. L., and Rhodes, G. (1991) Gene therapeutics. Nature 349, 351-352. (3) Marsh, M., and Helenius, A. (1989) Virus entry into animal cells. Adv. Virus Res. 36, 107-151. (4) Wu, G., and Wu, C. H. (1988) Evidence for targeted gene delivery to HepG2 hepatoma cells in uitro. Biochemistry 27, 887-892.

Haensler and Sroka (5) Wagner, E., Zenke, M., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. U.S.A. 87,34103414. (6) Wagner, E., Cotten, M., Mechtler, K., Kirlappos, H., and Birnstiel, M. L. (1991) DNA-bindingtransferrin conjugatesas functional gene-delivery agents: Synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety. Bioconjugate Chem. 2, 226-231. (7) Wu, G. Y., and Wu, C. H. (1988) Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 1462114624. (8) Wu, G. Y., Wilson, J. M., and Wu, C. H. (1989) Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in uiuo. J. Biol. Chem. 264, 16985-16987. (9) Wu, G. Y., Wilson, J. M., Shalaby,F., Grossman,M., Shafritz, D. A., and Wu, C. H. (1991) Receptor-mediated gene delivery in vivo-Partial correctionof genetic analbuminemiain nagase rats. J. Biol. Chem. 266, 14338-14342. (10) Wilson, J. M., Grossman, M., Wu, C. H., Roy Chowdhury, N., and Roy Chowdhury, J. (1992) Hepatocyte directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J. Biol. Chem. 267, 963-967. (11) Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Receptor-mediated endocytosisof transferrin-polycation conjugates: An efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A. 87, 3655-3659. (12) Cotten, M., Langle-Rouault, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation-mediatedintroduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87, 4033-4037. (13) Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., Curiel, D. T., and Birnstiel, M. L. (1992) High efficiency receptormediated delivery of small and large (48Kb) gene constructs using the endosome disruption activity of defective or chemically-inactivatedadenovirus particles. Proc. Natl. Acad. Sci. U.S.A. 89, 6094-6098. (14) Curiel, D. T.,Agarwal, S.,Romer, M. U., Wagner, E., Cotten, M., Birnstiel, M. L., and Boucher, R. C. (1992) Gene transfer to respiratory epithelial cells via the receptor-mediated endocytosis pathway. Am. J. Respir. Cell Mol. Biol. 6,247-252. (15) Huckett, B., Ariatti, M., and Hawtrey,A. 0. (1990) Evidence for targeted gene transfer by receptor-mediated endocytosis: Stable expression following insulin-directed entry of neo into HepG2 cells. Biochem. Pharmacol. 40, 253-263. (16) Rozenkrantz, A. A., Yachmenev, S. V., Jans, D. A., Serebryakova, N. V., Murav’ev, V. l., Peters, R., and Sobolev, A. (1992) Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct. Exp. Cell Res. 199,323-329. (17) Trubetskoy, V. S.,Tortchilin,V. P., Kennel,S. J.,andHuang, L. (1992) Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells. Bioconjugate Chem. 3, 323-327. (18) De Wet, J. R., Wood, K. V., De Luca, M., Helinsky, D. R., and Subramani, S. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell Biol. 7, 725737. (19) Bergeron, R. J., Stolovwitch,N. J.,and Porter, C. W. (1982) Reagents for the selective secondary N-acylation of linear triamines. Synthesis 689-692. (20) Bergeron, R. J., McGovern, K. A., Channing, M. A., and B u r t o n , P . S. ( 1 9 7 9 ) S y n t h e s i s of N 4 - a c y l a t e d Nl,N8-Bis(acyl)spermidines: An approach to the synthesis of siderophores. J. Org. Chem. 45, 1589-1592. (21) Chipowsky, S., and Lee, Y. C. (1973) Synthesis of l-thioaldoside5 having an amino group at the aglycon terminal. Carbohydr. Res. 31, 339-346. (22) Haensler, J., and Schuber, F. (1991) Influence of the galactosyl ligand structure on the interaction of galactosylated Iiposomes with mouse peritoneal macrophages. Glycoconjugate J. 8, 116-124.

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