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Bioconjugate Chem. 1994, 5, 312-315
Preparation of Vitamin &-Conjugated Peptides at the Amino Terminus and of Vitamin B6-Peptide-Oligonucleotide Conjugates Tianmin Zhu and Stanley Stein’ Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854. Received February 23, 1994”
A series of N-(4’-pyridoxyl)peptides has been made by standard Fmoc chemistry and a solid-phase coupling procedure. The last Fmoc group of the peptide was removed on the synthesizer, and the free amino group was then condensed with pyridoxal. The Schiff base formed was selectively reduced using sodium cyanoborohydride. The product was cleaved from the resin using a standard procedure. No deleterious effects were found when using the protected amino acids Fmoc-L-Ala, Fmoc-L-Arg(Pmc), Fmoc-L-Asp(OtBu), Fmoc-L-His(Trt), Fmoc-L-Ser(tBu), Fmoc-L-Thr(tBu), and Fmoc-L-Cys(Trt) for peptide synthesis. A vitamin Be-peptide-oligonucleotide conjugate could be synthesized using a cysteinyl peptide and a suitably activated oligonucleotide.
INTRODUCTION In contrast to attaining a pharmacologic response by binding to an extracellular receptor, certain compounds may require access to intracellular compartments. Cotransport of a compound as a conjugate with vitamin Bg has been shown to be an attractive option (I). Virtually all eukaryotic cells have a receptor-mediated transport system for vitamin Be, which also accepts secondary amine conjugates at position 4 of pyridoxal ( I ) . Following uptake into the cytosol, the conjugate can be enzymatically altered by phosphorylation and oxidation to release pyridoxal phosphate and the original amine-containing compound. In previous studies ( I ) , several amines were reacted with pyridoxal to form the Shiff base, which was subsequently reduced with sodium borohydride. These compounds were able to effectively compete for binding to the vitamin Be receptor and be taken up by cells in culture. We are interested in using vitamin Be to facilitate the cell uptake of peptides and peptide-oligonucleotide conjugates. A convenient solid-phase synthesis of N-(4’pyridoxy1)peptide at the amino terminus has been established. A series of N-(4’-pyridoxyl)peptideshas been made by standard Fmocl chemistry and a solid-phase coupling procedure prior to cleavage from the resin. It was then possible to covalently link a suitably activated oligonucleotide to the vitamin &-peptide conjugate using a previously developed procedure (2). EXPERIMENTAL PROCEDURES Reagents. N-a-Boc-L-ornithine and (benzotriazol-lyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) were from Bachem (Torrance, CA). Most peptide synthesis reagents were from Milligen/ Biosearch (Burlington, MA). Anisole, ethanedithiol, 1-hydroxybenzotriazole hydrate (HOBt), N- [ (9-fluorenylmethoxycarbonyl)oxylsuccinimide (Fmoc reagent), trifluoroacetic acid (TFA), pyridoxal hydrochloride, and sodium cyanoborohydride were from Aldrich (Milwaukee, @
Abstract published in Advance ACSAbstracts, June 15,1994. Abbreviations: Boc, tert-butyloxycarbonyl;BOP, (benzo-
triazol-l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; tBu, tert-butyl; OtBu, tert-butyl ester; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, hydroxybenzotriazole hydrate; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; TFA, trifluoroacetic acid; Trt, trityl. 1043-1ao219412905-0312to4.5010
WI). Water was purified by a Milli-Q Water System from was prepared Millipore. N-a-Boc-N-6-Fmoc-L-ornithine by the method described previously (3). Peptide Synthesis. The peptides were synthesized in a Milligen/Biosearch Model Excel1instrument. The N-aBoc-N-6-Fmoc-L-ornithinecartridge was packed with N-aBoc-N-6-Fmoc-L-ornithine (227 mg, 0.5 mmol), BOP reagent (221.3 mg, 0.5 mmol), and HOBt (67.5 mg, 0.5 mmol). The PAL support for peptide amide (0.1 mmol synthesis) was used to synthesize the carboxy-terminal amide, and Fmoc-L-Lys(Boc)PAC support was used for preparing the carboxy-terminal acid. Fmoc-L-aminoacid/ BOP + HOBt cartridges such as Fmoc-L-Ala, Fmoc-LArg(Pmc), Fmoc-L-Asp(OtBu), Fmoc-L-His(Trt), FmocL-Ser(tBu),Fmoc-L-Thr(tBu),and Fmoc-L-Cys(Trt) were used. The recommended conditions were followed, except that the coupling time for the ornithine derivative was extended to 90 min. The last protecting Fmoc group on the N-terminal residue of each peptide was removed. The peptides synthesized for these studies were (6-Orn),-CysNH2, where n = 1, 3, 8, 12, and Ala-Arg-His-Thr-AspTyr-Lys-OH. Solid-Phase Coupling of Pyridoxal to Peptide (Scheme 1 ). The peptide-resin in the synthesis cartridge, after removal of the final Fmoc protecting group, was removed from the machine and swelled with DMF for 10 min. Then 0.5 mmol of pyridoxal hydrochloride, dissolved in 5 mL of DMF and adjusted to pH 8 with 1 N NaOH, was added. The pyridoxal solution was added to the solid support cartridge by syringe, and the reaction was carried out overnight at room temperature. Excess pyridoxal was removed by syringe, and the solid support was washed three times with 5 mL of DMF and once with 5 mL of methanol. The solid support was swelled with DMF again and reacted with 0.5 mmol of sodium cyanoborohydride, dissolved in 5 mL DMF, and adjusted to pH 3-4 with 2 N HCl. The reduction reaction was at room temperature for 7 h (the yellow color of the solid support faded). The excess sodium cyanoborohydride was removed by syringe, and the solid support was washed three times with 5 mL of DMF and three times with 5 mL of methanol. The solid support was dried under vacuum, and the peptide was cleaved with 5 mL of TFA containing 20 pL of ethanedithiol for 3 h at room temperature. The solid support was filtered though a Pasteur pipet filled with glass wool and washed with 1 mL of TFA. The filtrate
0 1994 American Chemical Society
Preparation of Vitamin Be Conjugated Peptide
Bioconjugate Chem., Voi. 5, No. 4, 1994
Scheme 1. Solid-Phase Synthesis of Pyridoxylpeptide
313
Scheme 2. Synthesis of PyridoxylpeptideOligonucleotide Conjugate 3' CATITClTTA'IT
H2N-
I DMF 81 pH 8.0 H,C I
sH
ri.
1
NCNBH,
,
Pyridoxyl(S-Om).Cys-NH1 itpH34
t 0 II
3'
CATITCTITATT
fCH,-C- HN-
Fyridoxyl(60m).Cys-mz
I
t
I
TFA
R o I
II
R o I
II
HtCNH-CHC-[NHCHC-],,NH1
H,C%
k. was collected and blown to near dryness under nitrogen. The oily residue was precipitated by adding ethyl ether. The precipitate was filtered with a 10-15 M sintered glass filter and washed thoroughly with ethyl ether to obtain the dry product. Purification of Vitamin Ba-Peptide Conjugates. The conjugates were purified by HPLC on a Vydac (Hesperia, CA) 218TP1022, lO-pm, reversed-phase C-18 column (2.2 X 25 cm) using a gradient of 0-10 min 100% A and 10-45 min from 100% A to 75% A at a flow rate of 4 mL/min. Mobile phase A was 0.1% TFA in water, mobile phase B was 0.1 % TFA in acetonitri1e:isopropanol: water (70:20:10). The yield of vitamin &-peptides was about 90% by weight. The structure of each peptide was confirmed by fast atom bombardment mass spectrometry and one of them was by lH-NMR. Preparation of N-(4'-Pyridoxyl)peptide-Oligonucleotide Conjugates (Scheme 2). These conjugates could be prepared by reaction of an iodo group on the oligonucleotide and a thiol group on the peptide (2). Oligoand 2 mg nucleotide with 5' amino linker (4 units, of N-iodoacetoxysuccinimide,dissolved in 50 pL of DMSO, were mixed in 100pL of 0.1M sodium bicarbonate solution for 2 h. The unreacted N-iodoacetoxysuccinimideand byproducts were removed by anion-exchange chromatography on a Nucleogen 60-7 DEAE column (4 X 125 mm) from the Nest Group (Southboro, MA). Mobile phase A was 60% 20 mM sodium acetate, pH 6.5, and 40% acetonitrile. Mobile phase B was mobile phase A containing 0.7 M lithium chloride. The gradient was 100% A for 10 min, 100% A to 88% A for 20 min and 88% A to 50% A in 1 min. The flow rate was 1 mL/min. The eluent corresponding to the peak at 35 min, which was iodo-activated DNA (iodoacetyl-DNA), was collected. About 2 mg of pyridoxyl(&Orn)&ys-NH2 dissolved in
water was added into the iodoacetyl-DNA solution from the anion-exchange chromatography. The mixture was reacted for 1 2 h at room temperature and then put under vacuum to remove acetonitrile. The product, N-(4'pyridoxy1)peptide-oligonucleotide, was isolated by anionexchange chromatography using the same column and mobile phase, but the gradient was I% B/min. The flow rate was 1 mL/min. The final product was desalted by reversed-phase chromatography on a PRP-1 column (4.1 X 150 mm) from VWR (Piscataway, NJ) using a gradient of acetonitrile in 0.1 M trietkylammonium acetate, pH 9.7. After the buffer was removed under vacuum overnight, the conjugates were redissolved in water. The yield was 55% based on A260 (2.2 units). Polyacrylamide Gel Electrophoresis (PAGE). The pyridoxylpeptide-oligonucleotide and peptide-oligonucleotide conjugates were analyzed on a native 20% polyacrylamide gel. The running buffer was 40 mM MOPS and 10 mM sodium acetate (pH 7.0, adjusted with sodium hydroxide). The samples were suspended in 20 fiL of loading buffer (99% formamide), heated to 90 "C for 2 min, chilled on ice, and loaded on the gel. The pictures were taken by Polaroid 667 film under UV shadowing using a green filter from Eastman Kodak Co. (Rochester, NY). RESULTS
Vitamin B6 can be appended to the amimo-terminus of a peptide prior to cleavage from the solid support. The advantage of this approach is that only the amino-terminal amine is free to condense with pyridoxal, while the other potentially active side chain groups are still protected. The Shiff base was formed and then selectively reduced to the stable secondary amine with sodium cyanoborohydride. To test the utility of this approach, a trial of the synthesis was carried out to construct a pyridoxyl-&OmCys-NH:! conjugate. Excess pyridoxal and sodium cyanoborohydride were readily removed by filtration in the synthesis cartridge using a syringe. Following cleavage of the peptide from the resin and removal of protecting groups by TFA, the vitamin B6-containing peptide was analyzed by reversed-phase HPLC (Figure 1).The modified peptide eluted from the column as a single major peak, accounting for >93% of total peak area at 220 nm. Analysis of the peptide by FAB-MS (M 1)revealed the expected mass
+
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Zhu and Stein
Bioconjugate Chem., Vol. 5, No. 4, 1994
c 0.30
.0 c
F (I)
g
0.20
200
240
280
320
360
4
IO
Wavelenth (nm)
Figure 2. UV spectra of pyridoxylpeptide conjugates. B, pyridoxyl-6-Om-Cys-NH2; D, pyridoxyl(6-Orn)&ys-NH2;F, pyridoxyl(6-Orn)8Cys-NH2;H, pyridoxyl(6-Orn)&ys-NH2; J, pyridoxyl-Ala-Arg-His-Thr-Aps-Tyr-Lys-OH.
1
2
3
0 5 101520 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0
Time (min) Figure 1. HPLC chromatography of pyridoxyl-6-Om-Cys on a Vydac C18 column.
(calcd 386, fourld 386.2). The structure of the conjugate was also assigned on the basis of its lH-NMR spectrum for pyridoxyl-6-Om-Cys-NH2 (CD30D): 6 = 1.9-2.1 (b, 4H), 2.69 (s,3H, CH3), 2.85-3.0 (m, 2H), 3.15-3.2 (m, 2H), 3.95-4.1(b,lH),4.45-4.6 (m,3H),4.8 (s,2H),8.2 (s, 1H). These data indicate that the vitamin B6 group had been linked to the peptide in the expected way and remained stably attached and unmodified during synthesis, cleavage, and deprotection. The result showed that condensation can be performed under biphasic conditions. A series of similar isopeptides with different lengths of the ornithine chain was attempted and each gave the correct product accordingto FAB-MS (M + 1)pyridoxyl(6-Orn)&ys-NH, (calcd 614, found 614.3), pyridoxyL(6Orn)&ys-NH2 (calcd 1184, found 1184.5), pyridoxyL(6Orn)12-Cys-NH2(calcd 1641,found 1640.8). Next a model peptide with aliphatic, aromatic, basic, acidic, and hydroxyl-containingside chains, Ala-Arg-His-Thr-Asp-TyrLys, was attempted. The usual amino acid protecting groups, -tBu, -OtBu, -Trt and -Pmc were used. Vitamin B6-linked Ala-Arg-His-Thr-Asp-Tyr-Lyswas prepared and found to have the expected mass (M + 1) (calcd 1041, found 1041.5). Furthermore,the UV spectra demonstrated the presence of the pyridoxyl group (Figure 2). Thus, this method for coupling vitamin B6 to a peptide prior to cleavage from the solid support is generally applicable for the synthesis of vitamin Be-peptide conjugates. A model pyridoxylpeptide-oligonucleotideconjugate, pyridoxyl(6-Orn)&ys-CATTTCTTTATT,was synthesized by conjugation of the pyridoxylpeptide with the oligonucleotide. The purification was by anion-exchange chromatography. Excess pyridoxyl(6-Orn)&ys-NH2 was eluted within the void volume, and the N-(4’-pyridoxyl)peptide-oligonucleotide conjugate peak was at 30 min. This conjugate had the characteristic UV spectrum for both DNA and vitamin B6 (not shown). The conjugate was also analyzed by PAGE. The mobility of pyridoxylpeptide-oligonucleotide conjugate was retarded by the
Figure 3. Electrophoretic analysis of conjugates. Lane 1is 12mer DNA with 5’ amino linker. Lane 2 is (6-Orn)&ys-NH, (5’ 12-mer DNA). Lane 3 is pyridoxyl(6-0rn)8Cys-NH2(5’12-mer DNA).
positively charged peptide, although it migrated slightly faster then a similar peptide-oligonucleotide conjugate without the pyridoxyl group (Figure 3). DISCUSSION
Transmembrane delivery into cells of certain pharmacologic compounds may be achieved through utilization of unrelated transport mechanisms. According to this approach, a conjugate of the pharmacologic compound and the delivery vehicle is prepared. Generally,the vehicle represents a ligand that binds to a cell surface receptor which is internalized through an endocytic pathway. Examples include cellular uptake of ricin A-chain coupled to human chorionic gonadotropin (4),asialoglycoproteinpolylysine conjugates for delivery of DNA into hepatocytes (5), mannose 6-phosphate for uptake of low density lipoproteins (6), and cholera toxin binding subunit for transport of insulin (7). The problem of lysosomal entrapment of the pharmacologic agent has been addressed by the use of transferrin-adenovirus-polylysine conjugates for gene delivery (B), based on the lysosomal disruption ability of adenovirus.
Preparation of Vitamin BBConjugated Peptide
Cellular uptake of low molecular weight compounds, such as water-soluble vitamins, is by a process referred to as potocytosis, as opposed to endocytosis (9). This mechanism has been examined for folate uptake (IO),and it may also be applicable to vitamin Bg. McCormick and colleagues have suggested the utilization of the uptake system for several vitamins, including riboflavin, ascorbic acid, biotin, and vitamin B6 (I). In their studies on the vitamin Be adducts, it was suggested that positively charged groups should be placed near the pyridoxyl group to enhance receptor binding and internalization. Accordingly,the polyornithine bridge between the oligonucleotide and pyridoxyl portion of our conjugate may play a beneficial role. In conclusion, our procedure for conjugating vitamin B6 specifically at the amino-terminus of peptides, especially the possibility of reduction with tritiated cyanoborohydride, provides a foundation for studying the cellular uptake, intracellular localization, and metabolism of such conjugates. ACKNOWLEDGMENT
This work was funded by Gene Shears (Australia) Pty. Ltd. LITERATURE CITED (1) Zhang, Z., and McCormick, D. B. (1991) Uptake of N-(4’-
pyridoxy1)amines and release of amines by renal cells: A model for transporter-enhanced delivery of bioactive compounds. Proc. Natl. Acad Sci. U.S.A. 88, 10407-10410. (2) Zhu, T., Tung, C.-H., Dickerhof, W. A., Breslauer, K. J., and Stein, S. (1993) Preparation and Physical Properties of Conjugates of Oligodeoxynucleotides with Poly(6)ornithine Peptides. Antisense Res. Deu. 3, 349-356.
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(3) Zhu, T., Wei, Z., Tung, C.-H., Dickerhof, W. A, Breslauer, K. J., Georgopoulos, D. E., Leibowitz, M. J., and Stein, S. (1993) Oligonucleotide-Poly-L-ornithineConjugates: Binding to Complementary DNA and RNA. Antisense Res. Deu. 3,265275. (4) Oeltmann, T. N., and Heath, E. C. (1979) A Hybrid Protein Containing the Toxic Subunit of Ricin and the Cell-specific Subunit of Human Chorionic Gonadotropin. J. Biol. Chem. 254, 1028-1032. (5) Wu, G. Y., and Wu, C. H. (1987) Receptor-mediated in Vitro Gene Transformation by a Soluble DNA Carrier System. J. Biol. Chem. 262(10), 4429-4432. (6) Murray, G., and Neville, D. M. (1980) Mannose 6-Phosphate Receptor-mediated Uptake of Modified Low Density Lipoprotein Results in Down Regulation of HydroxymethylgiutarylCoA Reductase in Normal Familial Hypercholesterolemic Fibroblasts. J . Biol. Chem. 255(24), 11941-11948. (7) Roth, R. A., and Maddox, B. (1983) Insulin-Cholera Toxin Binding Unit Conjugate: A Hybrid Molecule With Insulin Biological Activity and Cholera Toxin Binding Specificity. J. Cell Phys. 115, 151-158. (8) Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D. T., and Birnstiel M. L. (1992) Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc. Natl. Acad Sci. U S A .89,6099-6103. (9) Anderson, R. G. W., Kamen, B. A., Rothberg, K. G., and Lacey, S. W. (1992) Potocytosis: Sequestration and Transport of Small Molecules by Caveolae. Science 255, 410-411. (10) Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A,, and Anderson, R. G. W. (1990) The Glycophospholipid-linked Folate Receptor Internalizes Folate Without Entering the Clathrin-coatedPit Endocytic Pathway. J. Cell Biol. 110,637649.
