464
Bloconjugate Chem. 1991, 2, 464-465
Preparation of Oligonucleotide-Peptide Conjugates Ching-Hsuan Tung, M. Jonathan Rudolph, and Stanley Stein* Center for Advanced Biotechnology and Medicine, and Chemistry Department, Rutgers University, 679 Hoes Lane, Piscataway, New Jersey 08854. Received August 9, 1991
A procedure for preparing oligonucleotide-peptide conjugates is presented. I t is based on appending a maleimide group to the oligonucleotide for selective coupling to the thiol side chain of a cysteine residue in the peptide. A convenient chromatographic purification procedure, based on Fmoc-on/ Fmoc-off, is described.
Conjugates of oligonucleotides with peptides having specific functions can be useful for various applications. Examples include the use of a nuclear transport signal peptide todirect intracellular trafficking ( I ) ,a hydrophobic peptide (2)or polylysine (3) to increase cell penetrability, and polylysine to provide multiple attachment sites for nonradioactive reporting probes ( 4 , 5). The tripeptide Gly-Gly-His has been demonstrated to be a metal chelator which can bind Cu(I1) or Ni(I1) (6, 7). This tripeptide has been conjugated to the N-terminus of the DNAbinding domain of the protein Hin recombinase. In the presence of Cu(I1) or Ni(I1) and a free-radical source, there is a sequence-specific cleavage of DNA via an oxidative degradation mechanism. Attachment of this tripeptide to an oligonucleotide might, therefore, provide a type of restriction enzyme based on sequence-specific hybridization. Synthesizing peptide-oligonucleotide conjugates directly on solid-phase peptide and DNA synthesizers has been reported (2,4). Unfortunately, the purine bases of adenine and guanine are labile to acidic conditions. Peptide amide bonds, as well as side chains of certain amino acids, can be partially hydrolyzed under alkaline conditions. Therefore, complete solid-phase synthesis of these conjugates on either a peptide synthesizer or a DNA synthesizer might be unsatisfactory due to side reactions during coupling cycles or cleavage from the solid support (I1. Postsynthesis conjugation is an alternative. A convenient and specific approach for conjugating an oligonucleotide to a peptide utilizes the reaction of a maleimide group with a thiol group. Similar conjugations of peptides or proteins through disulfide linkage have been reported (8-10). In our procedure, a primary, aliphatic amino group is incorporated at the 5’-terminus of the oligonucleotide at the last cycle of automated synthesis. The oligonucleotide is cleaved from the support and purified, and then a maleimide group is appended to the amino group. A residue of cysteine, which has a side-chain thiol, may be placed a t any position in the peptide during its synthesis. The peptide is, likewise, cleaved and purified. The maleimidyl oligonucleotide is then reacted in solution with the peptide. This approach differs from the recently published methods in which a thiol group is incorporated into the synthetic oligonucleotide (1, 11). That requires a more complicated thiolation step or detriylation step, and places the readily oxidizable thiol on the oligonucleotide, which is the limiting reagent for the final conjugation reaction. An additional feature of our process is the facile purification of the final product by two steps of chromatography of the Fmoc-on and Fmoc-off conjugates. The peptide amide (Fmoc-Gly-Gly-His-Cys-NH2) in Scheme I was synthesized on the PAL support by Fmoc 1043-1802/91/2902-0464$02.50/0
chemistry in a MilliGen/Biosearch Model Excel1 peptide synthesizer (Burlington, MA). After synthesis, the peptide with the last Fmoc group left on was cleaved from the solid support with 5 mL of TFA/anisole (95/5) and the solid support was removed by filtration. The filtrate was evaporated to near dryness under a stream of dry nitrogen and then precipitated from ethyl ether. The precipitate was collected by filtration and stored at room temperature. This peptide was used without further purification. Cysteine-containing peptides in the solid form are stable toward oxidation (12). The oligonucleotide used in these studies was an aminolinked 15-mer with the sequence of 5’ TAC TTG GGTTGG CTT 3’, which was synthesized by phosphoramidite methodology using a Model 380B DNA synthesizer (Applied Biosystems, Foster City, CA). A hexanolamine linker was coupled to the 5’-end of the 15-mer on the synthesizer. 3-Maleimidobenzoic acid sulfosuccinimido ester (SulfoMBS) (2 mg) (Pierce; Rockford, IL) and purified aminolinked oligonucleotide I (4 OD, A2m) were incubated in sodium bicarbonate solution (0.1 M, 100 p L ) for 30 min. The unreacted Sulfo-MBS and some byproducts were removed by anion-exchange chromatography. The chromatographic separations were performed on a Nucleogen 60-7 DEAE column (4 X 125 mm). Mobile phase A was 60% 20 mM NaOAc (pH 6.5) and 40% acetonitrile, and mobile phase B was mobile phase A containing 0.7 M LiCl. The gradient was 100% isocratic A for 10 min, 100% A to 88% A for 20 min, and 88% A to 50% A in 1min. The eluent a t 34 min was collected. The collected portion was then desalted by reverse-phase chromatography (Figure 1B). The chromatogram was quite clean, since the byproducts and unreacted Sulfo-MBS had been removed by ionexchange purification. Multiple peaks are commonly observed for derivatized oligonucleotides (13). All the peaks from 19 to 23 min were examined using the photodiode-array detector, and a UV spectrum characteristic of DNA was observed for each single peak. All peaks between 19 and 23 min were pooled and dried under vacuum, giving a 70% yield (2.8 OD, Azm). Fmoc-Gly-Gly-His-Cys-NH2 (3 mg) and maleimidobenzoyl oligonucleotide 11(2.8 OD, A2m) were allowed to react in phosphate buffer (0.1 M, pH 6.6, 200 p L ) and acetonitrile (100 pL) overnight. The reaction mixture was purified by reverse-phase chromatography. Because of the hydrophobicity of the Fmoc group, all the peaks between 19 and 23 min had disappeared, and a new broad peak was seen at 34 min (Figure 1C). Excess, unreacted peptide appeared at 42 min. Fmoc-peptide-oligonucleotide I11 was collected and dried under vacuum to give an 86% yield (2.4 OD, A2so). 0 1991 American Chemical Society
Technical Notes
Bioconlugete Chem., Vol. 2,
No. 8, 1991 466
Scheme I. Synthetic Scheme of Peptide-Oligonucleotide Preparation N a 0 3 s < N - 0 q
3
Oligonucieotide-Ny
0
YN5
3 Oligonucleotide-NH-
I
11
0 SH
I
‘d o
s I
Fmoc-Gly-Gly-His-dys-MI2 I11
Herein, we successfully coupled a metal-chelate peptide to an oligonucleotide through Sulfo-MBS. By Fmocon and Fmoc-off two-step purification, the product was easily prepared and purified. Further studies aimed a t sequence-specific cleavage are in progress.
II
LITERATURE CITED (1) Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio,
h
r 0
I
20
I
40
i
50min.
Figure 1. Reverse-phase HPLC chromatogram: (A) aminolinked oligonucleotide I, (B) maleimidyl oligonucleotide II; (C) Fmoc-on peptide-oligonucleotide 111;(D) Fmoc-off peptide-oligonucleotide IV. Reverse-phase separation was done on an EM LiChrospher 100RP-18, 5-rm column (4 X 125 mm). Mobile phase A was 95% 0.1 M TEAA buffer (pH 7.0) and 5% acetonitrile, and mobile phase B was 5% 0.1 M TEAA buffer (pH 7.0) and 95% acetonitrile. The gradient was 100%A for 5 min, 100% A to 50% A in 50 min. The flow rate was 1 mL/min.
The Fmoc group on the peptide-oligonucleotide conjugate was removed by treatment with piperidinel water (l:l, 100 pL) for 10 min. The piperidine solution was removed under vacuum. The residue was purified by reverse-phase chromatography. The major peak had shifted to 21 min (Figure 1D). The yield for this step was 88% (2.1 OD, Azm). The overall yield of this conjugation was 53%. The collected final product IV was identified by amino acid analysis. The ratio of G1y:His was 70:30, and cysteine was not measured.
F. (1991)Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence. Tetrahedron 47,4113-4120. (2) Juby, C. D., Richardson, C. D., and Brousseau, R. (1991) Facile preparation of 3’oligonucleotide-peptideconjugates. Tetrahedron Lett. 32, 879-882. (3) Leonetti, J.-P., Degols, G., and Lebleu, B. (1990) Biological activity of oligonucleotide-poly(L4ysine) conjugates: mechanism of cell uptake. Bioconjugate Chem. 1, 149-153. (4) Haralambidis, J., Duncan, L., and Tregear, G. W. (1987)The solid phase synthesis of oligonucleotidescontaining a 3’-peptide moiety. Tetrahedron Lett. 28, 5199-5202. (5) Haralambidis, J., Duncan, L., Angus, K., and Tregear, G. W. (1990)The synthesis of polyamide-oligonucleotide conjugate molecules. Nucleic Acids Res. 18, 493-499. (6) Mack, D. P., Iverson, B. L., and Dervan, P. B. (1988) Design and chemical synthesis of a sequence-specific DNA-cleaving protein. J. Am. Chem. SOC.110, 7572-7574. (7) Mack, D. P., and Dervan, P. B. (1990) Nickel-mediated sequence-specific oxidative cleavage of DNA by a designed metalloprotein. J. Am. Chem. Soc. 112, 4604-4606. (8) Gaur, P. K., Sharma, P., and Gupta, K. C. (1989) A simple method for the introduction of thiol group at 5’-termini of oligodeoxynucleotides. Nucleic Acids Res. 17, 4404. (9) Zuckermann, R., Corey, D., and Schultz, P. (1987) Efficient methods for attachment of thiol specific probes to the 3’-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 15,5305-5321. (10) Chu, B. C. F., and Orgel, L. E. (1988) Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds. Nucleic Acids Res. 16, 3671-3691. (11) Ghosh, S. S., Kao, P. M., McCue, A. W., and Chappelle, H. L. (1990) Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconjugate Chem. 1, 71-76. (12) Tsao, J., Lin, X., Lackland, H., Tous, G., Wu, Y., and Stein, S. (1991) Internally standardized amino acid analysis for determiningpeptide/carrierprotein coupling ratio. AnaLBiochen. 197, 137-142. (13) Agrawal, S.,and Zamecnik, P. C. (1990) Site specific functionalization of oligonucleotides for attaching two different reporter groups. Nucleic Acids Res. 18, 5419-5423.
