666
Bioconjugafe Chem. 1994,5,666-668
TECHNICAL NOTES Synthesis and Properties of Cholesteryl-ModifiedTriple-Helix Forming Oligonucleotides Containing a Triglycyl Linker Huynh Vu,*Theresa Schmaltz Hill, a n d Krishna J a y a r a m a n Triplex Pharmaceutical Corporation, 9391 Grogans Mill Road, The Woodlands, Texas 77380. Received June 2, 1994@
In order to enhance the nuclear uptake of triple-helix forming oligonucleotides (TFOs), a triglycylcholesterol group was attached to the 3’ end. The peptide unit was introduced as a “labile” linker with the aim of releasing the oligonucleotide from the endosomes by the action of peptidases after crossing the cell membrane. Cholesteryl-CPG (8) and -TentaGel (9) supports containing 2-[N(glycylglycylglycyl)aminolpropane-1,3-diol(GAP-3) linker were prepared and used for automated oligonucleotide synthesis. The synthesis, characterization, and stability of these compounds are described.
The permeability of the oligonucleotides across the cell membrane and into the nucleus plays a critical role in determining the cellular efficacy of antisense or antigene oligonucleotide ( I , 2 ) . Lipophilic end modifications of oligonucleotides have been shown to enhance the uptake and cellular effkacy (2-7). We have shown that cholesteryl-conjugated TFOs containing 3-aminopropyl glycerol (APG) linker (Figure 1)are taken up more efficiently than the unmodified or 3‘-amine modified TFOs (8). Fluorescence microscopy studies with these cholesterol-modified TFOs show that a major portion of them is still retained in the endosomes resulting in only a small enhancement in nuclear uptake (N. Chaudhary et al., unpublished results). The isolated yields in the preparation of these cholesteryl-modified TFOs containing APG linker were low, the first coupling being only -50%. The solubility of G-rich TFOs containing this end modification was also low. In order to increase the nuclear uptake and overcome the synthesis and solubility problems, we have designed and synthesized a 2-[N-(glycylglycylglycyl)aminolpropane-l,3diol (GAP-3) linker (Figure 2) for the attachment of cholesterol a t the 3‘ end. Another function of this group is to serve as a “labile” linker that will help in the release of the oligonucleotide from endosomes into the nucleus. The “labile” linker approach is well known in the prodrug strategy for the delivery of therapeutic molecules (9). An essential requirement of such a “labile”linker is that its linkage to cholesterol should be stable enough to enhance uptake into the cell and subsequently help in the release of the oligonucleotide from the endosomes by the action of peptidases. The hydrophobic cholesterol moiety is expected to be embedded in the endosomal membrane anchoring the oligonucleotide on the lumenal surface. We postulated that the tethered oligonucleotide along with the triglycyl moiety may then be flipped or transported into the cytosol by as yet a n undescribed mechanism. The peptidases which are abundant in the cytosol could cleave the oligonucleotide from the choles-
* To whom correspondence should be addressed. Tel: 713363-8761. Fax: 713-363-1168 Abstract published in Advance ACS Abstracts, September 15, 1994. @
81
0
d4-
d
51
k 2 / . N H C O
Figure 1. Cholesteryl-conjugated TFOs containing 3-aminopropyl glycerol (APG) linker. R1: oligonucleotide, Rz: -OH or -CPG, TentaGel support. 71
0 d-0-
A
r:
L H i O
R20 H
&
Figure 2. Cholesteryl-conjugated TFOs containing 2-[N-(glycylglycylglycyl)aminolpropane-1,3-diol(GAP-3)linker. R1: oligonucleotide, Rz: -OH or -CPG, TentaGel support.
terol moiety using the triglycyl group as a substrate. To test our hypothesis, a triglycyl linker (GAP-3 linker) was designed as a model compound. The GAP-3 linker was also designed to overcome the steric problems that resulted in low yield in the first coupling step of the APG linker containing oligonucleotides (8). The synthesis of the GAP-3 linker and the linked-oligonucleotides and preliminary data on their properties are described in this paper. The GAP-3 linker-cholesterol was introduced a t the 3‘ end by using solid supports 8 and 9 (Figure 3) for the synthesis of TFOs. Cholesteryl chloroformate (1) was reacted with the silylated glycylglycylglycine, followed by desilylation with 2% HC1 to give N-[(cholesteryloxy)carbonyllglycylglycylglycine (3,82%)(10).Compound 3 was coupled with 2-aminopropane-1,3-diol (4) by using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC) to give 2-[N-[N-(cholesteryloxycarbonyl)glycylglycylglycyl]amino]propane-l,3-diol(5, 85%) (11). Compound 5 was tritylated by 4,4‘-dimethoxytrityl chlo-
1043-1802/94/2905-0666$04.50/0 0 1994 American Chemical Society
Bioconjugate Chem., Vol. 5, No. 6, 1994 667
Technical Notes
i, ii, 82%
-
HO
iii, 85%
3 HO
5
v, 85% vi
DMTO
8
8, R: CPG-NH- (Loading: 52 pmole/gram) 9, R: TentaGel-NH- (Loading: 180 pmole/gram) Figure 3. (i) (a) 2, bis(trimethylsilyl)acetamide, DMF; (b) -20 "C, 1;(ii) 2% HCl; (iii) EDC, pyridine; (iv) pyridine, DMAP, EtsN, 4,4'-dimethoxytrityl chloride; (v) succinic anhydride, DMAP, pyridine; (vi)(a) TBTU, HOBT, DMF, N-ethylmorpholine, NHz-support; (b) acetic anhydride, DMAP.
ride in the presence of 4-(dimethylamino)pyridineand triethylamine to provide l-O-(4,4'-dimethoxytrity1)-2-[N-
[N-[(cholesteryloxy)carbonyl]glycylglycylglycyllamino]propane-1,3-diol (6, 50%) (12). The supports CPG (8, loading: 52 pmoYg, 64% loaded) and TentaGel (9, loading: 180 pmol/g, 86% loaded) were prepared by succinylation of compound 6 , followed by coupling of the succinate ( 7 ) (13)to the free amino group. The stability of the GAP-3 linker-cholesterol conjugate was determined before oligonucleotide synthesis was initiated. DMT-cholesteryl supports (8,9) were treated with various deprotection solutions: (hydrazine/MeOH; 1:3 v/v, 0.4 M NaOH in 75% MeOWwater, a t room temperature, overnight, or concd NH40H, 0.1 M NaOH aqueous solution, 56 "C, overnight) as well as the buffer conditions (1.5 M NaCl containing 15 mM NaOH, a t room temperature, overnight) used in the purification. The support was then filtered and washed thoroughly with dichloromethane, dich1oromethane:methanol 1:1 (v/v), and methanol to extract the hydrolyzed products from the support completely. The crude material was analyzed by thin layer chromatography (dichloromethane: methanol 1:9, v/v). The product bands were isolated by preparative TLC and characterized by lH-NMR. The compound, 1-0-(4,4'-dimethoxytrityl)-2-[N-[N-[(cholesteryloxy)carbonyllglycylglycylglycyllaminolpropane1,3diol (6) was shown to be fairly stable under the standard deprotection conditons (concentrated NH40H, 56 "C overnight). Only minor amounts of degraded products were detected. TLC analysis showed that treatment with 0.4 N NaOH overnight in methano1:water (3:1, v/v) solution at room temperature yielded cholesterol and unidentified products. The yield of cholesterol as judged by visual inspection of the TLC was -90%. On the basis of these stability studies, several 3' end cholesteryl-modified TFO sequences were synthesized on a 0.2-300 pmol scale using cholesteryl supports 8 and 9 on Applied Biosystems Models 380B, 39214, and/or MilliGen Models 8700 and 8800 with a coupling efficiency of >97%, including the first step. Several sequences of G-rich TFOs were synthesized using these supports. Cleavage and deprotection were carried out under standard conditions (concentrated NH.+OH,56 "C, overnight). Crude oligonucleotides were purified on a Pharmacia FPLC system by anion exchange chromatoghaphy on a Q-Sepharose column (1 cm x 10 cm) (14).Enzymatic digestion of the cholesteryl oligonucleotides by P1 nu-
cleasehacterial alkaline phosphatase gave the expected deoxynucleoside composition. Gel electrophoresis analysis of purified oligonucleotides after end labeling with 32PATP and using polynucleotide kinase showed two bands in the ratio of 7:3. The slower band (one unit slower than the oligonucleotides containing 3' end free amino group) contained cholesterol. Electrospray mass spectroscopy analysis of the slower band (on the gel) of a G-rich oligonucleotide, 21 mer, containing GAP-3-linker and cholesterol had a n observed mass of 7097.14 while the calculated mass was 7096.55. The faster moving band does not appear to contain cholesterol suggesting that some cholesterol is cleaved during the deblocking procedure. I t is not clear why the linker-cholesterol is unstable during deblocking of the oligonucleotide while it appears to be stable by itself before the oligonucleotide is attached. A possible explanation could be that on cleavage of the oligonucleotide-linker-cholesterol from the support, an hydroxyl group is liberated t h a t is in position to attack the neighboring phosphate group via the formation of a six-membered ring intermediate. However, on deblocking, the support containing linker cholesterol (compound 8 or 9) generated compound 6 which lacks the phosphate group. The above-mentioned pathway is, therefore, not possible. There was no loss of linker-cholesterol when the oligonucleotide was deblocked a t room temperature for 48 h. Under these conditions, the isobutyryl protecting group on the bases was also completely deprotected from the oligonucleotide. There was essentially only one band on purification by gel. Electrospray mass spectroscopy analysis of this band confirmed the presence of cholesterol on oligonucleotide. A comparison of binding affinities for TFOs containing 3'-propanolamine and 3'-cholesteryl GAP-3 linker modifications showed that cholesteryl attachment to the TFOs did not affect the binding significantly. A 3'-propanolamine TFO, 5'-GTGGTGGTGGTGTTGGTGGTGGTTTGGGGGGTGGGG-propanolamine-3') had a Kd of 5 x 10-lo M while the same sequence with 3' GAP-3 linkercholesteryl group 5'-GTGGTGGTGGTGTTGGTGGTGGTTTGGGGGGTGGGG-cholesterol-3' had also a Kd of 5 x M. Triplex formation was assessed using the gel shift assay, essentially as described (15). Preliminary uptake studies of TFOs containing GAP-3 linker and cholesterol showed a 2-5-fold enhancement in nuclear uptake and is in agreement with the expected enhancement in uptake using this approach.
668 Bioconjugate Chem., Vol. 5, No. 6 , 1994
The "labile" linker approach presented in this paper appears to be promising and may serve as a general and powerful tool for enhancing the nuclear uptake of oligonucleotides. ACKNOWLEDGMENT
The authors would like to thank the National Cancer Institute, SBIR Grant (No. 1R43 CA 61649-01, PI-K.J.) for supporting this work. We wish to thank Dr. Uhlmann, Hoechst AG, Germany, for carrying out the electrospray mass spectroscopy of the cholesterol containing oligonucleotides and N. Chaudhary for helpful discussions. LITERATURE CITED (1) Uhlmann, E., and Peyman, A. (1990) Antisense oligonucleotides: a new therapeutic principle. Chemical Rev. 90,
543-584. (2) Jaroszewski, J. W., and Cohen, J. S. (1991)Cellular uptake of antisense oligonucleotides Adv. Drug Design Res. 6, 235250. (3) de Smidt, P. C., Doan, T. L., deFalco, S., and Van Berkel, T. J. C. (1991)Association of antisense oligonucleotides with lipoproteins prolongs the plasma half-life and modifies the tissue distribution. Nucl. Acids Res. 19, 4695-4700. (4) Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-conjugated oligonucleotides: synyhesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Proc. Natl. Acad. Sci. U.S.A. 86, 6553-6556. (5) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligonucleotide conjugates Nucl. Acids Res. 18, 37773783. (6) Will, D. W., and Brown, T. (1992) Attachment of vitamin E derivatives to oligonucleotides during solid-phase synthesis. Tetrahedron Lett. 38, 2729-2732. (7) Ing, N. H., Beekman, J. M., Kessler, D. J., Murphy, M., Jayaraman, K., Zendegui, J. G., Hogan, M. E., O'Malley, B. W., and Tsai, M. (1993)In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplexforming oligonucleotide.Nucl. Acids Res. 21, 2789-2796. (8) Vu, H., Singh, P., Lewis, L., Zendegui, G. J.,and Jayaraman, K. (1993) Synthesis of cholesteryl supports and phosphoramidite for automated DNA synthesis of triple-helix forming oligonucleotides (TF'Os). Nucleosides Nucleotides 12,853-864. (9) H. Bundgaard, Ed. (1985) Design of Pro-Drugs, Elsevier, New York. (10) Compound 3: a white solid; mp 194-198 "C dec; 'H-NMR (DMSO-ds) 6 0.67 (5, 3H, -CH3, chol), 0.84 (9, 3H, -CH3, chol), 0.85 (s, 3H, -CH3, chol),0.91 (d, J = 6.4 Hz, 3H, -CH3,
Vu et al.
chol),0.98 (s,3H, -CH3, chol), 2.59-0.95 (m, 29H, chol), 3.66 (d, J = 5.8 Hz, 2H, -CO-CHzNH-, linker), 3.77 (d, J = 5.8 Hz,4H, 2(-COCHm-), linker), 4.34 (m, lH, -CHzCHCHz-, chol), 5.33 (b,s, lH, C=CH-, chol), 7.04, 7.98, and 8.03 (3 b,s, 3H, 3 (NH), linker), 12.30 (b,s, lH, -COOH, linker). Anal. Calcd for C34H~5N306(601.82): C, 67.86; H, 9.21; N, 6.98. Found: C, 67.97; H, 9.23; N, 6.91. (11) Compound 5: a white solid; mp 205-206 "C. 'H-NMR (DMSO-de) 6 0.65 (s, 3H, -CH3, chol), 0.84 (9, 3H, -CH3, chol), 0.85 (9,3H, -CH3, chol), 0.89 (d, J = 6.3 Hz, 3H, -CH3, chol), 0.97 (s,3H, -CH3, chol), 2.59-0.95 (m, 29H, chol),3.40 (t, J = 5.32 and 5.36 Hz, 4H, -CHzOH, linker), 3.63 (d, J = 5.28 Hz, 2H, -COCHzNH-, linker), 3.7 (t,J = 5.8 and 5.52 Hz,4H, 2(-COCH&'H-), linker), 3.75 (b s, lH, -CHzCHNH-, linker), 4.34 (m,lH, -CHzCHCHz-, chol), 4.59 (t, J = 5.52 and 5.48 Hz,2H, 2 (-OH)), 5.33 (b s, lH, C=CH-, chol), 7.22, 8.03, and 8.12 (3 b s, 3H, 3 (NH), linker), 7.47 (d, J = 7.9 Hz, lH, (NN),linker). Anal. Calcd for C37H6~N407(674.918): C, 65.85; H, 9.26; N, 8.30. Found: C, 66.09; H, 9.23; N, 8.16. (12) Compound 6: a white solid; mp 172-173 "C. lH-NMR (DMSO-de) 6 0.65 (s, 3H, -CH3, chol), 0.84 (s, 3H, -CH3, chol), 0.85 ( 8 , 3H, -CH3, chol),0.89 (d, J = 6.3 Hz, 3H, -CH3, chol), 0.97 (s,3H, -CH3, chol), 2.59-0.95 (m, 29H, chol),2.98 (m, 2H, CHzODMT), 3.51 (t, J = 5.4 and 5.36 Hz, 2H, -CHzOH, linker), 3.63 (d, J = 5.32 Hz, 2H, -COCHzNH-, linker), 3.74 (s, 6H, 2(CH30-)), 3.74 (m,4H, 2(-COCH2NH-), linker), 4.0 (m, lH, -CHzCHNH-, linker), 4.34 (m, lH, -CHzCHCHz-, chol), 4.59 (t, J = 5.52 and 5.48 Hz, lH, -OH), 5.33 (b s, lH, C=CH-, chol), 6.60-7.30 (m, 13H, DMT), 7.22, 8.03, and 8.11 (3 b s, 3H, 3 (NH), linker), 7.63 (d, J = 8.2 Hz, lH, (NH), linker). Anal. Calcd for CssHeoN409 (977.297): C, 71.28; H, 8.25; N, 5.73. Found: C, 71.27; H, 8.39; N, 5.73. (13) Compound 7: a white solid; mp 110-112 "C. 'H-NMR (DMSO-ds) 6 0.65 ( 8 , 3H, -CH3, chol), 0.83 ( 8 , 3H, -CH3, chol), 0.84 (s, 3H, -CH3, chol), 0.89 (d, J = 6.3 Hz, 3H, -CH3, chol), 0.96 ( 8 , 3H, -CH3, chol), 2.34 and 2.40 (m, m, 4H, -CHzCHz-, succinyl), 2.59-0.95 (m, 29H, chol), 3.0 (m, 2H, CHzODMT),3.62 (m, 2H, -COCHzNH-, linker), 3.73 (s,6H, 2(C&o-)), 3.76 (m, 4H, 2 (-COCHzNH-), linker), 4.08 (t,J = 4.68, 5.60 Hz, lH, -CHzCHNH-, linker), 4.20 (m, 2H, -CHzOH, linker), 4.31 (m, IH, -CHzCHCHz-, Chol), 5.32 (b s, lH, C=CH-, chol),6.88-7.38 (m, 13H,DMT), 7.38,7.87, 8.31, and 8.49 (4 b s, 4H, 4 (NH), linker). Anal. Calcd for C&I~401z+Hz0(1095.38): c, 67.97; H, 7.91; N, 5.11. Found: C, 68.36; H, 7.84; N, 5.12. (14) Murphy, M., Rieger, M., and Jayaraman, K. (1993) Largescale synthesis of triple-helix forming oligonucleotides using a controlled-poreglass support. Biotechniques 15,1004-1010. (15) Durland, R. H., Kessler, D. J., Gunnell, S., Duvic, M., Pettitt, B. M., and Hogan, M. E. (1991) Binding of triple helix forming oligonucleotidesto site in gene promoters. Biochemistry 30, 9246-9255.
