Application of the Favorskii Rearrangement to the Preparation of A-Norsteroids. Bill B. Smith, Harold R. Nace. J. Am. Chem. Soc. , 1954, 76 (23), pp 6119â6122. DOI: 10.1021/ja01652a076. Publication Date: December 1954. ACS Legacy Archive. Note: In
Mar 1, 2010 - School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, U.K. ... and subsequent addition of aldehydes provides a new route to enantioenriched homoallylic ... Journal of the American Chemical Society 2018 140 (7),
Application of the Peterson olefination to the preparation of substituted triafulvenes from methoxycyclopropenylium triflates. Hans H. Schubert, and Peter J. Stang.
A Theoretical Study of the Favorskii Rearrangement. Calculation of Gas-Phase Reaction Paths and Solvation Effects on the Molecular Mechanism for the ...
Apr 5, 2010 - natural logarithm (ln(kX/kH)). However, Hammett values are calculated on the basis of the logarithm to the base 10 (log(kX/. kH)). The correct ...
Apr 5, 2010 - 2010,. 132, 3793â3807]. Takeru Furuya, Diego Benitez,. Ekaterina. Tkatchouk, ... The correct values are F1 ) -0.19 (originally reported as. -0.45 ...
Bioconjugate Chem. 1994,5,373-370
Routine Preparation of Thiol Oligonucleotides: Application to the Synthesis of Oligonucleotide-Peptide Hybrids Nicholas J. Ede, Geoffrey W. Tregear, and Jim Haralambidis' Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. Received February 18, 1994"
Oligonucleotide-peptide hybrids have potential for use as antisense inhibitors of gene expression, with the peptide helping to increase the intracellular concentration of the active oligonucleotide. The preparation of such hybrids can be achieved by the coupling of thiol-derivatized oligonucleotides with maleimido-peptides. We have developed reliable methods for preparing 5'-thiol oligonucleotides in good yields using phosphoramidite chemistry and coupling 6-(tritylthio)hexyl phosphoramidite as the 5'-terminal residue. The use of highly pure thiol phosphoramidite as well as a manual iodine treatment after this coupling were found to be important. Oligonucleotide-peptide hybrids were prepared in high yield (855% ) by reacting freshly purified 5'-thiol oligonucleotides with peptides derivatized at their N-terminus with a maleimido functionality.
INTRODUCTION Synthetic oligonucleotides are useful tools in molecular biology as probes and as antisense inhibitors of gene expression (1-3). For the latter application, the oligonucleotide must enter the cell and bind to its target mRNA to inhibit its translation. However, oligonucleotides do not cross cell membranes readily, and it is thus difficult to achieve high intracellular concentrations. We have devised a novel series of oligonucleotide-peptide hybrid molecules in which the peptide segment is designed to enhance the ability of the oligonucleotide to enter the cell. In a previous report we described a total synthesis method of preparing oligonucleotide-peptide hybrids on a controlled pore glass (CPG) solid-phase support (4). The peptide is synthesized first, a derivatized linker attached, and the oligonucleotide assembled onto the linker by standard DNA synthesis methods. However, this method is unsuitable for the preparation of some oligonucleotidepeptide molecules in good yield. Therefore, we have investigated methods for the specific linking of peptides and oligonucleotides, prepared and purified separately. Conjugation of DNA and peptide synthons can be achieved if the 5'-terminus of the synthetic oligonucleotide is derivatized with a thiol group (5-9). The thiol group is introduced at the 5' terminus during the solid-phase synthesis procedure by reaction with commercially available thiol-linker phosphoramidites. Peptides can be synthesized separately containing the thiol-reactive maleimido group. Our initial attempts to conjugate thiol oligonucleotides to maleimido-peptides by this approach were not satisfactory. However, we have now developed improved procedures for the preparation of large amounts of thiol oligonucleotides and their conjugation to maleimidoderivatized peptides to give oligonucleotide-peptide hybrids in high yield. MATERIALS AND METHODS All melting points are uncorrected. lH NMR spectra were recorded at 300 MHz on a Brucker AM300 spectrometer. 3lP NMR spectra were recorded at 121.5 MHz on the same spectrometer. Amino acid analyses were Abstract published in Advance ACSAbstracts,May 15,1994. 1Q43-10Q2/94/29Q5-Q373$04.5Q/Q
performed on a Beckman System 6300 analyzer after the samples had been hydrolyzed in vacuo for 24 h at 130 "C, with HC1/0.1% phenol. High-performance liquid chromatography (HPLC) was performed on a Waters liquid chromatography system, consisting of a Waters 600 multisolvent delivery system with a variable-wavelength detector. The thiol-oligonucleotide and hybrid molecules were purified using one of the following Synchrom columns: RP CIS250 X 4.6 mm (column A), Cq 250 X 10 mm (column B), or CIS250 X 21.2 mm (column C). Buffer A was 0.1 M triethylammonium acetate (TEAA) in water, buffer B CHsCN, and a gradient of 0-5076 B over 30 min with flow rates of 1.5,3, or 10 mL/min for columns A, B, and C, respectively, were used with detection at 260 nm. Peptides were purified using a Vydac Cq 250 X 10 mm reversed-phasecolumn (cat. no. 214TP1010) buffer A water (0.1% TFA); buffer B CH3CN (0.1% TFA) with a flow rate of 3 mL/min and detection at 214 nm. The purity of both peptides was confirmed with a Vydac analytical Cla column, 250 X 4.6 mm (cat. no. 218TP54) buffer A water (0.1% TFA); buffer B CH&N (0.1% TFA) with a flow rate of 1.0 mL/min and detection at 214 nm. Flash chromatography was carried out using silica gel 60,4043 pm (230-400 mesh) (E. Merck cat. no. 9385) using solvent systems indicated in the text. Analytical thin-layer chromatography (TLC) was performed on Merck SG-60 precoated plastic plates. Chemicals. Unless otherwise stated, solvents were BDH analytical grade. Dimethylformamide (DMF) and trifluoroacetic acid (TFA)were of peptide synthesis grade (Auspep, Melbourne, Australia). Maleic anhydride was obtained from Pierce Chemicals, &alanine from BDH, dicyclohexylcarbodiimide (DCC),l-hydroxybenzotriazole (HOBt), trinitrobenzenesulfonic acid (TNBSA), thioanisole, and diisopropylethylamine (distilled from CaHz prior to use) from Fluka, N-Hydroxysuccinimide (NHS), (benzotriazol-l-y1oxy)trispyrrolidinophosphonium hexafluorophosphate (pyBOP), O-benzotriazolyl-N,N,N',N'tetramethyluronium hexafluorophosphate (HBTU) from Auspep, 4-methylmorpholine (NMM) from E. Merck, and triphenylmercaptan, 6-chlorohexanol, and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite from Aldrich Chemical. Succinimido 3-Maleimidopropanoate (3). The method of Nielsen and Buchardt (10)was adapted. Thus, 0 1994 American Chemical Society
Ede et al.
Bloconjugate Chem., Vol. 5, No. 4, 1994
Table 1. Characterization Data HPLC compd t R (min) peptide 1 27.94O 21.206 peptide 2
conjugate 9 conjugate 10
90e 78e 84f 85f
amino acid analysis Ala 3.03 (3)Leu 6.07 (6) Arg 2.90 (3) Asn 1.13 (1)Thr 2.00 (2) Ser 1.16 (1)Thr 4.07 (4) Pro 0.80 (1) Val 1.04 (1)Tyr 0.92 (1)Phe 0.88(1)Arg 1.99 (2) Ala 2.93 (3) Leu 6.10 (6) Arg 2.97 (3) Asn 1.06 (1)Thr 1.96 (2) Ser 1.00 (1)Thr 4.29 (4) Pro 0.92 (1) Val 0.92 (1)Tyr 1.00 (1)Phe 0.93 (1)Arg 1.92 (2)
oligonucleotide/ peptide ratio
0 Vydac analytical Cia, 250 x 4.6 mm, buffer A water (0.1% TFA); B CH3CN (0.1% TFA), &100% B over 30 min. * Vydac analytical Cia, 250 X 4.6 mm, buffer A water (0.1% TFA); B CHaCN (0.1% TFA), &50% B over 30 min. Synchrom C4 250 X 10 mm, buffer A (0.1 M TEAA); B CH3CN&50% B over 30 min. d Synchrom Cl* 250 X 4.6 mm, buffer A (0.1 M TEAA); B CHsCN &50% B over 30 min. e Based on weight of crude isolated peptide. f Based on amount of pure thiol oligonucleotide read at 260 nm.
/3-alanine (0.91 g, 10 mmol) was added to a solution of maleic anhydride (1.00 g, 10 mmol) in DMF (12 mL) and the mixture stirred for 2 h. The resulting solution was cooled in an ice bath, and N-hydroxysuccinimide (1.44 g, 12.5 mmol) was added followed by DCC (4.12 g, 20 mmol). After approximately 5 min the ice bath was removed and the solution stirred overnight. The resulting dicyclohexylurea (DCU) was filtered and the filtrate poured into water (60 mL) and extracted with CHzClz (3 X 20 mL). The organic phase was washed with water (20 mL), 5% NaHC03 (2 X 20 mL), and brine (20 mL) and dried (NazSod). The solution was filtered and the solvent evaporated (reduced pressure). The residue was dissolved in CHzClz (3 mL), andaddition of petroleum ether precipitated 3 as a white solid (1.10 g, 41%): mp 162-163 "C (lit. (10) 164166 "C). 6-(Tritylthio)hexanol (5). The method of Connolly and Rider (5) was adapted. Sodium hydroxide (0.88g, 22 mmol) was dissolved in water (5 mL) and added to a mixture of triphenylmercaptan (5.52 g, 20 mmol) in ethanol (30 mL) with stirring. Following this, 6-chlorohexanol (1.25 mL, 11mmol) was added and the mixture stirred at room temperature overnight. It was then filtered, the filtrate collected, and the solvent removed (reduced pressure). The residue was redissolved in CHzC12 (50 mL) and washed with water (3 X 30 mL). The organic extract was dried (NazS04)and evaporated (reduced pressure) to yield an oil which was flash chromatographed (CHCl3) to yield a clear oil which crystallized from ethedpetroleum ether to give 5 as a white solid (1.25 g, 30%): mp 68-69 "C (lit (11) 70-72 "C). No starting material could be detected by lH NMR: TLC (CHCl3) Rf = 0.30; lH NMR (CDC13) 7.40 (15H, m, ArH), 3.58 (2H, t, J = 6.58 Hz, OCH2),2.15 (2H, t, J = 7.25 Hz, SCH2),1.53-1.22 (8H, m, CH2). 6-(Tritylthio)hexanol, 2-Cyanoethyl N,N-Diisopropylphosphoramidite (6). The method of Connolly and Rider (5)was adapted. 6-(Tritylthio)hexano1(5)(0.70 g, 1.86 mmol) was coevaporated twice with 10% pyridine in CHzClz (10 mL) and dried overnight under vacuum. Under argon, distilled diisopropylethylamine (1.30 mL, 7.44 mmol) was added followed by CHzClz (5 mL). The resulting solution was cooled in an ice bath, and 2cyanoethyl N,N-diisopropylchlorophosphoramidite(1.04 mL, 4.65 mmol) was added (by syringe). After 2 h the solvent was evaporated by bubbling argon. The residue was dissolved in ethyl acetate (20 mL) and washed with 5% NaHC03 (2 X 20 mL) and water (2 X 20 mL). The organic phase was dried (Na2S04)and the solvent evaporated (reduced pressure) to yield a clear oil. The product was then purified according to the method of Sinha and Striepeke (11). A flash column was packed with silica gel using 25 % ethyl acetate in hexane containing 5 % pyridine. The column was first washed with one volume of 25% ethyl acetate in hexane. The crude product was loaded
dissolved in 50% ethyl acetate in hexane and the column eluted with 30% ethyl acetate in hexane to yield pure 6 as a clear oil (0.92 g, 86%): TLC (ethyl acetatdhexane, 50/50) Rf = 0.75; 31PNMR (CDzClz) 6 147.63. Peptide Synthesis. Continuous flow solid-phase peptide synthesis was carried out using a CRB manual Synthesizer on Pepsyn KlOO resin (CRB). An internal standard (glycine) and an acid-labile handle ((hydroxymethy1)phenoxyacetic acid, from Novabiochem) were attached before peptide synthesis was commenced. Fmoc amino acids were obtained from Auspep. Standard solidphase peptide synthesis protocols for Fmoc/t-Bu chemistry were employed (12). All couplings were carried out in DMF using a 3-fold excess of amino acid, HBTU (peptide l), or pyBOP (peptide 2) and HOBt. A 5-fold excess of NMM was used. The maleimido reagent 3, with an equimolar amount of HOBt, was coupled to the N-terminus of the resin-bound peptides to give (after cleavage, deprotection, and purification) peptides 1 and 2. The peptide-resins were cleaved at room temperature for 3 h with 95 % trifluoroacetic acid/5 % thioanisole. The cleavage mixture was filtered, and the volume of the filtrate was reduced to approximately 2 mL (reduced pressure). The crude peptides were precipitated by the addition of cold ether. After the peptides were stored overnight at 0 "C the ether was decanted and the peptides washed with fresh ether (3 X 20 mL). After the final decantation of ether, the peptides were dissolved in 25% aqueous CH3CN and freeze-dried. After dissolution of the peptides in 65% CH3CN (3 mL) they were purified by HPLC, using gradients of 0-100% B (peptide 1) or 0-50% B over 30 min (peptide 2). The purity of all peptides was confirmed by analytical CISHPLC (see Table 1for conditions) and amino acid analysis. Thiol Oligonucleotide Synthesis. Oligonucleotides 7 and 8 were synthesized on an Applied Biosystems 380A DNA Synthesizer using standard 8-cyanoethyl-protected phosphoramidites (Applied Biosystems, Foster City, CA) (13)on a 10 pmol scale. The thiol linker phosphoramidite 6 (50 mg, 85 pmol) was dissolved in the appropriate volume of acetonitrile for the program being used, e.g., 1.5 mL for a 10 pmol scale, and attached to a spare port on the DNA synthesizer. The amidite was then applied to the reaction column using a 10 pmol program. The coupling was followed by an acetonitrile wash and reverse flush, the column was removed immediately, and the resin was transferred to a sintered reaction vessel. It was then treated with iodine (0.05 M in THF:pyridine:water 7:1:2) for 30 s,washed with CH3CN (3 X 20 mL), and dried under a stream of argon. The oligonucleotide was immediately cleaved by treatment with 30% ammonia for 6 h and the resulting solution heated at 50 "C for 18 h to effect base deprotection. The ammonia was removed by a stream of argon and the resulting aqueous solution purified immediately by HPLC using column C. Fractions containing
Bioconjugate Chem., Vol. 5, No. 4, 1994
Technical Notes 6N;PEPTiDE
Scheme 1. Synthesis of Maleimido Reagent 3.
Figure 1. Oligonucleotide-peptide synthesis via the thiol oligonucleotide and maleimido-peptide conjugation reaction.
the pure trityl-ON oligonucleotide were pooled and freezedried. The trityl group was removed after purification according to published methods (5). Thus, trityl-ON oligonucleotide 7a (10 mg, 333 OD units) was dissolved in 0.1 M TEAA (20 mL), 1 M silver nitrate (2.1 mL) added, and the mixture vortexed and allowed to react for 30 min. Following this, 1M dithiothreitol (DTT, 2.5 mL) was added and the reaction mixture vortexed and allowed to react for a further 20 min. The yellow mixture was centrifuged and the supernatant collected. The precipitated silver salt was washed twice more with 0.1 M TEAA and centrifuged and the supernatants were pooled. The thiol oligonucleotide/DTT mixture can be stored frozen until the conjugation experiment. Oligonucleotide-Peptide Hybrid Synthesis. The previouslyprepared and purified peptide maleimides were reacted with thiol oligonucleotide immediately after removal of the excess DTT by HPLC. A sample synthesis follows. Trityl-ON thiol oligonucleotide 7a (3.0 mg) was deprotected as described in the previous section. Excess DTT was removed by HPLC (column B) to give pure thiol oligonucleotide 7b with a retention time of 15.24 min, 2.56 mg as read at 260 nm. The purified thiol oligonucleotide, still in HPLC buffer (-7 mL), was reacted immediately with peptide maleimide 1which was dissolved in 20% 0.1 M TEAA/acetonitrile (1mL). The molar ratio of peptide to oligonucleotide was 1O:l. The mixture was incubated at 37 OC for 2 h, after which time an analytical HPLC indicated complete reaction. The mixture was purified by HPLC (column B) immediately and dialyzed (Spectrapor 6 dialysis tubing, MW cutoff 2000) against 0.1 M NaCl (4 X 3 h) and water (4 X 3 h) to give the desired hybrid 9, retention time 29.19 min, 2.16 mg (85% yield) as read at 260 nm. Analytical data are shown in Table 1.
amidite 6. This reagent is available commercially and is coupled to the 5'-terminus of the oligonucleotide as the last coupling. The retardation of the resulting tritylcontaining oligonucleotides on RP HPLC facilitates purification. The trityl group is removed by reaction with silver nitrate and the product isolated by RP HPLC after treatment with dithiothreitol (DTT). The process has been reported to work well for short oligonucleotides (12mers) but yields decrease significantly for the preparation of longer thiol oligonucleotides (5, 8). In this report we describe the preparation of conjugates composed of either a 20mer sequence antisense to human immunodeficiencyvirus (HIV-1) (14)or a 20mer antisense to rat a-fetoprotein (rAFP) (15). The peptides that were conjugated to these oligonucleotides include the a-helical peptide 1 (16) and an F, receptor binding peptide 2 derived from the putative receptor binding site of rat IgG y2b (residues 289-302) ( I 7). The corresponding sequence in the human IgG c H 2 domain has high binding affinity for the Fc receptor (18). Preparation of maleimidopeptides. For the synthesis of peptides 1and 2, standard solid-phase peptide synthesis methodologyfor Fmoclt-Bu chemistry was employed (12).
RESULTS AND DISCUSSION
Our primary objective is to develop oligonucleotidepeptide hybrids as antisense agents. In order to prepare these hybrids efficiently, we have investigated methods for conjugating separately prepared peptides and oligonucleotides. The conjugation of a 5'-thiol oligonucleotide with a peptide containing a maleimide functionality at the N-terminus, reported by Eritja et al. (6),was a logical route to these hybrid molecules. The conjugation of these two motifs is shown in Figure 1. The conjugation of an oligonucleotide to a peptide can also be achieved by derivatizing the 5'-terminus of the oligonucleotide with a maleimido group and reacting this with a cysteinecontaining peptide (9). The incorporation of a thiol group into oligonucleotides is achieved commonly with 6-(tritylthio)hexylphosphor-
The maleimido reagent 3 was synthesized by adapting the method of Nielsen and Buchardt (10)(Scheme 1). We found it necessary to include a base wash to remove any uncyclized maleimido intermediate 4. The presence of
Ede et al.
Bioconjugate Chem., Vol. 5, No. 4, 1994
Scheme 2. Synthesis of Protected Thiol Linker Reagent 6. Ph3C-SH
RS-(CH2)6-O-P-GCG TAC TCA CCA GTC GCC GC
7a R=trityl 7b R=H PhSC-S-(CH2),yOH
RS-(CH2)6-O-P-CAT GGT TGC TGG CTG CTT CAT 0-
OCHpCHpCN 6 a
Key: (a) NaOH, EtOH; (b) CIP(OCH2CH2CN)N(iPr)2,
this byproduct was confirmed by lH NMR. The maleimide reagent 3, with an equimolar amount of l-hydroxybenzotriazole (HOBt), couples to the N-terminus of the peptide-resin within 20 min (as judged by the TNBSA test (19)). The crude (isolated peptide) yields of peptides 1and 2 were 90 and 78% respectively. For both peptides, purity of the crude cleaved product was good (285%) and amino acid analysis gave satisfactory ratios (Table l ) , including a peak for &alanine, derived from the maleimido reagent 3. The maleimido containing peptides are stable if stored dry. Preparationof Thiol-ContainingOligonucleotides. The successful preparation of large amounts (>1mg) of thiol oligonucleotides depends largely on the purity of the (trity1thio)hexyl phosphoramidite 6 and to a lesser extent on the workup procedure used. Most laboratories require only small amounts of thiol oligonucleotide for labeling studies and use the commercially available reagent. However if larger amounts are required, for example, for antisense studies, then it may be more economical to undertake synthesis of the reagent 6. The preparation of 6-(trity1thio)hexanol ( 5 ) and the corresponding phosphoramidite 6 was adapted from the method of Connolly and Rider (5) and is outlined in Scheme 2. We found that reacting approximately equimolar amounts of triphenylmercaptan with 6-chlorohexan01 resulted in substantial amounts of 6-chlorohexanol being retained in the final product. The product 5 and 6-chlorohexanol coeluted by TLC but the presence of the starting material could be clearly seen by IH NMR. To ensure complete conversion of 6-chlorohexanol to the product 5 a 2-fold excess of triphenylmercaptan was used. 1H NMR confirmed the purity of 5. The synthesis of the phosphoramidite was relatively straightforward, but the purity of this product is important to the success of thiol oligonucleotide synthesis. The purification method described by Sinha and Striepeke (11)was found to give the cleanest product. The purified phosphoramidite 6 was stored under argon at -20 OC. Our early attempts at thiol oligonucleotide synthesis were not successful. For the HIV-1 thiol oligonucleotide 7, the apparent “trityl ON” material 7a coeluted with unmodified “trityl OFF” oligonucleotide (this had been previouslysynthesized), suggestingthe absence of the trityl group. The isolated product (after treatment with silver nitrate and DTT) was not reactive to maleimido peptide
8a R=trityl 8b R=H
1, suggesting that the 5’-terminus did not contain a thiol functional group. Yields for the preparation of thiol oligonucleotidesutilizing phosphoramidite chemistry have been reported to be low ( 5 , 8 ) . Dimerization of the thiol oligonucleotide has been considered as a cause for the low yields, although attempts to reduce the dimer back to a reactive thiol with DTT were not successful (8). In the present study, treatment of the unreactive thiol oligonucleotide with DTT resulted in no change in maleimido peptide reactivity, suggesting that the low yields were not related to dimerization but more likely resulted from chemical modification of the 5’-thiol terminus. Phosphorus NMR spectroscopy of the amidite 6 used in the synthesis indicated that approximately 5 % hydrolysis had occurred. The amidite reagent was repurified, the homogeneity confirmed by 31PNMR, and the thiol oligonucleotide resynthesized. A hi.gh yield of “trityl ON” thiol oligonucleotide 7a was obtained, validating the importance of the purity of phosphoramidite 6. The yield is further improved by executing the final iodine oxidation manually. This improvesthe yield (for a lOpmol synthesis) by another 20-3076. This iodine oxidation was done manually to ensure that all the resin received an equally short treatment since, for a 10 pmol column, this is not possible when using the synthesizer. After manual oxidation the resin was washed with acetonitrile and cleaved with aqueous ammonia immediately. This results in substantial amounts (>60% ) of the required “trityl ON” thiol oligonucleotide. The exact effect of the impure (85-95% purity) phosphoramidite 6 on coupling efficiency is unclear. Even though HPLC analysis of the crude cleaved oligonucleotides indicates no “trityl ON” material, small-scale experiments on the prepared resin suggest otherwise. Addition of TFA to trityl oligonucleotide which is still resin-bound liberates a bright yellow color (presumably the trityl cation) into solution. In addition, after attachment of the tritylthio phosphoramidite 6 with no capping step, further nucleoside phosphoramidite couplings are completely negative. This is indicative of full coupling of the amidite 6. These results suggest that loss of trityl group occurs postsynthesis. Immediately following iodine oxidation, the resins were washed and dried before being treated with 30 % aqueous ammonia at room temperature for 6 h. The cleaved oligonucleotide was deprotected by heating at 55 “C overnight. Purification by preparative HPLC was always carried out immediately following resin cleavage and base deprotection. The HPLC-purified trityl S-oligonucleotides 7a and 8a were treated with silver nitrate to cleave the trityl group, followed by DTT to liberate the free thiol oligonucleotides. When working with larger
Bioconjugate Chem., Vol. 5, No. 4, 1994
18 2 0
Figure 3. Conjugation of thiol oligonucleotide 7b (retention time 15.24 min) with maleimido-peptide 1 to give conjugate 9 (retention time 29.13 min): A, purified 7b (no peptide);B, reaction mixture after 1.5 h.
t i m e (min.) Figure 2. Time course of the reaction between the dimer from thiol oligonucleotide 8b (19.80 min) and dithiothreitol to give monomer 8b (18.39 min): *, unknown impurity.
amounts of thiol oligonucleotide (>2-3 mg), dilution of the silver nitrate reaction mixture with 0.1 M TEAA (up to 25 mL) is essential to prevent precipitation of the thiol oligonucleotide silver salt (a white solid). Liberation of the free thiol oligonucleotide can be as low as 15% if the oligonucleotide is not in solution. The thiol oligonucleotide/DTT mixture can be stored until all or some of the thiol oligonucleotide is required. Preparation of the Oligonucleotide-Peptide Hybrids. Thiol oligonucleotides 7b and 8b were stored in the DTT solution and can be separated from excess DTT by HPLC. The peptide conjugation must be performed immediately following this step because the purified free thiol forms a dimer in the HPLC eluent within 1-2 h. This was evident from some early experiments with 8b in which the purified thiol oligonucleotide was freeze-dried in order to reduce the volume for the conjugation reaction. This resulted in no reaction with maleimido-peptides taking place. Addition of DTT to the purified unreactive thiol oligonucleotide resulted in conversion of the dimer back to the free thiol oligonucleotide. This is evidenced by a shorter HPLC retention time as shown in Figure 2. The dimer has a retention time of 19.80 min whereas the reduced monomer elutes at 18.39 min. Purification and reaction of this converted dimer (to free thiol oligonucleotide 8b) with maleimido peptide 2 resulted in full conjugation to give conjugate 10. The conjugate prepared from the reduced dimer had an identical HPLC retention time to conjugate prepared from thiol oligonucleotide that had not previously dimerized. The maleimido peptides (dissolved in 0.1 M TEAA/ CHsCN, 60:40) must be directly added to the HPLC eluent containing the thiol oligonucleotide, the volume of which has no significant effect on the conjugation reaction. In each oligonucleotide-peptide conjugation the peak corresponding to the starting oligonucleotide disappears and a new peak appears with a different retention time. The time required for complete reaction is dependant on the nature of the peptide. The conjugations of the thiol oligonucleotides 7b and 8b with the a-helical and Fc peptide maleimides 1 and 2, respectively, are shown in Figures 3 and 4. The oligonucleotide-peptide hybrids 9 and 10 (Figure 5 ) were obtained after HPLC purification and were characterized spectrophotometrically and by
18 20 18 20 - A
t i m e (min.) Figure 4. Time course of the reaction between thiol oligonucleotide 8b (retention time 16.28min) and maleimido-peptide 2 to give conjugate 10 (retention time 16.98 min).
'""9 N- CH2CH2 -$ 0 0
Figure 5. Oligonucleotide-peptide hybrid molecules prepared. HIV-1is the 20mer oligonucleotide from 7a and AFP the 2lmer oligonucleotidefrom 8a. The peptides are those derived from 1
amino acid analysis. The conjugation reactions have yields of greater than 8074, based on the amount of the starting thiol oligonucleotide. These yields include HPLC puri-
Bioconjug8te Chem., Vol. 5, No. 4, 1994
fication and dialysis. Results for the conjugation reactions are shown in Table 1. CONCLUSION
In this study, we have developed improved methods for preparing and reacting thiol oligonucleotides. The 6-(trity1thio)hexyl phosphoramidite must be pure in order to obtain any significant “trityl ON” product. Due to the inherent design of the automated DNA synthesizer, a manual iodine treatment of the resin is advisable to improve the yield of the tritylated oligonucleotide. The purified thiol oligonucleotide must be reacted with a thiol reactive molecule as soon as it is eluted from the HPLC column to avoid formation of the unreactive dimer. The protocol described here has been found to preceed efficiently for several other examples of oligonucleotide and peptide combinations and provides a practical and reliable method for the synthesis of hybrids in high yield. ACKNOWLEDGMENT
This work was supported by the Commonwealth AIDS Research Grants Council. The Howard Florey Institute is supported by an Institute block grant from the National Health and Medical Research Council of Australia. LITERATURE CITED (1) Agrawal, S. (1989) Antisense oligonucleotides as antiviral agents. TIBTECH 10, 152-158. (2) Crooke, S. T. (1992) Therapeutic applications of oligonucleotides. BIOITechnology 10, 882-886. (3) Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J. S., and Broder, S. (1987) Phosphorothioate analogues of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiencyvirus. Proc. Natl. Acad. Sci. U S A . 84, 7706-7710. (4) Haralambidis, J.,Duncan, L., Angus, K., and Tregear, G. W. (1990) The synthesis of polyamide-oligonucleotideconjugate molecules. Nucleic Acids Res. 18, 493-499. (5) Connolly, B. A., and Rider, P. (1985) Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specificprobes. Nucleic Acids Res. 13,4485-4502. (6) Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio, F. (1991)Synthesis of defined peptideoligonucleotide hybrids
Ede et al.
containing a nuclear transport signal sequence. Tetrahedron 47,4113-4120. (7) Li, P., Medon,P. P.,Skingle,D. C.,Lanser, J. A., and Symons, R. H. (1987) Enzyme-linked synthetic oligonucleotideprobes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens. Nucleic Acid Res. 15, 5275-5287. (8) Sinha, N. D., and Cook, R. M. (1988) The preparation and application of functionalized synthetic oligonucleotides: 111. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol. Nucleic Acids Res. 16, 2659-2669. (9) Tung, C.-H., Rudolph, M. J., and Stein, S. (1991)Preparation of Oligonucleotide-Peptide Conjugates. Bioconjugate Chem. 2,464-465. (10) Nielsen, O., and Buchardt, 0. (1991) Facile synthesis of reagents containing a terminal maleimido ligand linked to an active ester. Synthesis 819-821. (11) Sinha, N. D., and Striepeke, S. (1991) Oligonucleotideswith reporter groups attached to the 5’4erminus. Oligonucleotides and Analogues: A Practical Approach (F. Eckstein, Ed.) pp 185-210, Oxford University Press, New York. (12) Atherton, E., and Sheppard, R. C. (1989) Solid-phase synthesis: A practicle approach, IRL Press, Oxford, _peptide _ .. England.. (13) Beaucage, S. L., and Iyer, R. P. (1992) Synthesis of olieonucleotides bv the DhosDhoramidite ADDroach. Tetra. . .. hezron 48, 2223-2311. (14) Goodchild, J.,Agrawal, S., Civeira, M. P., Sarin, P. S., Sun, D., and Zamecnik, P.C. (1988) Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides. Proc. Natl. Acad. Sci. U.S. A. 85, 5507-5511. (15) Turcotte, B., Guertin, M., and Belanger, L. (1985) Rat alfetoprotein messenger RNA 5’-end sequence and glucocorticoid-suppressed liver transcription in an improved nuclear run-off assay. Nucleic Acids Res. 13, 2387-2398. (16) Lee, S., Mihara, H., Aoyagi, H., Kato, T., Izumiya, W.-J. O., Ito, A., Omura, Y., Uzu, S., and Nakajima, T. (1986) Effect of amphiphilic model peptides on biomembranes and mast cells. Peptide Chemistry 1985 (Y. Kiso, Ed.) pp 317-320, Protein Research Foundation, Osaka. (17) Bruggemann, M. (1988) Evolution of rat immunoglobulin gamma heavy-chain gene family. Gene 74, 473-482. (18) Sarmay, G., Benczur, M., Petranyi, E. K., Kahn, M., Stanworth, D. R., and Gergely, J. (1984) Ligand inhibition studies on the role of Fc receptors in antibody-dependent cellmediated cytotoxicity. Mol. Immunol. 21, 43-51. (19) Hancock, W. S., and Battersby, J. E. (1976) A new microtest for the detection of incomplete coupling reactions in solid phase peptide synthesis using 2,4,6-trinitrobenzenesulphonic acid. Anal. Biochem. 71, 260-264.