Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
UNIT 8.11
Lijing Fang,1 Zhengyin Pan,1 Paul M. Cullis,2 Glenn A. Burley,3 and Wu Su1 1
Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, People’s Republic of China 2 Department of Chemistry, University of Leicester, Leicester, United Kingdom 3 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom
In this unit, the fully automated solid-phase synthetic strategy of hairpin Py-Im polyamides is described using triphosgene (BTC) as a coupling agent. This automated methodology is compatible with all the typical building blocks, enabling the facile synthesis of polyamide libraries in 9% to 20% yield in C 2015 by John Wiley & Sons, Inc. 3 days. Keywords: Py-Im polyamide r DNA r minor groove r automated synthesis r solid-phase synthesis r triphosgene
How to cite this article: Fang, L., Pan, Z., Cullis, P. M., Burley, G. A., and Su, W. 2015. Pyrrole-imidazole polyamides: Automated solid-phase synthesis. Curr. Protoc. Nucleic Acid Chem. 63:8.11.1-8.11.14. doi: 10.1002/0471142700.nc0811s63
INTRODUCTION N-methylpyrrole-N-methylimidazole (Py-Im) polyamides are a class of synthetic ligands capable of recognizing pre-determined sequences of double-stranded DNA (dsDNA) by binding in the minor groove (Blackledge and Melander, 2013). This unit presents two methods for automated synthesis of Py-Im polyamides (1 and 2; Fig. 8.11.1) using a triphosgene coupling strategy. The Basic Protocol describes a procedure for coupling monomeric N-protected carboxylic acids (Fig. 8.11.2) on solid phase employing Boc chemistry. The Alternate Protocol uses Fmoc chemistry. Both protocols are facile and fully amenable for automation on a CS336X peptide synthesizer. The Support Protocol describes the preparation of Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin manually from Fmoc-hydrazinobenzoyl AM resin. CAUTION: Phosgene and CO2 gas are evolved during the coupling of each N-protected amino acid. It is highly recommended that the peptide synthesizer be placed in a fume hood and the waste gas be treated with aqueous NaOH solution (20%) to destroy the excess phosgene. Good laboratory safety practices should be observed at all times, including the use of safety goggles, a laboratory coat, and disposable gloves.
AUTOMATED SYNTHESIS OF Py-Im POLYAMIDES USING Boc CHEMISTRY
BASIC PROTOCOL
The automated synthesis of Py-Im polyamides is performed on a CS336X peptide synthesizer equipped with nitrogen gas, an air compressor, and a computer-controlled operation system. The synthesizer is programmed in the standard hardware configuration for DIC/HOBt (or HBTU/DIEA) protocols. Reagent position 1 is DMF, reagent position 2 is dry DMF, reagent position 3 is piperidine/DMF (20%), reagent position 4 is DCM,
Nucleic Acid Binding Molecules
Current Protocols in Nucleic Acid Chemistry 8.11.1-8.11.14, December 2015 Published online December 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142700.nc0811s63 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 8.11.1
Structure of Py-Im polyamides (1 and 2).
Figure 8.11.2
Building blocks for the synthesis of Py-Im polyamides.
reagent position 5 is TFA/phenol/H2 O (92.5:5:2.5), reagent position 6 is DIEA/dry DMF (10%), reagent position 7 is 2,4,6-collidine/dry THF (15%), and reagent position 8 is dry THF. Depending on the sequence of the Py-Im polyamide, the amino acid (AA) reservoirs are loaded with the monomers and activator in the corresponding slots on the carousel.
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
The synthesis of Py-Im polyamides is divided into six basic procedures: (1) deprotection; (2) monomer activation; (3) coupling; (4) terminal Im capping; (5) protecting group exchange; and (6) resin cleavage and purification. The first three procedures are iterative for each cycle of amino acid addition. Two successive coupling cycles are employed when coupling pyrrole amino acid to imidazole amines; all other couplings are performed with single coupling cycles. For a typical synthesis on resin (400 mg, 0.15 mmol/g), Bocamino acid (4 eq.) and BTC (2 eq.) are activated just prior to the coupling step and transferred to the pretreated resin to generate the corresponding polyamide. The progress of the coupling reaction can be monitored by stepwise HPLC analysis. The synthesis of Py-Im polyamide usually involves eight coupling cycles plus a protecting-group
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Figure 8.11.3 Synthetic scheme for automated synthesis of Py-Im polyamide 1. Reagents and conditions: (i) TFA/phenol/H2 O; (ii) Boc-Py-OH, BTC, collidine, DIEA; (iii) TFA/phenol/H2 O; (iv) Boc-Py-OH, BTC, collidine, DIEA; (v) TFA/phenol/H2 O; (vi) Boc-Im-OH, BTC, collidine, DIEA, HOAt; (vii) TFA/phenol/H2 O; (viii) Fmoc-D-Dab(Boc)-OH, BTC, collidine, DIEA, HOAt; (ix) TFA/phenol/H2 O; (x) Boc-Py-OH, BTC, collidine, DIEA; (xi) TFA/phenol/H2 O; (xii) Boc-PyOH, BTC, collidine, DIEA; (xiii) TFA/phenol/H2 O; (xiv) Boc-Py-OH, BTC, collidine, DIEA; (xv) TFA/phenol/H2 O; (xvi) Im-CCl3 , DIEA or Im-OH, PyBOP, DIEA; (xvi) 20% piperidine/DMF; (xvii) Boc2 O, DIEA; (xvii) 3,3 -diamino-N-methyl-dipropylamine, air, 90°C, 1 hr.
exchange step. It is then cleaved from the resin and purified by semi-preparative HPLC. The protocol is general and may be applied to a wide variety of Py-Im polyamides. The sequence of reactions is exemplified in the synthesis of a biologically relevant polyamide 1 (Fig. 8.11.3) targeting the 5 -WGWWCW-3 (where W = A/T) subset of the consensus androgen and glucocorticoid response element (Nickols and Dervan, 2007; Muzikar et al., 2009; Yang et al., 2013).
Materials Boc-Py-hydrazine resin (prepared manually from Boc-Py-OH and Fmoc-hydrazinobenzoyl AM resin, see Support Protocol)
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Dichloromethane (DCM, anhydrous) Trifluoroacetic acid (TFA, anhydrous) 92.5:5:2.5 (v/v/v) TFA/phenol/H2 O (see recipe) N,N-dimethylformamide (DMF; anhydrous, Sigma-Aldrich) 10% (v/v) N,N -diisopropylethylamine (DIEA) in dry DMF (see recipe) Tetrahydrofuran (THF; anhydrous, Sigma-Aldrich) 4-[(tert-butoxycarbonyl)amino]-1-methyl-1H-pyrrole-2-carboxylic acid (Boc-Py-OH, J&K Scientific) Triphosgene (BTC; Aladdin, cat. no. T103041) Nitrogen gas (N2 ) 15% 2,4,6-collidine in dry THF (see recipe) 4-tert-butoxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (Boc-Im-OH, J&K Scientific) 1-Hydroxy-7-aza-benzotriazole (HOAt; Aladdin, cat. no. H109328) Fmoc-(N-γ-Boc)-D-α, γ-diaminobutyric acid (Fmoc-D-Dab(Boc)-OH, Alfa Aesar) Im-CCl3 (prepared according to Masiukiewicza et al., 2005) 1-methyl-1H-imidazole-2-carboxylic acid (Im-OH, Sigma-Aldrich) Benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP; Aladdin, cat. no. P109336) 20% piperidine in DMF (see recipe) Di-tert-butyl dicarbonate (Boc2 O; Aladdin, cat. no. D106159) 3,3 -diamino-N-methyl-dipropylamine (Aladdin, cat. no. B105353) Methanol (MeOH; HPLC-grade, J&K Scientific) Diethyl ether (Et2 O, anhydrous) Acetonitrile (MeCN; HPLC-grade, J&K Scientific) CS Bio 336X peptide synthesizer (http://www.csbio.com) Air compressor Disposable syringe filter (Nylon 66, 0.22 μm; Jinteng) ULTIMAT 3000 Instrument (Dionex) ˚ ACE C18 column (10 × 250 mm, 5 μm, 300 A) Photodiode array detector capable of measuring absorbance Lyophilizer and freeze dryer vessels Centrifuge Shimadzu LC 20 with UV detector SPD-20 A ˚ Inertsil ODS-SP column (4.6 × 250 mm, 5 μm, 100 A) Additional reagents and equipment for deprotection procedure (Alternate Protocol) and HPLC (APPENDIX 3B) NOTE: To increase the solubility in THF, fine powders of Boc-Im-OH are obtained by dispersion of the commercial reagent (5 g) in H2 O (300 mL) via ultrasonication at 40°C followed by lyophilization. All other commercial reagents are used as received.
Deprotection 1. Insert the needle into an empty amino acid (AA) reservoir. 2. Load Boc-Py-hydrazine resin (400 mg, 0.15 mmol/g) in the reaction vessel (RV, 10 mL). 3. Wash the resin twice with DCM (5 mL, calibrated delivery from reagent bottle 4). Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Shake 2 min, then drain the RV.
4. Treat the resin twice with the mixture of 92.5:5:2.5 TFA/phenol/H2 O (5 mL, calibrated delivery from reagent bottle 5).
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Shake 2 min for the first treatment and 20 min for the second treatment, then drain the RV.
5. Wash the resin twice with DCM (5 mL, calibrated delivery from reagent bottle 4). Shake 2 min, then drain the RV.
6. Wash the resin with DMF (5 mL, calibrated delivery from reagent bottle 1). Shake 2 min, then drain the RV.
7. Wash the resin with 10% DIEA/dry DMF (5 mL from reagent bottle 6, bypassing the AA reservoir). Shake 2 min, then drain the RV.
8. Wash the resin with dry THF (to wash the needle, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
9. Wash the resin with DMF (5 mL from reagent bottle 1). Shake 2 min, then drain the RV.
10. Wash the resin with dry DMF (5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
11. Retract the needle and inject it in the following AA reservoir loaded with the monomer and BTC solids (using Advance_AA). The resin is ready for the coupling reaction. The same deprotection procedure is applied at the initiation of each coupling cycle.
Monomer activation Activation of Boc-Py-OH 12. Load a mixture of Boc-Py-OH (60 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir. 13. Deliver dry THF to the AA reservoir [1.25 mL from reagent bottle 8 bypassing amino acid metering vessel (MVAA)]. 14. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min (using the program Bubble/shake_1Min). 15. Deliver 15% 2,4,6-collidine in dry THF to the AA reservoir (1.25 mL from reagent bottle 7 bypassing MVAA). 16. Blend the resulting slurry by nitrogen bubbling for 2 min. 17. Deliver 10% DIEA/dry DMF to the AA reservoir (2.5 mL from reagent bottle 6 bypassing MVAA). 18. Blend the mixture by nitrogen bubbling for 1 min to form a clear pale yellow solution. 19. Transfer activated monomer from the AA reservoir to the RV for the coupling reaction. [bypassing the metering vessel (MV) and transfer vessel (TV)].
Activation of Boc-Im-OH 20. Load the mixture of Boc-Im-OH (60 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir.
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21. Load HOAt (34 mg, 0.25 mmol) into the following AA reservoir. 22. Activate the mixture of Boc-Im-OH and BTC as described in steps 13 to 18 to form a clear brown solution. 23. Transfer the activated monomer from the AA reservoir to TV. 24. Retract the needle from the first AA reservoir and inject it in the second one loaded with HOAt. 25. Deliver 10% DIEA/dry DMF solution (1.25 mL from reagent bottle 6) to the AA reservoir. 26. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min. 27. Transfer the solution from the AA reservoir to TV to mix it with the activated Im monomer. Wait for 3 min (using the program Shake_1Min).
28. Transfer the resulting solution from the TV to RV for the coupling reaction.
Activation of Fmoc-D-Dab(Boc)-OH 29. Load the mixture of Fmoc-D-Dab(Boc)-OH (109 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir. 30. Load HOAt (34 mg, 0.25 mmol) into the following AA reservoir. 31. Activate the monomer as described in steps 13 to 18 to form a clear brown solution. 32. Transfer the activated monomer to RV for the coupling reaction.
Monomer coupling 33. Shake RV loaded with the deprotected resin and the activated amino acid for 25 min (Shake_1Min), then drain it. 34. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 35. Wash the resin three times with DMF (5 mL from reagent bottle 1). The resin is ready for the next coupling cycle.
Terminal Im capping Usual procedure 36a. Load Im-CCl3 [57 mg, 0.25 mmol, prepared according to the literature method (Masiukiewicza et al., 2005)] in an AA reservoir. 37a. Deliver 10% DIEA/dry DMF (2 × 2.5 mL from reagent bottle 6) to the AA reservoir. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min. 38a. Transfer the solution from the AA reservoir to RV for the coupling reaction [bypassing the metering vessel (MV) and transfer vessel (TV)]. Shake RV for 40 min (Shake_1Min) then drain it.
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
39a. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 40a. Wash the resin three times with DMF (5 mL from reagent bottle 1).
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Alternative procedure for terminal Im capping 36b. Perform step 36a, above, using Im-OH (32 mg, 0.25 mmol) and PyBOP (130 mg, 0.25 mmol) instead of Im-CCl3 . 37b. Dissolve the mixture and transfer it to RV, employing steps 37a to 39a. 38b. Shake RV loaded with the deprotected resin and the amino acid for 120 min (Shake_1Min), then drain it. 39b. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 40b. Wash the resin three times with DMF (5 mL from reagent bottle 1).
Protecting-group exchange 41. Deprotection: Remove the Fmoc group of the γ-turn unit with 20% (v/v) piperidine in DMF following the “Deprotection Procedure” of the Alternate Protocol described below. 42. Perform step 36a using Boc2 O (115 μL, 0.50 mmol) instead of Im-CCl3 . 43. Dissolve the mixture and transfer it to RV employing steps 37a to 39a. 44. Shake RV loaded with the deprotected resin and Boc2 O for 15 min (Shake_1Min), then drain it. 45. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 46. Wash the resin three times with DMF (5 mL from reagent bottle 1). The resin is now ready for cleavage.
Resin cleavage and purification 47. Divide the resin from step 46 into four equal parts. 48. Add 3,3 -diamino-N-methyl-dipropylamine (100 μL) in DMF (200 μL) to one part and shake the mixture at 90°C for 1 hr. 49. Remove the resin by filtration through a syringe filter and wash it with MeOH (2 mL). 50. Remove the solvent under reduced pressure. 51. Add 10 volumes of cold diethyl ether to the residue and transfer the mixture into a 50-mL centrifuge tube, centrifuge 10 min at 3500 × g, then carefully decant and discard the supernatant. 52. Dissolve the light-yellow powder in 10% (v/v) MeCN/H2 O containing 0.1% (v/v) TFA and purify the crude peptide by semi-preparative RP-HPLC performed on ULTIMAT 3000 Instrument equipped with an ACE C18 column (10 × 250–mm, ˚ Measure UV absorbance using a photodiode array detector at 254 5 μm, 300 A). and 310 nm. Start the RP-HPLC gradient at 10% of B (MeCN), then increased to 100% of B over 30 min (A: 0.1% TFA in water). 53. Remove the solvent under reduced pressure and collect the pure product after lyophilization. Polyamide 1 was obtained as a pale yellow powder (3 mg, 15%). Analytical HPLC tr = 17.7 min. HRMS (ESI) m/z: calcd. for C62 H81 N22 O11 [M + H]+ 1309.6450, found 1309.6444.
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Stepwise HPLC analysis 54. Take a resin sample (4 mg) out during the coupling reaction (step 33), transfer to a 1.5-mL tube, and wash it twice by pipetting 1 mL DMF into the tube and removing the solvent after 1 min. 55. Treat the resin with 3,3 -diamino-N-methyl-dipropylamine (20 μL) and DMF (20 μL) at 90°C for 30 min. 56. Dilute the mixture with MeOH (100 μL) and filter. 57. Analyze the sample (20 μL) by analytical HPLC at 310 nm performed on the Shimadzu LC 20 with UV detector SPD-20 A using Inertsil ODS-SP column (4.6 × ˚ 250 mm, 5 μm, 100 A). The RP-HPLC gradient is started at 10% B (MeCN), then increased to 100% of B over 30 min (A: 0.1% TFA in water). ALTERNATE PROTOCOL
AUTOMATED SYNTHESIS OF Py-Im POLYAMIDES USING Fmoc-CHEMISTRY This alternative procedure for automated Py-Im polyamide synthesis uses Fmoc chemistry. First, the resin-bound polyamide is deprotected by piperidine/DMF. Then, the Fmoc-protected monomer is activated according to the programs described in the Basic Protocol and transferred to an RV for the coupling reaction. Two successive coupling cycles are employed when coupling pyrrole amino acid to imidazole amines (60 min each); all other couplings are carried out with a single-coupling cycle (40 min). Finally, the polyamide is capped with Im-OH or Im-CCl3 , cleaved from the resin, and purified by semi-preparative HPLC using the same procedures as described under “Terminal Im capping” and “Resin cleavage and purification” in the Basic Protocol. The sequence of reactions is exemplified in the synthesis of polyamide 2 (Fig. 8.11.4).
Additional Materials (also see Basic Protocol) 4-(Fmoc-amino)-1-methyl-1H-imidazole-2-carboxylic acid (Fmoc-Im-OH, Atomax Chemicals) Fmoc-4-amino-1-methylpyrrole-2-carboxylic acid (Fmoc-Py-OH, Atomax Chemicals) 4-(Fmoc-amino)butyric acid (Fmoc-GABA-OH; Aladdin, cat. no. F122242) Dimethylaminopropylamine (Aladdin, cat. no. D110909) Additional reagents and equipment for monomer activation (Basic Protocol) NOTE: To increase the solubility in THF, powders of Fmoc-Py-OH and Fmoc-Im-OH are obtained by dissolving the commercial reagents (2 g) in THF (300 mL), removing the solvents under reduced pressure, then drying in a vacuum oven at 50°C overnight.
Deprotection procedure 1. Insert the needle into an empty amino acid (AA) reservoir. 2. Load Fmoc-Py-hydrazine resin (400 mg, 0.15 mmol/g, prepared manually from Fmoc-Py-OH and Fmoc-hydrazinobenzoyl AM resin, see Support Protocol) into the reaction vessel (RV, 10 mL).
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
3. Treat the resin twice with the mixture of 20% piperidine/DMF (5 mL from reagent bottle 3). Shake 10 min for each treatment, then drain the RV.
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Figure 8.11.4 Synthetic scheme for automated synthesis of Py-Im polyamide 2. Reagents and conditions: (i) 20% piperidine/DMF; (ii) Fmoc-Py-OH, BTC, collidine, DIEA; (iii) 20% piperidine/DMF; (iv) Fmoc-Im-OH, BTC, collidine, DIEA, HOAt; (v) 20% piperidine/DMF; (vi) FmocPy-OH, BTC, collidine, DIEA; (vii) Fmoc-Py-OH, BTC, collidine, DIEA; (viii) 20% piperidine/DMF; (ix) Fmoc-GABA-OH, BTC, collidine, DIEA, HOAt; (x) 20% piperidine/DMF; (xi) Fmoc-Py-OH, BTC, collidine, DIEA; (xii) 20% piperidine/DMF; (xiii) Fmoc-Py-OH, BTC, collidine, DIEA; (xiv) 20% piperidine/DMF; (xv) Fmoc-Py-OH, BTC, collidine, DIEA; (xvi) 20% piperidine/DMF; (xvii) Im-CCl3 , DIEA, or Im-OH, PyBOP, DIEA; (xviii) dimethylaminopropylamine, air, 90°C, 1 hr. Nucleic Acid Binding Molecules
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4. Wash the resin twice with dry DMF (to wash the pipeline, 5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
5. Wash the resin twice with dry THF (to wash the needle, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
6. Wash the resin with DCM (5 mL from reagent bottle 4). Shake 2 min, then drain the RV.
7. Wash the resin with DMF (dry DMF not necessary in this step; 5 mL from reagent bottle 1). Shake 2 min, then drain the RV.
8. Wash the resin with dry DMF (5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
9. Retract the needle and inject it in the following AA reservoir loaded with the monomer and BTC solids (by Advance_AA). The resin is now ready for the coupling reaction. The same deprotection programs are applied at the initiation of each coupling cycle.
Monomer activation 10. Activate Fmoc-Py-OH, Fmoc-Im-OH, and Fmoc-GABA-OH according to the procedures in the Basic Protocol used to activate Boc-Py-OH, Boc-Im-OH, and FmocD-Dab(Boc)-OH, respectively. Monomer coupling 11. Shake RV loaded with the deprotected resin and the activated amino acid for 40 min (using the Shake_1Min program on the peptide synthesizer), then drain it. 12. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
13. Wash the resin three times with DMF (5 mL from reagent bottle 1). Shake 2 min, then drain the RV. The resin is ready for the next coupling cycle. Following procedures similar to “Terminal Im capping” and “Resin cleavage and purification” in the Basic Protocol, polyamide 2 was obtained after cleavage from the resin using dimethylaminopropylamine (2 mg, 12%). Analytical HPLC tr = 17.3 min. HRMS (ESI) m/z: calcd. for C55 H67 N20 O9 [M + H]+ 1151.5394, found 1151.5392. SUPPORT PROTOCOL
PREPARATION OF Boc-Py-HYDRAZINE RESIN AND Fmoc-Py HYDRAZINE RESIN Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin are prepared manually from BocPy-OH and Fmoc-hydrazinobenzoyl AM resin (Fig. 8.11.5).
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Additional Materials (also see Basic Protocol and Alternate Protocol) Fmoc-hydrazinobenzoyl AM resin (Novabiochem) Acetic anhydride (Ac2 O)
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Figure 8.11.5
Synthetic scheme for Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin.
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU; Aladdin, cat. no. H109327) 2,4,6-collidine (Aladdin, cat. no. T108942) N,N -diisopropylethylamine (DIEA) Solid-phase synthesis vessel 5-mL reaction vials (wholesale 5-mL clear glass vials with orifice reducers and plastic caps; Yanzhou United) Stirring bar and magnetic stirrer Rubber septa Round bottom flask (500 mL) Oil-type vacuum pump Preparation of Boc-Py-hydrazine resin 1a. Fit a two-necked, 500-mL round-bottomed flask with a solid-phase synthesis vessel and connect to a nitrogen line and an oil-type vacuum pump via rubber septa (to set up a manual solid-phase peptide synthesizer). Add Fmoc-hydrazinobenzoyl AM resin (400 mg, 0.66 mmol/g) to the manual solid-phase peptide synthesis vessel (10 mL). 2a. Swell the resin with CH2 Cl2 (3 mL) for 20 min, then drain it. 3a. Treat the resin twice with 20% piperidine in DMF (2 × 3 mL). Use 10 min for each treatment, then drain the resin.
4a. Wash the resin three times with DMF (not necessarily dry; 3 × 3 mL) and dry DMF (3 mL) by pipetting the solvent onto the resin and bubbling nitrogen through the mixture for 1 min, then draining the vessel under vacuum. 5a. Add Boc-Py-OH (29 mg, 0.12 mmol) and BTC (18 mg, 0.06 mmol) to a reaction vial (5 mL) equipped with a stirring bar. 6a. Add dry THF (1.5 mL) to the vial to dissolve the solids. 7a. Add [neat] collidine (95 μL, 0.72 mmol) dropwise to the vial.
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The reaction is violent, with the emission of CO2 accompanied by large amount of collidine hydrochloride precipitate.
8a. Stir the resulting slurry for 2 min. 9a. Add dry DMF (2.5 mL) and DIEA (200 μL, 1.20 mmol) successively to the vial to form a clear solution. 10a. Transfer this solution to the resin (from step 4a) and mix with nitrogen bubbling for 10 min. 11a. Repeat step 4a. 12a. Add Ac2 O (125 μL, 1.32 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) to the resin and mix them by nitrogen bubbling for 10 min. 13a. Repeat step 4a. The resin can be used as a solid support in the Basic Protocol at a substitution level of 0.15 mmol/g (meaning that the loading of the first amino acid is 0.15 mmol/g dry resin).
Preparation of Fmoc-Py-hydrazine resin 1b. Swell and remove Fmoc group of Fmoc-hydrazinobenzoyl AM resin (400 mg, 0.66 mmol/g) according to steps 1a to 4a of this protocol, above. 2b. Mix Fmoc-Py-OH (43 mg, 0.12 mmol), HATU (46 mg, 0.12 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) in a 5-mL reaction vial equipped with a stirring bar. Stir the mixture for 10 min. 3b. Transfer the solution to the resin and mix with nitrogen bubbling for 1 hr. 4b. Repeat step 4a. 5b. Add Ac2 O (125 μL, 1.32 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) to the resin and mix them with nitrogen bubbling for 10 min. 6b. Repeat step 4a. The resin can be used as a solid support in the Alternate Protocol at a substitution level of 0.15 mmol/g.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A.
2,4,6-Collidine in dry THF (15%) Mix 15 mL of 2,4,6-collidine (Aladdin, cat. no. T108942) with 85 mL of dry THF (THF; anhydrous, Sigma-Aldrich) in reagent bottle 7. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
DIEA in dry DMF (10%) Mix 10 mL of N,N-diisopropylethylamine (DIEA; Aladdin, cat. no. D109321) with 90 mL of dry DMF (DMF; anhydrous, Sigma-Aldrich) in reagent bottle 6. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks. Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Piperidine/DMF (20%) Mix 200 mL of piperidine with 800 mL of DMF in reagent bottle 3.
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All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
TFA/phenol/H2 O (92.5:5:2.5 v/v/v) Heat phenol in a water bath (60°C), then take 5 mL of phenol and mix it with 92.5 mL of TFA and 2.5 mL of H2 O in reagent bottle 5 in an ice-water bath. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
COMMENTARY Background Information We previously reported a facile and highly efficient solid-phase synthesis of hairpin DNA-binding polyamides using the costeffective BTC activating agent. This strategy was employed with great success to produce Py-Im polyamides in a 8% to 33% yield by manual solid-phase synthesis (Su et al., 2009). The limitation with current BTC-mediated coupling protocols is the strong exothermic nature of the reaction, resulting in the emission of CO2 accompanied by large amount of collidine hydrochloride precipitate (Falb et al., 1999; Thern et al., 2002). In order to render BTC couplings amenable to automation, methods were therefore required to mitigate the exothermicity and prevent clogging of the lines of the automated synthesizer by the precipitate. This unit describes a reproducible, highly efficient, and racemization-free procedure for BTC-mediated in situ activation of the amino acid. The key features of the procedure include: (1) the preactivation of the carboxylic acid is carried out with collidine in THF; and (2) the resulting precipitate is converted to the soluble DIEA hydrochloride salt by the addition of a DIEA/DMF solution (Fang et al., 2014). The monomer activation, coupling, and resin cleavage procedures are all optimized, allowing automated synthesis of a palette of hairpin Py-Im polyamides on a commercially available peptide synthesizer using BTC as the condensing agent for all coupling steps (Fang et al., 2015).
Critical Parameters and Troubleshooting To guarantee complete resin-to-solvent contact, the CS336X peptide synthesizer should be equipped with an air compressor to enable a pneumatically operated rotary actuator for fast, smooth and 180° inversion mixing. During the monomer activation steps, nitrogen is bubbled into the amino acid reservoir to blend the reaction mixture and facilitate the dissipation of CO2 and heat. The nitrogen flow
should be adjusted to avoid the loss of material and blockage of the needle. Anhydrous solvents are also important. The solvents should be taken from a freshly opened bottle of commercially prepared anhydrous solvent. It is best to purchase anhydrous solvents in the smallest-size bottle that will fit the requirements of the synthesis. The anhydrous solvents and solutions kept in the reagent bottles should be changed once every 2 weeks to avoid a drop in coupling efficiencies. The addition of one equivalent of HOAt to the BTC-activated Boc-Im-OH, Fmoc-ImOH, or Fmoc-D-Dab(Boc)-OH can dramatically accelerate their coupling rate with resinbound amines. Complete conversion can be observed by HPLC within 20 min. However, the combination of BTC/HOAt decreases the coupling rate of activated Boc-Py-OH or Fmoc-Py-OH with resin-bound amines. In the Basic Protocol, the mixture of BocIm-OH and BTC cannot be completely dissolved in dry THF even using the fine powder of Boc-Im-OH obtained after lyophilization. This will slightly affect the conversion of the amino acid to its corresponding acid chloride. However, this does not affect the coupling reaction, as a large excess of monomer is used. Because the Fmoc-Py-OH and Fmoc-Im-OH used in the Alternate Protocol have poor solubility and are less reactive than their Boc- protected counterparts, extended reaction times are necessary to drive the coupling reaction to completion. For better yield and crude purity of Py-Im polymides, two successive coupling cycles are employed when coupling pyrrole amino acids to resin-bound imidazole amines.
Anticipated Results The two protocols described in this unit enable the preparation of Py-Im polyamides in good yield and good crude purity. For polyamide 1, both protocols should give yields between 15% and 20% with a purity of 95%. The Basic Protocol is more suitable for preparation of polyamides containing difficult couplings such as the coupling of Boc-Py-
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OH, Boc-Im-OH, or Fmoc-D-Dab(Boc)-OH (γ-turn) to imidazole amines, and provides better yields than the Alternate Protocol.
Time Considerations
Fang, L., Yao, G., Pan, Z., Wu, C., Wang, H., Burley, G.A., and Su, W. 2015. Fully automated synthesis of DNA-binding Py-Im polyamides using a triphosgene coupling strategy. Org. Lett. 17:158161. doi: 10.1021/ol503388a.
To prepare Boc-Py-hydrazine resin for the Basic Protocol (Fmoc-Py-hydrazine resin for Alternate Protocol) manually as described in Support Protocol, and prepare Im-CCl3 according to the literature method, should take 1 day. The solid-phase synthesis of Py-Im polyamide on the CS336X peptide synthesizer should take 1 day. The estimated time to complete the resin cleavage, purification, and lyophilization is 1 day. Therefore, preparation of a hairpin Py-Im polyamide sequence using either Basic Protocol or the Alternate Protocol may take 3 days.
Masiukiewicza, E., Mrugala, D., and Rzeszotarska, B. 2005. An improved synthesis of 1-methyl2-trichloroacetylimidazole. Org. Prep. Proced. Int.: New J. Organ. Synth. 37:403-405. doi: 10.1080/00304940509354973.
Literature Cited
Su, W., Gray, S.J., Dondi, R., and Burley, G.A. 2009. Highly efficient synthesis of DNAbinding hairpin polyamides via the use of a new triphosgene coupling strategy. Org. Lett. 11:3910-3913. doi: 10.1021/ol9015139.
Blackledge, M.S. and Melander, C. 2013. Programmable DNA-binding small molecules. Bioorg. Med. Chem. 21:6101-6114. doi: 10.1016/j.bmc.2013.04.023. Falb, E., Yechezkel, T., Salitra, Y., and Gilon, C. 1999. In situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl) carbonate and its utilization for difficult couplings in solidphase peptide synthesis. J. Pept. Res. 53:507517. doi: 10.1034/j.1399-3011.1999.00049.x. Fang, L., Wu, C., Yu, Z., Shang, P., Cheng, Y., Peng, Y., and Su, W. 2014. Triphosgene-mediated couplings in the solid phase: Total synthesis of Brachystemin A. Eur. J. Org. Chem. 34:75727576. doi: 10.1002/ejoc.201403145.
Muzikar, K.A., Nickols, N.G., and Dervan, P.B. 2009. Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression. Proc. Natl. Acad. Sci. U.S.A. 106:1659816603. doi: 10.1073/pnas.0909192106. Nickols, N.G. and Dervan, P.B. 2007. Suppression of androgen receptor–mediated gene expression by a sequence-specific DNA-binding polyamide. Proc. Natl. Acad. Sci. U.S.A. 104:10418-10423. doi: 10.1073/pnas.0704217104.
Thern, B., Rudolph, J., and Jung, G. 2002. Total synthesis of the nematicidal cyclododecapeptide omphalotin A by using racemization-free triphosgene-mediated couplings in the solid phase. Angew. Chem., Int. Ed. 41:2307-2309. doi: 10.1002/1521-3773(20020703)41:13% 3c2307::AID-ANIE2307%3e3.0.CO;2-Y. Yang, F., Nickols, N.G., Li, B.C., Marinov, G.K., Said, J.W., and Dervan, P.B. 2013. Antitumor activity of a pyrrole-imidazole polyamide. Proc. Natl. Acad. Sci. U.S.A. 110:1863-1868. doi: 10.1073/pnas.1222035110.
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
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UNIT 8.11
Lijing Fang,1 Zhengyin Pan,1 Paul M. Cullis,2 Glenn A. Burley,3 and Wu Su1 1
Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, People’s Republic of China 2 Department of Chemistry, University of Leicester, Leicester, United Kingdom 3 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom
In this unit, the fully automated solid-phase synthetic strategy of hairpin Py-Im polyamides is described using triphosgene (BTC) as a coupling agent. This automated methodology is compatible with all the typical building blocks, enabling the facile synthesis of polyamide libraries in 9% to 20% yield in C 2015 by John Wiley & Sons, Inc. 3 days. Keywords: Py-Im polyamide r DNA r minor groove r automated synthesis r solid-phase synthesis r triphosgene
How to cite this article: Fang, L., Pan, Z., Cullis, P. M., Burley, G. A., and Su, W. 2015. Pyrrole-imidazole polyamides: Automated solid-phase synthesis. Curr. Protoc. Nucleic Acid Chem. 63:8.11.1-8.11.14. doi: 10.1002/0471142700.nc0811s63
INTRODUCTION N-methylpyrrole-N-methylimidazole (Py-Im) polyamides are a class of synthetic ligands capable of recognizing pre-determined sequences of double-stranded DNA (dsDNA) by binding in the minor groove (Blackledge and Melander, 2013). This unit presents two methods for automated synthesis of Py-Im polyamides (1 and 2; Fig. 8.11.1) using a triphosgene coupling strategy. The Basic Protocol describes a procedure for coupling monomeric N-protected carboxylic acids (Fig. 8.11.2) on solid phase employing Boc chemistry. The Alternate Protocol uses Fmoc chemistry. Both protocols are facile and fully amenable for automation on a CS336X peptide synthesizer. The Support Protocol describes the preparation of Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin manually from Fmoc-hydrazinobenzoyl AM resin. CAUTION: Phosgene and CO2 gas are evolved during the coupling of each N-protected amino acid. It is highly recommended that the peptide synthesizer be placed in a fume hood and the waste gas be treated with aqueous NaOH solution (20%) to destroy the excess phosgene. Good laboratory safety practices should be observed at all times, including the use of safety goggles, a laboratory coat, and disposable gloves.
AUTOMATED SYNTHESIS OF Py-Im POLYAMIDES USING Boc CHEMISTRY
BASIC PROTOCOL
The automated synthesis of Py-Im polyamides is performed on a CS336X peptide synthesizer equipped with nitrogen gas, an air compressor, and a computer-controlled operation system. The synthesizer is programmed in the standard hardware configuration for DIC/HOBt (or HBTU/DIEA) protocols. Reagent position 1 is DMF, reagent position 2 is dry DMF, reagent position 3 is piperidine/DMF (20%), reagent position 4 is DCM,
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Current Protocols in Nucleic Acid Chemistry 8.11.1-8.11.14, December 2015 Published online December 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142700.nc0811s63 C 2015 John Wiley & Sons, Inc. Copyright
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Figure 8.11.1
Structure of Py-Im polyamides (1 and 2).
Figure 8.11.2
Building blocks for the synthesis of Py-Im polyamides.
reagent position 5 is TFA/phenol/H2 O (92.5:5:2.5), reagent position 6 is DIEA/dry DMF (10%), reagent position 7 is 2,4,6-collidine/dry THF (15%), and reagent position 8 is dry THF. Depending on the sequence of the Py-Im polyamide, the amino acid (AA) reservoirs are loaded with the monomers and activator in the corresponding slots on the carousel.
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
The synthesis of Py-Im polyamides is divided into six basic procedures: (1) deprotection; (2) monomer activation; (3) coupling; (4) terminal Im capping; (5) protecting group exchange; and (6) resin cleavage and purification. The first three procedures are iterative for each cycle of amino acid addition. Two successive coupling cycles are employed when coupling pyrrole amino acid to imidazole amines; all other couplings are performed with single coupling cycles. For a typical synthesis on resin (400 mg, 0.15 mmol/g), Bocamino acid (4 eq.) and BTC (2 eq.) are activated just prior to the coupling step and transferred to the pretreated resin to generate the corresponding polyamide. The progress of the coupling reaction can be monitored by stepwise HPLC analysis. The synthesis of Py-Im polyamide usually involves eight coupling cycles plus a protecting-group
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Figure 8.11.3 Synthetic scheme for automated synthesis of Py-Im polyamide 1. Reagents and conditions: (i) TFA/phenol/H2 O; (ii) Boc-Py-OH, BTC, collidine, DIEA; (iii) TFA/phenol/H2 O; (iv) Boc-Py-OH, BTC, collidine, DIEA; (v) TFA/phenol/H2 O; (vi) Boc-Im-OH, BTC, collidine, DIEA, HOAt; (vii) TFA/phenol/H2 O; (viii) Fmoc-D-Dab(Boc)-OH, BTC, collidine, DIEA, HOAt; (ix) TFA/phenol/H2 O; (x) Boc-Py-OH, BTC, collidine, DIEA; (xi) TFA/phenol/H2 O; (xii) Boc-PyOH, BTC, collidine, DIEA; (xiii) TFA/phenol/H2 O; (xiv) Boc-Py-OH, BTC, collidine, DIEA; (xv) TFA/phenol/H2 O; (xvi) Im-CCl3 , DIEA or Im-OH, PyBOP, DIEA; (xvi) 20% piperidine/DMF; (xvii) Boc2 O, DIEA; (xvii) 3,3 -diamino-N-methyl-dipropylamine, air, 90°C, 1 hr.
exchange step. It is then cleaved from the resin and purified by semi-preparative HPLC. The protocol is general and may be applied to a wide variety of Py-Im polyamides. The sequence of reactions is exemplified in the synthesis of a biologically relevant polyamide 1 (Fig. 8.11.3) targeting the 5 -WGWWCW-3 (where W = A/T) subset of the consensus androgen and glucocorticoid response element (Nickols and Dervan, 2007; Muzikar et al., 2009; Yang et al., 2013).
Materials Boc-Py-hydrazine resin (prepared manually from Boc-Py-OH and Fmoc-hydrazinobenzoyl AM resin, see Support Protocol)
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Dichloromethane (DCM, anhydrous) Trifluoroacetic acid (TFA, anhydrous) 92.5:5:2.5 (v/v/v) TFA/phenol/H2 O (see recipe) N,N-dimethylformamide (DMF; anhydrous, Sigma-Aldrich) 10% (v/v) N,N -diisopropylethylamine (DIEA) in dry DMF (see recipe) Tetrahydrofuran (THF; anhydrous, Sigma-Aldrich) 4-[(tert-butoxycarbonyl)amino]-1-methyl-1H-pyrrole-2-carboxylic acid (Boc-Py-OH, J&K Scientific) Triphosgene (BTC; Aladdin, cat. no. T103041) Nitrogen gas (N2 ) 15% 2,4,6-collidine in dry THF (see recipe) 4-tert-butoxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (Boc-Im-OH, J&K Scientific) 1-Hydroxy-7-aza-benzotriazole (HOAt; Aladdin, cat. no. H109328) Fmoc-(N-γ-Boc)-D-α, γ-diaminobutyric acid (Fmoc-D-Dab(Boc)-OH, Alfa Aesar) Im-CCl3 (prepared according to Masiukiewicza et al., 2005) 1-methyl-1H-imidazole-2-carboxylic acid (Im-OH, Sigma-Aldrich) Benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP; Aladdin, cat. no. P109336) 20% piperidine in DMF (see recipe) Di-tert-butyl dicarbonate (Boc2 O; Aladdin, cat. no. D106159) 3,3 -diamino-N-methyl-dipropylamine (Aladdin, cat. no. B105353) Methanol (MeOH; HPLC-grade, J&K Scientific) Diethyl ether (Et2 O, anhydrous) Acetonitrile (MeCN; HPLC-grade, J&K Scientific) CS Bio 336X peptide synthesizer (http://www.csbio.com) Air compressor Disposable syringe filter (Nylon 66, 0.22 μm; Jinteng) ULTIMAT 3000 Instrument (Dionex) ˚ ACE C18 column (10 × 250 mm, 5 μm, 300 A) Photodiode array detector capable of measuring absorbance Lyophilizer and freeze dryer vessels Centrifuge Shimadzu LC 20 with UV detector SPD-20 A ˚ Inertsil ODS-SP column (4.6 × 250 mm, 5 μm, 100 A) Additional reagents and equipment for deprotection procedure (Alternate Protocol) and HPLC (APPENDIX 3B) NOTE: To increase the solubility in THF, fine powders of Boc-Im-OH are obtained by dispersion of the commercial reagent (5 g) in H2 O (300 mL) via ultrasonication at 40°C followed by lyophilization. All other commercial reagents are used as received.
Deprotection 1. Insert the needle into an empty amino acid (AA) reservoir. 2. Load Boc-Py-hydrazine resin (400 mg, 0.15 mmol/g) in the reaction vessel (RV, 10 mL). 3. Wash the resin twice with DCM (5 mL, calibrated delivery from reagent bottle 4). Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Shake 2 min, then drain the RV.
4. Treat the resin twice with the mixture of 92.5:5:2.5 TFA/phenol/H2 O (5 mL, calibrated delivery from reagent bottle 5).
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Shake 2 min for the first treatment and 20 min for the second treatment, then drain the RV.
5. Wash the resin twice with DCM (5 mL, calibrated delivery from reagent bottle 4). Shake 2 min, then drain the RV.
6. Wash the resin with DMF (5 mL, calibrated delivery from reagent bottle 1). Shake 2 min, then drain the RV.
7. Wash the resin with 10% DIEA/dry DMF (5 mL from reagent bottle 6, bypassing the AA reservoir). Shake 2 min, then drain the RV.
8. Wash the resin with dry THF (to wash the needle, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
9. Wash the resin with DMF (5 mL from reagent bottle 1). Shake 2 min, then drain the RV.
10. Wash the resin with dry DMF (5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
11. Retract the needle and inject it in the following AA reservoir loaded with the monomer and BTC solids (using Advance_AA). The resin is ready for the coupling reaction. The same deprotection procedure is applied at the initiation of each coupling cycle.
Monomer activation Activation of Boc-Py-OH 12. Load a mixture of Boc-Py-OH (60 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir. 13. Deliver dry THF to the AA reservoir [1.25 mL from reagent bottle 8 bypassing amino acid metering vessel (MVAA)]. 14. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min (using the program Bubble/shake_1Min). 15. Deliver 15% 2,4,6-collidine in dry THF to the AA reservoir (1.25 mL from reagent bottle 7 bypassing MVAA). 16. Blend the resulting slurry by nitrogen bubbling for 2 min. 17. Deliver 10% DIEA/dry DMF to the AA reservoir (2.5 mL from reagent bottle 6 bypassing MVAA). 18. Blend the mixture by nitrogen bubbling for 1 min to form a clear pale yellow solution. 19. Transfer activated monomer from the AA reservoir to the RV for the coupling reaction. [bypassing the metering vessel (MV) and transfer vessel (TV)].
Activation of Boc-Im-OH 20. Load the mixture of Boc-Im-OH (60 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir.
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21. Load HOAt (34 mg, 0.25 mmol) into the following AA reservoir. 22. Activate the mixture of Boc-Im-OH and BTC as described in steps 13 to 18 to form a clear brown solution. 23. Transfer the activated monomer from the AA reservoir to TV. 24. Retract the needle from the first AA reservoir and inject it in the second one loaded with HOAt. 25. Deliver 10% DIEA/dry DMF solution (1.25 mL from reagent bottle 6) to the AA reservoir. 26. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min. 27. Transfer the solution from the AA reservoir to TV to mix it with the activated Im monomer. Wait for 3 min (using the program Shake_1Min).
28. Transfer the resulting solution from the TV to RV for the coupling reaction.
Activation of Fmoc-D-Dab(Boc)-OH 29. Load the mixture of Fmoc-D-Dab(Boc)-OH (109 mg, 0.25 mmol) and BTC (37 mg, 0.125 mmol) into an AA reservoir. 30. Load HOAt (34 mg, 0.25 mmol) into the following AA reservoir. 31. Activate the monomer as described in steps 13 to 18 to form a clear brown solution. 32. Transfer the activated monomer to RV for the coupling reaction.
Monomer coupling 33. Shake RV loaded with the deprotected resin and the activated amino acid for 25 min (Shake_1Min), then drain it. 34. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 35. Wash the resin three times with DMF (5 mL from reagent bottle 1). The resin is ready for the next coupling cycle.
Terminal Im capping Usual procedure 36a. Load Im-CCl3 [57 mg, 0.25 mmol, prepared according to the literature method (Masiukiewicza et al., 2005)] in an AA reservoir. 37a. Deliver 10% DIEA/dry DMF (2 × 2.5 mL from reagent bottle 6) to the AA reservoir. Blend the mixture in the AA reservoir by nitrogen bubbling for 2 min. 38a. Transfer the solution from the AA reservoir to RV for the coupling reaction [bypassing the metering vessel (MV) and transfer vessel (TV)]. Shake RV for 40 min (Shake_1Min) then drain it.
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
39a. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 40a. Wash the resin three times with DMF (5 mL from reagent bottle 1).
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Alternative procedure for terminal Im capping 36b. Perform step 36a, above, using Im-OH (32 mg, 0.25 mmol) and PyBOP (130 mg, 0.25 mmol) instead of Im-CCl3 . 37b. Dissolve the mixture and transfer it to RV, employing steps 37a to 39a. 38b. Shake RV loaded with the deprotected resin and the amino acid for 120 min (Shake_1Min), then drain it. 39b. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 40b. Wash the resin three times with DMF (5 mL from reagent bottle 1).
Protecting-group exchange 41. Deprotection: Remove the Fmoc group of the γ-turn unit with 20% (v/v) piperidine in DMF following the “Deprotection Procedure” of the Alternate Protocol described below. 42. Perform step 36a using Boc2 O (115 μL, 0.50 mmol) instead of Im-CCl3 . 43. Dissolve the mixture and transfer it to RV employing steps 37a to 39a. 44. Shake RV loaded with the deprotected resin and Boc2 O for 15 min (Shake_1Min), then drain it. 45. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). 46. Wash the resin three times with DMF (5 mL from reagent bottle 1). The resin is now ready for cleavage.
Resin cleavage and purification 47. Divide the resin from step 46 into four equal parts. 48. Add 3,3 -diamino-N-methyl-dipropylamine (100 μL) in DMF (200 μL) to one part and shake the mixture at 90°C for 1 hr. 49. Remove the resin by filtration through a syringe filter and wash it with MeOH (2 mL). 50. Remove the solvent under reduced pressure. 51. Add 10 volumes of cold diethyl ether to the residue and transfer the mixture into a 50-mL centrifuge tube, centrifuge 10 min at 3500 × g, then carefully decant and discard the supernatant. 52. Dissolve the light-yellow powder in 10% (v/v) MeCN/H2 O containing 0.1% (v/v) TFA and purify the crude peptide by semi-preparative RP-HPLC performed on ULTIMAT 3000 Instrument equipped with an ACE C18 column (10 × 250–mm, ˚ Measure UV absorbance using a photodiode array detector at 254 5 μm, 300 A). and 310 nm. Start the RP-HPLC gradient at 10% of B (MeCN), then increased to 100% of B over 30 min (A: 0.1% TFA in water). 53. Remove the solvent under reduced pressure and collect the pure product after lyophilization. Polyamide 1 was obtained as a pale yellow powder (3 mg, 15%). Analytical HPLC tr = 17.7 min. HRMS (ESI) m/z: calcd. for C62 H81 N22 O11 [M + H]+ 1309.6450, found 1309.6444.
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Stepwise HPLC analysis 54. Take a resin sample (4 mg) out during the coupling reaction (step 33), transfer to a 1.5-mL tube, and wash it twice by pipetting 1 mL DMF into the tube and removing the solvent after 1 min. 55. Treat the resin with 3,3 -diamino-N-methyl-dipropylamine (20 μL) and DMF (20 μL) at 90°C for 30 min. 56. Dilute the mixture with MeOH (100 μL) and filter. 57. Analyze the sample (20 μL) by analytical HPLC at 310 nm performed on the Shimadzu LC 20 with UV detector SPD-20 A using Inertsil ODS-SP column (4.6 × ˚ 250 mm, 5 μm, 100 A). The RP-HPLC gradient is started at 10% B (MeCN), then increased to 100% of B over 30 min (A: 0.1% TFA in water). ALTERNATE PROTOCOL
AUTOMATED SYNTHESIS OF Py-Im POLYAMIDES USING Fmoc-CHEMISTRY This alternative procedure for automated Py-Im polyamide synthesis uses Fmoc chemistry. First, the resin-bound polyamide is deprotected by piperidine/DMF. Then, the Fmoc-protected monomer is activated according to the programs described in the Basic Protocol and transferred to an RV for the coupling reaction. Two successive coupling cycles are employed when coupling pyrrole amino acid to imidazole amines (60 min each); all other couplings are carried out with a single-coupling cycle (40 min). Finally, the polyamide is capped with Im-OH or Im-CCl3 , cleaved from the resin, and purified by semi-preparative HPLC using the same procedures as described under “Terminal Im capping” and “Resin cleavage and purification” in the Basic Protocol. The sequence of reactions is exemplified in the synthesis of polyamide 2 (Fig. 8.11.4).
Additional Materials (also see Basic Protocol) 4-(Fmoc-amino)-1-methyl-1H-imidazole-2-carboxylic acid (Fmoc-Im-OH, Atomax Chemicals) Fmoc-4-amino-1-methylpyrrole-2-carboxylic acid (Fmoc-Py-OH, Atomax Chemicals) 4-(Fmoc-amino)butyric acid (Fmoc-GABA-OH; Aladdin, cat. no. F122242) Dimethylaminopropylamine (Aladdin, cat. no. D110909) Additional reagents and equipment for monomer activation (Basic Protocol) NOTE: To increase the solubility in THF, powders of Fmoc-Py-OH and Fmoc-Im-OH are obtained by dissolving the commercial reagents (2 g) in THF (300 mL), removing the solvents under reduced pressure, then drying in a vacuum oven at 50°C overnight.
Deprotection procedure 1. Insert the needle into an empty amino acid (AA) reservoir. 2. Load Fmoc-Py-hydrazine resin (400 mg, 0.15 mmol/g, prepared manually from Fmoc-Py-OH and Fmoc-hydrazinobenzoyl AM resin, see Support Protocol) into the reaction vessel (RV, 10 mL).
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
3. Treat the resin twice with the mixture of 20% piperidine/DMF (5 mL from reagent bottle 3). Shake 10 min for each treatment, then drain the RV.
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Figure 8.11.4 Synthetic scheme for automated synthesis of Py-Im polyamide 2. Reagents and conditions: (i) 20% piperidine/DMF; (ii) Fmoc-Py-OH, BTC, collidine, DIEA; (iii) 20% piperidine/DMF; (iv) Fmoc-Im-OH, BTC, collidine, DIEA, HOAt; (v) 20% piperidine/DMF; (vi) FmocPy-OH, BTC, collidine, DIEA; (vii) Fmoc-Py-OH, BTC, collidine, DIEA; (viii) 20% piperidine/DMF; (ix) Fmoc-GABA-OH, BTC, collidine, DIEA, HOAt; (x) 20% piperidine/DMF; (xi) Fmoc-Py-OH, BTC, collidine, DIEA; (xii) 20% piperidine/DMF; (xiii) Fmoc-Py-OH, BTC, collidine, DIEA; (xiv) 20% piperidine/DMF; (xv) Fmoc-Py-OH, BTC, collidine, DIEA; (xvi) 20% piperidine/DMF; (xvii) Im-CCl3 , DIEA, or Im-OH, PyBOP, DIEA; (xviii) dimethylaminopropylamine, air, 90°C, 1 hr. Nucleic Acid Binding Molecules
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4. Wash the resin twice with dry DMF (to wash the pipeline, 5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
5. Wash the resin twice with dry THF (to wash the needle, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
6. Wash the resin with DCM (5 mL from reagent bottle 4). Shake 2 min, then drain the RV.
7. Wash the resin with DMF (dry DMF not necessary in this step; 5 mL from reagent bottle 1). Shake 2 min, then drain the RV.
8. Wash the resin with dry DMF (5 mL from reagent bottle 2). Shake 2 min, then drain the RV.
9. Retract the needle and inject it in the following AA reservoir loaded with the monomer and BTC solids (by Advance_AA). The resin is now ready for the coupling reaction. The same deprotection programs are applied at the initiation of each coupling cycle.
Monomer activation 10. Activate Fmoc-Py-OH, Fmoc-Im-OH, and Fmoc-GABA-OH according to the procedures in the Basic Protocol used to activate Boc-Py-OH, Boc-Im-OH, and FmocD-Dab(Boc)-OH, respectively. Monomer coupling 11. Shake RV loaded with the deprotected resin and the activated amino acid for 40 min (using the Shake_1Min program on the peptide synthesizer), then drain it. 12. Wash the resin twice with dry THF (to wash the needle and the AA reservoir, 2 × 2.5 mL from reagent bottle 8, bypassing the AA reservoir). Shake 2 min, then drain the RV.
13. Wash the resin three times with DMF (5 mL from reagent bottle 1). Shake 2 min, then drain the RV. The resin is ready for the next coupling cycle. Following procedures similar to “Terminal Im capping” and “Resin cleavage and purification” in the Basic Protocol, polyamide 2 was obtained after cleavage from the resin using dimethylaminopropylamine (2 mg, 12%). Analytical HPLC tr = 17.3 min. HRMS (ESI) m/z: calcd. for C55 H67 N20 O9 [M + H]+ 1151.5394, found 1151.5392. SUPPORT PROTOCOL
PREPARATION OF Boc-Py-HYDRAZINE RESIN AND Fmoc-Py HYDRAZINE RESIN Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin are prepared manually from BocPy-OH and Fmoc-hydrazinobenzoyl AM resin (Fig. 8.11.5).
Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Additional Materials (also see Basic Protocol and Alternate Protocol) Fmoc-hydrazinobenzoyl AM resin (Novabiochem) Acetic anhydride (Ac2 O)
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Figure 8.11.5
Synthetic scheme for Boc-Py-hydrazine resin and Fmoc-Py-hydrazine resin.
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU; Aladdin, cat. no. H109327) 2,4,6-collidine (Aladdin, cat. no. T108942) N,N -diisopropylethylamine (DIEA) Solid-phase synthesis vessel 5-mL reaction vials (wholesale 5-mL clear glass vials with orifice reducers and plastic caps; Yanzhou United) Stirring bar and magnetic stirrer Rubber septa Round bottom flask (500 mL) Oil-type vacuum pump Preparation of Boc-Py-hydrazine resin 1a. Fit a two-necked, 500-mL round-bottomed flask with a solid-phase synthesis vessel and connect to a nitrogen line and an oil-type vacuum pump via rubber septa (to set up a manual solid-phase peptide synthesizer). Add Fmoc-hydrazinobenzoyl AM resin (400 mg, 0.66 mmol/g) to the manual solid-phase peptide synthesis vessel (10 mL). 2a. Swell the resin with CH2 Cl2 (3 mL) for 20 min, then drain it. 3a. Treat the resin twice with 20% piperidine in DMF (2 × 3 mL). Use 10 min for each treatment, then drain the resin.
4a. Wash the resin three times with DMF (not necessarily dry; 3 × 3 mL) and dry DMF (3 mL) by pipetting the solvent onto the resin and bubbling nitrogen through the mixture for 1 min, then draining the vessel under vacuum. 5a. Add Boc-Py-OH (29 mg, 0.12 mmol) and BTC (18 mg, 0.06 mmol) to a reaction vial (5 mL) equipped with a stirring bar. 6a. Add dry THF (1.5 mL) to the vial to dissolve the solids. 7a. Add [neat] collidine (95 μL, 0.72 mmol) dropwise to the vial.
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The reaction is violent, with the emission of CO2 accompanied by large amount of collidine hydrochloride precipitate.
8a. Stir the resulting slurry for 2 min. 9a. Add dry DMF (2.5 mL) and DIEA (200 μL, 1.20 mmol) successively to the vial to form a clear solution. 10a. Transfer this solution to the resin (from step 4a) and mix with nitrogen bubbling for 10 min. 11a. Repeat step 4a. 12a. Add Ac2 O (125 μL, 1.32 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) to the resin and mix them by nitrogen bubbling for 10 min. 13a. Repeat step 4a. The resin can be used as a solid support in the Basic Protocol at a substitution level of 0.15 mmol/g (meaning that the loading of the first amino acid is 0.15 mmol/g dry resin).
Preparation of Fmoc-Py-hydrazine resin 1b. Swell and remove Fmoc group of Fmoc-hydrazinobenzoyl AM resin (400 mg, 0.66 mmol/g) according to steps 1a to 4a of this protocol, above. 2b. Mix Fmoc-Py-OH (43 mg, 0.12 mmol), HATU (46 mg, 0.12 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) in a 5-mL reaction vial equipped with a stirring bar. Stir the mixture for 10 min. 3b. Transfer the solution to the resin and mix with nitrogen bubbling for 1 hr. 4b. Repeat step 4a. 5b. Add Ac2 O (125 μL, 1.32 mmol), DIEA (200 μL, 1.20 mmol), and dry DMF (3 mL) to the resin and mix them with nitrogen bubbling for 10 min. 6b. Repeat step 4a. The resin can be used as a solid support in the Alternate Protocol at a substitution level of 0.15 mmol/g.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A.
2,4,6-Collidine in dry THF (15%) Mix 15 mL of 2,4,6-collidine (Aladdin, cat. no. T108942) with 85 mL of dry THF (THF; anhydrous, Sigma-Aldrich) in reagent bottle 7. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
DIEA in dry DMF (10%) Mix 10 mL of N,N-diisopropylethylamine (DIEA; Aladdin, cat. no. D109321) with 90 mL of dry DMF (DMF; anhydrous, Sigma-Aldrich) in reagent bottle 6. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks. Pyrrole-Imidazole Polyamides: Automated Solid-Phase Synthesis
Piperidine/DMF (20%) Mix 200 mL of piperidine with 800 mL of DMF in reagent bottle 3.
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All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
TFA/phenol/H2 O (92.5:5:2.5 v/v/v) Heat phenol in a water bath (60°C), then take 5 mL of phenol and mix it with 92.5 mL of TFA and 2.5 mL of H2 O in reagent bottle 5 in an ice-water bath. All the reagents and solutions stored in the reagent bottles can be kept under nitrogen for 2 weeks.
COMMENTARY Background Information We previously reported a facile and highly efficient solid-phase synthesis of hairpin DNA-binding polyamides using the costeffective BTC activating agent. This strategy was employed with great success to produce Py-Im polyamides in a 8% to 33% yield by manual solid-phase synthesis (Su et al., 2009). The limitation with current BTC-mediated coupling protocols is the strong exothermic nature of the reaction, resulting in the emission of CO2 accompanied by large amount of collidine hydrochloride precipitate (Falb et al., 1999; Thern et al., 2002). In order to render BTC couplings amenable to automation, methods were therefore required to mitigate the exothermicity and prevent clogging of the lines of the automated synthesizer by the precipitate. This unit describes a reproducible, highly efficient, and racemization-free procedure for BTC-mediated in situ activation of the amino acid. The key features of the procedure include: (1) the preactivation of the carboxylic acid is carried out with collidine in THF; and (2) the resulting precipitate is converted to the soluble DIEA hydrochloride salt by the addition of a DIEA/DMF solution (Fang et al., 2014). The monomer activation, coupling, and resin cleavage procedures are all optimized, allowing automated synthesis of a palette of hairpin Py-Im polyamides on a commercially available peptide synthesizer using BTC as the condensing agent for all coupling steps (Fang et al., 2015).
Critical Parameters and Troubleshooting To guarantee complete resin-to-solvent contact, the CS336X peptide synthesizer should be equipped with an air compressor to enable a pneumatically operated rotary actuator for fast, smooth and 180° inversion mixing. During the monomer activation steps, nitrogen is bubbled into the amino acid reservoir to blend the reaction mixture and facilitate the dissipation of CO2 and heat. The nitrogen flow
should be adjusted to avoid the loss of material and blockage of the needle. Anhydrous solvents are also important. The solvents should be taken from a freshly opened bottle of commercially prepared anhydrous solvent. It is best to purchase anhydrous solvents in the smallest-size bottle that will fit the requirements of the synthesis. The anhydrous solvents and solutions kept in the reagent bottles should be changed once every 2 weeks to avoid a drop in coupling efficiencies. The addition of one equivalent of HOAt to the BTC-activated Boc-Im-OH, Fmoc-ImOH, or Fmoc-D-Dab(Boc)-OH can dramatically accelerate their coupling rate with resinbound amines. Complete conversion can be observed by HPLC within 20 min. However, the combination of BTC/HOAt decreases the coupling rate of activated Boc-Py-OH or Fmoc-Py-OH with resin-bound amines. In the Basic Protocol, the mixture of BocIm-OH and BTC cannot be completely dissolved in dry THF even using the fine powder of Boc-Im-OH obtained after lyophilization. This will slightly affect the conversion of the amino acid to its corresponding acid chloride. However, this does not affect the coupling reaction, as a large excess of monomer is used. Because the Fmoc-Py-OH and Fmoc-Im-OH used in the Alternate Protocol have poor solubility and are less reactive than their Boc- protected counterparts, extended reaction times are necessary to drive the coupling reaction to completion. For better yield and crude purity of Py-Im polymides, two successive coupling cycles are employed when coupling pyrrole amino acids to resin-bound imidazole amines.
Anticipated Results The two protocols described in this unit enable the preparation of Py-Im polyamides in good yield and good crude purity. For polyamide 1, both protocols should give yields between 15% and 20% with a purity of 95%. The Basic Protocol is more suitable for preparation of polyamides containing difficult couplings such as the coupling of Boc-Py-
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OH, Boc-Im-OH, or Fmoc-D-Dab(Boc)-OH (γ-turn) to imidazole amines, and provides better yields than the Alternate Protocol.
Time Considerations
Fang, L., Yao, G., Pan, Z., Wu, C., Wang, H., Burley, G.A., and Su, W. 2015. Fully automated synthesis of DNA-binding Py-Im polyamides using a triphosgene coupling strategy. Org. Lett. 17:158161. doi: 10.1021/ol503388a.
To prepare Boc-Py-hydrazine resin for the Basic Protocol (Fmoc-Py-hydrazine resin for Alternate Protocol) manually as described in Support Protocol, and prepare Im-CCl3 according to the literature method, should take 1 day. The solid-phase synthesis of Py-Im polyamide on the CS336X peptide synthesizer should take 1 day. The estimated time to complete the resin cleavage, purification, and lyophilization is 1 day. Therefore, preparation of a hairpin Py-Im polyamide sequence using either Basic Protocol or the Alternate Protocol may take 3 days.
Masiukiewicza, E., Mrugala, D., and Rzeszotarska, B. 2005. An improved synthesis of 1-methyl2-trichloroacetylimidazole. Org. Prep. Proced. Int.: New J. Organ. Synth. 37:403-405. doi: 10.1080/00304940509354973.
Literature Cited
Su, W., Gray, S.J., Dondi, R., and Burley, G.A. 2009. Highly efficient synthesis of DNAbinding hairpin polyamides via the use of a new triphosgene coupling strategy. Org. Lett. 11:3910-3913. doi: 10.1021/ol9015139.
Blackledge, M.S. and Melander, C. 2013. Programmable DNA-binding small molecules. Bioorg. Med. Chem. 21:6101-6114. doi: 10.1016/j.bmc.2013.04.023. Falb, E., Yechezkel, T., Salitra, Y., and Gilon, C. 1999. In situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl) carbonate and its utilization for difficult couplings in solidphase peptide synthesis. J. Pept. Res. 53:507517. doi: 10.1034/j.1399-3011.1999.00049.x. Fang, L., Wu, C., Yu, Z., Shang, P., Cheng, Y., Peng, Y., and Su, W. 2014. Triphosgene-mediated couplings in the solid phase: Total synthesis of Brachystemin A. Eur. J. Org. Chem. 34:75727576. doi: 10.1002/ejoc.201403145.
Muzikar, K.A., Nickols, N.G., and Dervan, P.B. 2009. Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression. Proc. Natl. Acad. Sci. U.S.A. 106:1659816603. doi: 10.1073/pnas.0909192106. Nickols, N.G. and Dervan, P.B. 2007. Suppression of androgen receptor–mediated gene expression by a sequence-specific DNA-binding polyamide. Proc. Natl. Acad. Sci. U.S.A. 104:10418-10423. doi: 10.1073/pnas.0704217104.
Thern, B., Rudolph, J., and Jung, G. 2002. Total synthesis of the nematicidal cyclododecapeptide omphalotin A by using racemization-free triphosgene-mediated couplings in the solid phase. Angew. Chem., Int. Ed. 41:2307-2309. doi: 10.1002/1521-3773(20020703)41:13% 3c2307::AID-ANIE2307%3e3.0.CO;2-Y. Yang, F., Nickols, N.G., Li, B.C., Marinov, G.K., Said, J.W., and Dervan, P.B. 2013. Antitumor activity of a pyrrole-imidazole polyamide. Proc. Natl. Acad. Sci. U.S.A. 110:1863-1868. doi: 10.1073/pnas.1222035110.
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