Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis

Abstract

This protocol provides details for the preparation of nucleoside phosphoramidites with Dim (1,3-dithian-2-yl-methyl) and Dmoc (1,3-dithian-2-yl-methoxycarbonyl) as protecting groups, and a linker with Dmoc as the cleavable function; and using them for solid phase synthesis of sensitive oligodeoxynucleotide (ODN). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using the phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides and α-chloroamides, which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies, can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functionalities.

Basic Protocol: Synthesis, deprotection, cleavage, and purification of sensitive ODN.

Support Protocol 1: Synthesis of Dim-Dmoc nucleoside phosphoramidites.

Support Protocol 2: Preparation of CPG with a Dmoc linker.

Support Protocol 3: Synthesis of a phosphoramidite containing the sensitive alkyl ester group.

INTRODUCTION

Solid phase synthesis of oligodeoxynucleotide (ODN) requires the protection of the exo-amino group of nucleobases and the phosphate backbone, and reversible linking of the nascent ODN to a solid support. In traditional technologies, both the protection and linking are achieved using the acyl function. Deprotection and cleavage after the ODN is assembled are achieved under strongly basic and nucleophilic conditions such as concentrated NH₄OH and dilute KOCH₃. Although the traditional technologies have met the needs in many research areas, they are not suitable for the synthesis of ODNs that contain sensitive organic functional groups. These groups include but not limited to alkyl esters, aryl esters, thioesters, phosphate triesters, activated amides, N-aryl amides, alkyl halides, and α-chloroamides. They can co-exist with the functional groups of ODN, but cannot survive the harsh deprotection and cleavage conditions used in typical ODN synthesis technologies. Many efforts have been made to solve the problem (Avino and Eritja 1994, Hayakawa et al. 1990, Leisvuori et al. 2008, Matray and Greenberg 1994, Ohkubo et al. 2004, Virta 2009), but an ideal technology involving synchronized strategies for exo-amine and phosphate protection, and ODN linking to solid support to enable mild conditions for ODN deprotection and cleavage has not appeared. The new ODN synthesis technology described in this Unit aims to solve the problem (Halami et al. 2018, Lin et al. 2016, Shahsavari et al. 2019a, Shahsavari et al. 2019b). In this technology, the exo-amino groups of dA, dC and dG are protected with Dmoc (1,3-dithian-2-yl-methoxycarbonyl), and the hydroxyl group of the phosphoramidous acid in the dA, dC, dG and dT phosphoramidites, which is later converted to the phosphate diester linkage of ODN, is protected with Dim (1,3-dithian-2-yl-methyl). Linking the nascent ODN to solid support, for which CPG is used in this protocol, is achieved using Dmoc. The resulting Dim-Dmoc phosphoramidite monomers are 1a-d, and Dmoc linker is 2 (Figure 1). Using 1a-d and 2, the ODN synthesized can be represented as 3 (Figure 2). Deprotection and cleavage are achieved in two steps. First, the sulfides in Dim and Dmoc are oxidized with 0.4 M NaIO₄, which has a pH of 4. This converts 3 to 4. The pKa of H-2 in the 1,3-dithiane moiety of Dim and Dmoc is about 31. This high pKa is important to ensure the stability of the Dim and Dmoc protections and the Dmoc linker to prevent premature deprotection and cleavage. Oxidation of the sulfides lowered the pKa of H-2 drastically to about 12, which allows deprotection and cleavage under nearly neutral conditions. Second, the oxidized Dim and Dmoc groups and Dmoc linker are cleaved via β-elimination with 3% PhNH₂, which has a pH of 8 (Figure 2). This converts 4 to the fully deprotected ODN 5. Under these nearly neutral and non-nucleophilic deprotection and cleavage conditions, a wide range of sensitive groups such as esters, thioesters, activated amides, N-aryl amides, alkyl halides, and α-chloroamides can survive, and therefore the Dim-Dmoc ODN synthesis technology is well suited for the synthesis of sensitive ODNs. The technology has been demonstrated for the synthesis of ODNs that contain the sensitive alkyl esters, phenyl esters, thioesters, alkyl chlorides and 6-chloropurine (Halami et al. 2018, Lin et al. 2016, Shahsavari et al. 2019a, Shahsavari et al. 2019b). The sensitive ODNs were purified with trityl-on RP HPLC, and were characterized with MALDI-TOF MS and enzymatic digestion essay. It is envisioned that ODNs with many other sensitive groups can be synthesized and purified under similar conditions.

Figure 1.

Dim-Dmoc-phosphoramidites 1a-c, Dim-phosphoramidites 1d-f, and dT-Dmoc-CPG 2.

Figure 2.

Deprotection of ODN assembled with Dim-Dmoc phosphoramidites and Dmoc linker, and the structure of ODN 6.

The Basic Protocol describes a typical procedure for the synthesis, deprotection, cleavage, purification and characterization of sensitive ODNs using ODN 6 (Figure 2), which contains an ester group, as the example. Support Protocol 1 describes the synthesis of Dim-Dmoc nucleoside phosphoramidite monomers 1a-e. Support Protocol 2 describes the preparation of CPG-Dmoc linker 2. Support Protocol 3 describes the synthesis of the special phosphoramidite 1f. Phosphoramidites 1a-f and linker 2 are needed for the synthesis of ODN 6 in the Basic Protocol.

BASIC PROTOCOL

BASIC PROTOCOL TITLE

Synthesis, deprotection, cleavage, and purification of sensitive ODN

Introductory paragraph

This Protocol describes the synthesis, deprotection, cleavage, purification and characterization of sensitive ODNs using the Dim-Dmoc technology. The synthesis of the 20-mer ODN 6 (Figure 2), which contains an alkyl ester group, is used as an example for the description. Many other sensitive ODNs such as those containing aryl esters, thioesters, N-aryl amides, alkyl halides, and α-chloroamides can be synthesized similarly. The essential chemicals to enact the Dim-Dmoc technology are Dim-Dmoc phosphoramidites 1a-d, tagging phosphoramidite 1e and Dmoc-CPG linker 2 (Figure 1). For the synthesis of 6, the special phosphoramidite 1f (Figure 1) is needed to introduce the alkyl ester group. The synthesis can be conducted under typical conditions using the phosphoramidite chemistry with only minor modifications. One modification is to use 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (1g) instead of the typically used acetic anhydride for capping. The other is to tag the 5’-end of full-length ODN with a Tr (trityl) group instead of the commonly used DMTr group to assist RP HPLC purification. For this purpose, the phosphoramidite 1e is needed (Figure 1). After solid phase synthesis, ODN deprotection and cleavage are achieved by oxidation with NaIO₄ at pH 4 followed by β-elimination with PhNH₂ at pH 8 as described in the Introduction section (Figure 2). The ODN is then purified with trityl-on RP HPLC (Figure 3) and characterized with MALDI-TOF MS (Figure 4). The reason for using 1g instead of acetic anhydride for capping is to prevent cap-exchange, in which the Dmoc group on the exo-amines is replaced by an acetyl group. Once cap-exchange occurred, the exo-amine protection cannot be deprotected under the mild deprotection conditions used in the Dim-Dmoc technology. The reason for using 1e, which contains a 5’-Tr group, instead of a typical 5’-DMTr group to tag the 5’-end of full-length ODN is that the DMTr protection is unstable under the slightly acidic NaIO₄ oxidation conditions. The Dim-Dmoc technology has been reported previously (Shahsavari et al. 2019a).

Figure 3.

RP HPLC profiles of ODN 6. HPLC conditions: see Protocol.

Figure 4.

MALDI-TOF MS of ODN 6.

Materials

Dim-Dmoc phosphoramidites 1a-e (Support Protocol 1)

Dmoc-CPG linker 2 (Support Protocol 2)

Special phosphoramidite 1f (Support Protocol 3)

Drierite

Acetonitrile (anhydrous, Glen Research, Cat# 40–4050-50, or freshly distilled over CaH₂)

2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (1g, Sigma Aldrich, Cat# 305995)

Argon or nitrogen

Dichloromethane (DCM, anhydrous)

Dichloroacetic acid (DCA, 3% in DCM)

5-(Ethylthio)-1H-tetrazole solution (0.25 M in acetonitrile, Glen Research, Cat# 30–3140-57)

Iodine solution (0.02 M in THF/pyridine/H₂O v/v/v 70/20/10, Glen Research, Cat# 40–4330-57)

Sodium periodate (NaIO₄, 0.4 M solution, Sigma Aldrich, Cat# S1878)

Water (distilled or deionized)

Aniline (PhNH₂, 3% solution, Sigma Aldrich, Cat# 242284)

n-Butanol (nBuOH, molecular biology grade, 99%, Sigma Aldrich, Cat# 281549)

Acetic acid (AcOH, 80% solution, glacial, Sigma Aldrich, Cat# 695092)

Bottles that can fit to the DNA synthesizer to be used

Vacuum desiccator

Glass syringes (25 mL)

Syringe needles

Vacuum oil pump

Balance

Empty DNA synthesis column that can fit to the DNA synthesizer to be used

DNA synthesizer (e.g. MerMade 6)

Centrifuge tubes (1.5 mL)

Orbital shaker (can be substituted by a rotary evaporator for shaking)

Benchtop centrifuge

Pipette (100 μL, 500 μL, 1000 μL each)

Vacuum centrifuge concentrator

Vortex shaker

UV-Vis spectrometer

MALDI-TOF mass spectrometer

Freezer (−20 °C)

Additional reagents and equipment for HPLC (UNIT 10.5)

Protocol steps

Solid-phase synthesis of ODN 6

1. Dry phosphoramidites 1a-f in bottles that can fit to the DNA synthesizer in a desiccator over fresh Drierite under vacuum overnight.

2. Add dry acetonitrile to the bottles to prepare 0.1 M solutions of 1a-f.

3. Weigh 20 mg (0.52 μmol) Dmoc-CPG linker 2 (26 μmol/g loading) and pack into a synthesis column.

4. Prepare a 0.1 M solution of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (1g) in dry acetonitrile.

5. Load the following reagents needed for ODN synthesis onto a DNA synthesizer: argon, dry acetonitrile, dry DCM, 3% DCA in DCM, 0.25 M 5-(ethylthio)-1H-tetrazole in acetonitrile, and 0.02 M I₂ in THF/pyridine/H₂O (v/v/v 70/20/10).

6. Load the solutions of 1a-g and the synthesis column containing 2 onto the DNA synthesizer.

7. Perform automated synthesis of ODN 6 from the 3’-end with the dT at the 3’-end being introduced by 2, the alkyl chloride moiety being introduce by 1f, and the dT at the 5’-end being introduced by 1e under the conditions in Table 1. At the end of synthesis, do not perform detritylation.

   See UNIT 3.1 and other UNITs of Chapter 3 for additional details on solid-phase oligonucleotide synthesis.

8. Remove the synthesis column from the synthesizer.

Table 1.

ODN synthesis conditions.

Synthesis step	Detritylation	Coupling	Capping	Oxidation
Conditions	3% DCA, 90 s × 2	0.1 M 1a-e, or f, 0.25 M 5-(ethylthio)-1H-tetrazole, 60 s × 2	0.1 M 1g, 0.25 M 5-(ethylthio)-1H-tetrazole, 60 s × 3	0.02 M I2, 40 s

Deprotection and cleavage of ODN 6

1. Unpack the synthesis column and divide the CPG into five equal portions (~4 mg each).

2. Add 1 mL 0.4 M NaIO₄ solution (pH 4) into one portion of CPG in a 1.5 mL centrifuge tube.

3. Gently shake the centrifuge tube on an orbital shaker at room temperature for 3 hours.

4. Gently spin the centrifuge tube in a benchtop centrifuge to bring down the contents to the bottom of the tube.

5. Remove the supernatant with a pipette.

   If deprotection and cleavage are performed on all the CPG in one batch, make sure the amount of NaIO₄ is sufficient. This can be achieved by performing the oxidation 2 or 3 times.

6. Wash the CPG with 1 mL of water 4 times.

   This converts ODN 3 to 4 (Figure 2).

7. After the water for the last wash was removed via a pipette, add 1 mL 3% aniline solution (pH 8) to the CPG in the centrifuge tube.

8. Gently shake the centrifuge tube at room temperature for 3 hours.

9. Gently spin the centrifuge tube in a benchtop centrifuge to bring down the contents.

10. Transfer the supernatant with a pipette into a clean 1.5 mL centrifuge tube.

11. Wash the CPG with 100 μL water 2 times and transfer the washes into the same centrifuge tube.

    This converts ODN 4 to 5 (Figure 2), which is ODN 6 in this particular example of synthesis.

12. Concentrate the solution to about 100 μL in a centrifuge vacuum concentrator.

13. Add 900 μL nBuOH.

14. Vortex for 30 s, and spin in a benchtop centrifuge at about 14.5k rpm (14100 × g) for 5 min.

    ODN 6 is precipitated by nBuOH leaving excess aniline and small organic molecules from deprotection in the supernatant.

15. Carefully remove the supernatant with a pipette without disturbing the precipitated ODN 6.

Purification and analysis of ODN 6

1. Add 100 μL water into the centrifuge tube, vortex shortly to dissolve ODN 6.

2. Centrifuge the tube at about 14.5k rpm (14100 × g) for 3 minutes to bring down any accidental particles.

3. Inject 35 μL solution of ODN 6 into RP HPLC.

   RP HPLC profile is shown in Figure 3 (5’-Tr tagged, crude). HPLC conditions for all analyses and separations in this Protocol: Column, 250 × 3.20 mm, C-18, 5 μm diameter, 100 Å pore diameter; solvent A, 0.1 M triethylammonium acetate in 5% acetonitrile; solvent B, 90% acetonitrile; gradient, solvent B (0–45%) in solvent A over 60 min followed by solvent B (45–100%) in solvent A over 20 min; flow rate: 1.0 mL/min; detection, UV at 260 nm. For details of RP HPLC operation, see UNIT 10.5.

4. Pool the fractions that contain the Tr-on ODN 6, which has a retention time of about 40 min.

5. Concentrate to ~100 μL in a centrifuge vacuum concentrator.

6. Inject into RP HPLC.

   RP HPLC profile in Figure 3 (5’-Tr tagged, pure)

7. Add 1 mL 80% AcOH, and shake gently at room temperature for 3 hours.

8. Evaporate volatiles in a centrifuge vacuum concentrator.

9. Dissolve the de-tritylated ODN 6 in 100 μL water, and inject into RP HPLC.

   RP HPLC profile in Figure 3 (5’-Tr removed, before re-purification)

10. Pool the fractions that contain the Tr-off ODN 6, which has a retention time of about 21 min.

11. Evaporate volatiles in a centrifuge vacuum concentrator.

12. Quantify ODN 6 by measuring UV absorption at 260 nm

    See procedure in UNIT 10.3

13. Dissolve the ODN in 100 μL water, and inject into RP HPLC.

    RP HPLC profile in Figure 3 (5’-Tr removed, after re-purification)

14. Pool the fractions that contain the ODN, and evaporate volatiles in a centrifuge vacuum concentrator.

15. Analyze with MALDI-TOF MS and enzymatic digestion essay.

    MALDI-TOF MS in Figure 4.

    See procedures in UNIT 10.1 for MALDI-TOF MS and UNIT 10.6 for enzymatic digestion essay.

16. Store the remaining ODN 6 at −20 °C.

SUPPORT PROTOCOL 1

SUPPORT PROTOCOL TITLE

Synthesis of Dim-Dmoc nucleoside phosphoramidites

Introductory paragraph

This Protocol describes the synthesis of the phosphoramidites 1a-d, which are essential monomers for using the Dim-Dmoc technology for sensitive ODN synthesis. In addition, the synthesis of phosphoramidite 1e, which is needed for tagging the full-length ODN with the hydrophobic Tr group to assist RP HPLC purification, is also described. The routes for the synthesis are shown in Figure 5. The synthesis has been reported previously (Lin et al. 2016, Shahsavari et al. 2019a).

Figure 5.

Synthesis of Dim-Dmoc phosphoramidites 1a-e. Conditions: (a) (iPr₂N)₂PCl (9), DIPA, PhMe, rt. (b) (i) TMS-Cl, pyridine, rt; (ii) (1,3-dithian-2-ylmethyl)(4-nitrophenyl)carbonate (12), DMAP, pyridine, rt; (iii) NH₄OH, 0 °C. (c) 7, diisopropylammonium tetrazolide, DCM, rt. (d) (i) tBuMgCl, HMPA, THF, −78 °C to rt; (ii) 12, THF, −78 °C to rt. (e) (i) HF-pyridine, pyridine, rt; (ii) MeOSiMe₃, rt. (f) DMTr-Cl, pyridine, rt.

Materials

Argon or nitrogen

(1,​3-​Dithian-​2-​yl)​[dummy_junk]methanol (8, Aurora Fine Chemicals LLC)

Toluene (freshly distilled over CaH₂)

Diisopropylamine (99.95%, Sigma Aldrich, Cat# 386464, purified by redistillation)

Bis(diisopropylamino)chlorophosphine (9, 95%, Sigma Aldrich, Cat# 341347)

5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyadenosine (10, 99%, Sigma Aldrich, Cat# CH6371376672)

Pyridine (freshly distilled over CaH₂)

Trimethylchlorosilane (TMS-Cl) (99%, Sigma Aldrich, Cat# 386529)

(1,​3-​Dithian-​2-​yl-methyl)​(4-​nitrophenyl)​carbonate [12, Aquila Pharmatech LLC or synthesis (Barthels and Kunz 1982)]

4-(Dimethylamino)pyridine (DMAP) (99%, Sigma Aldrich, Cat# 107700)

Ammonium hydroxide (NH₄OH, concentrated, Sigma Aldrich, Cat# 338818)

Sodium bicarbonate solution (NaHCO₃, 5%)

Dichloromethane (DCM)

Na₂SO₄ (anhydrous, 99%, Sigma Aldrich, Cat# 239313)

Diisopropylammonium tetrazolide (ChemIimplex, 99%, Cat# 00951)

Dichloromethane (DCM, freshly distilled over CaH₂)

5’-O-(4,4’-Dimethoxytrityl)-2’-deoxycytidine (13, 99.5%, Sigma Aldrich, Cat# CH6371378860)

2’-​Deoxy-​3’,​5’-​O-​[1,​1,​3,​3-​tetrakis(1-​methylethyl)​-​1,​3-​disiloxanediyl]​-guanosine [15, Biosynth Carbosynth Ltd, or synthesis (Hargreaves, et al. 2015)]

Tetrahydrofuran (THF, freshly distilled over sodium benzophenone ketal)

Hexamethylphosphoramide (HMPA, 99%, Sigma Aldrich, Cat# H11602)

tert-Butylmagnesium chloride solution (tBuMgCl,1 M in THF, Sigma Aldrich, Cat# 364649)

Methanol (MeOH)

Ethyl acetate (EtOAc)

Ethylenediaminetetraacetic acid solution (EDTA, 0.15 M)

Sodium bicarbonate solution (NaHCO₃, saturated)

Hydrogen fluoride pyridine (HF-pyridine, HF ~70%, pyridine ~30%, Sigma Aldrich, Cat# 184225)

Methoxytrimethylsilane (MeOSiMe₃, 99%, Sigma Aldrich, Cat# 253006)

4,4′-Dimethoxytrityl chloride (DMTr-Cl, 95%, Sigma Aldrich, Cat# 100013)

5’-O-(4,4’-Dimethoxytrityl)thymidine (19, 98%, Sigma Aldrich Cat# 360139)

5’-O-Tritylthymidine (20, Sigma Aldrich, Cat# S859761)

Drying oven

2-Necked round-bottom flasks

Magnetic stirring bars

Magnetic stirring plate

Balance

Syringes

Syringe needles

Separatory funnel

Filter paper

Filter funnel

Rotary evaporator connected to a water aspirator

Cannula

Cannula filter (or cotton and copper wire)

Drying oven

Rubber septa

1-Necked round-bottom flasks

Thin-layer chromatography (TLC) plate (Silica gel 60 F254 glass plate)

UV lamp (254 nm)

Vacuum oil pump

Rotary evaporator connected to an oil pump

Additional reagents and equipment for column chromatography (Meyers, 2000)

Protocol steps

Synthesis of Dim-phosphitylation agent 7

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 1.57 g (10.48 mmol, 1.5 equiv) (1,3-dithian-2-yl)methanol (8) into the flask under positive argon pressure.

3. Add 25 mL freshly distilled toluene via a syringe.

4. Add 9.85 mL (69.9 mmol, 10 equiv) diisopropylamine via a syringe.

5. Add 2.80 g (10.48 mmol, 1.5 equiv) bis(diisopropylamino)chlorophosphine (9) under positive argon pressure.

6. Stir under argon at room temperature for 8 hours.

7. Stop stirring, and let the reaction mixture stand still for about 1 hour.

The mixture contains product 7 (theoretical amount 10.48 mmol, 1.5 equiv) and the diisopropylamine hydrochloride side product. The former is in the supernatant. The latter is insoluble and is precipitated at the bottom of the flask. Product 7 is not isolated and purified. It should be prepared separately, and used directly for the synthesis of each of the Dim phosphoramidites described below. The theoretical molar quantity of 7 to be prepared for each experiment should be calculated based on 1.5 equivalent 7 for 1.0 equivalent nucleoside or alcohol to be phosphitylated.

Synthesis of Dim-Dmoc phosphoramidite 1a

Converting 10 to 11

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 3.87 g (6.99 mmol, 1.0 equiv) 5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyadenosine (10) under positive argon pressure.

3. Add 50 mL pyridine via a syringe.

4. Add 1.87 mL (14.7 mmol, 2.1 equiv) TMS-Cl via a syringe into the flask.

5. Stir the mixture at room temperature for 30 minutes.

   TMS-Cl temporarily protects the 3’-OH of 10.

6. Add 3.10 g (9.83 mmol, 1.4 equiv) (1,3-dithian-2-ylmethyl)(4-nitrophenyl)carbonate (12) under positive argon pressure.

7. Add 0.30 g (2.45 mmol, 0.3 equiv) DMAP under positive argon pressure.

8. Stir at room temperature for 8 hours.

   The Dmoc group is introduced onto the exo-amino group of 10.

9. Cool the reaction mixture to 0 °C.

10. Add 10 mL water, and stir at 0 °C for 5 minutes.

11. Add 16 mL concentrated NH₄OH and stir at 0 °C for 30 minutes.

    The NH₄OH deprotects the 2’-OTMS group.

12. Pour the reaction mixture into a separatory funnel containing 100 mL 5% NaHCO₃.

13. Extract with 70 mL DCM for 3 times and combine the extracts.

14. Dry the DCM extracts over anhydrous Na₂SO₄, and filter.

15. Evaporate the filtrate to dryness under reduced pressure on a rotary evaporator, which gives a thick oily residue.

16. Purify the residue with flash column chromatography using SiO₂ (~150 g) as the stationary phase and hexanes/EtOAc/Et₂O/MeCN/MeOH/Et₃N (1:2:5:2:2:1) as the mobile phase.

    Compound 11: White foam (2.90 g, 3.98 mmol, 57%); m.p. 108.2–111.4 °C; TLC R_f = 0.47 (1:2:5:2:2:1 hexanes/EtOAc/Et₂O/MeCN /MeOH/Et₃N); ¹H NMR (400 MHz, CDCl₃) δ 1.89– 2.04 (m, 2H), 2.50–2.97 (m, 6H), 3.37 (d, J = 4 Hz, 2H), 3.72 (s, 6H), 4.09–4.16 (m, 1H), 4.16–4.19 (m, 1H), 4.52 (d, J = 4 Hz, 2H), 4.68–4.71 (m, 1H), 6.46 (t, J = 6 Hz, 1H), 6.73–6.75 (d, J = 8 Hz, 4H), 7.12– 7.35 (m, 9H), 8.13 (s, 1H), 8.66 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 25.4, 27.3, 40.2, 43.0, 55.2, 65.3, 72.2, 84.6, 86.2, 86.5, 113.1, 122.3, 126.9, 135.5, 141.4, 149.2, 158.4; HRMS (ESI) m/z calcd for C₃₇H₃₉N₅O₇S₂H [M+H]⁺ 730.2364, found 730.2366.

Converting 11 to 1a

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 2.55 g (3.50 mmol, 1 equiv) 11 under positive argon pressure.

3. Add 0.90 g (5.24 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

4. Add 60 mL freshly distilled DCM via a syringe.

5. Attach a cannula filter to one end of another oven-dried cannula (or wrap the cannula with cotton and secure with copper wire)

6. Insert the cannula to a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 5.24 mmol, 1.5 equiv; see the beginning of this Support Protocol for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the flask containing 11.

9. Transfer the supernatant containing 7 into the flask containing 11.

   The cannula filter or cotton is intended to minimize the transfer of the precipitated diisopropylamine hydrochloride side product into the flask containing 11. The setup is illustrated in Figure 6.

10. Stir the reaction mixture at room temperature for 8 hours.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate to dryness on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~120 g) as the stationary phase and hexanes/EtOAc/Et₃N (1:1:0.1) as the mobile phase.

    Compound 1a: White foam (2.40 g, 2.38 mmol, 68%); mixture of two diastereoisomers; R_f = 0.3 and 0.4 (SiO₂, 1:2 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.09–1.25 (m, 12H), 1.75–1.84 (m, 2H), 1.95–2.04 (m, 4H), 2.59–2.75 (m, 6H), 2.87–2.98 (m, 4H), 3.31–4.00 (m, 4H), 3.75 (s, 6H), 4.00 (t, J = 6.5 Hz, 0.5H), 4.05–4.18 (m, 1.5H), 4.21–4.27 (m, 0.5H), 4.30–4.39 (m, 0.5H), 4.55 (d, J = 7.1 Hz, 2H), 4.80–4.88 (m, 1H), 6.46 (t, J = 6.5 Hz, 1H), 6.74–6.77 (m, 4H), 7.14–7.30 (m, 7H), 7.36 (d, J = 11.9 Hz, 2H), 8.16 (s, 0.5H), 8.19 (s, 0.5H), 8.68 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 23.27 (d, J_cp = 2.6 Hz), 23.33 (d, J_cp = 2.0 Hz), 24.86, 24.92, 24.99, 25.8, 26.1, 27.6, 28.8 (d, J_cp = 11.5 Hz), 29.1 (d, J_cp = 14.5 Hz), 40.1 (d, J_cp = 14.8 Hz), 43.3, 43.4, 43.5, 45.45, 45.51, 47.2 (d, J_cp = 7.5 Hz), 47.7 (d, J_cp = 7.6 Hz), 55.5, 63.4, 63.7, 64.7 (d, J_cp = 13.8 Hz), 65.4 (d, J_cp = 18.5 Hz), 65.6, 73.9 (d, J_cp = 13.7 Hz), 74.0 (d, J_cp = 15.3 Hz), 84.8, 85.1, 85.9, 86.4, 86.6, 86.7, 113.3, 122.6, 127.0, 128.0, 128.3, 130.2, 135.78, 135.85, 141.6, 141.7, 144.67, 144.72, 149.2, 150.5, 151.06, 151.12, 152.8, 158.6; ³¹P NMR (162 MHz, CDCl₃) δ 149.4, 149.6; HRMS (ESI) m/z calcd for C₄₈H₆₂N₆O₈PS₄ [M+H]⁺ 1009.3249, found 1009.3255.

Figure 6.

The setup for transferring phosphitylation agent 7 using a cannula under inert atmosphere.

Synthesis of Dim-Dmoc phosphoramidite 1b

Converting 13 to 14

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 2.60 g (4.91 mmol, 1.0 equiv) 13 under positive argon pressure.

3. Add 50 mL pyridine via a syringe.

4. Add 1.87 mL (14.7 mmol, 3.0 equiv) TMS-Cl via a syringe.

5. Stir the mixture at room temperature for 30 minutes.

   TMS-Cl temporarily protects the 3’-OH of 13.

6. Add 3.10 g (9.82 mmol, 2.0 equiv) (1,3-dithian-2-ylmethyl)(4-nitrophenyl)carbonate (12) under positive argon pressure.

7. Add 0.30 g (2.45 mmol, 0.5 equiv) DMAP under positive argon pressure.

8. Stir at room temperature for 8 hours.

   The Dmoc group is introduced onto the exo-amino group of 13.

9. Cool the reaction mixture to 0 °C.

10. Add 10 mL water, and stir at 0 °C for 5 minutes.

11. Add 16 mL concentrated NH₄OH and stir at 0 °C for 30 minutes.

    The NH₄OH deprotects the 2’-OTMS group.

12. Pour the reaction mixture into a separatory funnel containing 100 mL 5% NaHCO₃.

13. Extract with 70 mL DCM for 3 times and combine the extracts.

14. Dry the DCM extracts over anhydrous Na₂SO₄ and filter.

15. Evaporate the filtrate to dryness under reduced pressure on a rotary evaporator to give a thick oily residue.

16. Purify the residue with flash column chromatography using SiO₂ (~120 g) as the stationary phase and hexanes/EtOAc/Et₂O/MeCN/MeOH/Et₃N (1:2:5:2:2:1) as the mobile phase.

    Compound 14: White foam (3.45 g, 4.91 mmol, 100%); m.p. 121.1–123.4 °C; R_f = 0.32 (1:2:5:2:2:1 hexanes/EtOAc/Et₂O/MeCN/MeOH/Et₃N); ¹H NMR (400 MHz, CDCl₃) δ 1.93–2.09 (m, 2H), 2.21–2.27 (m, 1H), 2.41 (br s, 1H), 2.62–2.96 (m, 5H), 3.37–3.41 (m, 2H), 3.79 (s, 6H), 4.09 (t, J = 6 Hz, 1H), 4.46–4.48 (m, 4H), 6.23 (t, J = 6 Hz, 1H), 6.83 (d, J = 8 Hz, 4H), 6.96 (d, J = 8 Hz, 1H), 7.20–7.39 (m, 9H), 8.23 (d, J = 8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 15.4, 27.3, 41.9, 42.8, 55.2, 62.6, 65.6, 70.7, 86.3, 86.9, 113.2, 127.0, 128.0, 128.1, 130.0, 135.3, 135.4, 135.4, 144.2, 158.6, 162.1; HRMS (ESI) m/z calcd for C₃₆H₃₉N₃O₈S₂H [M+H]⁺ 706.2251, found 706.2249.

Converting 14 to 1b

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 2.47 g (3.50 mmol, 1.0 equiv) 14 under positive argon pressure.

3. Add 0.90 g (5.24 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

4. Add 60 mL freshly distilled DCM via a syringe.

5. Attach a cannula filter to one end of an oven-dried cannula (or wrap the cannula with cotton and secure with copper wire).

6. Insert the cannula to a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 5.24 mmol, 1.5 equiv; see the beginning of this Support Protocol for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the flask containing 14.

9. Transfer the supernatant containing 7 into the flask containing 14.

   The cannula filter or cotton is intended to minimize the transfer of the precipitated diisopropylamine hydrochloride side product into the flask containing 14.

10. Stir the reaction mixture at room temperature for 8 hours.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate to dryness on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~80 g) as the stationary phase and hexanes/EtOAc/Et₃N (1:1:0.1) as the mobile phase.

    Compound 1b: White foam (1.79 g, 1.82 mmol, 52%); mixture of two diastereoisomers; R_f = 0.2 and 0.3 (SiO₂, 1:2 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.04–1.26 (m, 12H), 1.72–1.84 (m, 2H), 1.90–2.09 (m, 4H), 2.30–2.47 (m, 2H), 2.59–2.74 (m, 6H), 2.85–2.93 (m, 2H), 3.39–3.60 (m, 4H), 3.61–3.89 (m, 1H), 3.77 (s, 6H), 3.91–4.16 (m, 2H), 4.17–4.22 (m, 1H), 4.40–4.49 (m, 1H), 6.18–6.22 (m, 1H), 6.81 (d, J = 7.4 Hz, 4H), 7.18–7.29 (m, 7H), 7.7.39 (d, J = 7.6 Hz, 2H), 8.27–8.29 (m, 0.5H), 8.34–8.35 (m, 0.5H); ¹³C NMR (100 MHz, CDCl₃) δ 23.3 (d, J_cp = 2.2 Hz), 23.4 (d, J_cp = 1.6 Hz), 24.85, 24.89, 24.92, 24.98, 25.1, 25.7, 25.9, 26.1, 26.2, 27.5, 27.6, 28.7 (d, J_cp = 13.0 Hz), 29.1 (d, J_cp = 21.5 Hz), 41.2 (d, J_cp = 5.7 Hz), 41.5, 43.1, 43.4, 43.5, 45.4, 45.5, 47.1 (d, J_cp = 6.9 Hz), 47.7 (d, J_cp = 8.3 Hz), 55.5, 61.9, 62.4, 64.7 (d, J_cp = 19.9 Hz), 64.8 (d, J_cp = 18.5 Hz), 65.8, 65.9, 71.4 (d, J_cp = 9.3 Hz), 71.9 (d, J_cp = 10.1 Hz), 85.2 (d, J_cp = 7.3 Hz), 86.1, 87.0, 94.5, 113.4, 127.2, 128.1, 128.4, 130.2, 130.3, 135.5, 135.6, 135.7, 135.8, 144.3, 144.4, 144.9, 145.0, 151.9, 155.0, 158.7, 161.9, 162.0; ³¹P NMR (162 MHz, CDCl₃) δ 149.2, 149.5; HRMS (ESI) m/z calcd for C₄₇H₆₂N₄O₉PS₄ [M+H]⁺ 985.3137, found 985.3130.

Synthesis of Dim-Dmoc phosphoramidite 1c

Converting 15 to 16

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 2.57 g (5.04 mmol, 1.0 equiv) 15 under positive argon pressure.

3. Add 50 mL freshly distilled THF via a syringe.

4. Add 5 mL dry HMPA via a syringe.

5. Cool the mixture to −78 °C.

6. Add 15.1 mL tBuMgCl (1 M in THF, 15.1 mmol, 3.0 equiv) via a syringe.

7. Stir while warming to room temperature gradually.

8. Continue to stir at room temperature for 30 minutes.

9. Cool the mixture to −78 °C.

10. Prepare a solution of 3.97 g (12.6 mmol, 2.5 equiv) 12 in 25 mL freshly distilled THF in a round-bottom flask.

11. Transfer the solution of 12 into the flask containing the intermediate formed from 15 and tBuMgCl.

12. Stir at room temperature for 8 hours.

13. Add 8 mL MeOH to quench the reaction.

14. Evaporate all volatiles under reduced pressure on a rotary evaporator.

15. Dissolve the residue in 70 mL EtOAc in a separatory funnel, and wash the solution with 20 mL 0.15 M EDTA solution, 20 mL saturated NaHCO₃ and 30 mL brine.

16. Dry the EtOAc solution with anhydrous Na₂SO₄, filter, and concentrate the filtrate to dryness.

17. Purify the residue with flash column chromatography using SiO₂ (~50 g) as the stationary phase and CHCl₃/MeOH (19:1) as the mobile phase.

    This gives compound 16 as a white solid (1.52 g, 2.22 mmol, 44%).

Converting 16 to 17

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 0.57 g (0.83 mmol, 1.0 equiv) 16 under positive argon pressure.

3. Add 10 mL freshly distilled pyridine via a syringe.

4. Cool the mixture to 0 °C.

5. Add 325 μL (70%, 357 mg, 12.5 mmol, 15.0 equiv) HF-pyridine using a disposable glass pipette under moderate positive argon pressure.

   HF-pyridine and its vapor are highly toxic. The operation must be carried out in a well-ventilated hood.

6. Stir under argon at room temperature for 2 hours.

7. Add 1.72 mL (1.30 g, 12.5 mmol, 15.0 equiv) MeOSiMe₃ via a syringe dropwise over 2 minutes.

8. Stir at room temperature for 8 hours.

   Excess toxic HF is quenched by MeOSiMe₃.

9. Transfer the reaction mixture into a 1-necked round-bottom flask, and remove volatiles on a rotary evaporator under reduced pressure.

   This gives compound 17.

Converting 17 to 18

1. Add 8 mL freshly distilled pyridine to the flask, and evaporate the volatiles on a rotary evaporator under vacuum generated from an oil pump. Repeat the co-evaporation 2 more times.

2. Attach the round-bottom flask containing 17 (theoretical amount 0.83 mmol, 1.0 equiv) to an argon line.

3. Add 10 mL freshly distilled pyridine via a syringe.

4. Attach an oven-dried 2-necked round-bottom flask to an argon line.

5. Add 0.28 g (0.83 mmol, 1.0 equiv) DMTr-Cl under positive argon pressure.

6. Add 10 mL freshly distilled pyridine via a syringe.

7. Transfer the solution of DMTr-Cl in the 2-necked round-bottom flask to the 1-necked round-bottom flask that contains 17.

8. Stir the reaction mixture at room temperature under argon for 8 hours.

9. Pour the reaction mixture into a separatory funnel that contains 50 mL 5% NaHCO₃ and 50 mL DCM.

10. Separate the DCM layer, and extract the aqueous layer with 50 mL DCM. Repeat the extraction.

11. Combine the DCM extracts, dry over anhydrous Na₂SO₄, filter, and concentrate the filtrate to dryness.

12. Purify the residue with flash column chromatography using SiO₂ (~22 g) as the stationary phase and EtOAc/MeOH (9:1) as the mobile phase.

    Compound 18: White foam (501 mg, 0.66 mmol, 80% from 16); m.p. 159 – 161 °C; R_f = 0.2 (9:1 EtOAc/MeOH); ¹H NMR (400 MHz, CDCl₃) δ 1.94–2.06 (m, 2H), 2.48–2.59 (m, 2H), 2.63–2.96 (m, 4H), 3.27–3.41 (m, 2H), 3.68 (s, 6H), 4.10 (t, J = 8 Hz, 1H), 4.15–4.18 (m, 1H), 4.57 (d, J = 8 Hz, 2H), 4.87–4.91 (m, 1H), 6.22 (t, J = 6 Hz, 1H), 6.68 (dd, J = 9 Hz, 3 Hz, 4H), 7.07–7.33 (m, 9H), 7.68 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 10.8, 14.2, 25.4, 27.0, 40.1, 42.6, 45.8, 55.2, 60.4, 64.3, 65.7, 71.7, 84.3, 86.4, 86.5, 113.0, 120.9, 126.8, 135.7, 144.5, 148.4, 154.4, 155.8, 158.4; HRMS (ESI) m/z calcd for C₃₇H₃₉N₅O₈S₂H [M+H]⁺ 746.2313, found 746.2311.

Converting 18 to 1c

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 528 mg (0.70 mmol, 1.0 equiv) 18 under positive argon pressure.

3. Add 180 mg (1.05 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

4. Add 25 mL freshly distilled DCM via a syringe.

5. Attach a cannula filter to one end of an oven-dried cannula (or wrap the cannula with cotton and secure with copper wire)

6. Insert the cannula into a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 1.05 mmol, 1.5 equiv; see the beginning of this Support Protocol for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the flask containing 18.

9. Transfer the supernatant containing 7 into the flask containing 18.

   The cannula filter or cotton is intended to minimize the transfer of the precipitated diisopropylamine hydrochloride side product into the flask containing 18.

10. Stir the reaction mixture at room temperature overnight.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate to dryness on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~25 g) as the stationary phase and EtOAc/MeCN/Et₃N (8:1:1) as the mobile phase.

    Compound 1c: White foam (487 mg, 0.48 mmol, 68%); mixture of two diastereoisomers; R_f = 0.2 and 0.3 (SiO₂, 8:1:1 EtOAc/ACN/Et₃N); ¹H NMR (400 MHz, CDCl₃) δ 1.07–1.16 (m, 12H), 1.77–1.86 (m, 2H), 1.97–2.08 (m, 4H), 2.59–2.94 (m, 10H), 3.25–3.31 (m, 2H), 3.52–3.58 (m, 2H), 3.75 (s, 6H), 3.58–4.21 (m, 2.5H), 4.29–4.32 (m, 0.5H), 4.50 (d, J = 3.5 Hz, 1H), 4.52 (d, J = 3.4 Hz, 1H), 4.72–4.81 (m, 1H), 6.18–6.23 (m, 1H), 6.72–6.78 (m, 4H), 7.16–7.30 (m, 7H), 7.37 (d, J = 7.0 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.8 (s, 0.5H), 7.82 (s, 0.5H); ¹³C NMR (100 MHz, CDCl₃) δ 24.86, 24.91, 24.94, 24.98, 25.6, 26.1, 27.1, 28.88 (d, J_cp = 11.1 Hz), 29.16 (d, J_cp = 8.5 Hz), 39.9, 42.5, 43.4, 43.5, 47.2 (d, J_cp = 6.9 Hz), 47.6 (d, J_cp =7.4 Hz), 55.5, 63.6, 63.9, 64.8 (d, J_cp = 6.6 Hz), 65.0 (d, J_cp = 6.5 Hz), 66.0, 73.9 (d, J_cp = 11.1 Hz), 74.1 (d, J_cp = 16.5 Hz), 84.3, 84.4, 85.7 (d, J_cp = 6.6 Hz), 86.2 (d, J_cp = 2.9 Hz), 86.6, 113.3, 121.6, 127.0, 128.0, 128.3, 128.4, 130.18, 130.24, 135.8, 135.9, 137.4, 137.5, 144.6, 144.7, 146.3, 148.30, 148.32, 153.11, 153.13, 155.7, 158.6; ³¹P NMR (162 MHz, CDCl₃) δ 148.9, 149.6; HRMS (ESI) m/z calcd for C₄₈H₆₂N₆O₉PS₄ [M+H]⁺ 1025.3198, found 1025.3205.

Synthesis of Dim phosphoramidite 1d

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 3.81 g (6.99 mmol, 1.0 equiv) 19 under positive argon pressure.

3. Add 1.80 g (10.48 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

4. Add 60 mL freshly distilled DCM via a syringe.

5. Attach a cannula filter to one end of an oven-dried cannula (or wrap the cannula with cotton and secure with copper wire)

6. Insert the cannula into a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 10.48 mmol, 1.5 equiv; see the beginning of this Support Protocol for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the flask containing 19.

9. Transfer the supernatant containing 7 into the flask containing 19.

   The cannula filter or cotton is intended to minimize the transfer of the precipitated diisopropylamine hydrochloride side product into the flask containing 19.

10. Stir the reaction mixture at room temperature overnight.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate volatiles on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~220 g) as the stationary phase and hexanes/EtOAc/Et₃N (1:1:0.1) as the mobile phase.

    Compound 1d: White foam (5.07 g, 6.15 mmol, 88%); mixture of two diastereoisomers; R_f = 0.2 and 0.3 (SiO₂, 1:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.04–1.16 (m, 12H), 1.38 (s, 3H), 1.77–1.87 (m, 1H), 1.96–2.07 (m, 1H), 2.28–2.42 (m, 1H), 2.45–2.58 (m, 1H), 2.60–2.69 (m, 2H), 2.65–2.84 (m, 4H), 3.29–3.46 (m, 2H), 3.47–3.69 (m, 2H), 3.76 (s, 6H), 3.80–3.89 (m, 1H), 4.04–4.23 (m, 1H), 4.74–4.77 (m, 1H), 6.38 (t, J = 5.8 Hz, 1H), 6.81 (dd, J = 8.8, 3.2 Hz, 4H), 7.20–7.29 (m, 7H), 7.40 (d, J = 7.6 Hz, 2H), 7.60 (s, 0.5H), 7.63 (s, 0.5 H), 8.84 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 12.1, 24.81, 24.88, 24.95, 25.0, 26.1, 26.2, 28.8 (d, J_cp = 9.2 Hz), 29.0 (d, J_cp = 17.2 Hz), 40.5 (d, J_cp = 5.4 Hz), 40.6 (d, J_cp = 1.8 Hz), 43.4 (d, J_cp = 3.4 Hz), 43.5 (d, J_cp = 3.4 Hz), 47.1 (d, J_cp = 7.0 Hz), 47.8 (d, J_cp = 6.8 Hz), 55.5, 63.3, 63.7, 64.8 (d, J_cp = 18.2 Hz), 65.0 (d, J_cp = 18.9 Hz), 73.6 (d, J_cp = 15.6 Hz), 74.1 (d, J_cp = 15.2 Hz), 84.8, 85.0, 85.4 (d, J_cp = 6.7 Hz), 86.0 (d, J_cp = 2.8 Hz), 87.0, 87.1, 111.2, 113.4, 127.2, 128.1, 128.4, 130.4, 135.5, 135.6, 135.7, 136.0, 136.1, 144.5, 144.6, 150.4, 158.8, 164.0; ³¹P NMR (162 MHz, CDCl₃) δ 149.4, 149.6 ppm; HRMS (ESI) m/z calcd for C₄₂H₅₅N₃O₈PS₂ [M+H]⁺ 824.3168, found 824.3170.

Synthesis of Dim phosphoramidite 1e

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 3.39 g (6.99 mmol, 1.0 equiv) 20 under positive argon pressure.

3. Add 1.80 g (10.48 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

4. Add 60 mL freshly distilled DCM via a syringe.

5. Attach a cannula filter to one end of an oven-dried cannula (or wrap the cannula with cotton and secure with copper wire)

6. Insert the cannula into a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 10.48 mmol, 1.5 equiv; see the beginning of this Support Protocol for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the flask containing 20.

9. Transfer the supernatant containing 7 into the flask containing 20.

   The cannula filter or cotton is intended to minimize the transfer of the precipitated diisopropylamine hydrochloride side product into the flask containing 20.

10. Stir the reaction mixture at room temperature overnight.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate volatiles on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~210 g) as the stationary phase and hexanes/EtOAc/Et₃N (1:1:0.1) as the mobile phase.

    Compound 1e: White foam (4.64 g, 6.08 mmol, 87%); mixture of two diastereoisomers; R_f = 0.2 and 0.3 (SiO₂, 1:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.04–1.27 (m, 12H), 1.40 (s, 3H), 1.78–1.86 (m, 1H), 1.96–2.05 (m, 1H), 2.29–2.98 (m, 8H), 3.30–3.99 (m, 5H), 4.05–4.25 (m, 1H), 4.74–4.81 (m, 1H), 6.38 (t, J = 7.1 Hz, 1H), 7.18–7.35 (m, 9H), 7.36–7.45 (m, 6H), 7.56 (s, 0.5H), 7.60 (s, 0.5H), 9.11 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 12.1, 24.84, 24.88, 24.91, 24.95, 25.01, 26.1, 26.2, 28.8 (d, J_cp = 8.4 Hz), 29.0 (d, J_cp = 17.3 Hz), 40.4 (d, J_cp = 5.1 Hz), 40.6, 43.4, 43.5, 47.0 (d, J_cp = 7.2 Hz), 47.5 (d, J_cp = 7.4 Hz), 63.5, 63.9, 64.8 (d, J_cp = 17.9 Hz), 65.0 (d, J_cp = 18.4 Hz), 73.6 (d, J_cp = 15.3 Hz), 73.9 (d, J_cp = 14.4 Hz), 84.8, 85.0, 85.3 (d, J_cp = 6.7 Hz), 85.9, 87.55, 87.61, 111.1, 111.2, 127.5, 128.1, 128.9, 135.9, 136.0, 143.5, 143.6, 150.5, 164.1; ³¹P NMR (162 MHz, CDCl₃) δ 149.4, 149.7; HRMS (ESI) m/z calcd C₄₀H₅₁N₃O₆PS₂ [M+H]⁺ 764.2956, found 764.2960.

SUPPORT PROTOCOL 2

SUPPORT PROTOCOL TITLE

Preparation of CPG with a Dmoc linker

Introductory paragraph

This Protocol describes the preparation of the Dmoc-CPG linker 2, which is needed for sensitive ODN synthesis in the Basic Protocol. The routes for the synthesis are shown in Figure 7. The preparation has been reported previously (Lin et al. 2016).

Figure 7.

Preparation of Dmoc-CPG linker 2. Conditions: (a) (i) 1,3-Dithiane, nBuLi, THF, −78 °C to −40 °C; (ii) 21, THF, −78 °C. (b) Carbonyl diimidazole, CaH₂, PhMe, rt. (c) 19, DBU, PhMe, rt. (d) TBAF, THF, rt. (e) (i) Succinic anhydride, DMAP, pyridine, rt; (ii) lcaa-CPG, DCC, DMF, rt; (iii) Ac₂O, DMAP, pyridine, rt.

Materials

Argon or Nitrogen

1,3-Dithiane (97%, Sigma Aldrich, Cat# 157872)

Dry ice

Acetone

n-Butyl lithium (nBuLi, 1.7 M in pentane)

Tetrahydrofuran (THF, freshly distilled over sodium benzophenone ketal)

5-{[tert-​butyl(dimethyl)​silyl]​oxy}​pentanal (21, Sigma Aldrich, Cat# ADVH0430A737)

Ethanol

Ammonium chloride solution (NH₄Cl, saturated)

Ethyl acetate (EtOAc)

Sodium sulfate (Na₂SO₄, anhydrous)

Carbonyl diimidazole (97%, Sigma Aldrich, Cat# 21860)

Calcium hydride (CaH₂, 90%, Sigma Aldrich, Cat# 208027)

Toluene (freshly distilled over CaH₂)

5’-O-(4,4’-Dimethoxytrityl)thymidine (19, 98%, Sigma Aldrich, Cat# 360139)

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (98%, Sigma Aldrich Cat# 139009)

Tetrabutylammonium fluoride (TBAF, 1.0 M in THF, Sigma Aldrich, Cat# 216143)

Succinic anhydride (99%, Alfa Aesar, Cat# A12245)

4-(Dimethylamino)pyridine (DMAP, 99%, Sigma Aldrich, Cat# 107700)

Pyridine (freshly distilled over CaH₂)

Sodium bicarbonate solution (NaHCO₃, saturated)

Brine (NaCl solution, saturated)

Long chain alkyl amino CPG (lcaa-CPG, 107 μmol/g loading, 497 Å pore diameter, Prime Synthesis, Inc. or Glen Research)

Dimethylformamide (DMF, anhydrous, 99.8%, Sigma Aldrich, Cat# 227056)

N,N’-Dicyclohexylcarbodiimide (DCC, 99%, Sigma Aldrich, Cat# D80002)

Acetic anhydride (98%, Sigma Aldrich, Cat# 242845)

Methanol

Drying oven

2-Necked round-bottom flasks

Magnetic stirring bars

Magnetic stirring plate

Rubber Septa

Balance

Syringes

Syringe needles

1-Necked round-bottom flask

Cannula

Separatory funnel

Filter paper

Filter funnel

Rotatory evaporator connected to water aspirator

Thin-layer chromatography (TLC) plate (Silica gel 60 F254 glass plate)

UV lamp (254 nm)

Vacuum oil pump

Pipette (1000 μL)

Additional reagents and equipment for column chromatography (Meyers, 2000)

Protocol steps

Converting 21 to 22

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 3.25 g (27.1 mmol, 1.2 equiv) 1,3-dithiane under positive argon.

3. Add 50 mL freshly distilled THF via a syringe.

4. Cool the mixture to −78 °C.

5. Add 16.0 mL nBuLi (1.7 M in pentane, 27.1 mmol, 1.2 equiv) dropwise via a syringe over 5 minutes.

   nBuLi is highly pyrophoric. Extreme care and appropriate experience are required for its handling.

6. Stir while warming to −40 °C gradually over 30 minutes.

7. Continue to stir at −40 °C for 1 hour.

8. Cool to −78 °C.

9. Prepare a solution of 4.88 g (22.6 mmol, 1.0 equiv) 21 in 30 mL dry THF under argon.

10. Add the solution of 21 to the flask containing the intermediate formed from 1,3-dithane and nBuLi via a cannula.

11. Stir at −78 °C under argon for 30 minutes.

12. Quench the reaction by adding 1 mL EtOH.

13. Remove volatiles under reduced pressure on a rotary evaporator.

14. Partition the residue between 50 mL saturated NH₄Cl and 100 mL EtOAc.

15. Extract the aqueous phase with 50 mL EtOAc for 2 more times.

16. Combine the EtOAc extracts, dry over anhydrous Na₂SO₄, filter, and concentrate the filtrate to dryness under reduced pressure on a rotary evaporator.

17. Purify the residue with flash column chromatography using SiO₂ (~150 g) as the stationary phase and hexanes/EtOAc (9:1) as the mobile phase.

    Compound 22: Colorless oil (5.38 g, 16.0 mmol, 71%); R_f = 0.2 (9:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 0.02 (s, 6H), 0.86 (s, 9H), 1.38–1.48 (m, 1H), 1.49–1.59 (m, 4H), 1.77–1.84 (m, 1H), 1.89–1.98 (m, 1H), 2.01–2.10 (m, 1H), 2.43 (d, J = 3.6 Hz, 1H), 2.68–2.78 (m, 2H), 2.86–2.94 (m, 2H), 3.59 (t, J = 10.4 Hz, 2H), 3.80–3.85 (m, 1H), 3.88 (d, J = 6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ −5.3, 18.3, 22.1, 25.7, 26.0, 28.0, 28.5, 32.6, 33.8, 52.5, 63.0, 72.2; HRMS (ESI) m/z calcd for C₁₅H₃₂O₂S₂SiH [M+H]⁺ 337.1691, found 337.1695.

Converting 22 to 23

1. Attach a 2-necked round-bottom flask containing 2.16 g (6.42 mmol, 1.0 equiv) 22 and a magnetic stirring bar to an argon line.

2. Add 2.61 g (13.3 mmol, 2.1 equiv) carbonyldiimidazole under positive argon pressure.

3. Add 0.75 g (90% grade, 16.0 mmol, 2.5 equiv) CaH₂ under positive argon pressure.

4. Add 100 mL freshly distilled toluene via a syringe slowly over 5 minutes.

5. Stir the reaction mixture at room temperature under argon for 8 hours.

6. Remove the solid in the reaction mixture by filtration.

7. Concentrate the filtrate to dryness under reduced pressure on a rotary evaporator.

8. Purify the residue with flash column chromatography using SiO₂ (~90 g) as the stationary phase and hexanes/EtOAc (3:1) as the mobile phase.

   Compound 23: Thick oil (2.76 g, 6.42 mmol; 100%); R_f = 0.2 (3:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 0.01 (s, 6H, H-1), 0.82 (s, 9H), 1.39–1.56 (m, 4H), 1.80–2.08 (m, 4H), 2.67–2.78 (m, 2H), 2.84–2.95 (m, 2H), 3.57 (t, J = 4 Hz, 2H), 4.10 (d, J = 4 Hz, 1H), 5.28–5.33 (m, 1H), 7.04 (s, 1H), 7.40 (s, 1H), 8.12 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ −5.4, 18.2 21.7, 25.4, 25.8, 28.4, 28.6, 31.4, 32.2, 48.9, 62.4, 78.4, 117.2, 130.6, 137.2, 148.3; HRMS (ESI) m/z calcd for C₁₉H₃₄N₂O₃S₂SiH [M+H]⁺ 431.1858, found 431.1858.

Converting 23 to 24

1. Attach a 2-necked round-bottom flask containing 1.96 g (4.55 mmol, 1.0 equiv) 23 and a magnetic stirring bar to an argon line.

2. Add 3.72 g (6.83 mmol, 1.5 equiv) 19 under positive argon pressure.

3. Add 0.20 mL (0.21 g, 1.38 mmol, 0.3 equiv) DBU via a syringe.

4. Add 50 mL freshly distilled toluene via a syringe.

5. Stir the reaction mixture at room temperature under argon for 8 hours.

6. Evaporate volatiles under reduced pressure on a rotary evaporator.

7. Purify the residue with flash column chromatography using SiO₂ (~80 g) as the stationary phase and hexanes/EtOAc/Et₃N (3:1:0.2) as the mobile phase.

   Compound 24: White foam (3.14 g, 3.46 mmol, 76%); m.p. 81.2–82.6 °C; R_f = 0.45 (1:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 0.00 (s, 6H), 0.84 (s, 9H), 1.31 (s, 3H), 1.36–2.00 (m, 8H), 2.34–2.74 (m, 4H), 2.80–2.89 (m, 2H), 3.41–3.47 (m, 2H), 3.57 (t, J = 8 Hz, 2H), 3.75 (s, 6H), 3.94 (d, J = 8 Hz, 1H), 4.20 (s, 1H), 4.91–4.96 (m, 1H), 5.35 (d, J = 8 Hz, 1H), 6.43 (dd, J = 4 Hz, 8 Hz, 1H), 6.79 (d, J = 8 Hz, 4H), 7.18–7.31 (m, 8H), 7.31–7.33 (m, 1H), 7.54 (s, 1H), 8.08 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ −5.3, 11.6, 18.3, 21.7, 25.4, 26.0, 28.4, 28.6, 31.6, 32.3, 37.9, 48.9, 55.3, 62.7, 63.7, 78.8, 78.9, 84.0, 84.3, 87.2, 111.5, 113.3, 127.2, 128.0, 128.1, 130.1, 135.2, 135.3, 135.4, 144.2, 150.0, 154.3, 158.8, 163.3; HRMS (ESI) m/z calcd for C₄₇H₆₂N₂O₁₀S₂SiNa [M+Na]⁺ 929.3513, found 929.3497.

Converting 24 to 25

1. Attach a 2-necked round-bottom flask containing 1.47 g (1.62 mmol, 1.0 equiv) 24 and a magnetic stirring bar to an argon line.

2. Add 40 mL freshly distilled THF via a syringe.

3. Add 1.94 mL (1.0 M in THF, 1.94 mmol, 1.2 equiv) TBAF via a syringe.

4. Stir the reaction mixture at room temperature under argon for 8 hours.

5. Evaporate volatiles under reduced pressure on a rotary evaporator.

6. Purify the residue with flash column chromatography using SiO₂ (~35 g) as the stationary phase and hexanes/EtOAc (1:1) as the mobile phase.

   Compound 25: White foam (0.96 g, 1.21 mmol, 75%); m.p. 90.6–92.3 °C; R_f = 0.3 (1:3 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.35 (s, 3H), 1.41–1.63 (m, 4H), 1.69–1.81 (m, 2H), 1.89–2.08 (m, 2H), 2.37–2.44 (m, 1H), 2.56–2.75 (m, 3H), 2.84–2.93 (m, 2H), 3.42–3.51 (m, 2H), 3.61–3.65 (m, 2H), 3.77(s, 6H), 3.98 (d, J = 8 Hz, 1H), 4.24 (s, 1H), 4.97–5.01 (m, 1H), 5.34 (d, J = 4 Hz, 1H), 6.42 (t, J = 4 Hz, 8 Hz, 1H), 6.82 (d, J = 8 Hz, 4H), 7.20–7.34 (m, 8H), 7.34–7.37 (m, 1H), 7.58 (s, 1H), 8.78 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 11.6, 14.2, 21.0, 21.6, 25.4, 28.4, 28.6, 31.6, 32.1, 38.0, 48.9, 55.2, 60.4, 62.4, 63.7, 78.7, 79.1, 83.7, 84.4, 87.2, 111.6, 113.3, 127.2, 128.0, 128.1, 130.1, 130.1, 135.1, 135.2, 135.3, 144.2, 150.3, 154.2, 158.8, 158.8, 163.5; HRMS (ESI) m/z calcd for C₄₁H₄₈N₂NaO₁₀S₂ [M+Na]⁺ 815.2648, found 815.2636.

Converting 25 to 2

1. Attach a 2-necked round-bottom flask containing 100 mg (0.13 mmol, 1.0 equiv) 25 and a magnetic stirring bar to an argon line.

2. Add 50 mg (0.50 mmol, 3.8 equiv) succinic anhydride under positive argon pressure.

3. Add 30 mg (0.25 mmol, 1.9 equiv) DMAP under positive argon pressure.

4. Add 3 mL freshly distilled pyridine via a syringe.

5. Stir at room temperature under argon for 2 days.

6. Partition the reaction mixture between 15 mL EtOAc and 5 mL water.

7. Wash the organic phase with 5 mL saturated NaHCO₃ and 5 mL brine.

8. Dry the organic phase over anhydrous Na₂SO₄, filter and concentrate the filtrate to dryness under reduced pressure on a rotary evaporator.

9. Dry the residue under high vacuum generated by an oil pump for about 1 hour.

   Treating 25 with succinic anhydride and DMAP attaches the succinic anhydride to the hydroxyl group of 25 via the formation of an ester bond. The second carboxylic acid group resulted from the succinic anhydride is ready to react with the amino group of lcaa-CPG.

10. Attach the flask containing the residue (theoretical amount 0.13 mmol, 1.0 equiv) to an argon gas line.

11. Add 0.251 g (26.9 μmol, 0.2 equiv, 107 μmol/g loading, 497 Å pore size, Prime Synthesis, Inc.) lcaa-CPG.

12. Add 3 mL dry DMF via a syringe.

13. Add 0.11 mL (1.0 M in DCM, 0.10 mmol, 0.8 equiv) DCC via a syringe.

14. Shake the reaction flask manually for about 30 seconds.

15. Allow the reaction mixture to stand still at room temperature for 2 days.

    Occasional manual shaking can be helpful for the reaction but not required. Shaking the flask on a shaker can be helpful too. The reaction can also be performed in a peptide synthesis vessel. Stirring the reaction mixture using a magnetic stirring bar should be avoided, as that would grind the CPG into a fine powder.

16. Remove the supernatant with a pipette.

17. Wash the CPG with 3 mL dry pyridine for 5 times.

    The above steps attach the intermediate formed from 25 and succinic anhydride to the amino-functionalized CPG via the formation of an amide bond.

18. Attach the flask containing the CPG to argon line.

19. Add 5 mL solution of 0.1 M DMAP in dry pyridine and acetic anhydride (9:1, v/v) via a syringe.

20. Shake the reaction flask manually for about 30 seconds.

21. Allow the reaction mixture to stand still at room temperature for 2 days.

    Occasional manual shaking can be helpful for the reaction but not required.

22. Remove the supernatant with a pipette.

23. Wash the CPG with 3 mL pyridine for 5 times, 3 mL methanol for 3 times, 3 mL DMF for 3 times, and 3 mL acetone for 5 times.

    Treating the CPG with acetic anhydride and DMAP caps the unreacted amino groups on the CPG.

24. Dry the Dmoc-CPG linker 2 under high vacuum for 1 hour or longer.

25. Determine the loading of the CPG using the procedure in UNIT 3.2.

    26 μmol/g were found in this particular case, but it may vary from batch to batch.

26. Store the Dmoc-CPG linker 2 at −20 °C

SUPPORT PROTOCOL 3

SUPPORT PROTOCOL TITLE

Synthesis of a phosphoramidite containing the sensitive alkyl ester group

Introductory paragraph:

This Protocol describes the synthesis of an example phosphoramidite (1f) that contains the sensitive ester group. The phosphoramidite is used in the Basic Protocol for the description of the details regarding the synthesis of sensitive ODNs using the Dim-Dmoc technology. The route for the synthesis is shown in Figure 8. The synthesis has been reported previously (Shahsavari et al. 2019a, Shahsavari et al. 2019b).

Figure 8.

Synthesis of phosphoramidite 1f. Conditions: (a) EtOH, H₂SO₄ (cat), reflux. (b) DMTr-Cl, pyridine, 0 °C to rt. (c) 7, diisopropylammonium tetrazolide, DCM, rt.

Materials

Argon or nitrogen

10,​11-​Dihydroxyundecanoic acid (26, Sigma Aldrich, Cat# S489425)

Sulfuric acid (concentrated)

Ethanol

Sodium carbonate solution (Na₂CO₃, 5%)

Ethyl acetate

Sodium sulfate (Na₂SO₄, anhydrous)

Pyridine (freshly distilled over CaH₂)

4,4’-Dimethoxytrityl chloride (DMTr-Cl, 95%, Sigma Aldrich, Cat# 100013)

Dichloromethane (DCM)

Diisopropylammonium tetrazolide

Dichloromethane (DCM, freshly distilled over CaH₂)

Cannula

Cannula filter (or cotton and copper wire)

2-Necked round-bottom flasks

Drying oven

Magnetic stirring bar

Magnetic stirring plate

Reflux condenser

Balance

Syringes

Syringe needles

Glass pipettes (disposable)

Heating mantle

Heating mantle voltage controller

Rotary evaporator connected to a water aspirator

Thin-layer chromatography (TLC) plate (Silica gel 60 F254 glass plate)

UV lamp (254 nm)

Filter paper

Filter funnel

1-Necked round-bottom flasks

Cannula

Cannula filter (or cotton and copper wire)

Rubber septa

Vacuum oil pump

Additional reagents and equipment for column chromatography (Meyers, 2000)

Protocol steps

Converting 26 to 27

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line via a reflux condenser.

2. Add 2.0 g (9.16 mmol, 1.0 equiv) 26 under positive argon pressure.

3. Add 100 mL ethanol via a syringe.

4. Add 1 mL concentrated sulfuric acid using a glass pipette under positive argon pressure.

5. Heat the reaction mixture to reflux for 2 hours.

6. Cool the reaction mixture to room temperature.

7. Quench the reaction with 20 mL 5% Na₂CO₃.

8. Reduce the volume of the mixture to about 30 mL by evaporating the majority of ethanol under reduced pressure on a rotary evaporator.

9. Partition between 100 mL EtOAc and 50 mL 5% Na₂CO₃.

10. Wash the organic phase with 50 mL 5% Na₂CO₃ for 2 times.

11. Dry the organic phase over anhydrous Na₂SO₄, filter, and evaporate filtrate to dryness.

12. Purify the residue with flash column chromatography using SiO₂ (~50 g) as the stationary phase and hexanes/EtOAc (1:1) as the mobile phase.

    Compound 27: Colorless oil (1.72 g, 6.96 mmol, 76%); R_f = 0.2 (1:1 hexanes/EtOAc); ¹H NMR (400 MHz, CD₃OD): δ 1.22 (t, J = 7.1 Hz, 3H), 1.30 (s, 10H), 1.42–1.50 (m, 2H), 1.54–1.60 (m, 2H), 2.27 (t, J = 7.4 Hz, 2H), 3.28 (bs, 1H), 3.36–3.46 (m, 2H), 3.53 (bs, 1H), 4.08 (q, J = 7.1 Hz, 2H), 4.80 (s, 2H); ¹³C NMR (100 MHz, CD₃OD): δ 13.3, 24.8, 25.4, 28.9, 29.1, 29.3, 29.5, 33.2, 33.9, 60.1, 66.2, 72.0, 174.3; HRMS (ESI): m/z calcd for C₁₃H₂₇O₄ [M + H]⁺ 247.1909, found 247.1907.

Converting 27 to 28

1. Add 8 mL freshly distilled pyridine to the flask containing 185 mg (0.75 mmol, 1.0 equiv) 27, and evaporate the volatiles under vacuum. Repeat the co-evaporation 2 more times.

2. Attach the round-bottom flask containing 27 to an argon line.

3. Add 5 mL freshly distilled pyridine via a syringe.

4. Attach an oven-dried 2-necked round-bottom flask to an argon line.

5. Add 280 mg (0.83 mmol, 1.1 equiv) DMTr-Cl under positive argon pressure.

6. Add 5 mL freshly distilled pyridine via a syringe.

7. Transfer the solution of DMTr-Cl in the 2-necked round-bottom flask to the flask containing 27.

8. Stir the reaction mixture at room temperature under argon for 8 hours.

9. Pour the reaction mixture into a separatory funnel that contains 40 mL 5% Na₂CO₃ and 25 mL DCM.

10. Separate the DCM layer, and extract the aqueous layer with 10 mL DCM. Repeat the extraction.

11. Combine the extracts, dry over anhydrous Na₂SO₄, filter, and concentrate the filtrate to dryness.

12. Purify the residue with flash column chromatography using SiO₂ (~15 g) as the stationary phase and hexanes/EtOAc/Et₃N (3:2:0.25) as the mobile phase.

    Compound 28: yellow oil (408 mg, 0.74 mmol, 99%); R_f = 0.8 (1:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 1.20–1.28 (m, 10H), 1.35–1.45 (m, 2H), 1.53–1.63 (m, 2H), 2.26 (t, J = 7.3 Hz, 2H), 2.47 (bs, 1H), 2.98–3.04 (m, 1H), 3.13–3.17 (m, 1H), 3.74 (s, 6H), 4.10 (q, J = 7.1 Hz, 2H), 6.81 (d, J = 8.8 Hz, 4H), 7.14–7.19 (m, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 4H), 7.43 (d, J = 5.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 14.4, 25.1, 25.6, 29.31, 29.39, 29.5, 29.7, 33.6, 34.5, 55.3, 60.3, 67.8, 71.1, 86.2, 113.3, 126.9, 128.0, 128.3, 130.2, 136.3, 145.1, 158.6, 174.0; HRMS (ESI): m/z calcd for C₃₄H₄₄O₆Na [M + Na]⁺ 571.3035, found 571.3031.

Converting 28 to 1f

1. Attach an oven-dried 2-necked round-bottom flask with a magnetic stirring bar to an argon line.

2. Add 161 mg (0.94 mmol, 1.5 equiv) diisopropylammonium tetrazolide under positive argon pressure.

3. Dissolve 345 mg (0.63 mmol, 1.0 equiv) 28 in a round-bottom flask in 50 mL freshly distilled DCM under argon.

4. Transfer the solution of 28 into the 2-necked round-bottom flask via a cannula.

5. Attach a cannula filter to one end of another oven-dried cannula (or wrap the cannula with cotton and secure with copper wire)

6. Insert the cannula into a rubber septum.

7. Quickly replace the septum of the flask containing the Dim-phosphitylation agent 7 (theoretical amount 0.94 mmol, 1.5 equiv; see Support Protocol 1 for preparation) with the septum. The end of the cannula that has the cannula filter should be placed inside the flask containing 7.

8. Insert the other end of the cannula into the septum of the 2-necked round-bottom flask containing 28.

9. Transfer the supernatant containing 7 into the flask containing 28.

10. Stir the reaction mixture at room temperature overnight.

11. Transfer the reaction mixture into a 1-necked round-bottom flask and evaporate volatiles on a rotary evaporator.

12. Purify the residue with flash column chromatography using SiO₂ (~20 g) as the stationary phase and hexanes/EtOAc/Et₃N (9:1:0.5) as the mobile phase.

    Compound 1f: Colorless oil (412 mg, 0.50 mmol, 79%); mixture of two diastereoisomers; R_f = 0.6 and 0.7 (SiO₂, 3:1 hexanes/EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 1.05 (d, J = 6.8 Hz, 3H), 1.11–1.35 (m, 23H), 1.45–1.79 (m, 3H), 1.79–1.95 (m, 1H), 1.95–2.12 (m, 1H), 2.26 (t, J = 7.7 Hz, 1H), 2.263 (t, J = 7.5 Hz, 1H), 2.57–2.68 (m, 1H), 2.69–2.89 (m, 3H), 2.96 (q, J = 2.9 Hz, 1H), 3.06 (q, J = 5.8 Hz, 1H), 3.22 (q, J = 5.2 Hz, 1H), 3.22 (q, J = 5.0 Hz), 3.47–3.65 (m, 2H), 3.65–3.80 (m, 1H), 3.766 (s, 3H), 3.773 (s, 3H), 3.84–3.92 (m, 1H), 3.92–4.05 (m, 1H), 4.11 (q, J = 7.1 Hz, 2H), 4.10–4.21 (m, 1H), 6.78 (d, J = 11.7 Hz, 2H), 6.81 (d, J = 7.5 Hz, 2H), 7.13–7.21 (m, 1H), 7.24 (t, J = 7.9 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.35 (dd, J = 8.0, 1.6 Hz, 2H), 7.45 (d, J = 5.1 Hz, 1H), 7.46 (d, J = 5.2 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 14.6, 24.84, 24.86, 24.91, 24.94, 25.00, 25.04, 25.07, 25.11, 25.17, 25.31, 25.34, 25.39, 26.30, 26.34, 28.6 (d, J_cp = 7.0 Hz), 28.9 (d, J_cp = 9.0 Hz), 29.47, 29.51, 29.59, 29.72, 29.76, 29.91, 30.02, 33.76, 33.9 (d, J_cp = 6.3 Hz), 34.7, 43.2 (d, J_cp = 4.2 Hz), 43.4 (d, J_cp = 4.0 Hz), 46.9 (d, J_cp = 5.5 Hz), 47.3 (d, J_cp = 7.0 Hz), 55.5, 60.4, 64.9 (d, J_cp = 7.4 Hz), 65.1 (d, J_cp = 18.5 Hz), 66.3 (d, J_cp = 1.8 Hz), 66.4 (d, J_cp = 3.3 Hz), 73.7 (d, J_cp = 15.0 Hz), 74.3 (d, J_cp = 18.7 Hz), 85.9, 113.1, 126.7, 127.8, 128.45, 128.53, 130.30, 130.37, 136.6, 136.7, 145.3, 145.4, 158.4, 174.0; ³¹P NMR (162 MHz, CDCl₃) δ 149.0, 149.2; HRMS (ESI) m/z calcd for C₄₅H₆₇NO₇PS₂ [M+H]⁺ 828.4096, found 828.4099.

COMMENTARY

BACKGROUND INFORMATION

In the most widely used solid phase oligodeoxynucleotide (ODN) synthesis technologies, the exo-amino groups of nucleobases are protected with acyl groups such as acetyl, isobutyryl, and benzoyl groups; the internucleotide phosphate groups are protected with the 2-cyanoethyl group; and the growing ODN is anchored to a solid support using an ester linkage. After the synthesis is completed, deprotection and cleavage are achieved with concentrated ammonium hydroxide at elevated temperature. Under these strongly basic and nucleophilic conditions, many functional groups including common ones in organic chemistry such as ester, thioester, aryl amide, N-alkylpyridinium, alkyl halide and α-halo amide, cannot survive. However, ODNs that contain these groups, which we call sensitive groups, can be of interest to researchers for many reasons. For example, N-methylated or acetylated guanine, adenine and cytidine play important roles in the regulation of gene expression (Calle-Fabregat et al. 2020, Greenberg and Bourc’his 2019, Michalak et al. 2019, Urbano et al. 2019). N-Alkylpyridinium and N-imidazolium in DNA formed by reaction of DNA with alkylating agents have been proven to have various biological consequences (Brickner et al. 2019, Gillingham and Sauter 2017, Soll et al. 2017). Introduction of electrophilic groups into ODNs has the potential to lead to the discovery of new DNA cross-linking agents useful for antisense drug development and disease diagnosis (Ali et al. 2006a, Pande et al. 1999). Selective DNA cross-linking agents can also serve as molecular probes for the investigation of transit DNA-protein interactions using mass spectrometry (Bley et al. 2011, Rhee and Pugh 2011).

In order to overcome the challenge in the synthesis of ODNs that contain sensitive groups, significant efforts have been made, but an ideal technology remain elusive. The phenoxyacetyl and related groups have been used for the protection of exo-amino groups of nucleobases, which allows deprotection under significantly milder conditions including concentrated ammonium hydroxide at room temperature in two hours and dilute potassium methoxide in methanol at room temperature for four hours (Gillet et al. 2005, Schnetz-Boutaud et al. 2000). This method has helped to solve many problems, but ammonium hydroxide and potassium methoxide are still strong nucleophiles and bases, and many groups that are compatible with ODN cannot survive under the deprotection conditions. The palladium-cleavable allyl-based groups were studied for exo-amine protection, but excess palladium had to be used for the deprotection (Matray and Greenberg 1994, Spinelli et al. 2002). Because palladium is expensive, and more problematically, is challenging to remove from ODN product, the method is not ideal. The (p-nitrophenyl)ethyl (Npe) and (p-nitrophenyl)ethyloxycarbonyl (Npeoc) groups were also studied for exo-amino protection, but the need of strongly basic conditions for their removal may limit their applications (Eritja et al. 1992). In principle, more base-labile versions of Npe and Npeoc groups can be developed to alleviate the problem, but difficulty to prepare the phosphoramidite monomers and premature deprotection during ODN synthesis can be problematic. There are several reports on ODN synthesis without exo-amine protection (Ohkubo et al. 2004), but the challenge of achieving high levels of selective O-phosphitylation over N-phosphitylation may prevent the method for the synthesis of long ODNs. Besides addressing the problem of exo-amine protection, significant efforts were also made to develop linkers that can be cleaved under milder conditions, which is useful for sensitive ODN synthesis. For example, the UV-cleavable o-nitrobenzyl function has been studied for the purpose (Matray and Greenberg 1994). However, UV irradiation can cause ODN damage, which can limit the application of the linker. Besides developing protecting groups and linkers, many examples can be found in the literature on the synthesis of sensitive ODNs using post-synthesis modification and using enzymatic reactions (Ali et al. 2006b, Cowart and Benkovic 1991). The drawbacks of these methods include being not universally useful and complicated experimental procedures. In summary, in the literature a technology that synchronously uses protecting groups deprotectable under mild conditions for exo-amine and phosphate protection, and a linker cleavable under mild conditions for sensitive ODN synthesis has not appeared.

The Dim-Dmoc technology described in this protocol uses Dmoc for the protection of the exo-amino groups of dA, dC and dG; Dim for the protection of the hydroxyl group of the phosphoramidous acid of all the four nucleoside phosphoramidites; and Dmoc for linking the nascent ODN to solid support (Figure 1) (Halami et al. 2018, Lin et al. 2016, Shahsavari et al. 2019a, Shahsavari et al. 2019b). Using the technology, the ODN synthesized has the structure represented by 3 (Figure 2). Deprotection and cleavage can both be achieved under the same mild nearly non-nucleophilic and non-basic conditions – first, oxidization with sodium periodate at pH 4 at room temperature for 3 hours, and second, β-elimination with aniline at pH 8 at room temperature for 3 hours. Under these conditions, many sensitive functionalities that cannot survive deprotection and cleavage conditions used in traditional ODN synthesis technologies but can co-exist with ODN can survive, thus the Dim-Dmoc technology can be used to synthesize sensitive ODNs. We expect that the technology will find applications in many areas including developing new DNA and RNA cross-linking agents for applications such as antisense drug development, disease diagnosis, nucleic acid-protein interaction studies; and synthesizing sensitive model DNA analogs for studies such as DNA methylation and demethylation, and DNA damage and repair (Ali et al. 2006a, Bley et al. 2011, Brickner et al. 2019, Calle-Fabregat et al. 2020, Gillingham and Sauter 2017, Greenberg and Bourc’his 2019, Michalak et al. 2019, Pande et al. 1999, Rhee and Pugh 2011, Soll et al. 2017, Urbano et al. 2019).

CRITICAL PARAMETERS AND TROUBLESHOOTING

The yields for compounds 11 (57%), 1b (52%) and 16 (44%) are relatively low. For 11, besides adding sufficient amount of triethyl amine into the eluent to neutralize the residue acid in silica gel (5% or more), it is important to ran the flash column chromatography quickly so that the loss of the DMTr group of the compound can be minimized. For 1b, adding sufficient amount of triethyl amine to the eluent and running flash chromatography quickly are not only important for minimizing the loss of DMTr group, but also important to minimize the oxidation of the phosphoramidite to phosphoramidate and the hydrolysis of the phosphoramidite. For 16, to obtain the yield of 44% or higher, it is critical to remove as much HMPA as possible by vacuum evaporation. In our hands, removing HMPA by partition was not effective, and the remaining HMPA, which is highly polar, made column chromatography difficult. For all these compounds, if enough care is taken for the above mentioned manipulations, obtaining higher yields than what have been obtained is possible.

The Dim-Dmoc phosphoramidites 1a-c and Dim phosphoramidites 1d-f are as stable as typical nucleoside phosphoramidites such as those with acyl and 2-cyanoethyl protections widely used in traditional solid phase ODN synthesis. Like typical phosphoramidites, their purity is important for successful ODN synthesis. Particular attention should be paid to ensure dryness of the phosphoramidite solutions. For this, we suggest to dry the phosphoramidites over fresh Drierite in a desiccator under high vacuum overnight, and dissolve them in dry acetonitrile under argon and immediately attach the bottles containing them to the synthesizer to minimize the absorption of moisture by the solutions. In case that the synthesis yield is low due to impurities of the phosphoramidites, the use of phosphoramidite solutions with concentrations higher than 0.1 M could help as these phosphoramidites have high solubility in acetonitrile. Alternatively, increasing the number of coupling in each synthetic cycle can be used to alleviate the problem.

Oxidation of the Dim and Dmoc groups after ODN synthesis to convert 3 to 4 for deprotection and cleavage needs to be complete. Thus, sufficient equivalent of sodium periodate needs to be used. When performing deprotection and cleavage at scales larger than that in this Protocol, larger volumes of the sodium periodate solution should be used or the oxidation should be performed for more than one time. When doing this, shorter time for each oxidation can be used. There is no need of concern about oxidative DNA damage as we did not observe this even after allowing the oxidation to proceed at room temperature for 24 hours. After oxidation, it is important to completely wash away the excess sodium periodate and its reduced side product from the CPG. If washing is incomplete, sodium periodate may react with aniline used in the next step. It may also damage ODN once its amino groups are exposed after treating with aniline. After aniline treatment, the step for concentrating the solution should not be carried out to the extent of complete dryness as this may evaporate aniline (b. p. 184 °C/760 mmHg). If all aniline is evaporated, regeneration of the ODN deprotection side product 2-methylene-1,3-dithiane-1,3-dioxide from its aniline adduct may occur. This side product is a Michael acceptor and can react with the exo-amines of deprotected ODN. An alternative to avoid aniline evaporation is to use 4-aminobenzyl alcohol. 4-Aminobenzyl alcohol has a higher boiling point (171 °C/11 mmHg), and is less likely to be evaporated (Halami et al. 2018). Precipitation of ODN by nBuOH from aqueous solution is usually easy to do, but when the ODN synthesis yield is low, reducing the volume of solvents is important. In the Protocol, precipitating ODN from about 100 μL water/aniline solution by 900 μL nBuOH is suggested. When the ODN synthesis yield is low, the volumes should be cut in half.

UNDERSTANDING RESULTS

A successful synthesis of ODN using the Dim-Dmoc technology should give an RP-HPLC profile of crude Tr-on ODN like that shown in Figure 3 for ODN 6. The full-length ODN with a hydrophobic Tr group at its 5’-end should appear at around 35 minutes or later depending on the hydrophobicity of the sensitive group introduced using the technology. The failure sequences have no Tr group, and should appear before 28 minutes. The ratio of the area of the peak for the Tr-on full-length sequence over the area of the peaks for the failure sequences can be used to estimate the degree of success of the experiment. A higher ratio indicates a better synthesis and vice versa. In addition, peaks immediately before the peak for the Tr-on full-length sequence may indicate inefficient capping, which should not be common as capping with phosphoramidites is generally efficient (Fang et al. 2011, Pokharel et al. 2014). The peaks before the full-length peak may also indicate internucleotide phosphate backbone cleavage, which is possible if some of the Dim groups are not oxidized. The problem can be easily addressed by performing sodium periodate oxidation more than one time. Peaks immediately after the Tr-on full-length sequence may indicate incomplete deprotection of the Dim and Dmoc groups, which can be caused by incomplete β-elimination, or more likely incomplete oxidation. Both can be easily addressed. These peaks may also indicate the existence of the Michael adducts between deprotected ODN and the side product 2-methylene-1,3-dithiane-1,3-dioxide generated by deprotection of Dim and Dmoc groups. A method to address this problem has been discussed in the Critical Parameters and Troubleshooting section. While it is better to avoid all these potential peaks around the peak of full-length ODN, if they appeared, it is usually possible to remove them during RP-HPLC purification. A successful synthesis of ODN using the Dim-Dmoc technology also should give correct molecular mass of the ODN in MALDI-TOF MS analysis (UNIT 10.1). Further confirmation of the success of the ODN synthesis can be achieved by measuring UV absorption of the ODN (UNIT 10.3), and enzymatic digestion of the ODN followed by HPLC analysis of the resulting nucleosides (UNIT 10.6).

TIME CONSIDERATIONS

For the preparation of the Dim-Dmoc phosphoramidites 1a-e and linker 2, each step will take one to two days to set up the reaction and purify the product. Phosphoramidite 1f is a special one, and will not be needed by most researchers because they will most likely use the Dim-Dmoc technology to incorporate other sensitive groups into ODNs, and need a phosphoramidite containing a different sensitive group. As seen in Figures 5 and 6, for the synthesis of 1a-e and 2, a total of about 16 steps are needed. Therefore, if one researcher with expertise in organic synthesis is responsible for the project, the synthesis will need approximately one month to accomplish. Once the phosphoramidites and linker are ready, the ODN synthesis, cleavage and deprotection, purification and analysis can be accomplished within one week. More specifically, automated ODN synthesis can be set up and finish within a few hours. Cleavage and deprotection can be finished in 24 hours, and HPLC purification and MALDI MS analysis can be finished in one or two days.