Peptide–Oligonucleotide Conjugates: Catalytic Preparation in Aqueous Solution or On‐Column

Abstract

Peptide–oligonucleotide conjugates (POCs) are synthesized through a novel catalytic and sustainable approach. The amide coupling between peptide and oligonucleotide is facilitated by 1,4‐diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) as a stoichiometric activating agent. Two distinct methods are described for the POC synthesis: the first involves an on‐column conjugation in an anhydrous environment, while the second enables a post‐synthetic conjugation in an aqueous buffer. This last approach has been successfully extended to an iterative process, offering significant potential for the development of DNA‐encoded libraries.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: On‐column conjugation of an amino acid or of a peptide to an oligonucleotide

Support Protocol: Deprotection of the Boc‐protected amine of the lysine

Basic Protocol 2: In‐solution conjugation of an amino acid or of a peptide on an oligonucleotide

Basic Protocol 3: Iterative process for the preparation of a peptide–oligonucleotide conjugate

INTRODUCTION

Peptide–oligonucleotide conjugates (POCs) represent a promising strategy in therapeutic oligonucleotide development, combining the sequence‐specific targeting of oligonucleotides with the delivery and functional advantages of peptides. These conjugates can significantly improve cellular uptake, tissue targeting, and intracellular trafficking, which are key limitations of current oligonucleotide drugs. However, existing protocols for generating POCs often suffer from low efficiency, lack of site‐specificity, poor scalability, and high production costs. A novel streamlined approach to POC synthesis would therefore be of high interest to pharmaceutical developers, academic researchers, and manufacturers aiming to advance next‐generation RNA therapeutics with improved pharmacological profiles and clinical viability.

This article provides detailed procedures for the preparation of POCs linked with an amide bond using a catalytic and sustainable approach with 1,4‐diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) as activating agent. This approach has been applied to a large variety of peptides and oligonucleotides, providing an environmentally friendly alternative for POC synthesis, either by solid‐phase synthesis or post‐synthesis conjugation in solution. The coupling in solution has been also used in an iterative process with the successive incorporation of three different amino acids.

We provide protocols for the conjugation of an amino acid or a peptide to an oligonucleotide supported on CPG beads (see Basic Protocol 1) or in aqueous solution after cleavage of the support (see Basic Protocol 2). Complementary information for the synthesis of oligonucleotides by automated synthesis are also given. Finally, the preparation of peptide–oligonucleotide conjugates via an iterative process is described (see Basic Protocol 3).

STRATEGIC PLANNING

Both strategies, on controlled pore glass (CPG) or in solution (Fig. 1), require the synthesis of a 5'‐amino‐modified oligonucleotide. The standard phosphoramidite chemistry has been applied with incorporation of the 5'‐amino‐modifier C6 (C6‐MMT) at the 5' extremity (Beaucage & Caruthers, 2000). After MMT deprotection (TCA), the amino group could be immediately conjugated with an amino acid or a peptide on the solid support (Basic Protocol 1). Alternatively, conjugation can occur in solution after cleavage from the support of the oligonucleotide (Basic Protocol 2 or 3).

Figure 1.

Strategic planning of this work.

CAUTION: All reactions must be performed in a suitable fume hood with efficient ventilation. Safety glasses and reagent‐impermeable protective gloves should be worn.

Basic Protocol 1. ON‐COLUMN CONJUGATION OF AN AMINO ACID OR A PEPTIDE TO AN OLIGONUCLEOTIDE

This protocol describes the supported conjugation of either a single amino acid or a peptide to a 5’‐amino oligonucleotide. Starting from the commercially available–functionalized CPG beads, the protocol describes the synthesis of the oligonucleotide, followed by the conjugation reaction, cleavage from the support, purification, and analysis. One of the main advantages of solid‐supported synthesis is the ease of washing the CPG‐beads after coupling, resulting in a crude residue that is simpler to purify. Our conditions are smoother than classical methods in the literature and have been preferred for amide coupling on CPG‐beads (Graidia et al., 2024).

Materials

• DNA phosphoramidites (ChemGenes): 5’‐O‐(4,4’‐dimethoxytrityl)‐N‐protected‐2’‐deoxyribonucleoside‐3’‐O‐(2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidites, where the nucleobases are:

  • N ⁶‐benzoyladenin‐9‐yl (ChemGenes, ANP‐551)

  • N ²‐isobutyrylguanin‐9‐yl (ChemGenes, ANP‐553)

  • N ⁴‐benzoylcytosin‐1‐yl (ChemGenes, ANP‐552)

  • Thymin‐1‐yl (ChemGenes, ANP‐554)

• dT Synbase CPG 500/110S (deoxythymidine 3’‐Icaa CPG 500 Å) (Link Technologies, 2261)

• MMT‐amino C‐6 modifier phosphoramidite: 6‐(4‐monomethoxytritylamino)hexyl‐(2‐cyanoethyl)‐(N,N‐diisopropyl)‐phosphoramidite (ChemGenes, CLP‐1563)

• Argon (dry)

• Anhydrous acetonitrile (MeCN) (VWR Chemical, for DNA synthesis, water <10 ppm, filtered at 0.2 µm, packaged under nitrogen, VWRC85501.321)

• Dichloromethane (DCM) (Carlo Erba, anhydrous, stabilized with amylene, water ≤50 ppm, 442292000‐CER)

• Detritylation solution: 3% (w/v) trichloroacetic acid (TCA) in DCM (Biosolve Chimie SARL, Deblock TCA 3% in DCM, DNA synthesis, 04132402)

• Capping A solution: acetic anhydride in tetrahydrofuran (Glen Research, 40‐4012)

• Capping B solution: 10% (w/v) 1‐methylimidazole in tetrahydrofurane/pyridine (Glen Research, 40‐4110)

• Activator solution: 0.3 M 5‐benzylmercapto‐1H‐tetrazole (ChemGenes, 99.9%, water 20 ppm, RN‐1415) in anhydrous acetonitrile (VWR Chemical, for DNA synthesis)

• Oxidizer solution: 0.1 M iodine in tetrahydrofuran/water/pyridine (Glen Research, 40‐4330)

• Acid partners:

  • Fmoc‐Lys(Boc)‐OH (≥98%, Carbosynth, FF158761401)

  • Fmoc‐Val‐Arg‐Leu‐Pro‐Pro‐Pro‐OH (aka Fmoc‐VRLPPP‐OH, homemade)

  • Fmoc‐Pro‐Phe‐Gly‐OH (homemade)

  • Biotin (≥98.5%, Aldrich, 8.51209)

• 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) (97%, Aldrich, 375217)

• H₂O, deionized, Milli‐Q‐purified (Millipore, 18.2 MΩ.cm)

• 1,4‐diazabicyclo[2,2,2]octane (DABCO) (98%, Aldrich, 8034560100)

• N,N‐diisopropylethylamine (DiPEA) (99%, Thermo Scientific, A11801.AE)

• 30% (v/v) ammonium hydroxide, stored at 4°C (Carlo Erba, for analysis ACS, 419941)

• Acetonitrile (≥99.9%, HPLC gradient grade, Fisher Chemical, A/0627/17)

• THAP (matrix substance for MALDI‐MS, ≥99.5%, Aldrich, 05757)

• Ammonium citrate (98%, Aldrich, 374695)

• Empty DNA synthesizer bottles, oven‐dried, with rubber septa

• Disposable polypropylene syringes and needles

• Vacuum desiccator containing P₂O₅ and KOH

• Rotatory evaporator equipped with a vacuum pump

• Automatic DNA synthesizer (ABI 394)

• Synthesis reactor for 1‐µmol scale synthesis (empty synthesis columns‐TWIST, Glen Research)

• 2‐ml Eppendorf tubes (Fisherbrand 2.0‐ml MCT graduated natural)

• 0.6‐ml microcentrifuge tubes, Quali, clear (Kisker, G051)

• Micropipettes (for organic solutions, Gilson P100, and P1000; for aqueous solutions, Gilson P2, P10, P100, and P1000)

• Tips (for organic solutions, Gilson CP100 and CP1000; for aqueous solutions, Gilson D10, D200, and D1000)

• Thermoshaker (Thermal Shake lite VWR)

• 4‐ml screw‐cap pressure tubes, with O‐ring seal (cap screw 13‐mm black PP, white silicone/red PTFE septa, 1.5‐mm)

• 50°C oven

• Cotton

• 2‐ml Eppendorf tubes (Fisherbrand 2.0‐ml MCT graduated natural)

• SpeedVac concentrator (Thermo Scientific, Savant DNA 120)

• Lyophilizer

• HPLC vials, 100 PP vials N9 cone (Macherey‐Nagel)

• MALDI (Shimadzu Biotech)

• 1‐ml quartz cells for UV‐spectrophotometer

• UV‐1600PC spectrometer (VWR) with UV detection at 260 nm

• Additional reagents and equipment for automated oligonucleotide synthesis (Pon, 2001) and for analysis, purification, and characterization (Andrus & Kuimelis, 2001)

Synthesize the oligonucleotide by automated synthesis

For more information, see Ellington and Pollard (2001).

• 1
  Weigh the appropriate amount of each DNA phosphoramidite [(N+2) × 15 µmol, N being the number of incorporations of the base in the sequence] in an oven‐dried synthesizer glass bottle.

  • a. Cap the bottle with a rubber septum in which a disposable needle is inserted.
  • b. Put all flasks in desiccator, containing both P₂O₅ and KOH, under vacuum over 18 hr.
• 2Weigh 1 µmol dT‐3’Icaa CPG 500 Å in the synthesis reactor. Close the reactor tightly. Put it in the same desiccator for 18 hr.
• 3Open the desiccator under an argon‐atmosphere. Each DNA phosphoramidite is solubilized in a certain amount of anhydrous MeCN (0.2 ml per N+2, using a disposable polypropylene syringe and needle). The 5’‐MMT‐amino modifier phosphoramidite is directly solubilized in anhydrous MeCN in the original reaction flask to achieve a 0.1 M solution.

  Once solubilized, this solution can be stored in the freezer until next use. This dilution is necessary due to the high viscosity of this particular phosphoramidite.

• 4
  Install all the reagents on the specific ports of the ABI 394 synthesizer according to the producer's instructions.

  • a. Acetonitrile.
  • b. Dichloromethane.
  • c. Detritylation solution.
  • d. Both capping solutions (A and B).
  • e. Oxidizer solution.
  • f. Activator solution.
  • g. All phosphoramidite solutions (the four nucleosides and the amino modifier).
• 5Install the synthesis reactor on the appropriate position of the synthesizer.
• 6Enter the expected sequence on the computer.
• 7
  The synthesis cycle (trityl‐off mode) to be selected is the 1 µmol CE DNA where the time of reaction (“wait”) are as follows:

  • a. 30 s for the coupling (for the amino modifier phosphoramidite, the reaction time is increased to 60 s).
  • b. 25 s for the capping.
  • c. 15 s for the oxidation.
  • d. 65 s for detritylation.
    At the end of the synthesis, the final amine function will thus be deprotected, ready for conjugation.
• 8Once the synthesis is over, the reactor is removed from the instrument and dried under vacuum.

Conjugate the amino acid or peptide to the oligonucleotide on CPG beads

• 9Weigh the appropriate amount of the CPG‐beads and transfer to a microcentrifuge tube.

  Reactions were performed on 42 nmol scale (1 eq.).

• 10In another microcentrifuge tube, introduce a 50 mM solution of the acid partner in acetonitrile [N‐protected amino acid, or short peptide, 25 eq. for short oligonucleotides (6‐ and 12‐mers), 50 eq. for longer oligonucleotides (21‐mers)] with the micropipette and tip for organic solvent.

  For some peptides, solubility issues might occur. In that case, the solution was prepared with the same concentration, using a mixture of solvent MeCN/DMSO (9:1 v/v).

• 11To this solution, using the micropipettes and tips for organic solvents, add a 50 mM CDMT solution in MeCN/H₂O (9:1 v/v) (1.1 eq. towards the acid partner, e.g., 33 eq. for short oligonucleotides and 55 eq. for longer oligonucleotides) and a 5 mM DABCO solution in MeCN (0.05 eq. towards the acid partner, e.g., 1.5 eq. for short oligonucleotides and 2.5 eq. for longer oligonucleotides).
• 12Stir this mixture at 25°C in the thermoshaker for 30 min.
• 13Add this mixture to the CPG beads, using a micropropipette for organic solvent. Add a 100 mM solution of DiPEA in MeCN (2.2 eq. towards the acids, e.g., 66 eq. for short oligonucleotides and 110 eq. for longer oligonucleotides).
• 14Stir the resulting heterogeneous mixture in the thermoshaker at 25°C for 15 hr.
• 15Remove solvent and wash the CPG beads with 3 × 500 µl MeCN. Transfer the beads into a 4‐ml screw‐cap pressure tube, with O‐ring seal and add 1 ml of 30% hydroxide ammonium to the Eppendorf tube. Put the tube into a 50°C oven for 18 hr.
• 16Cool the mixture to room temperature. Filter through a polypropylene tip with cotton and collect the filtrate in a 2‐ml Eppendorf tube. Wash the beads with Milli‐Q water.
• 17Evaporate the ammonia in a Speedvac evaporator for 1 hr, then lyophilize.

Analyze and purify the conjugated oligonucleotide by RP‐HPLC

• 18Dissolve the residue in 500 µl Milli‐Q water. Remove 4 µl with the P10 micropipette and the D10 tip, transfer it to an HPLC vial, and add 96 µl Milli‐Q water with the P100 micropipette and the D200 tip. Perform analytical RP‐HPLC. Calculate the conversion of the reaction using the integration of the HPLC profile, in comparison to the integration of a pure sample of the starting material.
• 19
  Perform preparative RP‐HPLC. Our classical HPLC conditions are as follows:

  • a. For analytical HPLC (EC 75/4.5 Nucleodur 100‐3 C18 column), flow is 1 ml/min.
  • b. For preparative HPLC, flow is 4 ml/min.
  • c. Buffer A is 1% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • d. Buffer B is 80% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • e. Elution conditions, for both analytical or preparative HPLC, were either from 0% to 24% buffer B in 20 min, or from 0% to 56% buffer B in 20 min.

Analyze the conjugated oligonucleotide by mass spectrometry

• 20Remove 0.5 µl (with the P2 micropipette and the D10 tip) of the oligonucleotide solution and put it on a MALDI‐TOF MS plate. Add 0.5 µl (with the P2 micropipette and the D10 tip) of 2',4',6'‐trihydroxyacetophenone monohydrate (THAP) saturated matrix and ammonium citrate (0.1 M) as co‐matrix. Perform MALDI‐TOF MS measurement.

Determine the yield of the reaction

• 21Dilute lyophilized pure compounds into 500 µl. Remove 5 µl (with the P10 micropipette and the D10 tip) and bring up to 1 ml in a 1‐ml spectrophotometer cell (with the P1000 micropipette and the D1000 tip). Measure the absorbance at 260 nm. Calculate the concentration using the Beer–Lambert law and calculate the yield of the reaction.

  For all prepared compounds, the molar extinction coefficient of the conjugated peptide has been measured and was negligeable compared to the one of the oligonucleotides.

DEPROTECTION OF THE BOC‐PROTECTED AMINE OF THE LYSINE

In classical peptide chemistry, amine protecting groups are typically carbamates, such as Fmoc (base‐labile) or Boc (acid‐labile). Our procedures are designed to selectively and systematically remove the Fmoc group under mild basic conditions, while leaving the Boc group intact. This selectivity is particularly advantageous for oligonucleotide conjugation, as oligonucleotides are highly sensitive to strong acidic conditions, which can cause degradation or undesired chemical modifications. As a result, Boc deprotection is rarely used in this context. This Support Protocol illustrates the efficient Boc‐deprotection of the lysine in acidic medium (Usanov et al., 2018), which must be performed after Basic Protocol 1, steps 1 to 14.

Materials

• Acetonitrile (MeCN) (≥99.9%, HPLC gradient grade, Fisher Chemical, A/0627/17)

• Trifluoroacetic acid (TFA) (Carlo Erba, Pure, P0080212)

• Dichloromethane (DCM) (Fisher Chemical, 99%, laboratory reagent grade, D/1850/17)

• Dimethylformamide (DMF) (Honeywell Research Chemicals, >99.8%, 33120)

• Micropipettes (for organic solutions, Gilson P100, and P1000)

• Tips (for organic solutions, Gilson CP100 and CP1000)

• Thermoshaker (Thermal Shake lite VWR)

• Sigma 1‐13 centrifuge

• 1Remove solvent with the micropipette and tip for organic solvent and wash the CPG beads with 3 × 500 µl MeCN.
• 2Add 100 µl of a solution of 50% trifluoroacetic acid in DCM to the Eppendorf tube containing the CPG beads.
• 3Stir this mixture at 25°C in the thermoshaker for 1 min. Centrifuge 1 min at 11,340 × g, 25°C.

  This treatment should not last longer than 1 min due to the sensitivity of the oligonucleotides in acidic media.

• 4Using micropipettes and tips for organic solutions, remove solvent and wash the CPG beads with 3 × 500 µl DCM, then with 3 × 500 µl DMF, and finally with 3 × 500 µl MeCN.
• 5Continue from step 15 to step 21 to perform the deprotection off the CPG beads and analysis of the conjugated oligonucleotide.

Basic Protocol 2. IN‐SOLUTION CONJUGATION OF AN AMINO ACID OR OF A PEPTIDE ON AN OLIGONUCLEOTIDE

The solid‐supported strategy offers advantages of straightforward handling and simplified purification of the final POC. However, this approach is often limited by protecting group incompatibilities. In particular, peptide synthesis typically uses Fmoc/t‐Bu strategy, which is incompatible with iterative processes on CPG‐beads. To overcome these limitations, a solution‐phase approach has been developed and optimized under aqueous conditions, effectively avoiding such chemical incompatibilities. This protocol describes the conjugation of either a single amino acid or a peptide with a 5’‐aminomodified oligonucleotide. Starting from commercially available functionalized CPG beads, the protocol describes the synthesis of the oligonucleotide, its cleavage from the support and the conjugation reaction, followed by purification and analysis.

Materials

• DNA phosphoramidites (ChemGenes): 5’‐O‐(4,4’‐dimethoxytrityl)‐N‐protected‐2’‐deoxyribonucleoside‐3’‐O‐(2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidites, where the nucleobases are:

  • N ⁶‐benzoyladenin‐9‐yl (ChemGenes, ANP‐551)

  • N ²‐isobutyrylguanin‐9‐yl (ChemGenes, ANP‐553)

  • N ⁴‐benzoylcytosin‐1‐yl (ChemGenes, ANP‐552)

  • Thymin‐1‐yl (ChemGenes, ANP‐554)

• dT Synbase CPG 500/110S (deoxythymidine 3’‐Icaa CPG 500 Å) (Link Technologies, 2261)

• MMT‐amino C‐6 modifier phosphoramidite: 6‐(4‐monomethoxytritylamino)hexyl‐(2‐cyanoethyl)‐(N,N‐diisopropyl)‐phosphoramidite (ChemGenes, CLP‐1563)

• Argon (dry)

• Anhydrous acetonitrile (MeCN) (VWR Chemical, for DNA synthesis, water <10 ppm, filtered at 0.2 µm, packaged under nitrogen, VWRC85501.321)

• Dichloromethane (DCM) (Carlo Erba, anhydrous, stabilized with amylene, water ≤50 ppm, 442292000‐CER)

• Detritylation solution: 3% (w/v) trichloroacetic acid (TCA) in DCM (Biosolve Chimie SARL, Deblock TCA 3% in DCM, DNA synthesis, 04132402)

• Capping A solution: acetic anhydride in tetrahydrofuran (Glen Research, 40‐4012)

• Capping B solution: 10% (w/v) 1‐methylimidazole in tetrahydrofurane/pyridine (Glen Research, 40‐4110)

• Activator solution: 0.3 M 5‐benzylmercapto‐1H‐tetrazole (ChemGenes, 99.9%, water 20 ppm, RN‐1415) in anhydrous acetonitrile (VWR Chemical, for DNA synthesis)

• Oxidizer solution: 0.1 M iodine in tetrahydrofuran/water/pyridine (Glen Research, 40‐4330)

• 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) (Aldrich, 98%, 139009)

• H₂O, deionized, Milli‐Q‐purified (Millipore, 18.2 MΩ.cm)

• 30% (v/v) ammonium hydroxide, stored at 4°C (Carlo Erba, for analysis ACS, 419941)

• THAP (matrix substance for MALDI‐MS, ≥99.5%, Aldrich, 05757)

• Ammonium citrate (98%, Aldrich, 374695)

• Acid partner:

  • Fmoc‐Lys(Boc)‐OH (≥98%, Carbosynth, FF158761401)

  • Fmoc‐Pro‐Phe‐Gly‐OH (homemade)

  • Fmoc‐Val‐Arg‐Leu‐Pro‐Pro‐Pro‐OH (aka Fmoc‐VRLPPP‐OH, homemade)

  • Biotin (≥98.5%, Aldrich, 8.51209)

• Acetonitrile (≥99.9%, HPLC gradient grade, Fisher Chemical, A/0627/17)

• 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) (97%, Aldrich, 375217)

• 1,4‐diazabicyclo[2,2,2]octane (DABCO) (98%, Aldrich, 8034560100)

• Borate buffer (see recipe)

• N,N‐diisopropylethylamine (DiPEA) (99%, Thermo Scientific, A11801.AE)

• Ethyl acetate (Fisher Chemical, ≥99%, laboratory reagent grade, E/0850/21)

• Empty DNA synthesizer bottles, oven‐dried, with rubber septa

• Disposable polypropylene syringes and needles

• Vacuum desiccator containing P₂O₅ and KOH

• Rotatory evaporator equipped with a vacuum pump

• Automatic DNA synthesizer (ABI 394)

• Synthesis reactor for 1‐µmol scale synthesis (empty synthesis columns‐TWIST, Glen Research)

• 4‐ml screw‐cap pressure tubes, with O‐ring seal (cap screw 13‐mm black PP, white silicone/red PTFE septa, 1.5‐mm)

• 50°C oven

• Micropipettes (for organic solutions, Gilson P100, and P1000; for aqueous solutions, Gilson P2, P10, P100, P200, and P1000)

• Tips (for organic solutions, Gilson CP100 and CP1000; for aqueous solutions, Gilson D10, D200, and D1000)

• Cotton

• 2‐ml Eppendorf tubes (Fisherbrand 2.0‐ml MCT graduated natural)

• SpeedVac concentrator (Thermo Scientific, Savant DNA 120)

• Lyophilizer

• HPLC vials, 100 PP vials N9 cone (Macherey‐Nagel)

• MALDI (Shimadzu Biotech)

• 1‐ml quartz cells for UV‐spectrophotometer

• UV‐1600PC spectrometer (VWR) with UV detection at 260 nm

• Thermoshaker (Thermal Shake lite VWR)

• Additional reagents and equipment for automated oligonucleotide synthesis (Pon, 2001) and for analysis, purification, and characterization (Andrus & Kuimelis, 2001)

Synthesize the oligonucleotide by automated synthesis

• 1Start the synthesis with Basic Protocol 1, steps 1 to 6.
• 2
  The synthesis cycle (trityl‐off mode) to be selected is the 1 µmol CE DNA where the time of reaction (“wait”) are as follows:

  • a. 30 s for the coupling (for the amino modifier phosphoramidite, the reaction time is increased to 60 s).
  • b. 25 s for the capping.
  • c. 15 s for the oxidation.
  • d. 65 s for detritylation.

  At the end of the synthesis, the final amine function will thus be deprotected, ready for conjugation.

• 3Once the synthesis is over, remove the reactor from the instrument.
• 4Prepare a solution of 0.3 ml DBU in 1.7 ml of acetonitrile (anhydrous) using a disposable polypropylene syringe and needle, and load it in a 2‐ml syringe.
• 5Connect the syringe to the reactor and another empty 2‐ml syringe to the other side.
• 6Percolate the solution from one syringe to the other through the reactor for 3 min.
• 7Wash the CPG beads 3 times with 2 ml acetonitrile using a syringe.
• 8Install the reactor again on the ABI 394 synthesizer to perform the deprotection of the MMTr protecting group using TCA (using the classical conditions of deprotection) for 70 s. If the deprotection is not complete (orange traces remain), repeat this operation using TCA for <50 s.
• 9Wash the CPG beads with acetonitrile and remove the reactor from the synthesizer. Dry it under technical vacuum for 10 min.
• 10Transfer the beads into a 4‐ml screw‐cap pressure tube, with O‐ring seal.
• 11Add 1 ml of 30% hydroxide ammonium to the tube. Put the tube into a 50°C oven for 18 hr.
• 12Cool the mixture to room temperature. Filter through a polypropylene tip with cotton and collect the filtrate in a 2‐ml Eppendorf tube. Wash the beads with Milli‐Q water.
• 13Evaporate the ammonia in the Speedvac evaporator for 1 hr, then lyophilize.
• 14Dissolve the residue in 1000 µl Milli‐Q water (P1000 micropipette and the D1000 tip). Remove 4 µl with the P10 micropipette and the D10 tip, transfer it to an HPLC vial, and add 96 µl Milli‐Q water with the P100 micropipette and the D200 tip. Perform analytical RP‐HPLC.
• 15Remove 0.5 µl (with the P2 micropipette and the D10 tip) crude or pure oligonucleotide solution and deposit it on a MALDI‐TOF MS plate. Add 0.5 µl (with the P2 micropipette and the D10 tip) of 2',4',6'‐trihydroxyacetophenone monohydrate (THAP) saturated matrix and ammonium citrate (0.1 M) as co‐matrix. Perform MALDI‐TOF MS measurement.
• 16Remove 5 µl (with the P10 micropipette and the D10 tip) and bring volume to 1 ml (with the P1000 micropipette and the D1000 tip) in a 1‐ml spectrophotometer cell. Measure the absorbance at 260 nm. Calculate the concentration using the Beer–Lambert law and calculate the yield of the reaction.

  MALDI mass spectrometry experiment confirms the formation of the desired oligonucleotide. The HPLC indicates the retention time for the calculation of conversion of the next step. The absorbance measurement allows the quantifications to pool samples in different Eppendorf tubes for the next step.

Conjugate the amino acid, or the peptide, to the oligonucleotide in solution

• 17Pool the correct amount of oligonucleotide into a 2‐ml Eppendorf tube. Lyophilize.

  Reactions were performed on 20 nmol (1 eq.).

• 18In another 2‐ml Eppendorf tube, introduce a 100 mM solution of the acid partner in acetonitrile (HPLC grade) (N‐protected amino acid, or short peptide, 125 eq.).

  For some peptides, solubility issues might occur. In that case, the solution was prepared with the same concentration, using a mixture of solvent MeCN/DMSO (9:1 v/v).

• 19To this solution, using micropipettes and tips for organic solvent, add a 100 mM CDMT solution in MeCN/H₂O (9:1 v/v) (1.1 eq. towards the acid partner, e.g., 137.5 eq. towards the oligonucleotide) and a 10 mM DABCO solution in MeCN (0.1 eq. towards the acid partner, e.g., 12.5 eq. towards the oligonucleotide).
• 20Stir this mixture at 25°C in the thermoshaker for 30 min.
• 21Add this mixture to a solution of the oligonucleotide in borate buffer (250 M, pH 9.3, 100 µl). Add a 200 mM solution of DiPEA in MeCN (2.2 eq. towards the acids, e.g., 275 eq. towards the oligonucleotide).
• 22Stir the resulting mixture in the thermoshaker at 25°C for 2 hr.
• 23After 90 min of stirring, prepare a new solution of activated acid, as in steps 18 and 19. Stir this mixture at 25°C in the thermoshaker for 30 min.
• 24Add the fresh activated acid solution to the oligonucleotide mixture. Stir the resulting mixture in the thermoshaker at 25°C for 15 hr.
• 25Extract all organic residues three times with 100 µl ethyl acetate.
• 26Evaporate traces of ethyl acetate in a Speedvac evaporator for 15 min, then lyophilize.

Analyze and purify the conjugated oligonucleotide by RP‐HPLC

• 27Dissolve the residue in 200 µl Milli‐Q water (with the P200 micropipette and the D200 tip). Remove 10 µl (P10 micropipette and the D10 tip), transfer it to an HPLC vial, and add 90 µl Milli‐Q water (with the P100 micropipette and the D200 tip). Perform analytical RP‐HPLC. Calculate the conversion of the reaction using the integration of the HPLC profile, in comparison to the integration of a pure sample of the starting material.
• 28
  Perform preparative RP‐HPLC. Our classical HPLC conditions are as follows:

  • a. For analytical HPLC (EC 75/4.5 Nucleodur 100‐3 C18 column), flow is 1 ml/min.
  • b. For preparative HPLC, flow is 4 ml/min.
  • c. Buffer A is 1% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • d. Buffer B is 80% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • e. Elution conditions, for both analytical or preparative HPLC, were either from 0% to 24% buffer B in 20 min, or from 0% to 56% buffer B in 20 min.

Analyze the conjugated oligonucleotide by mass spectrometry

• 29Remove 0.5 µl (P2 micropipette and the D10 tip) crude or pure oligonucleotide solution and deposit it on a MALDI‐TOF MS plate. Add 0.5 µl (P2 micropipette and the D10 tip) of 2',4',6'‐trihydroxyacetophenone monohydrate (THAP) saturated matrix and ammonium citrate (0.1 M) as co‐matrix. Perform MALDI‐TOF MS measurement.

Determine the yield of the reaction

• 30Dilute lyophilized pure compounds into 200 µl (with the P200 micropipette and the D200 tip). Remove 10 µl (P10 micropipette and the D10 tip) and bring volume to 1 ml (with the P1000 micropipette and the D1000 tip) in a 1‐ml spectrophotometer cell. Measure the absorbance at 260 nm. Calculate the concentration using the Beer–Lambert law and calculate the yield of the reaction.

  For all prepared compounds, the molar extinction coefficient of the conjugated peptide has been measured and was negligeable compared to the coefficient of the oligonucleotides.

Basic Protocol 3. ITERATIVE PROCESS FOR THE PREPARATION OF A PEPTIDE–OLIGONUCLEOTIDE CONJUGATE

This protocol describes the iterative conjugation of Fmoc‐protected amino acids to a 12‐mer oligonucleotide. The beginning of the synthesis follows Basic Protocol 2, steps 1 to 26. Then, rather than being isolated, the compound is deprotected with piperidine, purified over NAP chromatography and submitted to a new coupling of another amino acid. We performed this synthesis to attach a tripeptide Pro‐Pro‐Phe on a 12‐mer oligonucleotide.

Materials

• DNA phosphoramidites (ChemGenes): 5’‐O‐(4,4’‐dimethoxytrityl)‐N‐protected‐2’‐deoxyribonucleoside‐3’‐O‐(2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidites, where the nucleobases are:

• N ⁶‐benzoyladenin‐9‐yl (ChemGenes, ANP‐551)

• N ²‐isobutyrylguanin‐9‐yl (ChemGenes, ANP‐553)

• N ⁴‐benzoylcytosin‐1‐yl (ChemGenes, ANP‐552)

• Thymin‐1‐yl (ChemGenes, ANP‐554)

• dT Synbase CPG 500/110S (deoxythymidine 3’‐Icaa CPG 500 Å) (Link Technologies, 2261)

• MMT‐amino C‐6 modifier phosphoramidite: 6‐(4‐monomethoxytritylamino)hexyl‐(2‐cyanoethyl)‐(N,N‐diisopropyl)‐phosphoramidite (ChemGenes, CLP‐1563)

• Argon (dry)

• Anhydrous acetonitrile (MeCN) (VWR Chemical, for DNA synthesis, water <10 ppm, filtered at 0.2 µm, packaged under nitrogen, VWRC85501.321)

• Dichloromethane (DCM) (Carlo Erba, anhydrous, stabilized with amylene, water ≤50 ppm, 442292000‐CER)

• Detritylation solution: 3% (w/v) trichloroacetic acid (TCA) in DCM (Biosolve Chimie SARL, Deblock TCA 3% in DCM, DNA synthesis, 04132402)

• Capping A solution: acetic anhydride in tetrahydrofuran (Glen Research, 40‐4012)

• Capping B solution: 10% (w/v) 1‐methylimidazole in tetrahydrofurane/pyridine (Glen Research, 40‐4110)

• Activator solution: 0.3 M 5‐benzylmercapto‐1H‐tetrazole (ChemGenes, 99.9%, water 20 ppm, RN‐1415) in anhydrous acetonitrile (VWR Chemical, for DNA synthesis)

• Oxidizer solution: 0.1 M iodine in tetrahydrofuran/water/pyridine (Glen Research, 40‐4330)

• 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) (Aldrich, 98%, 139009)

• H₂O, deionized, Milli‐Q‐purified (Millipore, 18.2 MΩ.cm)

• 30% (v/v) ammonium hydroxide, stored at 4°C (Carlo Erba, for analysis ACS, 419941)

• THAP (matrix substance for MALDI‐MS, ≥99.5%, Aldrich, 05757)

• Ammonium citrate (98%, Aldrich, 374695)

• Acid partner:

  • Fmoc‐Pro‐OH (99.92%, BLDPharm, BD8606)

  • Fmoc‐Phe‐OH (99.93%, BLDPharm, BD8605)

• 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) (97%, Aldrich, 375217)

• 1,4‐diazabicyclo[2,2,2]octane (DABCO) (98%, Aldrich, 8034560100)

• Borate buffer (see recipe)

• N,N‐diisopropylethylamine (DiPEA) (99%, Thermo Scientific, A11801.AE)

• Ethyl acetate (≥99%, Fisher Chemical, Laboratory reagent grade, E/0850/21)

• Piperidine (Sigma‐Aldrich, for synthesis, 8.22299)

• Acetonitrile (≥99.9%, HPLC gradient grade, Fisher Chemical, A/0627/17)

• Empty DNA synthesizer bottles, oven‐dried, with rubber septa

• Disposable polypropylene syringes and needles

• Vacuum desiccator containing P₂O₅ and KOH

• Rotatory evaporator equipped with a vacuum pump

• Automatic DNA synthesizer (ABI 394)

• Synthesis reactor for 1‐µmol scale synthesis (empty synthesis columns‐TWIST, Glen Research)

• 4‐ml screw‐cap pressure tubes, with O‐ring seal (cap screw 13‐mm black PP, white silicone/red PTFE septa, 1.5‐mm)

• 50°C oven

• Micropipettes (for organic solutions, Gilson P100, and P1000; for aqueous solutions, Gilson P2, P10, P100, and P1000)

• Tips (for organic solutions, Gilson CP100 and CP1000; for aqueous solutions, Gilson D10, D200, and D1000)

• Cotton

• 2‐ml Eppendorf (Fisherbrand 2.02ml MCT graduated natural)

• SpeedVac concentrator (Thermo Scientific, Savant DNA 120)

• Lyophilizer

• HPLC vials, 100 PP vials N9 cone (Macherey‐Nagel)

• MALDI (Shimadzu Biotech)

• 1‐ml quartz cells for UV‐spectrophotometer

• UV‐1600PC spectrometer (VWR) with UV detection at 260 nm

• Thermoshaker (Thermal Shake lite VWR)

• NAP chromatography (GE Healthcare, NAP‐25 columns Sephadex, G‐25 DNA grade)

• Additional reagents and equipment for automated oligonucleotide synthesis (Pon, 2001) and for analysis, purification, and characterization (Andrus & Kuimelis, 2001)

Synthesize the oligonucleotide by automated synthesis

• 1Start the synthesis as in Basic Protocol 2, steps 1 to 16.

Conjugate the Fmoc‐protected amino acid to the oligonucleotide in solution

• 2Pool the correct amount of oligonucleotide into a 2‐ml Eppendorf tube. Lyophilize.

  Reactions were performed on 100 nmol scale (1 eq.).

• 3In another 2‐ml Eppendorf tube, introduce a 100 mM solution of the Fmoc‐amino acid partner in acetonitrile (DNA synthesis) (125 eq.).
• 4To this solution, with micropipettes and tips for organic solvent, add a 100 mM CDMT solution in MeCN/H₂O (9:1 v/v) (1.1 eq. towards the acid partner, e.g., 137.5 eq. towards the oligonucleotide) and a 10 mM DABCO solution in MeCN (0.1 eq. towards the acid partner, e.g., 12.5 eq. towards the oligonucleotide). Stir this mixture at 25°C in the thermoshaker for 30 min.
• 5Add this mixture to a solution of the oligonucleotide in borate buffer (250 M, pH 9.3, 100 µl). Add a 200 mM solution of DiPEA in MeCN (2.2 eq. towards the acids, e.g., 275 eq. towards the oligonucleotide).
• 6Stir the resulting mixture in the thermoshaker at 25°C for 2 hr.
• 7After 90 min of stirring, prepare a new solution of activated acid, as in step 4. Stir this mixture at 25°C in the thermoshaker for 30 min.
• 8Add the fresh activated acid solution to the oligonucleotide mixture. Stir the resulting mixture in the thermoshaker at 25°C for 15 hr.
• 9Extract all organic residues three times with 100 µl ethyl acetate with micropipette P100 and tip D200 for organic solution.
• 10Add 1 ml of an aqueous solution of piperidine (10% v/v) to the oligonucleotide solution. Stir this mixture at room temperature for 2 hr.
• 11Extract all organic residues three times with 100 µl ethyl acetate.
• 12Purify the aqueous phase using a NAP‐chromatography. Collect and lyophilize the fractions.

  The NAP‐chromatography serves as steric exclusion purification: all benzofulvene, piperidine, ethyl acetate traces and inorganic salts are therefore removed to perform the next incorporation on a pure intermediate.

• 13Repeat steps 2 to 12 as many times as desired for the sequential introduction of Fmoc‐protected amino acids.

Analyze and purify the conjugated oligonucleotide by RP‐HPLC

• 14Dissolve the residue in 200 µl Milli‐Q water. Remove 10 µl, transfer it to an HPLC vial, and add 90 µl Milli‐Q water. Perform analytical RP‐HPLC. Calculate the conversion of the reaction using the integration of the HPLC profile, in comparison to the integration of a pure sample of the starting material.
• 15
  Perform preparative RP‐HPLC. Our classical HPLC conditions are as follows:

  • a. For analytical HPLC (EC 75/4.5 Nucleodur 100‐3 C18 column), flow is 1 ml/min.
  • b. For preparative HPLC, flow is 4 ml/min.
  • c. Buffer A is 1% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • d. Buffer B is 80% (v/v) MeCN in a solution of TEAAc (0.05 M).
  • e. Elution conditions, for both analytical or preparative HPLC, were either from 0% to 24% buffer B in 20 min, or from 0% to 56% buffer B in 20 min.

Analyze the conjugated oligonucleotide by mass spectrometry

• 16Remove 0.5 µl crude or pure oligonucleotide solution and deposit it on a MALDI‐TOF MS plate. Add 0.5 µl of 2',4',6'‐trihydroxyacetophenone monohydrate (THAP) saturated matrix and ammonium citrate (0.1 M) as co‐matrix. Perform MALDI‐TOF MS measurement.

Determine the yield of the reaction

• 17Dilute lyophilized pure compounds into 200 µl. Remove 10 µl and bring volume to 1 ml in a 1‐ml spectrophotometer cell. Measure the absorbance at 260 nm. Calculate the concentration using the Beer–Lambert law and calculate the yield of the reaction.

  For all prepared compounds, the molar extinction coefficient of the conjugated peptide has been measured and was negligeable compared to the coefficient of the oligonucleotides.

REAGENTS AND SOLUTIONS

Borate buffer

• For 10 ml:

• 8 ml H₂O, distilled

• 61.9 mg boric acid (0.1 M prepared from pure boric acid, Sigma, ≥99.5%, B6768)

• 43.9 mg sodium chloride (0.075 M prepared from pure sodium chloride, Carlo Erba, for analysis, 479689)

• 50.3 mg sodium tetraborate (0.025 M prepared from pure sodium tetraborate, Prolabo, R.P. Normapur)

• Bring volume up to 10 ml with distilled water

• Stir until boric acid is fully dissolved

• Adjust to pH 9.3 with 1 M NaOH

• Store up to 3 months at 4°C

  Homemade peptides have been prepared with SPPS, using classical peptide coupling conditions with PyBOP or HATU as coupling agent.

COMMENTARY

Background Information

Since the development of the first antisense oligonucleotide (ON) in the late 1970s by Zamecnik and Stephenson (1978), ON therapeutics have made rapid progresses in medicinal chemistry: from the first market launch in 1998 of the ON Fomivirsen to the actual 18 therapeutic ONs currently approved by the FDA and the 130 ONs that are in clinical evaluation (Egli & Manoharan, 2023). Relying on different therapeutic strategies (antisense ON, microRNA, small interfering RNA, or aptamers) (Roberts et al., 2020), these ONs have been increasingly used for treating diseases, often related with undruggable proteins (Lu et al., 2010; Thakur et al., 2022). However, their effectiveness is limited by poor cellular absorption, instability in biological fluids, and difficulties to reach the nucleus due to the numerous negative charges they carry (Boisguérin et al., 2015). To overcome these issues, extensive work has been performed by changing the structural core of the ON backbone, the nucleobase, or by conjugation with a vector (lipids or peptides). The bioconjugation of oligonucleotide to peptides has revealed excellent potential (Tung & Stein, 2000). Indeed, POCs have shown a strong improvement in delivery and stability for therapies like antisense oligonucleotides (Lebedeva, 2000), siRNAs (Lam et al., 2015), and aptamers (Li & Lan, 2015).

There are two main strategies for conjugating peptides to oligonucleotides, either sequentially, often by solid‐phase synthesis (Frieden et al., 2004; Wang et al., 2023), or post‐synthetically, usually in liquid phase (Klabenkova et al., 2021). Solid‐phase synthesis provides easier purification (Lönnberg, 2009) but faces compatibility issues due to peptide protecting groups (Klabenkova et al., 2021). In contrast, post‐synthetic methods circumvent these chemical incompatibilities and allow greater versatility in linker selection, frequently using cysteine residues, Click chemistry reactions (Honcharenko et al., 2019; Meyer et al., 2022), or oxime formation (Ghosh et al., 2004). Among the available conjugation techniques, linking peptides and oligonucleotides via an amide bond is particularly attractive, as it mimics natural peptide bonds. However, forming amides usually requires large amounts of traditional activating agents like HATU, DMTMM or EDC, especially in the field of DNA‐Encoded Library (DEL) technology (Fitzgerald & Paegel, 2021; Keller et al., 2022). Despite their efficiency, these reagents are not universally compatible with all amino acids and peptides. Consequently, the development of eco‐friendly and efficient methods for amide bond formation, compatible with oligonucleotide chemistry and adaptable to a wide range of peptides, remains a pressing challenge.

Recently, we reported a catalytic and sustainable approach for amide coupling applied to small organic molecules, using 1,4‐diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2,4‐dichloro‐6‐methoxy‐1,3,5‐triazine (DCMT) as substoichiometric activating agent. This method enables the synthesis of dipeptides efficiently, without racemization (Adler et al., 2023). We therefore set out to explore its applicability to the formation of POCs and we showcased its efficiency by using a similar catalytic system with DABCO and 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) (Gras et al., 2024).

Understanding Results

For this study, three 5’‐amino‐modified oligonucleotides have been synthesized. For each oligonucleotide, batches have been retained on the CPG beads for the solid‐phase coupling, while others have been cleaved off the support to yield fully deprotected oligonucleotides. The cleaved oligonucleotides were used for in‐solution conjugation without prior purification. HPLC and MALDI analysis are summarized in Figure 2.

Figure 2.

Non‐conjugated crude oligonucleotides.

Several amino acids and peptides were used to illustrate the efficiency of the method, with selected examples described below. For each case, both on‐column or in‐solution conditions were compared.

A first conjugation has been performed using a single amino acid, Fmoc‐Lys(Boc)‐OH, and a 12‐mer oligonucleotide (Fig. 3). HPLC profiles of the solid‐phase synthesis showed a clean reaction. After purification, the expected compound is isolated in a very good 65% yield. In contrast, the HPLC profile obtained from the coupling in solution of the Fmoc‐protected compound showed numerous side products, resulting either from the reaction conditions or from the fact that the starting oligonucleotide is used without being purified. The presence of the Fmoc moiety allows the compound to be more hydrophobic for easiest purification. After deprotection, the pure product is obtained in a good 38% yield.

Figure 3.

Conjugation of a single amino acid. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

Before cleavage off the CPG beads, the amine function of the lysine has been deprotected using the Support Protocol. The expected compound is obtained in a good 64% isolated yield (Figure 4), showcasing the possibility of introducing a Boc‐protected amine on the oligonucleotide without a loss in yield while deprotection.

Figure 4.

Deprotection of the Boc‐protected amine. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

Tripeptides have been introduced on a 21‐mer oligonucleotide analog of Fomivirsen, an antiviral oligonucleotide therapeutic (Fig. 5). Coupling was performed using Fmoc‐Pro‐Phe‐Gly‐OH, in good yields for solid phase synthesis, as well as in‐solution reaction. On that example, the crude on CPG‐beads contained impurities that have closed retention time with the expected POC. This explains the lower yield than usual. In‐solution reaction allowed the purification of the Fmoc‐protected POC, allowing an easier purification.

Figure 5.

Conjugation of a tripeptide. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

To showcase the utility of preparing POCs with better bioavailability, an hexapeptide monomer of the reported cell penetrating peptide SAP (Sweet Arrow Peptide) (Franz et al., 2016), has been conjugated to a 12‐mer oligonucleotide with success and easy purification on both CPG‐beads and in‐solution conditions (Fig. 6).

Figure 6.

Conjugation of a hexapeptide. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

As a final example on this conjugation, we decided to extend to other species beyond amino acids and peptides. Therefore, biotin has been successfully conjugated to a 12‐mer oligonucleotide (Fig. 7). Similar yields have been obtained on CPG‐beads and in solution, but purification was much easier from the solid‐phase synthesis, as depicted by the HPLC profile.

Figure 7.

Conjugation of biotin. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

Iterative steps were performed on a 12‐mer oligonucleotide to sequentially incorporate three amino acids. No further incorporations have been investigated. In this example, phenylalanine was added first, followed by two prolines. Between each coupling step, a short size‐exclusion chromatography was performed. A final preparative HPLC step was used to purify the resulting POC, achieving a comparable yield to that of the direct coupling between the ON and a tripeptide (Fig. 8).

Figure 8.

POC obtained after an iterative process. "[a]" All DNA samples were analyzed by analytic HPLC with an EC 75/4.5 Nucleodur 100‐3 C18 column, a variable gradient of buffer B (TEAAc 0.05 M + 80% MeCN) in buffer A (TEAAc 0.05M + 1% MeCN) at a flow of 1 ml/min over 20 min.

Critical Parameters and Troubleshooting

One of the most critical parameters is the amount of activated carboxylic acid. Two major issues were identified. First, both in solution and on CPG, the nucleophilic amine can either react on the carboxyl to yield the expected amide, or, it can irreversibly add on the triazinyl core to release the carboxylate. This problem was addressed by adding the activated carboxylic acid in two portions for the conjugation in‐solution, or by reducing the amount of activated acid on solid‐supported synthesis.

The other issue was the second acylation of the amine leading to the formation of an imide. This side reaction has been mainly observed on solid‐supported synthesis and has similarly been resolved by decreasing the amount of activated acid.

The good compromise between reactivity (e.g., decent conversion) and selectivity (e.g., no side products) was achieved by optimizing the amount of activated acid. For solid‐phase synthesis, 33 or 55 equivalents of the activated acid were used, depending on the length of the oligonucleotide. For in‐solution conjugation, 250 equivalents of the activated acid added in two portions proved the best efficiency.

A practical problem encountered here is the limited solubility of certain peptides in pure acetonitrile, typically tripeptides and longer sequences. To address this issue, the preparation of the peptide stock solution has been altered as followed: the calculated amount of peptide is weighed in an Eppendorf tube and 900 µl acetonitrile is added. If the peptide solubilizes, 100 µl acetonitrile is added to reach a total volume of 1 ml to reach the desired concentration of 50 mM. If the peptide does not dissolve, then 100 µl DMSO is added instead of acetonitrile. In our cases, this modification has consistently ensured complete solubilization.

Due to the sensitivity of ONs, the use of acid‐labile protecting groups is impossible, especially in solution. Consequently, the introduction of a Boc‐protected amine, such as lysine, must be performed on CPG‐beads. As detailed in the Understanding Results section, a Boc‐protected lysine has been introduced and deprotected very efficiently, without loss of yield, compared to the reaction without the Boc‐deprotection. To prevent ON degradation by TFA, it is essential to follow the specific conditions outlined in Support Protocol. The solid support facilitates the rapid and efficient removal of TFA, minimizing the risk of degradation.

The two methods described are complementary. Indeed, it is complicated to anticipate the HPLC profile of the crude and, consequently, whether purification will be straightforward. In general, POCs resulting from a solid‐phase synthesis are easier to purify, as depicted with the conjugation of biotin (Fig. 7).

For in‐solution conditions, it is advised to keep the Fmoc‐protecting group as it increases the lipophilicity and simplifies purification. After isolation, the Fmoc is removed by a treatment with piperidine (10% in water as described in Basic Protocol 3). A short second purification might be needed.

Time Considerations

The synthesis of a 5’‐amino‐modified oligonucleotide takes ∼0.5 days if the oligonucleotide remains on the CPG‐beads, or 1.5 days if it is cleaved from the support. The conjugation reaction itself requires 12 to 15 hr. Purification and analysis requires 1 full day due to the necessity to lyophilize the sample prior absorbance measurement. Overall, the iterative synthesis of a tripeptide–oligonucleotide requires 5 working days.

Author Contributions

Marion Gras: Data curation; formal analysis; investigation; writing—original draft. Michael Smietana: Funding acquisition; project administration; supervision; validation; writing—review and editing. Pauline Adler: Data curation; project administration; supervision; writing—original draft.

Conflict of Interest

The authors declare no conflict of interest.

Gras, M. , Smietana, M. , & Adler, P. (2025). Peptide–oligonucleotide conjugates: Catalytic preparation in aqueous solution or on‐column. Current Protocols, 5, e70154. doi: 10.1002/cpz1.70154

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.