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Synthesis and Application of Highly Reactive Amino Linkers for Functional Oligonucleotides

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Abstract
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Literature Cited

Abstract

Oligonucleotides are functionalized by conjugation with a variety of molecules, and aliphatic amino linkers have been frequently used as a tether for their modifications. This unit describes the syntheses and applications of novel amino linkers having a carbamate structure. Two major chemical properties of the primary amine are induced by the neighboring effect of the carbamate group, which are found to be optimum in an aminoethyl carbamate structure. First, the hydrophobic monomethoxytrityl group can be rapidly removed from the aminoethyl carbamate under very mild acidic conditions, while the deprotection is not completed in standard aliphatic amines even under high acid concentration. This significant feature enables the convenient purification of amino?modified oligonucleotides by using the hydrophobic interaction of the monomethoxytrityl group with a reverse?phase resin. Second, the introduction of the carbamate linkage reduces the pK a value of the neighboring primary amine, resulting in an increase in the conjugation yields with various functional molecules, such as those having active esters. The novel amino linkers that have an aminoethyl carbamate linkage indicate potent activity and are applicable for the preparation of various functional oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 48:4.48.1?4.48.23. © 2012 by John Wiley & Sons, Inc.

Keywords: oligonucleotide; amino linker; carbamate; purification; modification; array; conjugation

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Introduction
Basic Protocol 1: Preparation of ssR Linker Phosphoramidites
Basic Protocol 2: Synthesis and Purification of ssR‐Modified Oligonucleotides
Basic Protocol 3: Incorporation of Reporter Groups to Amino‐Modified Oligonucleotides
Commentary
Literature Cited
Figures
Tables

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Basic Protocol 1: Preparation of ssR Linker Phosphoramidites Materials

(S )‐(+)‐2,2‐Dimethyl‐1,3‐dioxolane‐4‐methanol, 98% ( S.1 ; Aldrich)
1‐(Chloromethyl)naphthalene, 90% ( S.2 ; Aldrich)
Toluene, anhydrous, drying with 4Å molecular sieves
1,4‐Dioxane, anhydrous, drying with 4Å molecular sieve
Potassium hydroxide (KOH)
Argon gas
Ethyl acetate (EtOAc)
Hexane
Brine (saturated aqueous sodium chloride; NaCl)
Sodium sulfate (Na 2 SO 4 ), anhydrous
Acetic acid
Ethanol (EtOH)
Silica gels: Wakogel C‐200 (particle size 75 to 150 µm, Wako Pure Chemical Industries) or Silica Gel 60 (particle size 105 to 210 µm, Nacalai Tasque)
Pyridine, anhydrous, drying with 4Å molecular sieves
p ‐Toluenesulfonyl chloride (p ‐TsCl)
Saturated aqueous sodium hydrogencarbonate (NaHCO 3 )
N ,N ‐Dimethylformamide (DMF), anhydrous, drying with 4 Å molecular sieves
Sodium azide (NaN 3 )
Ammonium chloride (NH 4 Cl)
Palladium‐activated carbon (Pd 10%), (Pd/C)
Hydrogen gas (H 2 )
4‐Methoxytrityl chloride (MMTCl)
Triethylamine (Et 3 N)
(R )‐(–)‐Glycidyl methyl ether ( S.5 ; Wako Pure Chemical Industries)
(S )‐(+)‐1‐Amino‐2‐propanol, 98% ( S.6 c ; Aldrich)
2‐Aminoethanol, 99% ( S.6 d ; Wako Pure Chemical Industries)
1,1′‐Carbonyldiimidazole
4‐Dimethylaminopyridine (DMAP)
6‐Amino‐1‐hexanol
N ,N ‐Diisopropylethylamine (redistilled), 99.5% (Sigma‐Aldrich)
2‐Cyanoethyl N ,N ‐diisopropylchlorophosphoramidite (Wako Pure Chemical Industries)
3‐Amino‐1‐propanol, 98% ( S.10 ; Wako Pure Chemical Industries)
Chloroform (CHCl 3 )
Dimethyl sulfoxide‐d 6 (DMSO‐d 6 ), 99.9% atom% D
Dichloromethane (CH 2 Cl 2 ), anhydrous, drying with 4 Å molecular sieves

50‐, 100‐, 200‐, 300‐, and 500‐mL round‐bottom flasks
Condenser
TLC, Merck silica gel 60F 254 precoated plates
254‐nm UV lamp (for TLC)
Rotary evaporator equipped with a diaphragm pump
Glass column with a diameter of 2.0 cm, 3.5 cm, 4.0 cm, 4.5 cm, and 5.0 cm
Vacuum oil pump
Celite pad

Additional reagent and equipment for TLC ( appendix 3D ) and column chromatography ( appendix 3E )

Basic Protocol 2: Synthesis and Purification of ssR‐Modified Oligonucleotides Materials

ssR Linker phosphoramidites ( S.9 a‐d and S.13 ; see protocol 1 )
Acetonitrile (CH 3 CN), anhydrous, drying with 3 Å molecular sieves
Standard 2′‐deoxynucleoside 5′‐O ‐(4,4′‐dimethoxytrityl) phosphoramidites (Glen Research):
- N ‐Benzoyl‐2′‐deoxyadenosine phosphoramidite
- N ‐Benzoyl‐2′‐deoxycytidine phosphoramidite or N ‐Acetyl‐2′‐deoxycytidine phosphoramidite
- N ‐Isobutylyl‐2′‐deoxyguanosine phosphoramidite
- Thymidine phosphoramidite
Argon gas
28% Aq. ammonia
2 M Triethylammonium acetate (TEAA), pH 7.0 (Glen Research)
Sterilized H 2 O
Trifluoroacetic acid
Buffer A: 5% CH 3 CN in 0.1 M TEAA, pH 7.0 (reverse‐phase HPLC)
Buffer B: 50% CH 3 CN in 0.1 M TEAA, pH 7.0 (reverse‐phase HPLC)
Buffer C: 20% CH 3 CN in H 2 O (ion‐exchange HPLC)
Buffer D: 20% CH 3 CN in 2 M ammonium formate (ion‐exchange HPLC)

DNA synthesizer
4‐mL screw‐capped glass vial
55°C incubator
Reverse‐phase open column (YMC C18, 500 mg)
Rotary evaporator equipped with a diaphragm pump
0.45 µm disposable filter
1.5‐mL microcentrifuge tubes
HPLC system with:
- Column: 3.9 × 150 mm µ‐Bondasphere (Waters) or 4.6 × 250 mm TSK‐gel DEAE‐2SW (TOSOH)
- Detector: 254 nm

Additional reagents and equipment for automated solid‐phase ONT synthesis ( appendix 3C ) and purification of ONTs (units 10.1 , 10.4 , 10.5 , 10.7 & 3.NaN )

Basic Protocol 3: Incorporation of Reporter Groups to Amino‐Modified Oligonucleotides Materials

5′‐amino‐modified ONT (X‐25: 5′‐X‐TCTTCCAAGCAATTCCAATGAAAGC, X = ssR, C6, C5; see protocol 2 )
1 M sodium phosphate buffer (pH 8)
Sterilized H 2 O
Fluorescein isothiocyanate (FITC; Dojindo)
N ,N ‐Dimethylformamide (DMF), anhydrous, drying with 4 Å molecular sieves
Biotin succinimidyl ester (Biotin‐NHS) (Dojindo)
2 M Triethylammonium acetate (TEAA), pH 7.0 (Glen Research)
Buffer A, C: 5% CH 3 CN in 0.1 M TEAA, pH 7.0 (reverse‐phase HPLC)
Buffer B: 50% CH 3 CN in 0.1 M TEAA, pH 7.0 (reverse‐phase HPLC)
Acetonitrile (CH 3 CN), anhydrous, drying with 3 Å molecular sieves
Argon gas
Cholesteryl chloroformate
4‐Dimethylaminopyridine (DMAP)
N ,N ‐Diisopropylethylamine (DIEA; redistilled), 99.5% (Sigma‐Aldrich)
Dichloromethane (CH 2 Cl 2 ), anhydrous, drying with 4 Å molecular sieves
28% Aq. ammonia
Buffer D: 100% CH 3 CN

NAP10 column (Amersham Pharmacia)
HPLC system with:
- Column: 3.9 × 150 mm µ‐Bondasphere (Waters)
- Detector: 254 nm
0.45‐µm Disposable filter
1.5‐mL microcentrifuge tubes
DNA/RNA synthesizer
4‐mL Screw‐capped glass vial
Rotary evaporator equipped with a diaphragm pump
Sintered glass funnel

Additional reagents and equipment for automated solid‐phase ONT synthesis ( appendix 3C ), and purification of ONTs (units 10.1 , 10.4 , 10.5 , 10.7 & 3.NaN )

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Figure 4.48.1 (A ) Structures of the 5′‐terminal amino linkers. Both C6 and C5 are commercially available linkers. (B ) Schematic drawing for chemical properties of the ssR linker. ONT and MMT indicate the oligonucleotide and monomethoxytrityl group, respectively.

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Figure 4.48.2 Scheme for the synthesis of ssR linker phosphoramidites (see ).

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Figure 4.48.3 Scheme for the synthesis of ssPro linker phosphoramidite (see ).

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Figure 4.48.4 Stability of the MMT group of ssH‐modified ONTs in aqueous buffers. The MMT‐protected ssH‐ONTs were incubated in 250 mM phosphate buffers at pH 6 (solid circles), pH 7 (solid diamonds), and pH 8 (solid triangles), followed by HPLC analyses.

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Figure 4.48.5 HPLC analyses of (A ) The ssH‐modified ONT and (B ) The C6‐modified ONT before and after open column purification. The ONT sequences are 5′‐X‐TCTTCCAAGCAATTCCAATGAAAGC (X‐25, X = ssH and C6).

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Figure 4.48.6 Intramolecular reactions of ssR‐modified ONTs under heated alkaline condition. (A ) Schematic drawing of the reactions. Aminohexyl product (C6 linker, P1) and cyclic carbamate (c‐carbamate) were generated by path 1. The urea form (P2) was generated by path 2. (B ) Percentages of the products (P1 and P2) of each ONTs after ammonia treatment at 60°C for 15 h. White and black bars indicate percentages of P1 and P2 products, respectively.

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Figure 4.48.7 Labeling reactions of X‐25 (X = C6, C5, and ssR) with functional molecules. (A ) Percentages of ONTs labeled with FITC. The reaction was carried out in 250 mM sodium phosphate buffer (pH 8) for 30 min. (B ) C6‐25 (solid diamonds), ssH‐25 (solid circle), and ssN‐25 (solid triangle) were labeled with biotin‐NHS in the presence of various concentrations of the bicarbonate buffer (pH 9 and 8). Percentages of the products are plotted against the buffer concentration.

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Figure 4.48.8 Anti ‐conformation structure of the ssR linker unit.

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