Expanding the Range of Methods for Obtaining Diverse Representatives of Sulfonyl Phosphoramidate Oligonucleotides

Abstract

Sulfonyl phosphoramidates are a class of internucleotide phosphate modifications that can be easily introduced during automatic solid-phase synthesis at the oxidation step using the Staudinger reaction with appropriate electron-deficient sulfonyl azides. Beyond altering the nature of the backbone, this class of modifications enables the introduction of various substituents within the structure of oligonucleotides. The existing method, which relies on a limited set of precursors, enables obtaining only a narrow range of representatives within this class. In this work, we report an approach for obtaining diverse representatives of the sulfonyl phosphoramidate class based on the introduction of commercially available building blocks with different functional groups into the structure of modification. The first proposed method involves the incorporation of chloroalkanesulfonyl azides, followed by the substitution of the chlorine atom with amine residues within the sulfonyl phosphoramidate derivative. Initial evaluation of a series of sulfonyl azides bearing terminal chlorine atoms on alkyl chains of varying lengths (C1–C3) led to the selection of 3-chloropropanesulfonyl azide as the most promising agent. A library of sulfonyl phosphoramidate derivatives bearing various amine residues, including bulky alkyl and polyamine moieties, was synthesized by using this azide. The introduction of reactive groups into a growing oligonucleotide chain enabled the multistep assembly of complex structures on a solid-phase support. In addition, the second method was proposed, involving the synthesis of sulfonyl azides already bearing amine residues for subsequent incorporation within oligonucleotides. This method can be implemented via three synthetic routes: introducing a lipophilic amine residue into the chloroalkanesulfonyl azide structure, synthesis of azides via sulfolactone ring-opening alkylation to obtain quaternized derivatives, and obtaining sulfamoyl azides. The developed methods are complementary, with one enabling the effective incorporation of functional residues that are difficult to introduce with the other two. The proposed approach enables a significant expansion of the set of sulfonyl phosphoramidate oligonucleotide derivatives by utilizing commercially available building blocks and straightforward reactions.

1. Introduction

Synthetic oligonucleotides have found numerous applications in various fields of molecular biology, biotechnology, and medicine, among which therapeutic application is of great interest. − To date, more than 20 oligonucleotide drugs have been approved by the US Food and Drug Administration (FDA), the vast majority of which are based on modified oligonucleotides. , Modifications provide the properties essential for the therapeutic application of nucleic acid including effective cellular uptake, resistance to enzymatic hydrolysis, and duplex stability. ,

Various strategies have been developed to introduce modifications at different positions within oligonucleotides, particularly within the nitrogenous bases, ribose moieties, and the internucleotide phosphate linkages. , Non-nucleotide modifiers are typically used to introduce functional fragments. In most cases, introducing modifications requires a laborious and costly process of obtaining the appropriate phosphoramidite reagents.

Of particular interest are modifications of the internucleotide phosphate group where the nonbridging oxygen atom is replaced by different substituents. Some of these modifications can be introduced specifically during the oxidation step of the phosphoramidite synthesis cycle. The key advantage of such an approach, based on introducing a modification using at an altered oxidation step, lies in its compatibility with standard synthesis protocols and reagent sets.

The most common modification introduced by this method is phosphorothioate. The method allows highly efficient yield of oligonucleotides up to a completely modified backbone. The phosphorothioate backbone enables efficient cellular uptake without the need for special transfection reagents due to its ability to bind with proteins. However, the nonspecificity of such interaction results in cytotoxicity.

In addition to phosphorothioate, other phosphate modifications introduced at the oxidation step have been developed, which enable not only an alteration of the nature of the backbone but also the attachment of various functional groups at the internucleotide phosphate group. However, these modifications are either limited in their compatibility with standard phosphoramidite monomers and require a special set of protective groups, such as boranophosphates, or require the use of H-phosphonate chemistry to achieve high incorporation efficiency, such as alkyl phosphoramidates.

In 2014, a new class of phosphate-modified oligonucleotides, phosphoryl guanidines, was proposed, and then an approach for obtaining them was developed based on oxidation of phosphite triesters by highly reactive electron-deficient azides via the Staudinger reaction, , which is fully compatible with the standard phosphoramidite protocol. As part of the further development, the approach was adapted to introduce other classes of internucleotide phosphate modifications. Among them was the previously proposed class of sulfonyl phosphoramidates. For the simplest representative of the class, methylsulfonyl (mesyl) phosphoramidate, the possibility of obtaining oligonucleotides with a completely modified backbone has been demonstrated, and therapeutic potential has been shown. − Sulfonyl phosphoramidates, in contrast to phosphoryl guanidines and previously discovered triazinyl phosphoramidates, retain negative charge and are capable of activating RNaseH, but are not prone to nonspecific interaction with proteins, unlike phosphorothioates. Such a class-related set of properties has enabled sulfonyl phosphoramidates to establish their own special niche in the field of nucleic acid chemistry. Now sulfonyl phosphoramidates together with phosphoryl guanidine and phosphorothioate modifications are regarded as a promising toolkit for the development of therapeutic oligonucleotides. −

It is worth noting that sulfonyl phosphoramidates represent an expandable class of modifications that enable the introduction of diverse functional groups in addition to backbone alteration. A number of various alkyl- and arylsulfonyl chlorides, the closest precursors of sulfonyl azides, already containing the corresponding substituents in their structure, are commercially available. Sulfonyl azides obtained from the appropriate chlorides in a one-step reaction have been used in a series of studies for the introduction of modifications. − However, the diversity of such reagents is still limited, and methods are currently being developed for the synthesis of sulfonyl azides bearing various functional groups and for the assembly of complexly modified oligonucleotide structures using sulfonyl phosphoramidate modifications. −

In this work, we report an approach based on the use of common precursors for obtaining various representatives of the sulfonyl phosphoramidate class, involving the introduction of commercially available building blocks with different functional groups into the structure of modification. This approach employs two methods: the first one involves the introduction of an azide precursor into the oligonucleotide followed by the attachment of substituents to the sulfonyl phosphoramidate group, and the second one is based on the use of presynthesized azides containing the target substituents.

2. Results

2.1. Chloroalkylsulfonyl Azides: Initial Evaluation of the Reactivity and the Possibility of Substituting Chlorine Atoms with Amine Residues within Oligonucleotide Derivatives under Final Deblock Conditions

To implement the first approach, which is based on incorporating an azide precursor within an oligonucleotide, commercially available sulfonyl chlorides bearing alkyl substituents of 1–3 carbon atoms with a terminal chlorine atom were selected (chloromethanesulfonyl chloride, 2-chloroethanesulfonyl chloride, 3-chloropropanesulfonyl chloride). The scheme involves introducing azides via the Staudinger reaction, followed by replacing the chlorine atom with amine residues bearing various substituents (Figure )

1.

Scheme for introducing modifications using sulfonyl azides bearing a terminal chlorine atom within the alkyl substituent: the Staudinger reaction (1) and nucleophilic substitution of chlorine with an amine residue within a sulfonyl phosphoramidate oligonucleotide derivative (2). R = 2-cyanoethyl residue.

Sulfonyl azides were synthesized by treating the corresponding chloride with an excess of sodium azide in acetonitrile, and solutions of the azides were then used without further purification. Since more harsh conditions and appropriate solvents are required to obtain alkylazides from the corresponding halides, it was assumed that only the sulfonyl chlorine atom would be substituted under these conditions. The Staudinger reaction was carried out under rather mild conditions in all cases: 1 h, 25 °C, 0.5 M solution, taking into account the high reactivity of electron-deficient sulfonyl azides with compact substituents. The alternative oxidation protocol via the Staudinger reaction was carried out in manual mode.

For the initial evaluation of the effectiveness of the Staudinger reaction and the subsequent chlorine substitution steps, no special amine treatment procedure was performed and the possibility of substitution during oligonucleotide cleavage from the solid-phase support was investigated (Table ). Two contrast conditions for the final deblock were used: mild (saturated aqueous ammonia solution, 25 °C, 30 min) and harsh (saturated aqueous methylamine solution, 55 °C, 30 min) (see Table ). It was assumed that under mild conditions, chlorine substitution would not occur, which would enable the effectiveness of the Staudinger reaction to be estimated. In contrast, it was expected that under harsh conditions, the substitution reaction would take place, thereby enabling amination at the final deblocking step. A series of experiments on octathymidylate system with sulfonyl modification positioned at the closest to the 5′-end internucleotide phosphate (5′-T*TTTTTTT-3′) were conducted to minimize the possible side reactions.

1. Series of Oligonucleotides Obtained Using Chloroalkylsulfonyl Chlorides for Initial Evaluation of Azide Reactivity and the Possibility of Substituting Chlorine Atoms with Amine Residue Derivatives under Final Deblock Conditions .

code	azide	conditions of the cleavage	R group	conversion	M r(theor)	M r(exp)
OD1	A1	NH3 (aq.), 30 min, 25 °C	Cl-CH2–CH2–CH2–	∼95%	2509.4	2509.8
OD2	A1	MeNH2 (aq.), 30 min, 55 °C	H3C-NH–CH2–CH2–CH2–	∼95%	2504.5	2503.3
OD3	A1	MeNH2 (aq.), 30 min, 25 °C	Cl-CH2–CH2–CH2–	∼95%	2509.4	2509.4
OD4	A1	NH3 (aq.), 20 h, 55 °C	H2N–CH2–CH2–CH2–	∼95%	2490.5	2490.8
OD5	A2	NH3 (aq.), 30 min, 25 °C	N3 –CH2–CH2–	∼85%	2502.4	2501.4
OD6	A3	NH3 (aq.), 30 min, 25 °C	CH2CH-	∼90%	2459.4	2459.2
OD7	A3	MeNH2 (aq.), 30 min, 55 °C	H3C-NH–CH2–CH2–	∼80%	2490.5	2490.4
OD8	A4	NH3 (aq.), 30 min, 25 °C	Cl-CH2–	∼95%	2481.4	2481.4
OD9	A4	MeNH2 (aq.), 30 min, 55 °C	Cl-CH2–	∼95%	2481.4	2481.2
OD10	A1	NH3 (aq.), 30 min, 25 °C	Cl-CH2–CH2–CH2–	∼95%	2509.4	2509.4

^aThe Staudinger reaction conditions: 0.5 M solution of the corresponding azide in acetonitrile, 1 h, 25 °C. Sequences: 5′-T*TTTTTTT-3′ for OD1-OD9; 5′-TTTTTTT*T-3′ for OD10. The conversion values were calculated from the peak areas in HPLC profiles (Figures S1 and S2) and relate to the major products of modification introduction. For PAGE, see Figure S4. For mass spectra, see Supporting Information, Figures S5 and S8. R₁2-cyanoethyl residue.

^bSubstituent within the major sulfonyl phosphoramidate product.

2.1.1. Application of 3-Chloropropanesulfonyl Azide

First, the introduction of a modification using 3-chloropropanesulfonyl azide (A1, compound in which the chlorine atom is furthest from the sulfonyl group, C3-azide) in the synthesis of a set of test octathymidilates was investigated (Figure and Table , OD1-OD4).

2.

Obtaining 3-chloropropylsulfonyl phosphoramidate and conversion of the resulting derivative under various deblocking conditions. Staudinger reaction conditions: 0.5 M solution of 3-chloropropanesulfonyl azide (A1) in acetonitrile, 1 h, 25 °C. Final deblock conditions: OD1conc. aq. ammonia solution, 30 min, 25 °C; OD2conc. aq. methylamine solution, 30 min, 55 °C; OD3conc. aq. methylamine solution, 30 min, 25 °C; OD4conc. aq. ammonia solution, 20 h, 55 °C. R₁2-cyanoethyl residue.

The HPLC profiles of reaction mixtures (Figure a) show that derivatives obtained at different deblocking conditions have distinguished mobilities: the retention time of the major product in the reaction mixture of the OD1 oligonucleotide treated at mild conditions is slightly higher than in the case of OD2 treated at harsh conditions. It should be noted that the bifurcation of peaks in each case is due to the presence of two diastereomers of phosphate-modified oligonucleotides having distinguished retention times.

3.

(a) RP HPLC profiles of the reaction mixtures of oligonucleotides OD1 and OD2. The acetonitrile gradient from 0 to 50% in 15 min in 0.02 M TEAA buffer. (b) Electrophoretic mobility of the reaction mixtures of oligonucleotides OD1 and OD2 in 15% PAGE. 1unmodified octathymidylate control (T8); 4modified octathymidylatoligonucleotide 5′-T*TTTTTTT-3′ bearing a phosphoryl guanidine modification (1,3-dimethylimidazolidin-2-ylidene phosphoramidate). Z = total charge of oligonucleotides. The chemical structures relate to the major products of modification introduction. For full PAGE, see Supporting Figure S3.

The PAGE analysis (Figure b) showed that the mobility of oligonucleotide OD1 (Lane 2) decreased slightly compared to the unmodified octathymidylate control T8 (Lane 1), since the sulfonyl phosphoramidate group, like native phosphate, is negatively charged, while the size of the molecule slightly increased due to the introduced modification. For the OD2 derivative, mobility decreased significantly, indicating the presence of a positively charged amino group in its structure, which led to a decrease in the total charge of the oligonucleotide. We used an octathymidylate bearing a single uncharged phosphoryl guanidine modification (Figure b, PG, Lane 4) as a control of mobility for the modified oligonucleotide with reduced total negative charge by one (Z = −6) compared to T8 (Z = −7). The similar electrophoretic mobility of oligonucleotides OD2 and PG confirms that both of them have a total charge Z = −6. The slight difference in the PAGE mobility between OD2 and PG, both having equal charges, is due to the disparity in the sizes of modifications. Based on the data obtained, it can be concluded that the Staudinger reaction proceeded with high efficiency, and in the case of OD1, the chlorine atom remained in the modification structure, whereas in the case of OD2, a methylamine residue was incorporated at an efficiency close to quantitative. These observations were confirmed by ESI mass analysis (Table ).

The further experiments, where the conditions of the final deblock step were varied, showed that under treatment with aqueous methylamine at room temperature (Table , OD3), substitution of the chlorine atom did not occur at all, as in the case of ammonia solution. Meanwhile, to achieve quantitative substitution of the chlorine atom with ammonia, much harsher conditions were required than for methylamine, 20 h at 55 °C (Table , OD4), whereas at treatment for 3 h, only 50% substitution was achieved (oligonucleotide OS1, see Supporting, Table S1 and Figures S9–S10). It can be explained by the significantly lower nucleophilicity of ammonia compared to methylamine.

2.1.2. Application of 2-Chloroethanesulfonyl Azide and 2-Azidoethanesulfonyl Azide

The next step was to study the reactivity of the 2-chloroethanesulfonyl azide (C2-azide). In contrast to the synthesis of C3-azide (A1), treatment of 2-chloroethanesulfonyl chloride with an excess of sodium azide resulted in substitution of both sulfonyl and alkyl chlorine atoms, obtaining 2-azidoethanesulfonyl azide (Figure , A2). This process can be explained by the high reactivity of the chlorine atom in the β-position relative to that of the acceptor sulfonyl group.

4.

Obtaining 2-azidoethanesulfonyl azide (A2) and 2-chloroethanesulfonyl azide (A3), and conversion of the resulting sulfonyl phosphoramidate derivatives under various deblocking conditions. Staudinger reaction conditions: 0.5 M solution of the corresponding azide in acetonitrile, 1 h, 25 °C. Final deblock conditions: OD5conc. aq. ammonia solution, 30 min, 25 °C; OD6conc. aq. ammonia solution, 30 min, 25 °C; OD7conc. aq. methylamine solution, 30 min, 55 °C. R₁2-cyanoethyl residue.

In the synthesis of model octathymidylate (OD5), it was shown that the Staudinger reaction proceeded with high efficiency, with the participation of the more reactive sulfonyl azido group of the obtained diazide (A2). The alkyl azido group, as expected, did not undergo substitution either under mild deblocking conditions (see Table , OD5, for HPLC and PAGE analysis see Supporting, Figures S1 and S4) or even during harsh methylamine treatment (oligonucleotide OS2, see Supporting, Table S1 and Figures S9 and S10).

In order to replace only the sulfonyl chlorine atom in 2-chloroethanesulfonyl chloride, the reaction with 1 equiv of sodium azide was carried out at cooling, thereby obtaining 2-chloroethanesulfonyl azide (A3, C2-azide).

As with the C3-azide (A1), transformations of modifications in model octathymidylates obtained with the use of C2-azide were studied under two contrast conditions of the final deblock step (Table , OD6 and OD7). According to the ESI mass analysis of reaction mixtures, the Staudinger reaction proceeded with high efficiency. Under harsh conditions (OD7), a major product was a derivative bearing a methylamine residue, and a vinyl sulfonyl derivative as a minor byproduct was found. Meanwhile, under mild deblocking conditions (OD6) vinyl sulfonyl derivative was the major product (Table and Figure , OD6).

Such a set of products obtained in different conditions could be explained by the tendency of 2-chloroethyl sulfonyl phosphoramidate to eliminate HCl with the formation of a vinyl sulfonyl fragment containing a conjugated system of bonds. This meets the literature data about the elimination of β-chloroalkylsulfonyl compounds under basic conditions. , Upon treatment with methylamine, this compound may react as a Michael donor in the nucleophilic addition reaction. , Therefore, C2 reagents such as 2-chloroethanesulfonyl azide and 2-azidoethanesulfonyl azide could be efficiently applied in the oxidation step, but the possibility of vinyl derivative formation should be taken into account during planning the amination step.

2.1.3. Application of Chloromethanesulfonyl Azide

Next, we moved on to studying chloromethanesulfonyl azide (C1-azide). Even with an excess of sodium azide, no substitution of the alkyl atom with chlorine occurred during the synthesis of azide from chloromethanesulfonyl chloride (Figure ). The obtained azide exhibited a high efficiency in the Staudinger reaction. However, the substitution of the chlorine atom did not proceed under either mild (OD8) or harsh (OD9) deblocking conditions. Analyzing the literature data, we found that halogen atoms at the α position to the sulfonyl group demonstrate abnormally low reactivity in nucleophilic substitution reactions. This is attributed to the hindrance of nucleophile attack in the SN2 reaction, caused by the tetrahedral sulfonyl group bearing two oxygen atoms with high electron density. This explains the absence of substitution, even under harsh deblocking conditions.

5.

Obtaining chloromethanesulfonyl azide (A4) and conversion of the resulting sulfonyl phosphoramidate derivative under deblocking conditions. Staudinger reaction conditions: 0.5 M solution of chloromethanesulfonyl azide in acetonitrile, 1 h, 25 °C. Deblocking conditions: OD8conc. aq. ammonia solution, 30 min, 25 °C; OD9concentrated aqueous methylamine solution, 30 min, 55 °C. R₁2-cyanoethyl residue.

After the incorporation of modifications within the internucleotide phosphate closest to the 5′ end of the model octathymidylate was successfully performed, their stability during the cycle of oligonucleotide synthesis was investigated. For this purpose, the modifications were introduced into the position closest to the 3′-end of the model octathymidylate (5′-TTTTTTT*T-3′, where * is the position of sulfonyl phosphoramidate modification). These experiments showed that modifications introduced using all of the obtained azides (A1, A3, A4 bearing terminal chlorine atoms as well as A2 bearing terminal azide group) are stable under oligonucleotide synthesis conditions (see Table , OD10 and Supporting, Table S1 and Figures S9 and S10, oligonucleotides OS3-OS5).

Based on the data obtained, 3-chloropropanesulfonyl azide (A1) was selected as the most promising compound for further research due to the simplicity of its synthesis, good predictability of the substitution process, and the absence of side reactions.

2.2. Obtaining a Library of Sulfonyl Phosphoramidate Derivatives via the Substitution of Chlorine Atom with Various Amines in a 3-Chloropropanesulfonyl Phosphoramidate Precursor

3-Chloropropanesulfonyl azide, selected after the initial evaluation, was used for obtaining an octathymidlilate (5′-T*TTTTTTT-3′) library of sulfonyl phosphoramidate derivatives bearing various amine residues. The introduction of amines within the oligonucleotide derivative was carried out after the Staudinger reaction as a separate amination step using solutions of the appropriate amines. After detritylation, the oligonucleotides were cleaved from the solid-phase support using aq. ammonia under mild conditions to avoid substitution of unreacted chlorine at the final deblock step (Figure a).

6.

(a) Amination step: substitution of chlorine atom with an amine residue in a 3-chloropropylsulfonyl phosphoramidate precursor before oligonucleotide cleavage from the solid-phase support. R = −CH₂–CH₂–CN. (b) The library of sulfonyl phosphoramidate derivatives bearing various amine residues.

Preliminary experiments (oligonucleotide OS6, see Supporting Information, Table S2 and Figure S26) demonstrated that even when using the compact and highly nucleophilic secondary amine, piperidine, harsh conditions were necessary to achieve a high yield of substituted product: 50 °C, 20 h, 30% v/v acetonitrile solution.

Subsequently, these conditions were used for the introduction of the most compact alkylamines and diamines (Table , Conditions a). For the incorporation of more hindered alkylamines, aromatic amines, and tri- or polyamines, the reaction time was increased to 40 h (Table , Conditions b). Special conditions were selected for amines that are solid and insoluble in acetonitrile: 30 mg in an appropriate solvent (Table , Conditions c-f).

2. Library of Derivatives Obtained via the Substitution of a Chlorine Atom with Various Amines in a 3-Chloropropylsulfonyl Phosphoramidate Precursor .

code	amine	conditions	conversion	M r(theor)	M r(exp)
OL1	Propylamine	a	∼60%	2532.5	2532.4
OL2	Butylamine	b	∼70%	2546.5	2546.4
OL3	Methylbutylamine	b	∼70%	2560.5	2560.4
OL4	Piperidine	a	∼90%	2558.5	2557.4
OL5	Dodecylamine	c	∼60%	2658.6	2658.6
OL6	N-Methyldodecylamine	c	∼60%	2672.7	2673.1
OL7	Diethylamine	b	∼40%	2546.5	2546.4
OL8	Dibutylamine	b	∼40%	2602.6	2602.4
OL9	Benzylamine	b	∼95%	2580.5	2580.4
OL10	Aniline	b	∼50%	2566.5	2566.4
OL11	Piperazine	d	∼95%	2559.5	2559.4
OL12	N-Methylpiperazine	a	∼95%	2573.5	2573.4
OL13	Ethylenediamine	e	∼95%	2533.5	2533.4
OL14	1,3-diaminopropane	a	∼95%	2547.5	2547.4
OL15	1,4-diaminobutane	a	∼95%	2561.5	2561.3
OL16	N,N′-dimethylethylenediamine	a	∼95%	2561.5	2561.4
OL17	3-(dimethylamino)-1-propylamine	a	∼90%	2575.5	2575.7
OL18	3,3′-iminobis(N,N-dimethylpropylamine)	b	∼90%	2660.6	2660.6
OL19	3,3′-diaminodipropylamine	b	∼90%	2604.6	2604.4
OL20	Tris(2-aminoethyl)amine	f	∼90%	2921.7	2921.6
OL21	Spermine	f	∼90%	2977.8	2977.6
OL22	3-Methylamino-1-propanol	a	∼95%	2562.5	2562.4
OL23	Morpholine	a	∼95%	2560.5	2560.4
OL24	Imidazole	g	∼40%	2541.5	2541.4

^aStaudinger reaction conditions: 0.5 M solution of 3-chloropropanesulfonyl azide in acetonitrile, 1 h, 25 °C. Sequence 5′-T*TTTTTTT-3′. The conversion values were calculated from the peak areas in HPLC profiles (Figures S11–S14) and relate to the desired products of amine introduction. For PAGE, see Figures S15–S17. For mass spectra, see Supporting Information, Figures S18–S25. Conditions for the introduction of amine residue: (a) 30% vol. in ACN, 20 h, 50 °C; (b) 30% vol. in ACN, 40 h, 50 °C; (c) 30 mg in 100 μL toluene, 40 h, 50 °C; (d) 30 mg in 100 μL CHCl₃, 20 h, 50 °C; (e) 30% vol. in toluene, 20 h, 50 °C; (f) 30% vol. H₂O, 20 h, 50 °C; (g) 15 mg in 100 μL ACN + 1 equiv DIPEA.

First, amines bearing alkyl substituents of varying lengths were introduced. As can be seen (Table ), conversion declines as the size of the alkyl substituents increases, most notably for secondary amines with two bulky alkyl moieties. For example, the incorporation efficiency of methyldodecylamine (OL6) bearing one bulky substituent was around 60%, whereas for dibutylamine (OL8) bearing two less bulky substituents, the conversion is even lower (∼40%). In the case of dihexylamine (oligonucleotide OS7, see Supporting Table S2 and Figure S26), the target product was not obtained in a significant amount. It is worth noting that the best conversion, close to quantitative, was achieved for the compact secondary amine piperidine (OL4).

Moreover, benzylamine (OL9) and aniline (OL10) were introduced. The incorporation efficiency of aniline was low due to the reduced nucleophilicity of aromatic amines, whereas for benzylamine, it was comparable to that of compact alkylamines.

Next, incorporation of di- and triamines bearing primary, secondary, and tertiary amine groups was performed, in order to increase the number of protonatable groups introduced per modification. Analyzing the conversion values, it can be noticed that diamines generally react better than monoamines of comparable size, for example, propylamine (OL2) compared to diaminopropane (OL14), while both were introduced under condition A (Table ). We can assume that increased reactivity of diamines is due not only to the presence of multiple reaction centers in the used amine, but also to the fact that extra amino group may stabilize the resulting transition state. For the bulkier triamines (OL18, OL19), an increase in conversion was also observed relative to alkylamines, for example, for 3,3′-iminobis­(N,N-dimethylpropylamine) (OL18) compared to the sterically similar dibutylamine (OL8).

Polyamines (tris­(2-aminoethyl)­amine and spermine) bearing 4 amino groups were also introduced. Acetonitrile solutions of these amines were found to significantly cleave the oligonucleotide from the solid-phase support, resulting in almost complete loss of the product during the amination step. Therefore, introducing these amines was carried out at the final deblocking step using aqueous solutions, as was previously done for methylamine introduction but under harsh conditions (20 h, 50 °C). During the process, the amine was incorporated, and the oligonucleotide was cleaved from the solid-phase support. In this way, derivatives bearing tris­(2-aminoethyl)­amine and spermine residues (OL20, OL21) were obtained with a high efficiency.

The total charges of the obtained oligonucleotide derivatives bearing cationic groups were determined by PAGE (see Figures S15–S17), compared with the mobility of control oligonucleotides containing 1 or 2 methylaminopropylsulfonyl phosphoramidate groups. It was shown that even the introduction of simple alkylamine residues resulted in the formation of zwitterionic derivatives, in which the positively charged amino group neutralizes the negatively charged sulfonyl phosphoramidate under electrophoresis conditions.

For diamines, it was found that when the amino groups were spaced by 2 or 3 methylene units, only one of them was protonated under PAGE conditions, regardless of the nature of the amino groups (primary, secondary). When the distance was increased to 4 methylene groups, both amino groups (diaminobutane, OL15) were protonated, providing a total positive charge of the internucleotide phosphate unit. A similar phenomenon occurred in the case of tri- and polyamines. For example, only two of the three amino groups were protonated within the 3,3′-iminobis­(N,N-dimethylpropylamine) residue (OL18) under electrophoretic conditions. This can be explained by the weakening of the basicity of neighboring amino groups, resulting in low second-stage pK _a. Additional experiments were also performed on the incorporation of other diamines with a distance of 2–3 carbon atoms between amino groups, differing in the set and size of substituents (see Supporting Table S2 and Figures S26, S27, and S29), and in all cases, one amino group was protonated.

Moreover, an amino alcohol, 3-methylamino-1-propanol (OL22), and morpholine (OL23) were introduced. In the case of OL22, protonation was observed as expected, whereas the morpholine residue, the only one among the introduced amines, was not protonated at all since the basicity of the amine group was weakened by the electronegative oxygen atom.

In addition, we made attempts to introduce a tertiary amine and N-heterocycles to obtain quaternized nitrogen derivatives as substituents (oligonucleotides OS14-OS16) (see Supporting Information, Table S2 and Figures S27 and S29). However, the introduction of triethylamine and pyridine did not yield the desired product; only N-methylimidazole was introduced at low conversion. Detailed analysis of the mass spectrum (Supporting Figure S30) showed that the reaction mixture in the case of N-methylimidazole, in addition to the desired quaternized derivative and unsubstituted chlorine derivative, contained a product of elimination bearing an alkene moiety. Based on this observation, we conducted an experiment (oligonucleotide OS18, see Supporting Information, Table S2 and Figures S28–S30) in which the 3-chloropropane derivative was treated with the non-nucleophilic base DIPEA to achieve elimination while avoiding substitution. It was shown that the reaction mixture contained the desired alkene product. However, the conversion of elimination was low even at harsh conditions. The significantly lower elimination tendency of the 3-chloropropyl fragment, in contrast to 2-chloroethyl, is well anticipated.

In view of the low efficiency in obtaining the quaternized N-methylimidazole derivative, we attempted to incorporate an unsubstituted imidazole residue. To deprotonate imidazole, the introduction was carried out in the presence of DIPEA. The desired product was obtained at a higher conversion (∼40%) than the N-methylimidazole derivative. It is also worth noting that, as in the case of morpholine (OL23), the imidazole residue within the resulting derivative was not protonated, which is explained by its low basicity. In addition, attempts were made to introduce tetramethylguanidine. However, treatment with this reagent led to degradation of the oligonucleotide (oligonucleotide OS17, see Supporting Table S2 and Figures S27 and S29).

Thus, the developed method based on the substitution of a chlorine atom with various amines in a 3-chloropropylsulfonyl phosphoramidate precursor at the distinct amination step allowed the straightforward yield of a library of sulfonyl phosphoramidate derivatives bearing different substituents.

2.3. Multistep Oligonucleotide Functionalization Using 3-Chloropropyl and 2-Azidoethyl Sulfonyl Phosphoramidate Precursors

In addition to the direct introduction of various substituents that affect the properties of the resulting sulfonyl phosphoramidate derivatives, the incorporation of new reactive groups enabling further functionalization of the oligonucleotide is of great interest.

First, it is worth noting the introduction of primary or secondary amino groups, which can be subsequently used in a wide range of electrophilic reactions, in particular for acylation. A number of derivatives in the obtained library (see above) contain such groups. Taking into account the good yields of the obtained oligonucleotide OL16, for further functionalization, N,N′-dimethylethylenediamine was selected. Upon incorporation, one amino group displaces chlorine and becomes tertiary, whereas the other secondary amino group remains capable of acylation.

We carried out the acylation directly after the amination step by treating the obtained product with the appropriate halogen anhydride, prior to the final deblocking step (Figure ). It was shown that in the case of octanoyl chloride (Table , OF1), a high conversion of acylation was achieved under relatively mild conditions. Next, we performed a series of additional experiments in which we varied the used halogen anhydride and incorporated bulky acyl residues (C10–C14) as well as compact acetyl. In addition, we carried out the acylation using incorporated piperazine residue (OL11) instead of N,N′-dimethylethylenediamine (oligonucleotides OS20-OS25, see Supporting Information, Table S3 and Figures S35–S38). In all cases, the acylation proceeded at high efficiency.

7.

Multistep oligonucleotide functionalization using chloroalkylsulfonyl phosphoramidate precursors. (a) Acylation of amino groups and addition of phosphoramidites to a hydroxyl group within the methylamino-1-propanol residue. (b) Reactions of the 2-azidoethane precursor: azide–alkyne cycloaddition and reduction of azido group by triphenylphosphine. (c) Structures of the branched oligonucleotide OF2 and the conjugate OF3 obtained via CuAAC reaction.

3. Oligonucleotide Derivatives Obtained via Multistep Functionalization Using Chloroalkylsulfonyl Phosphoramidate Precursors .

code	azide	second step of functionalization	3d step of functionalization	conversion	M r(theor.)	M r(exp.)
OF1	A1	N,N′-dimethylethylenediamine	Octanoyl chloride	∼90%	2687.6	2688.6
		Conditions: A	Conditions: C
OF2	A1	3-Methylamino-1-propanol	Oligonucleotide branching	∼90%	4997.4	4995.0
		Conditions: A	Conditions: D
OF3	A2	CuAAC reaction		∼90%	4841.8	4841.9
OF4	A2	Triphenylphosphine		∼90%	2476.4	2476.2
		Conditions: B
OF5	A2	Triphenylphosphine	BDP630/650-X-NHS-ester	∼60%	3021.6	3021.6
		Conditions: B

^aIn all cases, Staudinger reaction conditions: 0.5 M solution of the corresponding azide in acetonitrile, 1 h, 25°C. Sequence of OF1 and OF2. 5′-T*TTTTTTT-3′. Sequences of branched oligonucleotides OF3 and OF4: See Figure . The conversion values were calculated from the peak areas in HPLC profiles (see Supporting Information, Figure S31) and relate to the desired products of multistep functionalization. For PAGE, see Figure S32. For mass spectra, see Supporting Information, Figures S33 and S34. Conditions of functionalization: A30% vol. solution in ACN, 20 h, 50 °C; B0.5 M solution in ACN, 20 h, 50 °C; C(2 M solution in ACN + 1 equiv DIPEA),1 h, 40 °C; Daddition of thymidylate phosphoramidite monomers to the OH group at methylaminopropanol residue.

Another important group for introduction is the free hydroxyl group. It can serve as an additional 5′-end and be coupled with various phosphoramidite nucleotide or non-nucleotide reagents, resulting in nonlinear oligonucleotide structures. For this purpose, we used a derivative bearing a 3-methylamino-1-propanol residue (OL22) with an inert tertiary amino group and a free hydroxyl group. A preliminary experiment demonstrated the possibility of coupling thymidine phosphoramidite to the OH group in the N-methylaminopropanol residue (oligonucleotide OS26, see Supporting Table S3 and Figures S35 and S37). The coupling efficiency was slightly lower than when attaching to the oligonucleotide 5′–OH, but increasing the time led to higher coupling yields and allowed us to obtain a branched oligonucleotide structure with a high conversion (OF3, Figure , and Table ). In addition, a non-nucleotide phosphoramidite, bearing fluorescent label 6-FAM, was also successfully attached to the incorporated extra hydroxyl group (oligonucleotide OS27, see Supporting Information, Table S3 and Figures S35 and S37).

In addition to hydroxy and amino groups, azido groups are commonly used in oligonucleotide chemistry. This application is based on conjugation via the CuAAC reaction. It has been shown above (Figure ) that 2-azidoethanesulfonyl azide (A2) can be easily obtained from 2-chlorethanesulfonyl chloride. This compound undergoes the Staudinger reaction via the electron-deficient sulfonyl azide group, while the electron-donating alkyl azide group remains intact within the resulting 2-azidoethylsulfonyl phosphoramidate derivative. We performed a model experiment in which we conjugated two oligonucleotides via an azide–alkyne cycloaddition reaction (OF3). One of the oligonucleotides was modified with azide A2 and contained an azide group, while the second was synthesized using the commercially available 3′-alkyne modifier CPG. Conjugation of these oligonucleotides proceeded almost quantitatively (Table ).

It has also been shown that the alkyl azido group in the oligonucleotide structure can be successfully converted to an amino group. For this purpose, treatment with triphenylphosphine followed by hydrolysis at the final deblocking step was carried out, resulting in the formation of a derivative containing a terminal -NH₂ group with high conversion (OF4). This primary amino group can be used for further oligonucleotide functionalization. We attached to this group a fluorescent dye in the form of activated NHS ester (Table , OF5). In contrast to the acylation by acyl chlorides (OF1), which was carried out before the final deblock step with the oligonucleotide product immobilized on the solid-phase support, in this case, we performed the reaction after the final deblock step.

Thus, the proposed method enables one not only to introduce various functional substituents but also to implement a multistep assembly of complex modified oligonucleotides by performing a set of simple reactions such as amination, azide-amine reduction, acylation, and CuAAC conjugation.

2.4. Synthesis of Sulfonyl Azides Bearing Amine Residues for Obtaining the Corresponding Sulfonyl Phosphoramidate Derivatives

The method based on the introduction of an azide precursor within the oligonucleotide, followed by the incorporation of amine residues (Section ), has shown promising results. However, this method has been demonstrated to be less effective in some cases. Therefore, we decided to apply another method based on the synthesis of azides bearing the corresponding amine residues for expanding the synthetic capabilities of our approach.

2.4.1. Synthesis of Sulfonyl Azide Bearing a Bulky Alkylamine Residue

It was shown (see Section ) that achieving an acceptable conversion rate when introducing the bulky amines within the 3-chloropropylsulfonyl phosphoramidate derivative required harsh conditions and long reaction times (e.g., 40 h at 50 °C for OL6 to achieve 60% conversion). Therefore, it was decided to synthesize a sulfonyl azide bearing a bulky methyldodecylamine residue and incorporate it into the oligonucleotide, taking into consideration that the Staundinger reaction, even with bulky substituted sulfonyl azides, does not require as harsh conditions as nucleophilic substitution of an aliphatic chlorine atom.

We selected C2-azide (A3) instead of the C3-azide (A1) as a precursor for obtaining the sulfonyl azide with methyldodecylamine residue, as it has a more active chlorine atom. The sulfonyl phosphoramidate derivative with 2-chloroethyl substituent turned out to be elimination-prone under final deblock conditions (see Section , OD6-OD7). However, for the substitution of the chlorine atom within the C2-azide, a reported procedure can be employed. , This route involves the use of a mild base (K₂CO₃) and a catalyst (KI), allowing the reaction to proceed under mild conditions. This is crucial to avoid undesired processes, including elimination, and as a significant advantage, it prevents heating in the presence of the azide group. We adopted this procedure to obtain a sulfonyl azide bearing N-methyldodecylamine residue (Figure a).

8.

Synthesis of sulfonyl azides bearing amine residues. (a) Synthesis of sulfonyl azide bearing N-methyldodecylamine residue (A5) from 2-chloroethansulfonyl azide. (b) Scheme of obtaining sulfonyl azides from 1,3-propane sultone and a library of obtained azides.

The resulting azide (A5) was slightly less reactive than its compact chloroethanesulfonyl precursor and was incorporated under harsher conditions (0.5 M solution in acetonitrile, 3 h, 40 °C). A conversion of about 60% was achieved (Table ), which is comparable to the efficiency of nucleophilic substitution of 3-chloropropanesulfonyl oligonucleotide derivative with methyl dodecylamine, while the conditions were significantly milder.

4. Library of Sulfonyl Phosphoramidate Derivatives Obtained Using Sulfonyl Azides Bearing Amine Residues .

code	azide	Staudinger reaction conditions	conversion (HPLC)	M r(theor.)	M r(exp.)
OX1	A5	0,5 M in ACN, 3 h, 40 °C	∼60%	2658.6	2658.6
OX2	A6	0,5 M in ACN, 1 h, 25 °C	∼90%	2555.5	2554.4
OX3a	A7a	0,5 M in ACN, 1 h, 25 °C	∼90%	2574.5	2574.5
OX3b	A7b	0,5 M in ACN, 1 h, 25 °C	∼90%
OX4	A8	0,5 M in ACN, 1 h, 25 °C	∼80%	2552.5	2552.4
OX5	A9	0,5 M in ACN, 1 h, 25 °C	∼80%	2558.5	2558.4
OX6	A10	0,5 M in ACN, 1 h, 25 °C	∼60% (mixture)	2672.7	2672.6
OX7	A11	0,5 M in ACN, 1 h, 25 °C	∼90%	2588.5	2588.4

^aSequence 5′-T*TTTTTTT-3′. The conversion values were calculated from the peak areas in HPLC profiles (Figures S39 and S40) and relate to the desired product of modification introduction. For PAGE, see Figure S41. For mass spectra, see Supporting Information, Figures S42–S44.

2.4.2. Synthesis of Sulfonyl Azides Bearing Quaternary and Tertiary Amine Moieties from 1,3-Propane Sultone

The first method, involving substitution within the sulfonyl phosphoramidate precursor (Section ), proved to be ineffective for one particular application: the incorporation of tertiary amine residues. Therefore, we decided to obtain sulfonyl azides bearing quaternized amine moieties and adopted for this purpose a reported synthetic route.

The ring-opening of sultone with a set of tertiary amines was carried out under solvent-free conditions by mixing the reagents in equivalent amounts. The resulting zwitterionic sulfonic acids were treated with oxalyl chloride to obtain sulfonyl chloride and then converted to the corresponding azide without isolation and purification. Azides bearing aromatic heterocyclic moieties (N-methylimidazole, pyridine) and a triethylamine moiety were obtained (A6-A8, Figure b). For azide A7, which carries a pyridine residue, a modified version of the synthetic scheme was also performed, involving the synthesis from the commercially available sulfonic acid 3-(1-pyridinio)-1-propanesulfonate (A7b). In this case, the chlorine counterion was replaced with hexafluorophosphate to increase the solubility of the resulting azide in aprotic solvents. All of the obtained azides bearing quaternary moieties (A6-A8) were introduced at high conversions (Table , OX1-OX4).

Next, the same synthetic scheme was used for the incorporation of secondary amines as an alternative route for the method listed in Section . As in the case of tertiary amines, the reagents were mixed in equimolar amounts. The azide-bearing piperidine residue (A9) was successfully incorporated (oligonucleotide OX5), with a small amount of a byproduct found in the reaction mixture. In the case of methyldodecylamine (oligonucleotide OX6), a byproduct was obtained in an amount comparable to the desired product. As shown by mass spectrometric analysis (see Figure S43), in the case of secondary amines, exhaustive alkylation of secondary amines with sulfolactone occurred, resulting in the formation of a quaternized derivative bearing an additional sulfonyl group (see Figure S45). This process was more significant in the case of the methyldodecylamine, while piperidine mainly reacted with sultone in a 1:1 ratio, forming the desired product with a tertiary amine moiety.

In addition, the synthesis of azide bearing a tetramethylguanidine residue and its incorporation was successfully performed using this method (Table , oligonucleotide OX7). In contrast to secondary amines, no excessive alkylation occurred, which is consistent with the reported data on the synthesis involving ring-opening reaction of 1,3-propane sultone with tetramethyl guanidine.

Thus, it was shown that the approach including alkylation with 1,3-propane sultone enables efficient obtaining sulfonyl phosphoramidate derivatives bearing quaternized amine moieties and guanidine derivatives and can be considered as a useful tool for the incorporation of certain functional groups, complementing the first method based on the substitution of 3-chloropropane sulfonyl precursor.

2.5. Synthesis of Sulfamoyl Azides Bearing Amine Residues for Obtaining the Corresponding Sulfamoyl Phosphoramidate Derivatives

In addition to the synthesis of C1–C3 azides, which starts from appropriate sulfonyl chlorides bearing an alkyl substituent with a terminal chlorine atom (see Section ), it was also decided to obtain C0 azide. This can be achieved using sulfuryl chloride as a starting material. This compound (Figure a) can be considered as an analogue of these sulfonyl chlorides with a zero-length alkyl linker. Sulfuryl chloride contains two equal chlorine atoms. When treated with one equivalent of sodium azide, it can be converted into monosubstituted sulfuryl chloroazide (Figure b). As this azide is explosive and may contain traces of significantly more explosive sulfuryl diazide, it was not considered for the direct introduction of modifications within oligonucleotides.

9.

Scheme of obtaining sulfamoyl azides from sulfuryl chloride and a library of obtained azides. aSulfuryl chloride; bsulfuryl chloroazide; csulfamoyl azide.

However, the chlorine atom in this compound can be substituted with amines to obtain significantly less explosive sulfamoyl azides, a subclass of sulfonyl azides (Figure c). In contrast to its aliphatic counterparts in C1–C3 azides, this reaction proceeds easily under mild conditions, since the chlorine atom is directly bound to the acceptor sulfo group. The introduced amino group as a part of the sulfamoyl azide no longer exhibits the properties of a free amine in contrast to amino groups in the products of substitution reaction of common precursors obtained using C1–C3 azides. This structural alteration not only simplifies the synthesis process compared to the nucleophilic substitution reaction and prevents side reactions but also significantly facilitates the purification procedure of the target compounds from the starting amine.

A set of sulfamoyl azides bearing various residues was obtained (Figure , A12-A15). Studies on butanesulfamoyl azide (A12) have demonstrated that under mild conditions (0,5 M, 1 h, RT) the incorporation efficiency of this azide did not exceed 50% (Table , OA1). In contrast, a comparably sized 3-chloropropanesulfonyl azide studied in this work (Table , OD1) was incorporated at high efficiency under the same mild conditions as well as butanesulfonyl azide according to literature data. The decrease in the reactivity of sulfamoyl azides can be explained by the presence of a directly bound amino group, which reduces the acceptor properties of the sulfonyl group. Thus, to achieve better conversion values for the Staudinger reaction, we obtained a set of sulfamoyl phosphoramidate derivatives under more harsh conditions (0.5 M, 3 h, 40 °C) (Table , OA2-OA4). Sulfamoyl azides bearing butylamine (OA2), N-methylpiperazine (OA3), and tetramethylguanidine (OA4) residues show almost quantitative yields (>95%).

5. Library of Sulfonyl Phosphoramidate Derivatives Obtained Using Sulfonyl Azides Bearing Amine Residues .

code	azide	Staudinger reaction conditions	conversion (HPLC)	M r(theor.)	M r(exp.)
OA1	A12	0,5 M in ACN, 1 h, 25 °C	∼50%
OA2	A12	0,5 M in ACN, 3 h, 40 °C	∼95%	2504.5	2504.6
OA3	A13	0,5 M in ACN, 3 h, 40 °C	∼95%	2531.5	2531.6
OA4	A14	0,5 M in ACN, 3 h, 40 °C	∼95%	2546.5	2545.4
OA5	A15	0,5 M in ACN, 1 h, 25 °C	∼80%	2499.6	2499.4

^aSequence 5′-T*TTTTTTT-3′. The conversion values were calculated from the peak areas in HPLC profiles (Figure S46) and relate to the desired product of modification introduction. For PAGE, see Figure S47. For mass spectra, see Figures S48 and S49.

It is worth noting that the obtained imidazolesulfonyl azide (Figure , A15) bearing an acceptor imidazole moiety instead of donor amine residues was incorporated with high efficiency even under mild conditions (Table , OA5). Meanwhile, substitution of the imidazole moiety did not occur at the final deblock step, which demonstrates the stability of the imidazolesulfonyl phosphoramidate modification.

Thus, despite the sulfuryl chloroazide not being used for direct introduction of the modification within oligonucleotides, the proposed synthetic route enabled production of another sulfonyl subclass, sulfamoyl phosphoramidate oligonucleotides, bearing not only common linear aliphatic substituents but also some special residues such as guanidine and imidazole as a direct part of the modification structure.

2.6. Obtaining Hetero-Oligonucleotides

Following the successful modification of oligothymidylates, we extended our studies to include model hetero-oligonucleotides. Using 3-chloropropaneulfonyl azide, a series of oligonucleotides bearing various amine residues was obtained, with the single modifications introduced at various positions within the heterosequence: closest to the 5′-end, to the 3′-end, and in the middle (Table , OH1-OH12). For introducing modification to the 3′-end and in the middle, after completion of the Staudinger reaction, synthesis of the oligonucleotide chain was continued, and the amination was performed after completion of the synthesis of the oligonucleotide chain. In addition, hetero-oligonucleotides containing single modifications at various positions in the chain were obtained using azide A11b, bearing a quaternized pyridine residue (Table , OH13-OH15).

6. Series of Hetero-Oligonucleotides Carrying a Single Modification at Various Positions .

code	oligonucleotide sequence, 5′-3′	azide	amine, conditions	conversion
OH1	G*CGCCAAACA	A1	Methylamine, c	∼90%
OH2	G*CGCCAAACA	A1	Piperidine, a	∼90%
OH3	G*CGCCAAACA	A1	Morpholine, a	∼90%
OH4	G*CGCCAAACA	A1	3,3′-iminobis(N,N-dimethylpropylamine), b	∼90%
OH5	GCGCCAAAC*A	A1	Methylamine, c	∼90%
OH6	GCGCCAAAC*A	A1	Piperidine, a	∼90%
OH7	GCGCCAAAC*A	A1	Morpholine, a	∼90%
OH8	GCGCCAAAC*A	A1	3,3′-iminobis(N,N-dimethylpropylamine), b	∼90%
OH9	GCGCCA*AACA	A1	Methylamine, c	∼90%
OH10	GCGCCA*AACA	A1	Piperidine, a	∼90%
OH11	GCGCCA*AACA	A1	Morpholine, a	∼90%
OH12	GCGCCA*AACA	A1	3,3′-iminobis(N,N-dimethylpropylamine), b	∼90%
OH13	G*CGCCAAACA	A11b		∼90%
OH14	GCGCCA*AACA	A11b		∼90%
OH15	G*CGCCAAACA	A11b		∼90%

¹Condition of the Staudinger reaction: for OH1-OH12:0.25 M solution of 3-chloropropanesulfonyl azide in acetonitrile, 1 h, 25 °C. For OH15-OH17:0.25 M solution of azide A11b, 1 h 25 °C (manual introduction). The conversion values were calculated from the peak areas in HPLC profiles (Figures S50–S52). For PAGE, see Figures S53–S54. Conditions for the introduction of amine residues: a30% v/v in ACN, 20 h, 50 °C; b30% v/v in ACN, 60 h, 50 °C; cconc. aq. methylamine solution, 30 min, 55 °C.

As can be seen, the conversion values did not decrease compared to those of oligothymidylates.

Thus, it was shown that the efficiency of introducing the modifications remains stable upon moving from oligothymidilates to hetero-oligonucleotides and varying the position of the modification in the oligonucleotide chain. This creates opportunities to advance methods toward the automated incorporation of modifications and the production of oligonucleotides with partially or completely modified backbones.

3. Discussion

Currently, the Staudinger reaction is garnering significant attention as a promising tool for obtaining phosphate-modified oligonucleotides. ,,− Sulfonyl phosphoramidates together with phosphoryl guanidine and phosphorothioate modifications have been established as a promising tool for the development of therapeutic oligonucleotides. −

However, if the modification is considered not only as a tool for changing the backbone but also as a means for introducing diverse functional groups, it becomes highly desirable to obtain a broad range of representatives of the sulfonyl phosphoramidate class. This work is devoted to overcoming the limitation imposed by the narrow set of commercially available sulfonyl chlorides by developing a versatile approach for incorporating various functional residues into the sulfonyl phosphoramidate backbone.

We applied a general approach to obtain diverse representatives of the sulfonyl phosphoramidate class based on the incorporation of amines as commercially available building blocks within the reactive precursors via an alkylation reaction. As part of the approach, two methods were implemented: the first one involves introduction of amine residues within an oligonucleotide sulfonyl phosphoramidate derivative, while the second one is based on introducing amine residues in the process of azide synthesis. Since a much wider variety of amines is commercially available compared to sulfonyl chlorides, employing them as functionalized building blocks provides a significant expansion of the range of sulfonyl phosphoramidates.

Generally, the nucleophilic substitution reaction of halogen with an amine residue has limited application in organic synthesis, especially in the case of primary and secondary amines, since excessive alkylation occurs, and a mixture of products is formed. The key advantage of the first method is that the alkyl halide is a part of the oligonucleotide immobilized on a solid-phase support, while the amine is in solution. This configuration drastically reduces the probability of a single amine molecule reacting with multiple alkyl halide sites and thus enables the introduction of various amine residues within oligonucleotide derivatives without side processes.

For the implementation of the first method, choroalkanesulfonyl azides with C1–C3 alkyl chains were tested. It was shown that the α-chlorine atom obtained during the Staudinger reaction C1-precursor was deactivated, whereas a β-chlorine in the C2-precursor is overactivated and prone to elimination during the amination step. However, the C2-sulfonyl chloride can be easily converted into C2-diazide (A2), which is also of interest as a tool for the incorporation of the azide group. The best results were achieved in the case of 3-chloropropanesulfonyl azide (A1), where the terminal chlorine possesses sufficient reactivity for substitution without leading to side reactions. Therefore, we used this azide for obtaining a library of sulfonyl phosphoramidate derivatives bearing various amine residues.

For specific synthetic tasks where the first method proved to be less effective, a complementary method was employed, based on incorporating desired amine residues directly into sulfonyl azide. In each case, appropriate synthetic routes were used. Thus, for the incorporation of a bulky secondary amine, C2-azide with activated chlorine under mild conditions was used, which prevented undesired excessive alkylation. Using for this purpose a stronger alkylation reagent, 1,3-propane sultone, resulted in excessive alkylation. However, for obtaining quaternized derivatives, in the case where exhaustive alkylation was our purpose, sultone was successfully applied.

The developed approach enables the acquisition of a wide range of sulfonyl phosphoramidate derivatives bearing various substituents, thereby providing the use of modification for the conjugation of oligonucleotides with diverse functional residues. This can be achieved by introducing substituted amines that can potentially modulate the properties of the oligonucleotide. In particular, the incorporation of amines bearing long alkyl chains can be used for obtaining lipophilic derivatives.

Moreover, the introduction of amine residues bearing reactive groups (−NH₂, −OH, −N₃) enables the attachment of various reagents, offering an alternative to commonly used specialized controlled pore glasses (CPGs) or non-nucleotide phosphoramidites (e.g., amino modifiers, branching agents, and azido modifiers). Our method uses a standard set of reagents to achieve the introduction of such groups via phosphate modification, involving multistep assembly of modified oligonucleotide constructs. The set of performed model experiments demonstrates the diverse application areas of the developed tool, including assembly on a solid-phase support, as in the cases of acylation and branching, as well as the conjugation in solution, as in the cases of click-reaction and attachment of the dye.

In addition to conjugation with different moieties, the developed approach can be considered as a tool for altering the oligonucleotide backbone. In the future, we are planning to extend our research toward automated introduction of modification and obtaining multiply- or fully modified oligonucleotides. In contrast to the simplest representatives such as mesyl phoshoramidate, where the properties of oligonucleotides are determined by the nature of sulfonyl phosphoramidate modifications, for the modifications developed in this work, the substituent’s nature is a factor of comparable importance.

In particular, introducing alkylamine residues (for example, methylamine, piperidine) results in the formation of the zwitterionic modification. Certain representatives bearing polyamine residues not only neutralize the modified phosphate group but also provide a total positive charge of the nucleotide unit. While cationic modifications are frequently introduced in a form of conjugates like Zip Nucleic Acid, , our approach utilizes phosphate modifications to precisely tune not only total backbone charge but also charge distribution over the backbone structure. Our future plans are to study the synthesis and properties of fully modified sulfonyl phosphoramidate oligonucleotides with zwitterionic or cationic backbones. Oligonucleotides with such backbones possess unique properties, such as enhanced cellular uptake and triplex formation. At the same time, we expect that our modifications will retain the fundamental properties of the sulfonyl phosphoramidate class, such as the ability to activate RNaseH. This ability was demonstrated for some zwitterionic and cationic oligonucleotides. Therefore, we assume that a positive charge in the substituents would not significantly inhibit the RNaseH activity.

To conclude, the proposed strategy, based on the use of common precursors and the introduction of amines as commercially available building blocks, significantly expanded the range of representatives of the sulfonyl phosphoramidate class and enabled the incorporation of various functional groups. The proposed methods, based on a set of simple reactions, can be helpful for a wide range of challenges in nucleic acid chemistry. These include modifying the backbone nature to obtain zwitterionic and cationic oligonucleotides as well as conjugation with various functional moieties, such as lipophilic residues, dyes, and fluorophores. Owing to its versatility, this approach may find various applications and greatly broaden the potential of the sulfonyl phosphoramidate class.

4. Materials and Methods

The following reagents and solvents were used in this study: 2-chloroethanesulfonyl chloride, 3-chloropropanesulfonyl chloride, N-methyldodecylamine (Thermo Fischer, USA); 1,3-propane sultone (TCI, Japan); sulfuryl chloride, aqueous methylamine, propionic anhydride, N,N-diisopropylethylamine, N-methylbutylamine, dodecylamine, piperazine, ethylenediamine, 1,3-diaminopropane, tris­(2-aminoethyl)­amine, methylaminopropanol, triphenylphosphine, acetyl bromide (Acros Organics, USA); sodium azide, butylamine, piperidine, imidazole, octanoyl chloride, dibutylamine, N-methylpiperazine, oxalyl chloride (Sigma-Aldrich, USA); teramethylguanidine (Merck, Germany); N-methylimidazole (Roth, Germany); spermine (Fluka, Switzerland); chloromethanesulfonyl chloride, 3-(1-pyridinio)-1-propanesulfonate, diethylamine, benzylamine, aniline, 1,4-diaminobutane, N,N′-dimethylethylenediamine, 3-dimethylamino-1-propylamine, 3,3′-iminobis­(N,N-dimethylpropylamine), 3,3′-diaminodipropylamine, morpholine (Macklin, China); acetonitrile, pyridine, triethylamine, dichloromethane, tetrahydrofuran, dimethylformamide (Panreac, Spain); aqueous ammonia, toluene, chloroform (Reakhim, Russia).

For TLC, DC-Alufolien Kieselgel 60 F254 plates (Merck, Germany) were used. For column adsorption chromatography, a Kieselgel 60 sorbent (particle size 0.06–0.20 mm, pore size 60 Å; Merck, Germany) was used.

¹H NMR spectra were recorded on a Spinsolve 80 spectrometer (Magritek, New Zealand) at 80 MHz.

Registration of IR spectra of synthesized azido-triazine modifiers was performed on a Varian 640-IR spectrometer (Varian, USA) at the Chemical Research Center for Collective Use of the Siberian Branch, Russian Academy of Sciences, at the Vorozhtsov Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences.

4.1. Organic Synthesis

¹H NMR spectra were recorded on a Spinsolve 80 NMR spectrometer (Magritek, Germany; 80 MHz). All moisture-sensitive reactions were performed under an argon atmosphere.

For NMR spectra, see Figures S55–S62.

4.1.1. 3-Chloropropanesulfonyl Azide (A1)

To a stirred solution of 2-chloropropanesulfonyl chloride (91 μL, 0.75 mmol) in acetonitrile (1.5 mL) was added sodium azide (98 mg, 1.5 mmol). The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter and was used without further purification.

¹H NMR (80 MHz, CDCl₃, δ, ppm): 3.71 (m, 4H, S-CH ₂ -CH₂–CH ₂ -), 2.37 (m, 2H, S-CH₂–CH ₂ -).

IR: N₃-group -2150 cm^–1.

4.1.2. 2-Azidoethanesulfonyl Azide (A2)

To a stirred solution of 2-chloroethanesulfonyl chloride (52 μL, 0.5 mmol) in acetonitrile (1 mL) was added sodium azide (65 mg, 1 mmol). The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter and used without further purification.

¹H NMR (80 MHz, CDCl₃, δ, ppm): 3.52 (t, 2H, S-CH₂–CH ₂ -), 3.87 (t, 2H, S-CH ₂ -).

IR: N₃-group -2150 cm^–1.

4.1.3. 2-Chloroethanesulfonyl Azide (A3)

To a stirred solution of 2-chloroethanesulfonyl chloride (52 μL, 0.5 mmol) in acetonitrile (1 mL) at 0 °C was added sodium azide (32 mg, 0.5 mmol). The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter and used without further purification.

¹H NMR (80 MHz, CDCl₃, δ, ppm): 4.03 (t, 2H, S-CH ₂ -CH ₂ -).

IR: N₃-group -2150 cm^–1.

4.1.4. Chloromethanesulfonyl Azide (A4)

To a stirred solution of chloromethanesulfonyl chloride (75.4 μL, 0.5 mmol) in acetonitrile (1 mL) was added sodium azide (65 mg, 1 mmol). The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter and used without further purification.

¹H NMR (80 MHz, CDCl₃, δ, ppm): 4.79 (s, 2H, −CH₂Cl)

4.1.5. 2-(Dodecylmethylamino)­ethanesulfonyl Azide (A5)

To a stirred 0.5 M solution of 2-chloroethanesulfonyl azide in acetonitrile (0.5 mmol, 1 mL) at 0 °C, potassium carbonate (138 mg, 0.1 mmol), potassium iodide (catalytic amount), and a solution of N-methyldodecylamine (100 mg, 0.5 mmol) in toluene (200 μL) were added successively. The reaction mixture was stirred for 3 h at room temperature and then centrifuged to remove insoluble solids. The crude product was purified by column chromatography (10% ethanol and 1% triethylamine in dichloromethane). The resulting product was dissolved in acetonitrile (500 μL) and held at −18 °C for 20 h. The precipitate was filtered off to give the resulting compound as a white solid (63 mg, yield 41%).

¹H NMR (80 MHz, CDCl₃, ppm): 3.49 (t, 2H, S-CH ₂ -), 2.90 (t, 2H, S-CH₂–CH ₂ -), 2.28 (m, 5H, N-CH ₃ + N-CH ₂ -(CH₂)₁₀-CH₃), 1.25 (m, 20H, N–CH₂–(CH ₂ ) ₁₀-CH₃), 0.87 (t, 3H, N–CH₂–(CH₂)₁₀-CH ₃ )

4.1.6. 3-(Azidosulfonylpropyl)-1-methylimidazolium Chloride (A6)

To stirred N-methylimidazole (200 μL, 2.5 mmol) was added 1,3-propane sultone (220 μL, 2.5 mmol) dropwise at 0 °C. The reaction mixture was stirred for 40 min at room temperature. The resulting solid was washed with diethyl ester 3 times to remove unreacted compounds and dried under a vacuum. Next, the solid was suspended in dichloromethane (6 mL), and oxalyl chloride (640 μL, 7.5 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature and then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (325 mg, 5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid (295 mg, yield 40%)

¹H NMR (80 MHz, DMSO-d6, δ, ppm): 9.31 (s, 1H, NCH-), 7.93 (d, 2H, NCH₂–N-CHCH-), 4.47 (t, 2H, S-CH₂–CH₂–CH ₂ -), 4.01 (m, 5H, NCH-N-CH ₃ + S-CH ₂ -CH₂), 2.48 (m, 2H, S-CH₂–CH ₂ -).

4.1.7. 3-(Azidosulfonylpropyl)­pyridinium Chloride (A7a)

To stirred pyridine (200 μL, 2.5 mmol) was added dropwise 1,3-propane sultone (220 μL, 2.5 mmol) at 0 °C. The reaction mixture was stirred for 40 min at room temperature. The resulting solid was washed with diethyl ester 3 times to remove unreacted compounds and dried under vacuum. Next, the solid was suspended in dichloromethane (6 mL), and oxalyl chloride (640 μL, 7.5 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature, then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (325 mg, 5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 235 mg, yield 36%.

¹H NMR (80 MHz, DMSO-d6, δ, ppm): 9.14 (d, 2H, -CH-N-CH-), 8.65 (t, 1H, N–CH-CH-CH-), 8.18 (t, 2H, -CH-CH-N–CH-CH-), 4.80 (t, 2H, S-CH₂–CH₂–CH ₂ -), 3.86 (m, 2H, S-CH ₂ -), 2.50 (m, 2H, S-CH₂–CH ₂ -)

4.1.8. 3-(Azidosulfonylpropyl)­pyridinium Hexafluorophosphate (A7b)

3-(1-Pyridinio)-1-propanesulfonate (500 mg, 2.5 mmol) was suspended in dichloromethane (6 mL), and oxalyl chloride (640 μL, 7.5 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature; next, potassium hexafluorophosphate (550 mg, 3 mmol) was added. The reaction mixture was stirred for 20 h at room temperature, centrifuged to remove insoluble matter, and then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (325 mg, 5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 250 mg, yield 26%.

¹H NMR (80 MHz, DMSO-d6, δ, ppm): 9.14 (d, 2H, -CH-N-CH-), 8.65 (t, 1H, N–CH-CH-CH-), 8.18 (t, 2H, -CH-CH-N–CH-CH-), 4.80 (t, 2H, S-CH₂–CH₂–CH ₂ -), 3.90 (m, 2H, S-CH ₂ -), 2.50 (m, 2H, S-CH₂–CH ₂ -)

4.1.9. 3-(Azidosulfonylpropyl)-N,N,N-triethylaminium Chloride (A8)

To stirred triethylamine (350 μL, 2.5 mmol) was added 1,3-propane sultone (220 μL, 2.5 mmol) dropwise at 0 °C. The reaction mixture was stirred for 40 min at room temperature. The resulting solid was washed with diethyl ester 3 times to remove unreacted compounds and dried under vacuum. Next, the solid was suspended in dichloromethane (6 mL), and oxalyl chloride (640 μL, 7.5 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature, then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (325 mg, 5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 670 mg, yield 94%.

¹H NMR (80 MHz, DMSO-d6, δ, ppm): 3.83 (t, 2H, S-CH ₂ -), 3.25 (m, 8H, N-(CH ₂ -)), 2.07 (m, 2H, S-CH₂–CH ₂ -), 1.19 (t, 6H, N-(CH₂–CH ₃ )).

4.1.10. 3-(Azidosulfonylpropyl)­piperidinium Chloride (A9)

To a stirred solution of piperidine (250 μL, 2.5 mmol) in dichloromethane (6 mL) was added 1,3-propane sultone (220 μL, 2.5 mmol) dropwise at 0 °C. The reaction mixture was stirred for 20 h at room temperature and then evaporated. The resulting solid was washed with diethyl ester 3 times to remove unreacted compounds and dried under vacuum. Next, the solid was suspended in dichloromethane (6 mL), and oxalyl chloride (640 μL, 7.5 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature, then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (325 mg, 5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 256 mg, yield 38%.

¹H NMR (80 MHz, DMSO-d6, δ, ppm): 3.82 (t, 2H, S-CH ₂ -), 2.78 (m, 6H, N-(CH ₂ ) ₃ -), 2.07 (m, 2H, S-CH₂–CH ₂ -), 1.54 (m, 6H, N-(CH₂–CH ₂ )₂-CH ₂ -)

4.1.11. 3-(Azidosulfonylpropyl)­methyldodecanaminium Chloride (A10)

To a stirred solution of N-methyldodecylamine (250 μL, 1 mmol) in toluene (6 mL), 1,3-propane sultone (88 μL, 1 mmol) was added dropwise at 0 °C. The reaction mixture was stirred for 20 h at 45 °C and then evaporated. Next, the solid was suspended in dichloromethane (6 mL), and oxalyl chloride (257 μL, 3 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature, then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (130 mg, 2 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 271 mg, yield 71%.

¹H NMR (80 MHz, CDCl₃, ppm): 0.95 (m, 3H, N–CH₂–(CH₂)₁₀-CH ₃ ), 1.34 (s, 20H, N–CH₂–(CH ₂ ) ₁₀-CH₃), 2.59 (m, 2H, S-CH₂–CH ₂ -), 2.82 (s, 3H, N-CH ₃ ), 3.14 (m, 4H, -CH ₂ -N-CH ₂ -(CH₂)₁₀-CH₃), 3.93 (m, 2H, S-CH ₂ -).

4.1.12. 3-(Azidosulfonylpropyl)­bis­(dimethylamino)­methyleneaminium Chloride (A11)

To a stirred solution of tetramethylguanidine (627 μL, 5 mmol) in ethyl acetate (6 mL) was added dropwise 1,3-propane sultone (438 μL, 5 mmol) at 0 °C. The reaction mixture was stirred for 20 h at room temperature. The resulting precipitate was filtered out to remove unreacted compounds, washed with diethyl ester 3 times, and dried under vacuum to obtain 260 mg (1 mmol) of sulfonic acid.

Next, the sulfonic acid was suspended in dichloromethane (6 mL), and oxalyl chloride (260 μL, 3 mmol) and catalytic dimethylformamide were added subsequently while stirring. The reaction mixture was stirred for 20 h at room temperature, then evaporated to remove excess oxalyl chloride. The resulting solid was dissolved in acetonitrile (6 mL), and sodium azide (100 mg, 1.5 mmol) was added while stirring. The reaction mixture was stirred for 20 h at room temperature, then centrifuged to remove insoluble matter, and evaporated to give the resulting compound as a yellow solid 253 mg, yield 17%.

¹H NMR (80 MHz, CDCl₃, ppm): 3.69 (t, 2H, S-CH ₂ -), 3.43 (t, 2H, S-CH₂–CH₂–CH ₂ -N), 3.00 (s, 12H, N–CH₃), 2.44 (t, 2H, S-CH₂–CH ₂ -CH₂–N).

Caution! Synthesis of azides A12-A15 proceeds via formation of highly explosive sulfuryl chloroazide. One should not evaporate or heat the solution of sulfuryl chloroazide before conversion to a sulfamoyl azide.

4.1.13. Butanesulfamoyl Azide (A12)

To a stirred solution of sulfuryl chloride (243 μL, 3 mmol) in acetonitrile (5 mL) was added sodium azide (200 mg, 3 mmol) at 0 °C. The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove the insoluble matter. Next, to the resulting solution, butylamine (563 μL, 5.7 mmol) was added while stirring at 0 °C. The reaction mixture was stirred for 3 h at room temperature. Then, the reaction mixture was centrifuged to remove insoluble matter, evaporated, and then dissolved in dichloromethane (30 mL) and washed with 1 M hydrochloric acid (2 × 30 mL), 10% sodium bicarbonate (30 mL), and brine (30 mL). The organic layer was dried over sodium sulfate and evaporated to give the resulting product as a white powder (92 mg, yield 17%).

¹H NMR (80 MHz, CDCl₃, δ, ppm): 0.93 (t, −CH₂–CH ₃ , 3H), 1.17–1.70 (m, 4H, NH–CH₂–CH ₂ -CH ₂ -), 3.04 (t, 2H, -NH-CH ₂ -).

4.1.14. (N-Methylpiperazine)­sulfonyl Azide (A13)

To a stirred solution of sulfuryl chloride (243 μL, 3 mmol) in acetonitrile (5 mL), sodium azide (200 mg, 3 mmol) was added at 0 °C. The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter. Next, to the resulting solution, N-methylpiperazine (570 μL, 5.1 mmol) was added while stirring at 0 °C. The reaction mixture was stirred for 3 h at room temperature. Then, the reaction mixture was centrifuged to remove insoluble matter and evaporated. The crude product was purified by column chromatography (3% triethylamine in ethanol) to give the resulting product as a white powder (300 mg, yield 49%).

¹H NMR (80 MHz, DMSO-d⁶, δ, ppm): 2.19 (s, 3H, −CH₃), 2.31–2.52 (m, 8H, −CH₂–CH₂).

4.1.15. (N,N,N′,N′-Tetramethylguanidine)­sulfonyl Azide (A14)

To a stirred solution of sulfuryl chloride (122 μL, 1.5 mmol) in acetonitrile (5 mL) was added sodium azide (98 mg, 1.5 mmol) at 0 °C. The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter. Next, to the resulting solution was added tetramethylguanidine (360 μL, 2.9 mmol) while stirring at 0 °C. The reaction mixture was stirred for 3 h at room temperature. Then, the reaction mixture was centrifuged to remove insoluble matter and evaporated. The crude product was purified by column chromatography (10% triethylamine in ethanol) to give the resulting product as colorless crystals (267 mg, yield 80%).

4.1.16. Imidazolesulfonyl Azide (A15)

To a stirred solution of sulfuryl chloride (243 μL, 3 mmol) in acetonitrile (5 mL), sodium azide (200 mg, 3 mmol) was added at 0 °C. The reaction mixture was stirred for 20 h at room temperature. The mixture was centrifuged to remove insoluble matter. Next, to the resulting solution, imidazole (408 mg, 6 mmol) was added with stirring at 0 °C. The reaction mixture was stirred for 3 h at room temperature. Then, the reaction mixture was centrifuged to remove insoluble matter, evaporated, then dissolved in ethyl acetate (30 mL), and washed with 10% sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried over sodium sulfate and evaporated to give the resulting product as a colorless liquid (278 mg, yield 55%).

¹H NMR (80 MHz, CDCl₃, δ, ppm): 7.19 (s, 1H, N–CHCH-N), 7.33 (s, 1H, N-CHCH-N), 8.00 (s, 1H, N–CHN-).

4.2. Oligonucleotide Synthesis

Standard phosphoramidite solid-phase synthesis of all modified and unmodified oligonucleotides containing phosphodiester linkages (PO) was carried out on an ASM-800 DNA/RNA synthesizer (Biosset, Novosibirsk, Russia). Oligonucleotides were synthesized at a 0.2 μmol scale using standard commercial 2-cyanoethyl deoxynucleoside phosphoramidites and CPG solid supports (Glen Research, USA).

For the introduction of modifications via the Staudinger reaction, the following procedure was performed. The corresponding monomer was coupled without the steps of capping, oxidation, and deprotection. The reactor was removed from the synthesizer. The solid-phase support (CPG) was transferred from the reactor into a plastic tube and treated with a solution of a modifier azide (Staudinger reaction). After completion of the modification, the CPG was washed with acetonitrile, and then additional treatments were performed if necessary. After washing, the CPG was placed into the reactor, and all the following procedures were performed according to the standard protocol of automatic solid-phase phosphoramidite synthesis.

Detailed information about the synthesis of different oligonucleotide series is given below.

4.2.1. OD Series

Conditions of the Staudinger reaction: 0.5 M solution of the corresponding azide (see Table ) in acetonitrile, 25 °C, 1 h. For azides A2, A3, and A4, 10% v/v DIPEA was added to the reaction mixture to neutralize acidic media. No additional treatment was performed. For conditions of the final deblock, see Table .

4.2.2. OL Series

Conditions of the Staudinger reaction: 0.5 M solution of azide A1 in acetonitrile, 25 °C, 1 h. For oligonucleotides OL1-OL19, OL22-OL24, after completion of the Staudinger reaction, the CPG was washed with acetonitrile, and then the corresponding solution of amine was added (for condition, see Table ). Solutions of the amines were preliminarily dried by treatment over sodium hydroxide powder for 20 h. The conditions of the final deblock were as follows: aqueous ammonia, 30 min, 25 °C.

For oligonucleotides OL20, OL21, and OL 13, amination was performed during the final deblock stage.

OL12Ethylenediamine 30% vol. in toluene, 20 h, 50 °C. After cleavage, the solution was removed, the CPG was dried, and the resulting oligonucleotide was washed with water.

OL20Tris­(2-aminoethyl)­amine 30% vol in water, 20 h, 50 °C; OL21, 20 mg of spermine in water, 20 h, 50 °C. After cleavage, OL20 and OL21 were precipitated by 2% lithium perchlorate solution in acetone, and the removal of the DMTr group was performed using 80% acetic acid.

4.2.3. OF Series

OF1Conditions of the Staudinger reaction: 0.5 M solution of azide A1 in acetonitrile, 25 °C, 1 h. After completion of the reaction, the CPG was washed with acetonitrile, and then N,N′-dimethylethylenediamine was added (for condition, see Table ). After washing with acetonitrile, octanoyl chloride was added (for condition, see Table ).

Conditions of the final deblock: aqueous ammonia, 30 min, 25 °C.

OF2Conditions of the Staudinger reaction: 0.5 M solution of azide A1 in acetonitrile, 25 °C, 1 h. After completion of the reaction, the CPG was washed with acetonitrile, and then N-methylaminopropanol was added (for condition, see Table ). After washing, the CPG was placed into the reactor, and automated solid-phase phosphoramidite synthesis was continued using a modified program with an extended time of coupling (20 min)

OF3An oligonucleotide bearing sulfonyl phosphoramidate modification was synthesized. Conditions of the Staudinger reaction: 0.5 M solution of azide A2 in acetonitrile, 25 °C, 1 h. Another oligonucleotide was synthesized using alkyne-modified CPG (Lumiprobe, Russia). Conditions of the final deblock: aqueous ammonia, 15 min, and 55 °C.

Then, CuAAC reaction between two oligonucleotide chains was performed using a copper-containing buffer and sodium ascorbate solution (Lumiprobe, Russia) according to the manufacturer’s protocol.

OF4Conditions of the Staudinger reaction: 0.5 M solution of azide A2 in acetonitrile, 25 °C, 1 h. After completion of the reaction, the CPG was washed with acetonitrile, and then a solution of triphenylphosphine was added (for condition, see Table ). Conditions of the final deblock: aqueous ammonia, 30 min, 25 °C.

OF5Conditions of the Staudinger reaction: 0.5 M solution of azide A2 in acetonitrile, 25 °C, 1 h. After completion of the reaction, the CPG was washed with acetonitrile, and then a solution of triphenylphosphine was added (for condition, see Table ). Conditions of the final deblock: aqueous ammonia, 30 min, 25 °C.

To an aqueous solution of the obtained oligonucleotide, BDP630/650-X-NHS-ester (Lumiprobe, Russia) was added, and the reaction was performed according to the manufacturer’s protocol (0,006 M ester in DMSO, 3h, 35 °C)

4.2.4. OA and OX Series

Conditions of the Staudinger reaction: 0.5 M solution of the corresponding azide (Tables and ) in acetonitrile, 40 °C, 3 h for oligonucleotides OA1, OA3-OA6, and OX1-OX6; 0.5 M solution of the corresponding azide in acetonitrile, 25 °C, 1 h for oligonucleotide OA2. No additional treatment was performed. Conditions of the final deblock: aqueous ammonia, 15 min, 55 °C.

4.2.5. OH Series

Conditions of the Staudinger reaction: 0.5 M solution of the corresponding azide (see Table ) in acetonitrile, 25 °C, 1 h. For oligonucleotides OH1-OH12, after completion of the Staudinger reaction, the synthesis was continued according to the standard protocol to obtain the full oligonucleotide chain. Then, before final detritylation, the oligonucleotides were treated with the corresponding amine solutions (Table ). Solutions of the amines were preliminarily dried by treatment over sodium hydroxide powder for 20 h. After completion of the amination, the final DMTr group was removed according to the standard protocol. Conditions of the final deblock: aqueous methylamine, 1 h, 55 °C.

4.3. Oligonucleotide Analysis

Oligonucleotides were analyzed by RP HPLC on a Milikhrom A02 chromatograph using a ProntoSIL-120–5-C18 column (2 × 75 mm²; Ekonova, Russia) at a flow rate of 200 μL/min or on an Agilent 1200 HPLC system (USA) using a Zorbax SB-C18 5 μm column (4.6 × 150 mm²; Agilent, USA) at a flow rate of 2 mL/min. The analysis was performed in a linear gradient of acetonitrile 0–50% or 0–90% in 20 mM triethylammonium acetate, pH 7.0. The analyzed products were detected at a wavelength of 260 nm.

Electrophoretic analysis was performed in 15% PAAG, 8 M urea, gel with 0.1 M Tris-borate, pH 8.3, as running buffer. The oligonucleotide bands were visualized in a gel by staining with Stains-All (Sigma-Aldrich, Germany).

ESI mass analysis was performed in the Joint Center for Collective Use of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences. An Agilent G6410A mass spectrometer (USA) was used in negative ion mode. The samples were prepared by dissolving oligonucleotides in 20 mM hexafluoroisopropanol-triethylamine buffer in 60% aqueous acetonitrile at a concentration of 0.1 mM. The volume of the samples was 10 μL. Analysis was carried out using 80% aqueous acetonitrile as an eluent at a flow rate of 0.1 mL/min and using standard device settings. Molecular masses were calculated using experimental m/z values obtained for each sample.

The data underlying this study are available in the published article and its online Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11209.

• HPLC, PAGE profiles and undeconvoluted ESI mass spectra for oligonucleotides, additional list of modified oligonucleotides, including structures of modifications, and ¹H NMR spectra for azides (PDF)

†.

S.A.Z. and E.G.S contributed equally to this work. S.A.Z.: Formal analysis, investigation, methodology, validation, writingoriginal draft, writingreview and editing. E.G.S.: Formal analysis, investigation, writingoriginal draft, writingreview and editing. M.S.K.: Conceptualization, data curation, formal analysis, funding acquisition, project administration, supervision, writingoriginal draft, writingreview and editing.

The authors declare no competing financial interest.