Introduction

The installation of desirable chemical stimulus-responsive functionalities into bio(macro)molecules (e.g., nucleic acids and proteins) is important for the development of useful molecular tools in the fields of chemical and synthetic biology [1, 2]. The chemical approaches used to introduce stimulus-responsive groups into oligonucleotides (ONs) often include pre-modification of a stimulus-responsive moiety into a constituent monomer molecule (nucleotide) prior to the construction of a target ON. The pre-modification methods are generally based on chemical synthesis of the constituent monomer and the subsequent solid-phase synthesis of ONs, which enables site-selective introduction of stimuli-responsive groups into ONs. Alternatively, post-modification of a stimulus-responsive group after the construction of the ON has been less explored [3,4,5,6,7,8,9,10]. In fact, the majority of previously reported stimuli-responsive ONs have been constructed using pre-modification approaches [7,8,9]. Nevertheless, the chemical synthesis of long ONs is associated with technical limitations. Hence, the development of new chemistry-based post-modification methods for the preparation of stimuli-responsive ONs, as well as extension of the scope of existing approaches, is desirable [6].

Diazo compounds have been effectively applied in post-modification of bio(macro)molecules with modest to good selectivity [11, 12]. For instance, photo-caged nucleotides, such as caged adenosine triphosphate (ATP) [13], were constructed by reacting diazo compounds with phosphonate groups of the corresponding nucleotides. Subsequently, the same method was applied in a pioneering work involving the addition of diazo compounds into plasmid DNA to enable photo-controlled gene expression using photo-caged groups introduced randomly into plasmid DNA (e.g., 4,5-dimethoxy-2-nitrophenylethyl (DMNPE)-diazo or 6-bromo-4-diazomethyl-7-coumarin (Bhc)-diazo; Fig. 1a) [14,15,16]. More recently, Kala et al. demonstrated the selective introduction of a DMNPE group into terminal phosphate (monoester) groups instead of internal phosphodiester groups of double-stranded RNA (siRNA) using diazo compounds (DMNPE-diazo) [17], enabling photo-controlled RNA interference. Moreover, by employing the same method, the same group subsequently reported the installation of an additional azide handle, which could be modified via a click reaction [18]. Raines and Dennis investigated the reactivity of diazo compounds toward carboxylate and phosphate groups [19, 20] and determined that the reactivity depended on the pH as well as the presence and type of organic solvent. Therein, they putatively proposed that the nascent ion paired salt formed by the diazonium and oxo-anion species in a solvent cage by Coulombic forces plays an important role in their selective modification (O-alkylation) [20].

Fig. 1
figure 1

a Chemical structures of aryl diazomethanes, including 4-nitrophenyl diazomethane (4NB-diazo), investigated in this study. b Reaction scheme showing post-modification of an oligonucleotide (ON) bearing a 5′-phosphate group (ON1-5′p) using 4NB-diazo to introduce a reduction-responsive functionality

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In this study, we describe the construction of a reduction-responsive ON by a post-modification method using a diazo compound bearing a 4-nitrobenzyl moiety (4NB-diazo) as the reduction-responsive cleavable group (Fig. 1b). We envisioned that this post-modification approach can enable the effective preparation of ON-based reduction-responsive molecular tools directly from ONs bearing phosphate groups, which can be useful in the fields of chemical and synthetic biology [1, 2, 21, 22]. To the best of our knowledge, the reactivity of this diazo compound toward biomolecules containing phosphate groups, including ONs, as well as the reduction responsiveness of the obtained ONs, has not been previously explored.

Results and discussion

To introduce the 4-nitrobenzyl (4NB) group as a reduction-responsive removable moiety in ONs employing a post-modification approach, we first prepared 4NB-diazo (Fig. 1a) as the Bamford–Stevens reaction intermediate [23, 24] (Scheme S1). Briefly, according to a previously described method, we used a sterically hindered aromatic sulfonyl hydrazide (i.e., 2-mesitylenesulfonyl hydrazide) to synthesize the corresponding sulfonyl hydrazone precursor (Figs. S1, S2), which was converted to 4NB-diazo using 1,1,3,3-tetramethylguanidine (TMG) as the base (caution! diazo compounds are presumed to be highly toxic and potentially explosive) [25]. In the present study, instead of utilizing less sterically hindered p-toluenesulfonyl hydrazide, 4NB-diazo was efficiently prepared by employing 2-mesitylenesulfonyl hydrazide under milder conditions (data not shown). Owing to the instability and the potential explosive nature of the compound, the diazomethane derivative 4NB-diazo, which was obtained as a pale brown powder, was used without further purification. Notably, 1H nuclear magnetic resonance (NMR) analysis confirmed the identity and acceptable purity of 4NB-diazo (Fig. S3).

We subsequently studied the stability of 4NB-diazo under aqueous conditions. Because aryl diazomethane derivatives exhibit characteristic absorption bands at a wavelength of ~400 nm, monitoring the decrease in this absorbance using ultraviolet–visible spectroscopy enabled us to evaluate their stability. The half-lifetimes (τ) for 4NB-diazo at pH 7.0 and 6.0 under aqueous conditions were evaluated to be 40 and 11 min, respectively (Fig. S4A). Rapid decomposition of 4NB-diazo within ~10 s was noted at pH 5.0. A decrease in the half-lifetime of this compound under acidic pH is consistent with previous reports [19]. However, it is noteworthy that under the experimental conditions used in this study, the half-lifetimes for 4NB-diazo were relatively longer than those for 2NB-diazo (9.7 and 3.7 min at pH 7.0 and 6.0, respectively; Fig. S4B). In general, there is a tradeoff between stability and reactivity. Nevertheless, the half-lifetimes for 4NB-diazo compared to those for 2NB-diazo were deemed satisfactory for the subsequent investigation of the reactivity of 4NB-diazo toward the phosphate groups in ONs.

To evaluate the post-modification of ONs by 4NB-diazo, we employed an ON bearing a 5′-phosphate group (5′-pCCCTAGTTAGCCATCTCCC-3′ [19 nt]), hereafter referred to as ON1-5′p, where “p” denotes a phosphate group introduced at the 5′ position (Fig. 2a). The sequence itself was not important in this study, but it was designed to contain all four bases. Various amounts of freshly prepared dimethyl sulfoxide (DMSO) stock solutions of 4NB-diazo were added to an aqueous buffer solution of ON1-5′p (63 µM). Ion pair (triethylammonium acetate was used as the ion-pairing reagent, see Supplementary Information for the experimental details) reversed-phase high-performance liquid chromatography was performed to analyze the reaction mixture composed of an aqueous buffer (13 mM Tris-HCl containing 1.3 mM EDTA, pH 7.5) and DMSO in a 4:1 (v/v) ratio. As shown in Fig. 2b, the peak (tR = 12.8 min) corresponding to ON1-5′p significantly decreased 3 h after the addition of 4NB-diazo (50 and 75 eq.; Fig. 2b_vi, v). Concurrently, the appearance of a new peak (tR = 18.7 min) was observed. On the basis of the area of the peak, the consumption of ON1-5′p was evaluated to be 81%. The peak at tR = 24.2 min detected in the HPLC chart of the reaction mixture containing 4NB-diazo (Fig. 2b_viii) in the absence of ONs was attributed to 4-nitrobenzyl alcohol, a hydrolyzed product of 4NB-diazo, which was confirmed by comparison with an authentic sample of the compound (data not shown). To assign the new peak at tR = 18.7 min, we conducted matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. As shown in Fig. 2c_ii, the obtained spectrum exhibited a peak corresponding to ON1-5′pNB (5′-pNBCCCTAGTTAGCCATCTCCC-3′). Accordingly, the HPLC peak shift from 12.8 to 18.7 min was attributed to the increased hydrophobicity of ON due to the introduction of the 4NB group. The control experiments revealed that the ON without the 5′-phosphate moiety (ON1) only showed a negligible change in the HPLC chart under the same reaction conditions (Fig. 2b_vii). This result further supported our hypothesis that 4NB-diazo mainly reacted with the 5′-phosphate group of ON1-5′p and not the internal phosphodiester [17], affording ON1-5′pNB. These outcomes are comparable to the results obtained in previous studies of 2NB-diazo [19].

Fig. 2
figure 2

a Post-modification of an ON bearing a 5′-phosphate group (ON1-5′p) with 4NB-diazo. b Ion pair (IP) reversed-phase high-performance liquid chromatography (RP-HPLC) traces for ON1-5′p (i) before and (ii–v) 3 h after the addition of 4NB-diazo (ii: 10 eq., iii: 20 eq., iv: 50 eq., and v: 75 eq.), ON1 (vi) before and (vii) 3 h after the addition of 4NB-diazo (75 eq.), and  (viii) 4NB-diazo without the addition of either ON1-5′p or ON1 (detection wavelength = 260 nm; see Supplementary Information for the experimental details). c Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis of (i) ON1-5′p and (ii) ON1-5′pNB (3-hydroxypicolinic acid [3-HPA] was used as the matrix in the negative detection mode). Post-modification reaction conditions: [ON1-5′p] = 63 µM, [4NB-diazo] = 3–19 mM (10–75 eq. against ON1-5′p), aqueous buffer (13 mM Tris-HCl containing 1.3 mM EDTA, pH 7.5):DMSO = 4:1 (v/v), ambient temperature

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We subsequently investigated the reduction-responsive propensities of ON1-5′pNB obtained after HPLC purification (the isolated yield was evaluated to be ca. 20%). We employed Na2S2O4 (7.0 mM as the final concentration in the medium) as the chemical reducing agent capable of reducing nitro groups [7, 8]. As shown in Fig. 3b_ii, the peak at tR = 18.7 min attributed to ON1-5′pNB almost completely disappeared within 2 h following the addition of Na2S2O4. In addition, a new peak at tR = 12.8 min, which corresponded to the original ON1-5′p, was detected. To assign this peak, we conducted MALDI-TOF-MS measurements, which clearly demonstrated a peak assigned to ON1-5′p (Fig. 3c_ii). No severe decomposition of the ONs was detected, which was evidenced by the lack of apparent new peaks in the analyzed retention time range. These outcomes were consistent with previously reported findings for Na2S2O4-induced reduction of ONs without severe decomposition [7, 8]. Moreover, a control experiment involving the addition of the same amount of Na2S2O4 to ON1-5′p (Fig. 3b_iv, v) showed no noticeable changes in the peak. As shown in Fig. 3b_iii, only marginal peak changes were observed in the HPLC chart for ON1-5′pNB upon the addition of reduced glutathione (7.0 mM in the medium). This highlighted the selectivity of ON1-5′pNB toward the reducing species [26]. Overall, the results obtained in the present study demonstrated that recovery of the original ON1-5′p can indeed be induced by the selective reduction-responsive function of ON1-5′pNB.

Fig. 3
figure 3

a Reduction responsiveness of ON1-5′pNB to regenerate ON1-5′p. b IP RP-HPLC traces for ON1-5′pNB (i) before and 2 h after the addition of (ii) Na2S2O4 and (iii) glutathione and ON1-5′p (iv) before and 2 h after the addition of (v) Na2S2O4 (detection wavelength = 260 nm; see Supplementary Information for further details). c MALDI-TOF-MS spectra for (i) ON1-5′p and (ii) ON1-5′pNB following the reduction reaction (3-HPA, negative mode). Reduction conditions: [ON1-5′pNB] = 3.5 µM, [Na2S2O4] = 7.0 mM or [glutathione] = 7.0 mM, 10 mM Tris-HCl (pH 7.5), ambient temperature

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Conclusion

In summary, we successfully constructed a reduction-responsive ON via  a post-modification approach using a nitrophenyl diazomethane derivative. This post-modification approach is applicable to phosphate groups typically at the 5′- and/or 3′-end positions but not to internal phosphodiester groups of ONs; thus, the consequences of this approach might be selective but minimal. Nevertheless, the presence of a free 5′-phosphate group in ONs is crucial in several biological events, such as the resection of DNA ends by an exonuclease [26] and mRNA cleavage by Ago2 [27], suggesting that it might be possible to realize the reduction-responsive control of such biological events. Moreover, the developed simple protocol enabled the facile preparation of reduction-responsive ONs, which can be applied in the fields of chemical and synthetic biology. Further optimization of the process (e.g., improvement of the isolated yield) is necessary, but the described reduction-responsive functionality offers the possibility of constructing unique ON-based medicines because hypoxia-related conditions can trigger the reduction of nitroaromatics [28, 29]. Further research on this topic is currently ongoing in our laboratory.