Systematic Structural Tuning Yields Hydrazonyl Sultones for Faster Bioorthogonal Protein Modification

Abstract

We report the synthesis of a series of hydrazonyl sultones (HS) containing an ortho-CF₃ group, a five- or six-membered sultone ring, and a varying N-aryl substituent, and characterization of their aqueous stability and reactivity toward bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) in a 1,3-dipolar cycloaddition reaction. To avoid purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields with optimized reaction conditions. The X-ray crystal structure analysis of two HS revealed that the partially negative-charged fluorine atoms in CF₃ electrostatically shield the electrophilic nitrile imine (NI) center from a nucleophilic attack, underpinning their extraordinary aqueous stability. In addition, the N-aryl substituents further modulate HS reactivity and stability, with the electron-rich six-membered HS displaying excellent aqueous stability and increased cycloaddition reactivity. The utility of these improved HS reagents was demonstrated through fast and selective modification of a BCNK-encoded nanobody with second-order rate constants as high as 1500 M⁻¹ s⁻¹ in phosphate-buffered saline−ethanol (9:1), representing the fastest HS−BCN ligation reported in the literature.

Graphical Abstract

A robust HS: Systematic studies of substituent effect yield hydrazonyl sultones (HS) that permit autonomous access of highly reactive nitrile imines through tautomerization to afford faster bioorthogonal modification of a nanobody carrying a strained alkyne, bicyclo[6.1.0]non-4-yn-9-ylmethanol.

Introduction

Tautomerization entails the reversible interconversion among two or more isomeric structures through the proton migration. This process is controlled by several factors including temperature,^[1] solvent,^[2] pH,^[3] and substituents^[4]. Ring-chain tautomerization, a subclass of tautomerism, encompasses the interconversion between the cyclic and acyclic forms, which significantly impacts molecular properties. The ring-chain tautomerism is particularly important in biological chemistry; e.g., six tautomers of D-glucose are present in aqueous solution, including two straight chain forms, two pyranose forms, and two furanose forms.^[5] Factors such as ring size and neighboring groups may affect the stability of the ring−chain tautomers.^[6]

Nitrile imines (NI) are 1,3-dipoles known for their outstanding reactivity in cycloaddition reactions with alkenes and alkynes in various solvents including water to form the valuable pyrazolines and pyrazoles, respectively.^[7] Owing to their high reactivity, NI are typically masked in the stable tetrazole forms, which upon photoirradiation release NI in situ.^[8] To prolong NI half-lives in water, two strategies have been successfully developed: 1) the use of steric shielding groups; ^[9] and 2) the use of CF₃ as an electrostatic shielding group.^[10] While these strategies are useful in vitro, photoactivation would be difficult to implement in animal studies because light penetration into tissues is limited. Recently, we reported the autonomous access of highly reactive NI through the relatively stable hydrazonyl sultones (HS) via the HS−NI tautomerization.^[11] While we have examined the effects of sultone ring size and C-phenyl ring substituents on HS stability and reactivity, the effects of ortho-CF₃ and N-aryl substituents have not been investigated.

To gain a better understanding of the structure-stability-reactivity relationship among HS, here we report the synthesis of a series of five- and six-membered HS carrying an ortho-CF₃ group and a varying N-aryl substituent, and characterization of their stability in aqueous media and their reactivity toward BCN in a cycloaddition reaction (Figure 1). We also obtained two HS X-ray crystal structures, shedding a light on how the ortho-CF₃ group stabilizes the highly reactive NI tautomers. Furthermore, four six-membered HS displayed significantly faster reaction kinetics than the best HS reported previously in selective modification of a BCN-encoded nanobody in aqueous medium.

Figure 1.

Optimizing HS structures for faster bioorthogonal modification of a BCNK-encoded nanobody.

Results and Discussion

Recently, we reported the synthesis of hydrazonyl sultones by irradiating 2,5-diaryltetrazoles bearing a neighboring sulfonic acid.^[11] However, purification of the sulfonic acid-containing tetrazoles proved challenging due to their high polarity. To address this issue, we decided to protect the sulfonic acid group in the sulfonate form and remove the protecting group prior to photolysis without purification. In addition, since installation of CF₃ group at the ortho-position on C-aryl ring has been shown to increase the stability of 5-membered HS and enhance the cycloaddition reactivity,^[11] we prepared a series of HS containing either five- or six-membered sultone, an ortho-CF₃ substituent at C-phenyl ring, and a varying N-aryl substituent.

Synthesis of ortho-sulfonate diaryltetrazoles

The synthesis of 2,5-diaryltetrazoles 3a-j bearing a protected phenyl benzenesulfonate is depicted in Scheme 1. Starting from the commercially available 2-fluoro-6-(trifluoromethyl)benzonitrile, we first converted ortho-fluorine to phenyl benzenesulfonate 1 using a three-step procedure: (i) thiolation with sodium sulfide in DMF to yield the thiophenol intermediate; (ii) conversion to sulfonyl chloride through oxychlorination;^[12] (iii) quenching with phenol to generate phenyl benzenesulfonate. Since cycloaddition reaction of NaN₃ with the sterically hindered benzonitrile 1 was sluggish, we employed ZnCl₂ as a Lewis acid catalyst and TMEDA to improve solubility in toluene at 95°C. This modified condition produced 2H-tetrazole 2 in 81% yield. The tetrazole was then subjected to Cu₂O-catalyzed coupling^[13] with a range of aryl boronic acids to generate ortho-CF₃ and ortho-sulfonate-substituted 2,5-diaryltetrazoles 3a-I in 31−82% yields.

Scheme 1.

Synthesis of 2,5-diarytetrazoles containing ortho-CF₃ and ortho-sulfonate groups.

The synthesis of diaryltetrazoles 10a-j bearing a neopentyl-protected ortho-isopropylsulfonate is depicted in Scheme 2. To begin, we prepared benzonitrile 4 following a literature procedure.^[14] Since the cyano group was unstable during reduction of the neighboring methyl ester, we decided to install the 2H-tetrazole first, which was then protected by a cumyl group to give tetrazole 5 in 51% yield. We selected the cumyl group because: 1) it exhibits greater stability than the trityl group under strong acidic conditions; 2) it can be easily removed using TFA in the presence of TES, whereas the tert-butyl group proved to be challenging to remove. Tetrazole 5 was converted to benzyl bromide 6 under standard reduction and bromination conditions in 51% yield. Synthesis of sulfonate 7 was achieved in 60% yield following the same three-step sequence as previously except that neopentyl was chosen as a protecting group because of its superior stability under both basic and acidic conditions.^[15] The addition of the geminal dimethyl groups was accomplished through sequential methylations: the first one using NaH as a base; and the second using a stronger base LiHMDS, affording tetrazole 8 with 82% yield. After removing the cumyl group, 2H-tetrazole 9 intermediate was subjected to Cu₂O-catalyzed coupling with a variety of arylboronic acids to generate 2,5-diaryltetrazoles 10a-h and 10j in 14−82% yields. In addition, tetrazole 10i was derived from 10h through partial oxidation followed by reductive amination with morpholine in an overall 85% yield.

Scheme 2.

Synthesis of 2,5-diarytetrazoles containing ortho-CF₃ and ortho-isopropylsulfonate groups. ^a Derived from 10h in two steps: (i) Dess-Martin periodinane; (ii) morpholine, NaBH₃CN, AcOH.

Synthesis of HS from diaryltetrazoles

The five-membered hydrazonyl sultones (HS) were obtained by hydrolyzing the phenyl sulfonates under basic conditions followed by photolysis (Scheme 3). Notably, a 254-nm handheld UV lamp was used for photo-irradiating tetrazoles 3f and 3g because they display blue-shifted UV-Vis spectra due to the presence of an electron-withdrawing group on the N-phenyl ring.^[16] HS with electron-deficient and -neutral aryl rings such as 11c, 11f, 11g, and 11h were obtained in moderate-to-high yields, whereas those with electron-rich aryl rings such as 11a, 11d, and 11e were obtained in poor-to-moderate yields. The lower yields can be attributed to NI dimerization as the electron-rich NI possess high reactivity and tend to dimerize.^[7] Separately, the six-membered hydrazonyl sultones (HS) were obtained by deprotecting the neopentyl group in tetrazoles 10a-g, 10i, and 10j with KF in trifluoroethanol at 110 °C followed by photolysis. Interestingly, contrary to five-membered HS, six-membered HS carrying the electron-rich aryl rings such as 11j, 11k, and 11r were obtained in moderate-to-excellent yields (Scheme 3), whereas HS 11l, 11n, and 11o containing the electron-deficient N-aryl rings were obtained in lower yields.

Scheme 3.

Synthesis of HS from 2,5-diarytetrazoles. PG: protecting group. Conditions for deprotection: KOH, EtOH/H₂O, reflux (5-membered HS); KF, TFE, 110 °C (6-membered HS). Conditions for photolysis: 302 or 254 nm, EtOH/EtOAc (5-membered HS) or THF/EtOAc (6-membered HS). ^a Isolated yield for combined steps of (i) KOH, EtOH/H₂O, reflux; (ii) AcCl, pyridine; (iii) photoirradiation. ^b Isolated yield for combined steps of (i) KF, TFE, 110 °C; (ii) NaOH, H₂O; (iii) photoirradiation.

Characterization of HS stability and reactivity

Among the five-membered HS, 11a-c and 11g were found to be most stable with a half-life (t_1/2) of ~1.6 hours (Table 1). In contrast, 11f showed a shorter t_1/2 of 0.59 hour, attributable to a greater NI fraction in tautomerization equilibrium due to the lower pKa of the N-H. Compound 11i emerged as the most unstable HS with t_1/2 of 0.46 hour, likely a result of facilitated deprotonation of the N-H by the neighboring OH group, which was supported by ¹H NMR data showing a downfield shift of the N-H from typical 7−8 ppm to ~10 ppm, thereby shifting the HS−NI tautomeric equilibrium towards the unstable NI tautomer.

Table 1.

Stability and BCN reactivity of HS in aqueous media

HS	t1/2 (h)a	k2 (M−1 s−1)b
11a	1.59 ± 0.04	2540 ± 260
11b	1.53 ± 0.01	1990 ±190
11c	1.63 ± 0.01	1710 ± 180
11d	1.07 ± 0.01	1610 ± 100
11e	1.35 ± 0.02	1430 ± 140
11f	0.59 ± 0.01	2630 ± 130
11g	1.64 ± 0.01	2060 ± 80
11h	0.95 ± 0.04	2830 ± 450
11i	0.464 ± 0.003	739 ± 59
11j	8.6 ± 2.1	81 ± 3
11k	8.9 ± 1.2	117 ± 3
11l	14.3 ± 2.1	69 ± 0.9
11m	11.4 ± 5.8	47 ± 2
11n	16.0 ± 6.9	8.2 ± 0.4
11o	3.9 ± 0.6	112 ± 6
11p	4.5 ± 0.4	99 ± 4
11q	7.6 ± 2.2	3.5 ± 0.3
11r	4.4 ± 2.3	172 ± 5

^aFor stability assays, absorbance of 0.5 mL solution of HS (100 μM 11a-i in 1:1 PBS−ACN; 50 μM 11j-r 9:1 PBS−EtOH) was monitored at 353 −388 nm.

^bFor BCN reactivity assays, absorbance of 0.5 mL solution of 20 μM HS and 200–500 μM BCN (11a-i in 1:1 PBS−ACN; 11j-r in 9:1 PBS−EtOH) was monitored at 353 – 388 nm. See Supporting Information for details.

For six-membered HS, we performed the stability assays in PBS− ethanol (9:1, pH 7.4) because they displayed substantially higher stability than five-membered HS in aqueous media. The t_1/2 values range from 3.9 to 16 hours (Table 1). Contrary to five-membered HS, six-membered HS carrying electron-deficient aryl rings (11l-n) showed higher stability than those carrying electron-rich rings (11j-k and 11p-r). One exception is 11o possessing the electron-deficient fluoropyridine ring showing lower stability, indicating that the stability is not solely determined by the electronic effect. For reactivity studies, we selected BCN because of its exceptional in vivo stability and genetic encodability in protein systems.^[17] We surmise that the N-aryl substituents affect HS reactivity and stability through two mechanisms: (i) altering the position of the HS−NI tautomerization equilibrium, and (ii) modifying energy level of the corresponding NI.^[18] Essentially all five-membered HS (11a-h) displayed high reactivity toward BCN, with second-order rate constants (k₂) ranging from 1430 to 2830 M⁻¹ s⁻¹ (Table 1). Compound 11i gave the slowest reaction in this series with k₂ value of 739 ± 59 M⁻¹ s⁻¹, presumably due to the steric hindrance of the proximal hydroxymethyl group. The reactivity trend in the six-membered HS series (11j-n, 11p, 11r) generally mirrors the intrinsic reactivity trend of the corresponding NI (Table 1), with the electron-rich NI likely possessing higher HOMO energy.^[18] Notably, one of the least stable HS, 11r, yielded the fastest reaction (k₂ = 172 ± 5 M⁻¹ s⁻¹), in agreement with our recent observation that HS reactivity is inversely proportional to its stability.^[11]

Structural studies of HS

Compared to HS without the CF₃ group (Table S1),^[11] our new series of HS displayed greater stability (up to 11 times longer t_1/2) and increased reactivity toward BCN (up to 3.0 times greater k₂). To understand structural basis of this enhanced stability−reactivity profile, we obtained crystal structures of HS 11d and 11k (Figure 2) and found that fluorine atoms tilt toward the top and bottom of the benzene ring (see side view). This rotamer conformation acts as a requisite electrostatic shield ^[10] to prevent the attack of a nucleophile such as water to the NI electrophilic center (C7 in 11d, C8 in 11k; see top view).

Figure 2.

Crystal structures of 11d and 11k: left, top view; tight, side view. The structural diagrams containing anisotropic displacement ellipsoids are drawn at the 50% probability level.

From the side view, 11d adopts a coplanar arrangement between C-phenyl ring and N-naphthyl ring, which is energetically very favorable for sultone ring rupture, leading to a greater amount of reactive NI in the tautomerization equilibrium and thus higher BCN reactivity.^[11] In contrast, the hydrazonyl group in 11k is twisted out of plane with respect to the hydrazine ring. This high-energy geometry would increase the activation barrier for the sultone ring rupture and decrease the amount of reactive NI in the tautomerization equilibrium, which in turn slows down the cycloaddition. These structural differences underpin the divergent reactivity profiles for the five- and six-membered HS.

Bioorthogonal modification of nanobody NB1

Nanobodies, also known as heavy-chain-only antibodies (VHHs),^[19] offer a powerful modality for the development of precision diagnostics and therapeutics.^[20] Compared to the conventional immunoglobulin IgG, nanobodies possess small size (~15 kDa), excellent solubility and stability,^[21] and greater tissue penetration.^[22] The use of robust display technologies and large-scale production in bacteria have made nanobodies extremely versatile for various clinical applications.^[23] To harness HS reactivity for selective nanobody modification, we selected NB1—a prototypical nanobody that binds to GFP^[24]—and substituted Val-4 next to CDRs with bicyclononyne-lysine (BCNK) via genetic code expansion. The NB1-V4BCNK mutant was obtained at a yield of 6.5 mg/L, and incubated with four selected HS analogs, along with HS-14 as a benchmark, in PBS−EtOH (9:1; Figure 3a). The reaction progress was monitored by QTOF-LC/MS (Figure 3b). We found that HS showed 5.6−11 times faster reactions with NB1-V4BCNK than with BCN under the same conditions, with 11r and 11 k giving the fastest reaction and the largest rate enhancement within the experimental error (Figure 3c). We attributed this rate enhancement to the microenvironment of BCN on NB1 surface, which can either recruit HS from bulk solvent to increase effective local concentration or help to shift the tautomerization equilibrium to the reactive NI form, or both. Remarkably, regardless of the N-aryl ring structure, all tested HS exhibited faster reactions with BCN than HS-14, a compound identified previously for its excellent stability and reactivity,^[11] both in solution and on NB1 surface (Figure 3c), suggesting that the rate acceleration is derived primarily from the otho-CF₃ group that helps to shift the tautomerization equilibrium to the reactive NI form.

Figure 3.

Bioorthogonal modification of a BCNK-encoded nanobody via HS−BCN ligation. (a) Reaction scheme. (b) Time courses of chemical modification of NB1-V4BCNK by HS reagents monitored by QTOF-LC/MS. (c) Tabulated data comparing reaction kinetic constants of HS with BCN to those with NB1-V4BCNK.

Conclusions

In summary, we have synthesized a series of HS bearing a five- or six-membered sultone ring, an ortho-CF₃ substituent, and a varying N-aryl substituent. The challenge of purifying high-polarity intermediates during the synthesis was overcome using the proper protecting groups. We have further characterized the stability and reactivity of the new HS analogs in aqueous solution. Specifically, the five-membered HS exhibit lower aqueous stability but higher BCN reactivity in PBS−ACN (1:1), whereas the six-membered HS display higher aqueous stability (up to 16 hours in t_1/2) but lower BCN reactivity in a competitive PBS−EtOH (9:1) solvent. Among six-membered HS, compound 11r exhibited the fastest reaction with BCN (k₂ = 172 ± 5 M⁻¹ s⁻¹) in PBS−EtOH (9:1). The crystal structures of HS 11d and 11k provided insights into the electrostatic shielding provided by the ortho-CF₃ group on aqueous stability of HS, and the lower cycloaddition rate of the six-membered HS compared to the five-membered HS. The bioorthogonal reactivity of HS toward BCN was assessed using a BCN-encoded nanobody, with all selected HS yielding faster reactions than the benchmark HS-14. Compounds 11r and 11k gave the fastest cycloaddition reaction (as fast as 1500 M⁻¹ s⁻¹), 10 times faster than HS-14. The present study underscores the importance of understanding the HS structure−stability−reactivity relationship, and highlights how subtle structural changes lead to significantly improved bioorthogonal reactivity in protein systems.

Experimental Section

Bioorthogonal modification of NB1-V4BCNK by hydrazonyl sultones.

In a 0.6-mL Eppendorf tube, a solution of 5 μM NB1-V4BCNK and 50 μM of hydrazonyl sultone in 250 μL PBS, pH 7.4, was incubated at room temperature. At a pre-specified timepoint, a 25-μL reaction mixture was aliquoted to a 50-μL solution containing 0.5 mM BCNK in PBS to quench the remaining hydrazonyl sultone. The resulting mixture was analysed by QTOF-LC/MS. The conversions were calculated based on the areas under curve in LC/MS traces.

Deposition Numbers 2256240 (11d) and 2256241 (11k) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.