Further enhancement of the clickability of doubly sterically-hindered aryl azides by para-amino substitution

Abstract

Introduction of an amino group at the para position of doubly sterically-hindered aryl azides significantly enhances the reactivity with cyclooctynes. The distortability of the azido group is synergistically enhanced by the para-electron-donating group and two bulky ortho substituents, which increases the HOMO energy level and provoke the steric inhibition of resonance, respectively.

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The click reaction,^1,2 particularly the catalyst-free, strain-promoted azide–cycloalkyne cycloaddition (SPAAC),^3,4 is one of the most reliable methods for conjugating molecules, having wide applications in broad research fields such as materials chemistry⁵ and chemical biology.⁶ To make the method more practical, a wide variety of useful cyclic alkynes with improved properties such as higher azidophilicity, lower hydrophobicity, and synthetic easiness have been developed.⁴ In contrast, little attention has been paid to increase the reactivity of the azide.⁷

In the course of our studies on azide chemistry,⁸ we found that 2,6-disubstituted phenyl azides show remarkably enhanced clickability in the reaction with strained alkynes such as Sondheimer diyne (1),^9,10 despite the steric hindrance.^8d From experimental and computational studies, we concluded that this seemingly contradictory phenomenon could be attributed to the increased distortability of the azido group elicited by the steric inhibition of resonance with the aromatic ring (Fig. 1). Compared to the electronic effects observed for the clickability of para-substituted phenyl azides (e.g., 2aversus2b), doubly sterically-hindered substrates such as 2,6-diisopropylphenyl azide (2c), in which the resonance is canceled to some extent, showed remarkably high reactivity. This finding enabled the facile preparation of trifunctional molecules by three sequential azido-type-selective triazole forming reactions using a triazido platform molecule bearing three different types of azido groups.^8g In this study, with the aim of enhancing the selectivity of the click conjugation, the effect of para substituents on the clickability of 2,6-diisopropylphenyl azides was examined.

Fig. 1 Double-click reaction of Sondheimer diyne (1) with aryl azides 2.^8dk: second-order rate constant.

We firstly examined the reactivity of 2,6-diisopropylphenyl azides 2d–f bearing para-bromo, -iodo, and -pinacolatoboryl group, respectively, with diyne 1 (Table 1). Despite the different electronegativity of the three groups, little difference in clickability was observed between these azides (entries 2–4) and 2,6-diisopropylphenyl azide (2c) (entry 1).

Table 1 Clickability of para-halogenated or -borylated 2,6-diisopropylphenyl azides

Entry	R	2	k (M^−1 s^−1)	k rel	Yield (%)	trans/cis
a From ref. 8d. b pin = pinacolato.
1^a	H	2c	0.67	1	95	96/4
2	Br	2d	0.65	0.97	Quant	95/5
3	I	2e	0.62	0.93	Quant	95/5
4	Bpin^b	2f	0.51	0.76	79	96/4

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We then turned our attention to the resonance effect that would be induced by the para substituent. For an efficient comparison, we prepared two types of 2,6-diisopropylphenyl azides 2g–k and 2l–o bearing a variety of para-aryl¹¹ or -amino¹² groups by the palladium-catalyzed cross-coupling reactions of 4-bromo-2,6-diisopropylaniline (4), followed by azidation of the resulting anilines via diazotization (Scheme 1). In addition, by means of the azido-type-selective cycloaddition^8g of diazide 5 with acetylacetone catalyzed by potassium carbonate,¹³para-triazolyl-substituted 2,6-diisopropylphenyl azide 2p was prepared (Scheme 2).

Scheme 1 Preparation of azides 2g–o.

Scheme 2 Preparation of azide 2p.

The effect of para-aryl substituents on the clickability of 2,6-diisopropylphenyl azides was found quite limited in the reaction with diyne 1 (Table 2, entries 2–6). The reaction rates for azides 2g–j, bearing different aryl groups, i.e., phenyl, 2,6-xylyl, 4-(trifluoromethyl)phenyl, and 4-methoxyphenyl, respectively, were almost the same or smaller than that for azide 2c (entries 2–5), whereas 2k, which has an electron-rich 4-(dimethylamino)phenyl group, reacted slightly faster than 2c (entry 6).

Table 2 Clickability of para-aryl- and -amino-substituted 2,6-diisopropylphenyl azides

Entry	R	2	k (M^−1 s^−1)	k rel	Yield (%)	trans/cis
a From ref. 8d. b In acetonitrile.
1	H	2c	0.67^a (0.37)^b	1 (1)^b	95^a	96/4^a
2	Ph	2g	0.74	1.1	Quant	96/4
3	2,6-Me2C6H3	2h	0.75	1.1	81	96/4
4	4-F3CC6H4	2i	0.47	0.70	Quant	95/5
5	4-MeOC6H4	2j	0.72	1.1	Quant	96/4
6	4-Me2NC6H4	2k	0.83	1.2	88	96/4
7		2l	1.4	2.1	Quant	95/5
8		2m	1.6	2.4	77	96/4
9		2n	1.7	2.5	Quant	96/4
10		2o	3.5 (2.1)^b	5.2 (5.8)^b	Quant	95/5
11		2p	0.53	0.79	Quant	99/1

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In marked contrast to the limited effect of the aryl, halogeno, and boryl groups, introduction of an amino group at the para position of 2,6-diisopropylphenyl azide significantly enhanced the clickability (Table 2, entries 7–11). For example, azides 2l–o bearing the para-amino groups reacted with 1 more than twice faster than 2c (entries 7–10). In particular, 2,6-diisopropyl-4-pyrrolidinophenyl azide (2o) showed the highest clickability, reacting more than five times faster than 2c (entry 10) and approximately 400 times faster than phenyl azide (2a). The enhanced clickability of azide 2o was also observed in the reaction with diyne 1 in acetonitrile, although the reaction rate decreased slightly compared to that in methanol (entry 10 versus entry 1). The reaction of azide 2p with 1 was rather slower than that of 2c, indicating that the triazolyl group was not effective in accelerating the reaction (entry 11).

Introduction of an amino group at the para position of phenyl azide also enhanced the clickablity of substrates without ortho substituents, as demonstrated in the reaction with diyne 1 (Table 3). For example, 4-morpholinophenyl azide (2q) showed slightly higher clickablity than 4-methoxyphenyl azide (2b) (entry 3 versus entry 2). The introduction of a morpholino group at the para position of phenyl azide elicited a 3.4-fold acceleration of the reaction (entry 3 versus entry 1). 4-Azidoaniline (2r), which has an unsubstituted amino group, also showed a comparable reactivity to that of the methoxy congener 2b (Table 3, entry 4 versus entry 2). Intriguingly, this accelerating effect was completely canceled by salt formation of the amino group of 4-azidoaniline, which resulted in the deceleration of the reaction (entry 5).

Table 3 Clickability of para-substituted phenyl azides

Entry	R	2	k (M^−1 s^−1)	k rel	Yield (%)	trans/cis
a From ref. 8d. b In acetonitrile.
1	H	2a	8.8 × 10^−3^a (6.4 × 10^−3)^b	1 (1)^b	94^a	43/57^a
2	MeO	2b	3.3 × 10^−2^a (1.8 × 10^−2)^b	3.8 (2.7)^b	89^a	36/64^a
3		2q	(2.2 × 10^−2)^b	(3.4)^b	Quant	37/63
4	H2N	2r	2.7 × 10^−2	3.1	Quant	36/64
5	ClH·H2N	2s	6.5 × 10^−3	0.74	Quant	35/65

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Frontier molecular orbital (FMO) analysis of azide 2c and para-amino-substituted 2,6-diisopropylphenyl azides 2m–o revealed a good correlation between the HOMO energy levels and the second-order rate constants (k) (Table 4). It was found that the reactivity of azides with diyne 1 increased with the rise of the HOMO energy. This result suggests that the electron-donating amino group at the para position reduced the energy gap between the HOMO of the aryl azide and the LUMO of diyne 1, which resulted in the enhancement of the cycloaddition. Indeed, azide 2o, which exhibited the highest HOMO energy due to the presence of the highly electron-donating pyrrolidino group, proved to be the most reactive among the azides examined.¹⁴

Table 4 FMO analysis of azides 2c and 2m–o

Entry	ArN3	HOMO (eV)	LUMO (eV)	k (M^−1 s^−1)	k rel
a Second-order rate constant (k) for the reaction of 1 with aryl azides 2 conducted in methanol at 25 °C (from Table 2).
1	2c	−6.19	−0.75	0.67	1
2	2m	−5.29	−0.65	1.6	2.4
3	2n	−5.16	−0.58	1.7	2.5
4	2o	−4.85	−0.44	3.5	5.2

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To gain further mechanistic insight into the enhanced clickability of para-amino-substituted phenyl azides, the transition state (TS) structure for the first cycloaddition of 1 with 2o was obtained by a density functional theory (DFT) (B3LYP/6-31G(d)) method¹⁵ and compared with those obtained for the reactions with 2a and 2c (Table 5). The activation energy for the reaction of 1 with 2o was lower than that with 2c, which was in good agreement with the experimental result. The corresponding distortion–interaction analysis¹⁶ indicated that, in accord with our previous report,^8d the enhanced clickability of 2o mostly rested on the enhanced distortability of the azido group (2a: 17.4 kcal mol⁻¹; 2c: 15.1 kcal mol⁻¹; 2o: 13.8 kcal mol⁻¹). We previously explained that the higher clickability of 2c compared to that of 2a was elicited by the steric inhibition of resonance: i.e., the two bulky ortho substituents of 2c forced the azido group to twist out of the plane of the benzene ring, which partially released the azido from the strong conjugation. In this case, however, the calculated rotational angle (θ) of the azido group from the aromatic plane for 2o (θ = 61.2°) was similar to that for 2c (θ = 58.2°) (Fig. 2), indicating that the steric inhibition of resonance alone cannot be invoked to rationalize the higher clickability of 2o compared to that of 2c. Taken together, the FMO and the distortion–interaction analyses suggest that the clickability of doubly sterically-hindered aryl azides bearing an amino group at the para position stemmed from the enhanced distortability of the azido group, which was synergistically provoked by the steric inhibition of resonance and the raised HOMO energy level.

Table 5 DFT analysis of the cycloaddition between azides and diyne 1^a

Reactants	2a + 1^b		2c + 1^b		2o + 1
a Distortion, interaction, and activation energies for the first cycloaddition obtained at the B3LYP/6-31G(d) are shown in kcal mol^−1. b From ref. 8d. c Energy required to distort the geometry of each reactant to the transition state (TS). d Interaction energy between the distorted fragments at the TS. e Energy differences of each fragment between the optimized and the TS geometries.
Distorted reactants^c	+17.4	+3.1	+15.1	+3.1	+13.8	+2.7
Distortion energy^c (ΔE^‡d)	+20.4		+17.8		+16.5
Interaction energy^d (ΔE^‡i)	−9.0		−8.9		−8.6
Activation energy^e (ΔE^‡)	+11.4		+8.9		+7.9

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Fig. 2 Global minima on the potential energy surface of aryl azides 2c and 2o obtained by DFT (B3LYP/6-31G(d)) method. (A) Overhead view of 2c. (B) Side view of 2c. (C) Overhead view of 2o. (D) Side view of 2o. θ indicates the rotational angle of the azido group from the aromatic plane.

The clickability enhancement by para-amino substitution of sterically-hindered aryl azide was also observed in the SPAAC reactions using other cycloalkynes that are widely used for bioconjugation. The most reactive azide 2o in the reaction with diyne 1 also showed significantly higher reactivity in the reactions with a bicyclo[6.1.0]non-4-yne (BCN)⁴ⁱ derivative (k = 0.92 M⁻¹ s⁻¹) in MeOH than 2c (k = 0.20 M⁻¹ s⁻¹). The reaction of a 1 : 1 mixture of 2o and 2c with a dibenzoazacyclooctyne (DIBAC)^4g derivative afforded 2o- and 2c-derived cycloadducts at a ratio of 3.3 : 1.¹⁷

The enhanced clickability of the aryl azide elicited by the para-amino group enabled performing a sequential double-click reaction using diazido compound 6 with excellent azido-type selectivity (Scheme 3). Thus, the reaction of 6 with the strained alkyne 7 took place exclusively at the sterically-hindered para-amino-substituted aryl azido group, despite the presence of another phenyl azido group with sufficient reactivity, and the subsequent copper-catalyzed azide–alkyne cycloaddition with terminal alkyne 8 afforded bistriazole 9 in high yield.

Scheme 3 Azido-type selective double-click reaction of diazide 6.

In summary, we have found that the introduction of an amino group at the para position of doubly sterically-hindered aryl azides significantly enhances their clickability in the reaction with strained alkynes. The azide is synergistically activated by the electron-donating para-amino group and two bulky ortho substituents, with the former increasing the HOMO energy level of the azide, thereby enhancing its ynophilicity, and the latter inhibiting the resonance between azido and phenyl groups, which enhances the distortability of the azido group. This finding allowed for the sequential double-click conjugation of a diazido compound with excellent efficiency, which would be useful for the preparation of multifunctionalized molecules using a multiazido platform molecule bearing a para-amino-2,6-disubstituted aryl azide unit. Further studies to develop more reactive azides for their application to the preparation of multifunctional molecules are ongoing.

This work was supported by AMED under Grant Numbers JP18am0101098 (Platform Project for Supporting Drug Discovery and Life Science Research, BINDS) and JP18am0301024 (the Basic Science and Platform Technology Program for Innovative Biological Medicine); JSPS KAKENHI Grant Numbers JP15H03118 and JP18H02104 (B; T. H.), JP16H01133 and JP18H04386 (Middle Molecular Strategy; T. H.), JP17H06414 (Organelle Zone; T. H.), JP26350971 (C; S. Y.), and JP17K13266 (Young Scientist B; Y. N.); the Cooperative Research Project of Research Center for Biomedical Engineering; and the Naito Foundation (S. Y.).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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17. See the ESI† for details.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, characterization for new compounds including NMR spectra. See DOI: 10.1039/c8cc05791e

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