Click with a Boronic Acid Handle: A Neighboring Group-assisted Click Reaction that Allows Ready Secondary Functionalization

Abstract

The feasibility of a neighboring boronic acid-facilitated facile condensation of an aldehyde is described. This reaction is bio-orthogonal, complete at room temperature within minutes, and suitable for bioconjugation chemistry. The boronic acid group serves the dual purpose of catalyzing the condensation reaction and being a handle for secondary functionalization.

Graphical Abstract

A novel boronic acid facilitated cyclization reaction was developed. This reaction reaches completion at room temperature within minutes and is suitable for coupling and bioconjugation chemistry.

Click chemistry describes reactions that are fast and versatile, require mild conditions, and achieve high yields after minimum purification effort.¹ A number of reactions in this category has been reported. Some examples are the well-known copper-catalyzed and strain promoted/copper-free azide-alkyne cycloaddition reactions,² tetrazine and strained alkyne/alkene Diels-Alder type of reactions,³ and the Staudinger–Bertozzi ligation.⁴ These types of reactions have found wide-spread utility in a number of applications including bioconjugation, drug delivery, development of libraries of compounds, materials chemistry, and DNA modifications.⁵ In many such applications, the ability to undergo a secondary reaction will be very useful.⁶ The idea of orthogonal click-click reactions is also reflected in our earlier effort in tuning reaction rates for staged labeling.^3c A recent and very significant report of protecting strained alkynes with Cu(I) also allows for sequential click reactions.⁷ Incorporation of the azido/alkyne chemistry and cyanobenzothiazole/1,2-aminothiol chemistry pairs into a single molecule would also allow for sequential click reactions and thus staged labeling.⁸ Herein, we report a simple system, which allows for sequential reactions by utilizing a neighboring boronic acid-mediated click cyclization. The boronic acid functional group can then be used for a secondary modification reaction under mild conditions. The initial cyclization is very fast, complete within minutes in excellent yields (up to > 95 %), does not need an external catalyst, and uses mild/inert conditions. The key aspects of the developed methodology are 1) the reaction is promoted through activation of the aldehyde by an ortho-positioned boronic acid; 2) the reaction is tolerant of various substituents on the formyl aromatic ring; and 3) the benzothiazole product carries a boronic acid moiety that can be used as a handle for further modifications using a large number of well-established conditions.⁹

In the process of searching for new bioorthogonal click reactions, we are interested in condensations involving aldehydes. It is well known that aldehydes can undergo facile condensation reactions with compounds that have vicinal nucleophiles. Aldehyde condensations with aryl diamines, aminobenzothiols, aminophenols, and cysteine have been previously reported.¹⁰ For example, aminobenzothiols and aminophenols have been condensed with an aldehyde in the presence of ZnO nanoparticles^10c or in toluene at refluxing temperature; and similar reactions using cysteine analogs took 6–15 hours to complete at room temperature.^10a Recently, Ai and colleagues reported a facile oxidative condensation of aldehydes with o-phenylenediamine analogs under acid catalysis.¹¹ This was used for protein conjugation for various applications. Such reactions are also similar to the cyanobenzothiazole-cysteine condensation reactions.^{8a, 8b, 12} We are interested in taking advantage of this class of reactions by developing mild and facile conjugation chemistry that occurs without the use of acid. In doing so, we were interested in exploring the ability of a boronic acid group to function as a Lewis acid. Our lab has had a long-standing interest in boronic acid chemistry, exploring its applications in sensing,¹³ catalysis,¹⁴ drug design,¹⁵ and imaging¹⁶ as well as its behavior in mass spectrometry.¹⁷ It is well known that a Lewis base/nucleophile in a 1,5-relationship with an arylboronic acid can donate its lone pair electrons to the boron open shell. This has been shown with amino and hydroxyl groups.¹⁸ Our lab has also shown that an aldehyde carbonyl oxygen can also engage in such coordination and intermolecular aldehyde-boronic acid interactions facilitate aldehyde reduction.^14b In addition, it has been previously reported that o-formyl boronic acids can easily form imines with an amine under physiological conditions.¹⁹ Recently, Gillingham and co-workers reported oxime condensation with o-formylphenylboronic acid as a linking tool for various chemical biology applications.²⁰ This could very well be due to the ability for boronic acid to catalyze the reaction through coordination. Another piece of evidence seems to suggest that the boron atom in an o-formyl arylboronic acid tends to stay in the tetrahedral form, suggesting coordination.^15b For all these reasons, we envisioned that a boronic acid group ortho to the aldehyde would be able to interact with the aldehyde oxygen atom and thus promote its acid-mediated cyclization reaction. Thus, we examined whether o-formylphenylboronic acid would undergo facile cyclization reactions with compounds containing vicinal nucleophiles at room temperature (Table 1).

Table 1.

Design of boronic acid facilitated “click” reaction.

Compound	X	Y	R	Isolated Yield (%)
1	NH2	OH	2-B(OH)2	-
2	NH2	NH2	2-B(OH)2	-
3	NH2	SH	2-B(OH)2	85
4	NH2	SH	3-B(OH)2	-
5	NH2	SH	4-B(OH)2	-
6	NH2	SH	H	10

We first tried the reaction with phenylenediamine. Much to our surprise, the reaction generated a complex mixture. We did not examine the detailed composition of the reaction mixture because it was clear that this was not going to be a successful click reaction. However, all indications are that polymerization and cross-linking are possible side reactions. With this in mind, we were interested in using a structural moiety that has only one of the nucleophiles being “divalent.” In such a case, polymerization becomes not possible. With this consideration, we studied the same reaction with o-aminophenol and o-aminobenzothiol. With o-aminophenol, the cyclization product was not isolated. However, when the reaction was conducted with o-aminobenzothiol, it was very fast (Tables 1 and 2). We hypothesize that the open shell of the boron atom can serve as Lewis acid and coordinate with the oxygen of the adjacent aldehyde group in facilitating the first step of the reaction (Scheme 1). Furthermore, we reasoned that the boron atom could also coordinate with the nitrogen of the imine intermediate and thus facilitate the second step of the reaction as well (Scheme 1). In order to examine whether the cyclization was indeed facilitated by the O-B interaction that afforded the enhanced reactivity of the aldehyde group, we examined the same reactions using m- and p-formylphenylboronic acids and benzaldehyde. Not surprisingly, reacting aminobenzothiol and benzaldehyde without the boronic acid moiety gave no more than 10% of the cyclized product 6. Furthermore, the reaction did not reach completion even after 24 h. Indeed, when the boronic acid is not positioned ortho to the aldehyde functional group, no such cyclization happened (Table 1). Thus, the presence of a boronic acid moiety at a position ortho to the aldehyde was crucial to the reaction. The reaction was also examined using cysteine and homocysteine as naturally occurring molecules with two adjacent nucleophiles. However, no new spots or consumption of the starting material was observed by TLC within one hour of stirring at room temperature; consequently, the reaction was not further examined. Thus, a system with two adjacent nucleophiles is needed for the click-like cyclization with o-formyphenylboronic acid to occur.

Table 2.

Scope of the developed cyclization reaction.

Compound	R1	R2	Conversion (%)	Isolated Yield (%)
7	4-Mea	Ha	100	95
8	4-OCF3a	Ha	100	97
9	3-OBna	Ha	100	60
10	4-Fa	Ha	100	78
11	3-OMea	Ha	100	95
12	Ha	CIb	100	84
13	4-Mea	CIb	100	75
14	3-OBna	CIb	100	77
15	4-Fa	CIb	100	52
16	3-OMea	CIb	100	74
17	Ha	CF3c	100	56
18	4-Mea	CF3c	100	79
19	4-OCF3a	CF3c	100	56
20	3-OBna	CF3c	100	74
21	4-Fa	CF3c	100	68
22	3-OMea	CF3c	100	94

^a1 equivalence of the reagent used.

^b2 equivalence of the reagent used.

^c1.2 equivalence of the reagent used.

Scheme 1.

Proposed mechanism (R₁ = H, Me, OMe, OBn, OCF₃, F; R₂ = H, Cl, CF₃).

In order to examine the scope of this reaction, six o-formylbenzylboronic acids, with various substitutions on the aromatic ring, were examined for their reactivity against three different o-aminobenzothiols (Table 2). The substituents did not seem to have much of an effect on the reaction outcome. The difference in the isolated yield of the oxidized final products was largely due to purification issues as TLC indicated full conversion in all reactions. The purification issues arise from the challenge of separating the cyclized intermediate 30 and the oxidized final product 31 (Scheme 1).

It should be noted that the reaction sequence also involves oxidation, which we assume is air-oxidation because no other reagent was added. The oxidation step appears to be much slower compared to the click-like cyclization reaction. Addition of one equivalent of DDQ and stirring for 30 min substantially increased the oxidation rate and isolated yields (data not shown). However, the use of harsh oxidizing reagents is undesirable and simple overnight air oxidation resulted in the desired fully oxidized products (example: compounds 7, 8 11, 22, Table 2). This is also consistent with the condensation work by others that air-oxidation would drive the reaction to produce the fully aromatized system.²¹

To further explore the scope of the reaction, commercially available 2-amino-4-(trifluoromethyl)thiophenol and 2-amino-4-chlorothiophenol were selected. One of these reagents introduces an electron withdrawing group in the nucleophilic system and the other a slightly electron donating group that can potentially be used as a functionalization site (Table 2, compounds 12–22).

Although aminobenzothiol has two nucleophilic groups, we propose that imine formations happens first (26, Scheme 1), followed by intramolecular cyclization driven by 1) boronic acid activated imine (28) and 2) energetically favorable five-membered ring formation (29, Scheme 1). As previously mentioned, the presence of a boronic acid at a position ortho- to the aldehyde not only is crucial for the cyclization reaction, but also provides a handle for further modifications of the synthesized benzothiazole.

The functionalization of the synthesized boronic acid benzothiazoles can easily be achieved through a number of coupling methods for various applications.^{8a, 8b, 12} For example, molecules can be conjugated to the benzothiazole via Suzuki coupling or the Chan – Lam reaction (Scheme 2). In one such test, we conjugated biotin to the cyclization product benzothiazole. This can be extremely useful for the evaluation of biologically active compounds in binding studies such as SPR, QCM, QCM-D, and ELISA studies. Therefore, this new neighboring group assisted benzaldehyde cyclization reaction can be a versatile tool for various applications.

Scheme 2.

The use of the boronic acid as a handle for further modifications.

Conclusions

In summary, a facile neighboring group assisted cyclization reaction between formylphenylboronic acid and amino thiol has been developed. The reaction achieves full conversion at room temperature without the use of harsh conditions. The benzothiazole products can easily be modified through various boronic acid-based coupling methodologies for different applications such as bioconjugation, biolabeling, and fluorescent tagging, thus giving two sequential facile reactions.

In addition, benzothiazole compounds have shown a wide array of biological activities including anticancer,²² anti-inflammation,²³ antiviral,²⁴ antidiabetic,²⁵ antimicrobial²⁶ and anticonvulsant²⁷ activities. This class of compounds is perhaps most useful in the area of diagnostics where it has been clinically used for amyloid fibrils imaging for Alzheimer’s disease detection.²⁸ More recently, benzothiazole analogs have been utilized in studying the biologically important and naturally occurring gasotransmitter molecules, hydrogen sulfide²⁹ and carbon monoxide.³⁰ Consequently, the new chemistry described here has a wealth of synthetic applications as well.