Synthesis and cycloaddition reactions of strained alkynes derived from 2 , 2 ’-dihydroxy-1 , 1 ’-biaryls

A series of strained alkynes, based on the 2,2'-dihydroxy-1,1'-biaryl structure, were prepared in a short sequence from readily-available starting materials. These compounds can be readily converted into further derivatives including examples containing fluorescent groups with potential for use as labelling reagents. The alkynes are able to react in cycloadditions with a range of azides without the requirement for a copper catalyst, in clean reactions with no observable side reactions.


Introduction
The use of highly reactive strained alkynes, typically within eight-membered rings, 1 in cycloaddition reactions with azides is now a well-established reaction with numerous applications in materials chemistry and in bioconjugation applications. 2 Such reagents are ideal for these applications because the cycloaddition reactions take place spontaneously and without the need for a catalyst to be addedin contrast to the reactions of unstrained terminal alkynes with azides in which case a copper-based catalyst is generally required. 3 Widely adopted cyclooctyne reagents such as 1-3 and their derivatives ( Fig. 1), [4][5][6] are highly reactive, and can be used at the low concentrations which are often required in bioconjugation applications, particularly for in vivo reactions. 7 In applications where the concentration of reagents is more typical of synthetic reactions e.g. 0.01-0.5 M, and on larger scales, less reactive larger-ring molecules, which can be prepared through a short synthetic sequence, have also proven to be synthetically valuable reagents. 8 Earlier and less reactive cyclooctynes remain synthetically important, for example (2-cyclooctyn-1-yloxy)acetic acid (a derivative of 'OCT') was the subject of a successful multigram scale up optimisation study reported in 2018. 5d Some highly strained derivatives are also prone to addition of thiols. 5e In a recent paper, we reported the synthesis and applications of a class of strained alkyne based on the 10-membered structure 4, derived from 2,2′-dihydroxy-1,1′-biaryl compounds. 9 The unfunctionalised compound, 8, 13-dioxatricyclo [12.4.0.02,7]octadeca-1 (14), 2,4,6,15,17-hexaen-10-yne (dioxabiaryldecyne) 4 and its close derivatives are readily prepared in one step through the reaction of 2,2′-biphenol with but-2-yne-1,4-diyl bis (4-methylbenzene)sulfonate in the presence of potassium carbonate. 9 Before our studies, the reactions of alkynes such as 4 with azides had not been reported, and just three papers could be identified which reported the synthesis of the same heterocyclic structure. 10 In addition, we demonstrated that reagents such as 4 and its derivatives react with azides, without the need for a Cu catalyst, at rates similar to unfunctionalised cyclooctyne, although lower than the most reactive and recently reported strained alkynes. Significantly, although longer reaction times are required than would be the case for reagents such as 1-3, our alkynes reacted with azides in clean reactions with no visible decomposition when followed by 1 H-NMR. We also reported the synthesis of acid 5 and the activated ester 6 derivatives and demonstrated applicability to bio- Fig. 1 Strained alkynes 1-3, dioxabiaryldecyne 4 and its derivatives 5 and 6. † Electronic supplementary information (ESI) available: General experimental details, synthesis of intermediates 7, 13 11, 12 12, 12 13, 19 34, 20 and compounds 29-31 and 41-45, 1 H and 13 C NMR spectra, graphs of conversion/time, fluorescence spectra, functionalisation of amino-loaded beads and X-ray crystallographic data. CCDC 1852221, 1852222 and 1852224. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8ob01768a. The research data supporting this publication can be accessed at http://wrap.warwick.ac.uk/. conjugation through its attachment to a number of peptides and one protein in in vitro studies. 9 Following our report, another group reported the preparation of some of the same derivatives, as well as N-containing heterocyclic variants, together with a comprehensive molecular modelling study to explain the enhanced reactivity of the reagents. 11 This group also demonstrated that the dioxabiaryldecynes do not rapidly undergo reactions with thiols. 11 In this paper we report the synthesis of a series of functionalised analogues of the strained alkyne structure 4, in as little as two steps, from readily available and inexpensive starting materials, their subsequent functionalisation and representative applications to a number of cycloaddition reactions with several azides.
Both the Pd-catalysed coupling and the cyclisation to form aldehydes 15 and 16 worked more efficiently for the product containing a methoxy group adjacent to the strained alkyne, giving a product in unoptimised but acceptable yield in each case. In the case of the transformation of aldehyde 12 to 16, we followed the reaction over time using chiral HPLC, which resolved the two non-interconverting enantiomers of product and allowed the conversion to be monitored over time (see ESI † for HPLC details and graph of conversion over time). The X-ray crystallographic structures of aldehyde 16 (Fig. 2) and ketone 17 (Fig. 3) revealed the strained nature of the alkyne within the constrained ring.
Alkyne 16 could also be reduced to the alcohol 19 using sodium borohydride, which gave a clean product, however attempts to reduce substrate 15, lacking the methoxy group, to 18 gave a complex mixture of products, for reasons that are not clear. 14 The strained alkynes prepared in this project are stable solids at rt which can be stored for months without significant decomposition. However a thermal gravimetric analysis (TGA) was carried out in order to examine their stability at higher temperatures. Aldehyde 16 exhibited a drop of ca. 10% mass around 180°C which may be associated with the loss of CvO from the aldehyde, followed by a gradual mass loss of just over 20% as the temperature was raised to 600°C. The TGA analysis of the previously reported methyl ester of acid 5 was stable to ca. 300°C then gradually lost ca. 40% of its mass as the temperature was increased to 600°C (see ESI †).
Scheme 1 Synthesis of aldehyde-functionalised CBD strained alkynes 15-17 and alcohol 19.  Given the improved synthesis of the methoxy-substituted aldehyde 16 over 15, we focussed our studies on the former reagent. Its reaction with a range of functionalised azides was studied (Scheme 2) and in each case the reactions were followed over time, using 1 H NMR to monitor the cyclisations of a 1 : 1 mixture of reagents in solution; the spectra are in the ESI. † The reaction of 16 with benzylazide 20 was also carried out in MeCN, the product 21 being isolated in 84% yield. In all cases, the cycloadditions proceeded smoothly, with no obvious accompanying decomposition of reagents. Conversions (by NMR) and yields (isolated products) are given in Scheme 2. In all cases the products were formed as inseparable regioisomeric mixtures in ca. 1 : 1-3 : 2 ratios. Benzylazide gave a clean product 21 of addition, in analogy with previous reactions. 9,11 An azide attached to a red dye, disperse red, 22 15a gave a red product 23 from the cycloaddition, which was carried out at 0.128 M, 9 days at rt (95% conversion, 76% isolated yield). An azide containing a PEG-2000 chain, 24, also added cleanly to the strained alkyne 16, and the product in this case (25) was characterised by GPC as well as by NMR, revealing the expected increase to the molar mass of the polymeric product (ESI †). This was gratifying as the reagent concentration (0.025 M) in this example was lower than for other cycloadditions. The cycloaddition of coumarin azide 26 gave a highly fluorescent product 27 (see ESI †) as has been reported previously for this class of reagent. 15b,c For improved solubility, deuterated acetonitrile was used as the solvent, and the reaction at 0.11 M proceeded to ca. 80% conversion to 27. Although long reaction times are required relative to the more reactive strained alkynes such as 1-3, the benefits of the catalyst-free conditions and clean cycloadditions make these reagents potentially valuable for the preparation of materials for biological applications.
The addition of benzyl azide to alcohol 19 to give adduct 28 as a 3 : 2 regioisomeric mixture of products ( Fig. 4) proceeded at a similar rate (0. 17 M, 5 d at rt, 97% yield, ESI †) indicating that the functional group has minimal influence on the rate of the cycloaddition, probably because of the separation from the alkyne.
The aldehyde group on 15 and 16 permits their functionalisation with other reagents. The reaction of 15 with benzylhydroxylamine in MeOH overnight at 45°C gave oxime ether 29 in 66% isolated yield. The formation of oxime ethers represents a valuable method for functionalisation due to their high stability and ease of preparation. 16 Also, notably, reductive amination with benzylamine led to the synthesis of aminecontaining derivatives 30 and 31. The reaction of methoxy-substituted 30 with benzylazide was found to proceed at a similar rate to aldehyde-containing reagents 16 (ESI †). It was gratifying that these functionalisations could be completed without damaging the strained alkyne group.
The treatment of amine-functionalised polystyrene beads with 16 and sodium cyanoborohydride was followed by reaction of the functionalised beads 32 with disperse red azide 22. After washing, the strong red colour of the dye remained on the beads 33 (Scheme 3). As a control reaction, stirring the solution of red dye-azide 22 with unfunctionalised beads gave only lightly coloured beads after washing, indicating that the cycloaddition had taken place on the dioxabiaryldecyne reagent on the beads (ESI †).
Other reagents were prepared through reactions of the aldehyde, notably fluorescent groups. The reductive amination of 16 with the amine-functionalised dansyl reagent 34 resulted in formation of 35 (Scheme 4A), which showed strong fluorescent behaviour upon irradiation. A number of BoDIPY derivatives 36-38 were also prepared through the direct reaction of pyrroles with the aldehyde and BF 3 in good yield (Scheme 4B). 17 Again, the ability to functionalise aldehyde 16 with a variety of reagents, without damaging the strained alkyne, is noteworthy. The X-ray crystallographic structure of 38 (Fig. 5) revealed the strained nature of the alkyne but also that the BoDIPY component was orientated almost perpendicular to the connected arene ring, presumable with restricted rotation about the connecting C-C bond. This accounts for the observed differences in chemical shifts of the groups attached to the heterocyclic rings of the BoDIPY unit in each of 36-38, which will be in sharply different diastereotopic environments.
The fluorescence spectra for compounds 35-38 are given in the ESI. † However the strong and contrasting fluorescence behaviour of the BoDIPY dyes 36-38 is sharply illustrated by their response to UV irradiation. Compound 36 and 37 both show strong fluorescence upon irradiation whereas 38 gives a weaker response (ESI †).
The addition of benzylazide to BoDIPY derivative 36 was tested and worked efficiently to give two regiosiomers 39 and 40 in a 1 : 1 ratio (Fig. 6). In this case, we were able to separate the isomers by flash chromatography and independently characterise them. We have not unambiguously established which regiosiomer is which, of the two possibilities, however on the basis of the positions of the methylene groups in the 13 C-NMR spectra compared to previous examples, we have tentatively assigned them as shown in Fig. 6 (see ESI †).
Further derivatives were also prepare from the corresponding alcohol 19 using a variety of coupling methods (Fig. 7). These included a biotin-containing reagent 41 which was formed through formation of an ester bond to biotin in one step.
It was also possible to attach a group through a carbamate i.e. 42, using N,N′-disuccinimidyl carbonate (DSC) as a coupling agent to attach alcohol 19 to form the dansyl amine derivative 34. 7d,18 Finally, from the alcohol, the direct reaction with an isocyanate could also be employed to create a derivative

Paper
Organic & Biomolecular Chemistry with a carbamate linkage i.e. 43. Formation of derivatives from the acid 5 was also investigated; the in situ formation of isocyanate from acid 5 using diphenylphosphoryl azide and trapping with MeOH gave carbamate derivative 44 through a method that could be used for future functionalisation. Acid 5 was also linked using EDC·HCl to create the disperse-red functionalised 45. These results (Fig. 7) illustrate the range of methods which can be employed to functionalise the strained alkynes.
In conclusion, we have prepared a selection of derivatives, including fluorescently-labelled variants, of a new class of strained alkyne, which benefit from ease of synthesis from readily available and inexpensive starting materials through a short sequence of reactions. We have demonstrated that this class of alkyne undergoes uncatalysed cycloaddition reactions with azides with minimal decomposition or side product formation. Studies of the applications of these reagents are ongoing and further results will be published in due course.

Alkyne 16
This compound is novel.

Alkyne alcohol 19
This compound is novel. NaBH 4 (15 mg, 0.41 mmol, 1.0 eq.) was added carefully at 0°C to a stirring solution of 16 (0.12 g, 0.41 mmol, 1.0 eq.) in methanol (10 mL) under a nitrogen atmosphere and the reaction was left for 1 hour to react at rt. The methanol was removed under vacuum and the residue was redissolved in ethyl acetate (15 mL). The organic extracts were washed with sat. NH 4 Cl (15 mL) and then brine (15 mL

Ketoalkyne 17
This compound is novel.

Cycloadduct 21
This compound is novel.

Fluorescent coumarin dye cycloadduct 27
This compound is novel.

01Dansyl amine alkyne 35
This compound is novel.
To a solution of alkyne 16 (100 mg, 0.340 mmol, 1.0 eq.) in DCM (22 mL) was added 3-ethyl-2,4-dimethylpyrrole (87.3 mg, 0.1 mL, 0.71 mmol, 2.1 eq.), at which point the solution became red, TFA (3.9 mg, 2 µL, 34 µmol, 0.1 eq.) was added, resulting in the formation of a green solution which was stirred for 3 h, during which time it returned to a red colour. The solution was then washed with a saturated solution of NaHCO 3 (20 mL), turning the organic layer yellow, and brine (20 mL). The organic layer was then dried over MgSO 4 , which was subsequently removed by filtration, and the solvent was evaporated. The residue was then dissolved in toluene (12.3 mL) and a suspension of DDQ (84 mg, 0.37 mmol, 1.1 eq.) in toluene (6 mL) was added, resulting in the solution turning purple. The mixture was then stirred for 1 h before TEA (141 mg, 0.20 mL, 1.4 mmol, 4.1 eq.) was added along with BF 3 ·Et 2 O (290 mg, 0.25 ml, 2.04 mmol, 6.0 eq.) and the mixture was refluxed at 75°C for 45 min. The mixture was then cooled before being filtered through a silica plug eluted with DCM. The resultant solution was then concentrated under vacuum to afford the crude product. The product was purified by column chromatography using an eluent of 2 : 8 EtOAc : pet. ether to afford the pure product 36 as a red/green metallic solid (128 mg, 0.225 mmol, 66%). (Found (ESI) [M + Na] 2962,2928,2868,1536,1454,1315,1182,972 and 960 cm −1 ; δ H (500 MHz, CDCl 3 ) 7.38 (1H, t, J 7.5, ArH),

BoDIPY strained alkyne 38
This compound is novel.