A Method for Synthesizing Partially Substituted Cucurbit[n]uril

A novel approach to cucurbituril synthesis is described where partial substitution is introduced into cucurbit[n]uril. The identification of homologues (and their substitution) in reaction mixtures is achieved by a combination of ESMS and the use of the molecular probes (guests) 1,4-dioxane and 1,9-octanediamine. A unique symmetrical hexamethylcucurbit[3,3]uril, the major product, was isolated and characterized.


Introduction
The cucurbit[n]uril, Q[n], are an interesting new family of molecular hosts [1] that are both chemically and thermally robust. They have limited solubility in aqueous solvents, or organic solvents such as formic acid, acetic acid [2], trifluoroacetic acid and trifluoroethanol [3]. For more than ten years decamethylcucurbit [5]uril stood as the only fully substituted cucurbituril [4]. Recently the fully substituted cyclohexanocucurbit[n]uril, Cy n Q[n], where n = 5 and 6, have been reported and these have some organic solvent solubility [5]. While this latest report is encouraging for the possible achievement of other fully substituted cucurbiturils, the limiting factor in the synthesis of fully substituted Q[n] appears to be that substituted glycolurils have a propensity to form the smaller cucurbituril homologues. In the case of methyl substituted Q[n], Me 10 Q [5] is the only product formed, whereas for cyclohexano substituted Q[n], Cy 5 Q [5] occurs as the major product, >80%. Access to the interesting and potentially more valuable [1] fully substituted higher homologues is restricted. Given that there are only two examples in the literature [4,5] of fully substituted cucurbiturils and despite significant efforts in our laboratories [6,7], the synthesis of various fully substituted Q[n], bearing the same substitution on every glycoluril unit appears difficult. The smallest homologue consistently forms as the major product when fully substituted Q[n] are successfully synthesized.
We wish to report a novel approach to cucurbituril synthesis where partial substitutio n can be introduced into Q[n]. To aid in the discussion of the cucurbiturils of this type, we propose that the original nomenclature [4] be expanded from cucurbit[n]uril, Q[n], to substituted cucurbit[s,u]uril, SQ [s,u], to accommodate the substitution, where s = the number of substituted glycolurils and u = the number of unsubstituted glycolurils [6]. The prefix S describes the substitution carried.
Recently the partially substituted Q [6], diphenylcucurbit[1,5]uril has been synthesized and isolated in moderate yield [8]. Here we describe a method for introducing higher orders of substitution suitable as a model for the development of partially substituted Q[n] that includes functionality. In addition, chemical manipulation is possible where a range of partially substituted Q[n] homologues (n = 5 -7) can be synthesized with the major size being n = 6. We examined the synthesis of partially substituted Q[n] building on our previous experiences with the formation of Q[n] [9] and in particular relying on identification of products by ESMS.

Results and Discussion
A successful approach was derived from the demonstrated self condensation of dimethyltetracyclic ether (3) in 8 M HCl which produces the known decamethylcucurbit [5]uril, Me 10 Q[5] [9]. In the formation of Me10Q [5] from 3 there are two mole equivalents of methylene linkers more than are required and these are regenerated as formaldehyde. We reasoned that if one mole equivalent of glycoluril (1) were added to 3, which carries the linkers, then incorporation of the dimethylglycoluril moiety into the oligomer and subsequent condensation to a partially substituted Q[n], was probable. By this method, under acid catalysis there are sufficient linkers to achieve Q[n] of partial substitution without the addition of formaldehyde. ESMS spectra showed the reaction to be successful. We have established that Q[n] dissolved in a solvent in the presence of CsCl show ESMS spectra with characteristic patterns of mass ions of the parent ion plus combinations of the alkali metal ions and its counter ion, such as (P+2Cs + )/2, P+Cs + and P+Cs + 2Cl - [9]. The ESMS spectra of the crude reaction mixture of the above reaction, showed ions attributable to Q[n] of partial methyl substitution of sizes n = 5-7, Table 1. The relative ion intensities of the sizes (s + u) 5, 6 and 7, were cone voltage dependent and therefore did not accurately reflect the proportions in the reaction mixture. To establish the relative ratios of the sizes, we investigated the mixture by 1 H-NMR spectroscopy ( Figure 1a) using two molecular guests as probes. 1,4-Dioxane in CsCl/D 2 O has previously been used to identify small amounts of Q [6] in reaction   Significantly, neither of these two molecule guests showed any encapsulation in Q [5] and were therefore considered suitable as probes to identify Q[6]-and Q [7]-sized cavities in the product mixture of the partially substituted Q[n]. Dioxane is encapsulated inside the cavity of Q [6] and the slow exchange results in a sharp signal of bound dioxane at 2.71 ppm, upfield of free dioxane at 3.56 ppm. The upfield shift is characteristic of encapsulation [1a,1d,9]. Unsubstituted Q [7] also binds dioxane but exchanges more quickly giving a broad weak upfield signal between 2.45 and 2.96 ppm. Adding dioxane to the crude reaction mixture of methyl substituted cucurbit [s,u]  Using a similar method except with 1,8-octanediamine in 5M DCl/D 2 O, the proportion of Q[7]-sized cavities was calculated to be 10-15% of the total reaction mixture. This was achieved by comparing the integral of 1,8-octanediamine α methylene protons to the integral of the same set of methylene cucurbituril resonances as above. This diamine, in excess, forms a 1:1 complex with unsubstituted Q [7] with slow exchange. Characteristic upfield resonances at 2.55(αCH 2 ), 0.77(βCH 2 ), 0.46 and 0.38 ppm appear for the diamine methylenes when they are inside the cavity. The unbound diamine resonances occur as a set of three peaks at 2.81, 1.47, and 1.13 ppm. In contrast, when encapsulated in Q [6], the αCH 2 of this diamine moves downfield [10] as they fall into the shielding zone of the portal, allowing clear distinction. Multiple sets of bound signals as found with dioxane were not observed although some broadening was apparent. The relatively clean 1 H-NMR spectrum of the crude reaction mixture (Figure 1a) indicates that substitution can be achieved with high conversion to Me 2s Q[s,u] while also maintaining a significant proportion of the higher homologues where s + u = 6 and 7.  If condensation is purely a random process there are ten possible combinations for Me 2s Q[s,u], where s + u = 6, within the four basic groups detected by ESMS (Figure 3). It was anticipated however, that the condensation would not be random and that condensation between 1 and 3 would occur in an alternate manner as shown in Scheme 1. Clearly, two methyl substituted glycolurils can condense adjacent to each other, as indicated by the major ions that identify Me 6 Q[3,2] as a single isomer or a mixture of two possible isomers. In this case the odd number of glycoluril units required to form a Q[5]-sized product precludes the possibility of an alternative mechanism, other than at least two substituted glycolurils condensing adjacent to each other. Given that Me 6 Q[3,3] is comprised of an even number of glycoluril moieties an alterna te condensation appeared probable. Chromatography on cation exchange resin and recrystallization of enriched fractions of Q[6]-sized products from HCl/dioxane gave the complex dioxane@Me 6

Scheme 1
Recently we have developed synthetic methods that can be used to induce a degree of control over the cucurbituril homologue distribution, either by using different acids, by changing reactant concentrations [9], or by adding alkali metal salts [11]. In this work we report preliminary findings that show similar effects on the distribution of partially substituted cucurbituril homologues, Me 2s Q [s,u].
LiCl added to reaction mixtures was found to increase the proportion of Me 2s Q[s,u], where s + u = 6, to 76%, a 20% improvement. The proportion of s + u = 7 was virtually unaffected, remaining at 10%, and s + u = 5 was decreased to 15%. Increasing the concentration of reactants in the absence of LiCl did not have an anticipated effect of increasing the proportion of higher homologues [9]; rather these conditions had an effect on the number of Me substitution combinations. This was evident in the increased number of bound dioxane signals found in the 1 H NMR spectrum of the product mixture, 7 signals instead of 4 ( Figure 2b). This result indicates a change in the assemble of glycoluril units during condensation to Me 2s Q [s,u]. It is probable that under these conditions there is increased opportunity for the dimethylglycoluril moiety or s units, to condense immediately adjacent to each other instead of a repetitive alternate series of condensations of 1 and 3 (Scheme 1).
LiCl was found not only to increase the proportion of Q[6]-sized products but also to affect assembly in favor of the repetitive alternate series of condensations of 1 and 3 . Two changes were evident: the bound dioxane experiment showed an increased signal intensity at 2.64 ppm in the 1 H NMR spectrum, and the proportion of Q Ideally a technique such as preparative HPLC would be a valuable asset in the isolation of individual components of reaction mixtures in this difficult area of partially substituted Q[n]. Preliminary studies with HPLC indicate that this is possible using a C18 reverse phase column, provided the mixture has been separated into Q[5]- [7] size groups on cation exchange resin prior to HPLC. Solvent gradients of formic acid/water/acetonitrile, of 20/80/0% to 5/25/70%, initially elute the low orders of partially substituted Q[n]. The higher orders of substituted Q[n] elute with increases in the proportion of acetonitrile. The HPLC fractions were analyzed by ESMS. The major product, the symmetrical Me6Q [3,3], 4c, can be isolated by this method, but the yield is difficult to quantify. The HPLC conditions have yet to be optimized and problems regarding detection remain, however the method shows great promise and is likely to be applicable to other substituted and partially substituted Q[n] systems.
The isomers 4a and 4b (also of the formula of Me6Q [3,3]), were not detected during the isolation and purification of the symmetrical Me 6 Q [3,3], 4c. The alternate condensation (Scheme 1) is most likely to be controlled by the kinetics of the reaction. The condensation between the methylene carbons of the ether ring of 3 (or oligomers) and the urea nitrogen of 1 is probably faster than the displacement of the two methylene carbons, a process necessary to allo w condensation between two adjacent methyl substituted glycoluril moieties. However, this displacement has been demonstrated by the condensation of 3 to form Me 10 Q[5] [9] and in the synthesis of partially substituted Q[n], by the formation of products such as Me 6 Q [3,2] or Me8Q [4,2]. Even with these side reaction processes 4c occurs as the major product with or without LiCl present. From a LiCl template reaction [11] the isolated yield of 4c was 10%. However, the yield in the mixture was ca 47% when calculated by considering the proportion of the integral attributable to bound dioxane in 4c (2.64ppm) as a fraction of the percent of Q[6]-sized products. A calculation based on the ratio of the ions of Q [6]-sized products in the ESMS spectrum also gave a similar yield.
Significantly an attempt to synthesize Me 2s Q[s,u] by direct acid catalyzed condensation of 1 and 2 in the presence of formaldehyde (as in a conventional Q[n] synthesis) was less successful; a major portion of the product mixture was unsubstituted Q[n]. This result reflects a competitive reaction where 1 reacts with formaldehyde to form an oligomer, and finally Q[n], which is faster than the incorporation of 2 into the oligomer. The favored condensation of 1 with itself might also help to explain the formation of products such as Me 4 Q [2,4], where at least two unsubstituted glycoluril moieties condense adjacent to each other. This could occur by two pathways, either by regeneration of formaldehyde by hydrolysis of the ether ring of 3, or by transfer of a methylene linker to the unsubstituted glycoluril mioety by a hydrolytic disconnection of oligomers. The red dashed lines in Scheme 1 illustrate this type of disconnection for structures 5 and 6 [9,12]. Me 6 Q[3,3], (4c), a new type of cucurbituril, was found to be soluble in the polar organic solvents acetonitrile, DMSO and trifluoroethanol, however always requiring the addition of some water. The solubility was 7.5g / 100 mL (1:1 acetonitrile/water) and similar solubility in trifluoroethanol with the addition of 5-10% water. This compound is insoluble without the addition of water. It is clear that organic solvent solubility can be achieved even with partial substitution. Organic solvent solubility opens the possibility of a broader range of molecular guest binding capabilities. Cucurbiturils are particularly interesting as unique molecular hosts, with low polarizability [1a].

Conclusions
Significant insights have been gained in this study toward developing a method for the synthesis of partially substituted cucurbit[s,u]uril, SQ[s,u], while maintaining the ability to synthesize substantial proportions of the higher homologues s + u = 6 and 7. In addition a degree of regiochemical control has been achieved. This method for introducing substitution i nto cucurbiturils gives the potential for solubility manipulation and functionalisation through substituted glycolurils carrying functional groups. Alternative substituted glycoluril combinations are under investigation together with the refinement of HPLC as a purification method for partially Q[n].

Procedure
The tetracyclic diether (3) [13] (1.043gm, 4.04mmol) and the glycoluril (2) (574 mg, 4.04 mmol) were combined together and ground into a homogeneous powder. To this we added LiCl (170 mg, 4.0 mmol) and 36% HCl (6.5 mL) and the mixture stirred at room temperature for approx 10 min at which point all solids had dissolved. Heating to 100 °C for 2 hr resulted in a translucent red-brown solution which was cooled and the acid evaporated in vacuo to give a light brown solid. This was soluble in aqueous salt solutions, TFA and mineral acid solutions. The crude product was dissolved in 0.5 M HCl/90% formic acid, 1:1 and applied to a column of cation exchange resin. Eluting with the same solvent gave fractions in the order of methyl substituted Q [5], [6] and [7] respectively. Fractions were analyzed by ESMS, and 1 H-NMR spectroscopy using a dioxane probe. Dioxane in >2 mole equivalents in 0.2 M CsCl/D2O binds with Q [6] in an integral ratio of 1:0.5, Q [6]:Dioxane. The fractions containing Me 2s Q[s,u] where s+u=6 were evaporated in vacuo and the residue dissolved in a small volume of 36% HCl, followed by the addition of a large excess of dioxane. The colorless crystalline product was collected by filtration, dissolved in acetonitrile/water and evaporated to dryness. This process was repeated 3 times to give 145 mg of 4c, (10% yield).