To enable our search for a C2'-selective acylation of AmB, we first protected the C3' amine and C41 carboxylic acid as the corresponding phenylacyl amide30
and methyl ester.39
With the goal of biasing acylation towards the C2' position, we then selectively masked the hydroxyl groups at C13, C3/C5, and C9/C11 as a methyl ketal28,40
-methoxybenzylidene acetals, respectively30,41
(see Supplementary Information
). Collectively these manipulations provided scalable access to derivative 1
, possessing five unprotected secondary hydroxyl groups. Of these remaining sites, steric considerations suggested that the C2'-hydroxyl group might be the most accessible to an acylating reagent.
When 1 was exposed to a standard set of acylating conditions [4-dimethylaminopyridine (DMAP), one equivalent of acetic anhydride (2a), diisopropylethylamine (DIPEA) in THF], a complex mixture containing many different mono-, bis-, and tris-acylated products was observed by HPLC (, entry 1). Analysis of this product mixture revealed that it contained only 2% of the C2' monoacylated product 3a.
We thus attempted to achieve site-selective acylation at C2' via screening a large collection of commercial lipase enzymes under a wide range of reaction conditions and using many different acyl donors. However, while some encouraging results were obtained, these enzymatic reactions suffered from low conversions, lack of scalability, and/or poor reproducibility.
An established strategy for enhancing site-discrimination is to increase the steric bulk of the acylating reagent,1,42,43
thereby increasing its sensitivity towards subtle differences in the local steric environments of different alcohols appended to the substrate. Following this approach, we evaluated a series of anhydrides with increasing steric bulk. However, little improvement in site-selectivity was observed with propionic (Supplementary Information
) or isobutyric anhydride (2b
) (, entry 2), and no conversion was observed for pivalic anhydride (Supplementary Information
). Thus, steric modifications of the anhydride donors were unable to improve site-selectivity with this substrate.
Intrigued by reports that changing the counterion of acylpyridinium complexes can also impact site-selectivity,16,44
we next surveyed the analogous series of sterically modified acyl chlorides. Treatment of 1
with acetyl chloride (2c
) again provided a complex mixture of acylated products (, entry 3), further demonstrating that the site-discriminating features of 1
were subtle, and if there was an inherent preference for reactivity at C2', then this preference was small. Pivaloyl chloride led to no reactivity. Encouragingly, however, when we increased the steric bulk of the acyl chloride donor in the form of isobutyryl chloride 2d
, we observed the somewhat selective formation of the major product 3b
(, entry 4, 48% site-selectivity). Although 3b
could not be separated from this complex mixture of products via standard silica gel chromatography, carefully optimized preparative HPLC provided a few milligrams of purified material. Characterization by multidimensional 1
H NMR analysis and high resolution mass spectrometry established that 3b
was monoacylated at C2' (Supplementary Information
Albeit an important step forward, we were unable to develop a practical process for purifying intermediate 3b on larger scale. Thus, it was ultimately not possible to transform this moderately site-selective acylation into a preparatively useful process. Faced with the need to substantially improve this site-selectivity, we considered the hypothesis that electronic tuning of the acyl donor might have an impact. As shown in , the Hammond postulate predicts that increasing the electron–richness of the acyl donor will increase the product-like nature of the transition state of rate-limiting acyl transfer. As a result, the site-discriminating interactions between the acyl donor and the polyol substrate should be magnified. This, in turn, should lead to larger differences in the activation energies for acylations of different hydroxyl groups and thus greater site-selectivity.
To test this hypothesis, we alternatively employed electronically tunable para-substituted benzoyl chlorides as acyl donors under otherwise identical reaction conditions. The electron-deficient p-nitrobenzoyl chloride (2e) provided only a modest 39% site-selectivity for formation of the corresponding C2' acylated product 3c (, entry 5). In contrast, simply switching to the much more electron-rich p-N,N-dimethylaminobenzoyl chloride (2f) donor provided the desired C2' acylated product 3d with an outstanding site-selectivity of 72% (, entry 6).
To systematically evaluate whether this effect is primarily attributable to the electronic nature of the acyl donor, we performed a Hammett study45
with a series of sterically similar para
-substituted benzoyl chlorides. Importantly, control experiments confirmed that for all of these donors acyl transfer was irreversible and the rate of background acylation in the absence of DMAP was negligible (Supplementary Information
). Thus, the ratio of site-isomers (C2'/other) is attributable to kinetic selectivity for acyl transfer from the corresponding acylpyridinium complexes to one hydroxyl group versus the others. In turn, with a lack of correction for the minor contributions of multiple acylations noted, this ratio of site isomers is a function of the difference in energies of the transition states of the corresponding acylation reactions (ΔΔG‡
As predicted by the analysis in , C2' site-selectivity progressively decreased as the electron-withdrawing capacity of the para-substituent increased (). A Hammett plot of log[ratio of site-isomers (C2'/other)] vs. σpara revealed a linear correlation with a negative slope (rho = −0.395) (). A complementary prediction of the analysis presented in is that the reaction rate should also exhibit a linear correlation with σpara, but in the opposite direction. Specifically, as the electron-withdrawing character of the substituent on the aryl ring increases, the reaction rate should also increase. We tested this prediction directly by determining the relative initial rates for the same five reactions and in fact observed a linear and positive correlation between log(initial rate) and σpara (). Combining these experiments, a plot of C2' selectivity vs. initial rate () also revealed a linear correlation, as collectively predicted by the Hammond analysis presented in . These results demonstrate that electronic tuning of reagents can have a substantial impact on site-selective functionalization of a complex small molecule substrate, and that this effect is consistent with the Hammond postulate.
Analysis of acylation reactions with para-substituted benzoyl chlorides
With this concept established, an interesting observation from our earlier studies caused us to further question whether electronic tuning might also contribute to another frontier challenge in the area of site-selectivity, i.e., the development of reagent based site-divergent functionalization reactions. Specifically, although neither reaction was highly site-selective, we noted that acylations with isobutyryl chloride 2d
(, entry 4) and the corresponding anhydride 2b
(, entry 2) produced different outcomes.16,44
As described above, the major product derived from 2d
is mono-acylated at C2'. In contrast, HPLC purification and multidimensional NMR characterization of the mixture of products formed from anhydride 2b
revealed a nearly stoichiometric mixture of derivatives mono-acylated at C4' and C15 (Supplementary Information
We recognized that electronically modified derivatives of benzoic anhydrides would translate into concomitant electronic tuning of both the acylpyridinium ion intermediate and its associated carboxylate counterion. Because both of these components are thought to play a role during rate-limiting acyl transfer,25–27
we anticipated that electronic tuning of these anhydrides might also have a substantial impact on site-selectivity. Due to the combinatorial and potentially competing nature of the effects, however, the specific outcome was difficult to predict in this case. When we reacted 1
with either the electron-rich p
-tertbutylbenzoic anhydride 2g
t-Bu = −0.20) or its electron-deficient counterpart p
-nitrobenzoic anhydride 2h
, a very interesting pair of stereodivergent acylation reactions were observed. Specifically, with the electron-rich anhydride 2g
site-selective acylation of the C4' hydroxyl group is the primary pathway yielding 4a
as the major product (, entry 1). In contrast, utilization of the electron-poor anhydride 2h
caused a remarkable turnover in site-selectivity, with a new major product 5b
resulting from selective acylation at C15 (, entry 2). It has been demonstrated that chiral catalysts can be used to achieve reagent-based site-divergent functionalizations of complex small molecules.1,6,8–11
Importantly, we note that all of the site-divergent functionalizations shown in this work were achieved using only achiral reagents. Thus, electronic tuning has the potential to provide a highly complementary alternative approach for reagent-based site-divergent functionalizations of complex small molecule substrates.
Having established electronic tuning as a strategy for the development of site-selective functionalization reactions, we returned to the initial goal of selectively modifying the C2' position of AmB. A survey of various electron-rich benzoyl donors revealed that p-tertbutylbenzoyl chloride provided an optimized combination of C2' site-selectivity (66%), conversion (68%), and ease of purification of the monoacylated product 3e by standard silica gel chromatography. Importantly, both this reaction and chromatographic purification proved to be readily scalable, providing more than 3 grams of purified 3e (45% isolated yield) from a single run. Thus, electronic tuning can transform a minimally site-selective reaction into a highly selective and preparatively useful process.
With efficient and scalable access to monoacylated derivative 3e in hand, unique exposure and subsequent functionalization of the C2' hydroxyl was readily achieved according to the plan outlined in . Specifically, as shown in , concomitant protection of the four remaining hydroxyl groups as the corresponding diethylisopropylsilyl (DEIPS) ethers was followed by facile cleavage of the aryl ester at C2' with KCN in MeOH to yield 6. As demonstrated with this transformation, another important benefit of the electronic tuning approach is that it allows relatively mild conditions to be employed to achieve deacylation. This stands in contrast to the much more forcing conditions typically required to remove sterically encumbered acyl groups, which can lead to competitive decomposition of complex small molecule substrates.
Selective functionalizations at the C2' position of AmB
, having a uniquely exposed hydroxyl group at C2', has proven to be a highly versatile intermediate. For example, despite the presence of the very sensitive polyene macrolide core, efficient deoxygenation at C2' to form 7
was achieved via nucleophilic displacement of the axial C2'-hydroxyl group to generate the equatorial iodide46
followed by a novel AgOAc-mediated reductive deiodination with NaBH4
Alternatively, epimerization at C2'34
to yield 8
was readily achieved using standard Mitsunobu conditions. This approach also provides unique access to AmB-small molecule conjugates48
linked via the C2' hydroxyl group. For example, a molecule of ergosterol was tethered to 6
via simple esterification with acid chloride intermediate 9
to form the novel heterodimer 10
, which is reminiscent of the predicted structure of the AmB-ergosterol complex ().