RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 61/713,072, filed October 12, 2012.

BACKGROUND OF THE INVENTION

Structurally complex small molecules have an extraordinary capacity to perform a wide range of useful functions. Accessing structural derivatives of these compounds, however, represents a major bottleneck in efforts to understand and/or optimally harness this capacity. Site-selective functionalization represents a frontier synthesis strategy with outstanding potential for addressing this limitation. Site-selective acylation has emerged as a particularly promising approach for accessing derivatives of polyhydroxylated natural products, with the capacity for exceptional step-efficiency relative to total synthesis. Current strategies for achieving selectivity in this context include modifying the steric and/or stereochemical features of the acylating reagents, or utilizing lipase enzymes. However, suboptimal site selectivities too often limit the preparative utility of this approach. New strategies for maximizing site selectivity or enabling the development of reagents that can override substrate bias to achieve site-divergent functionalizations stand to address these important limitations and thereby have a major impact on small molecule science.

SUMMARY OF THE INVENTION

An aspect of the invention is a method of site-selective functionalization of a substrate molecule having a plurality of potential sites for functionalization. The method comprises the steps of:

selecting, based on its electron richness, a functionalizing reagent for reaction with at least one potential site for functionalization of a substrate molecule having a plurality of potential sites for functionalization; and

reacting the functionalizing reagent with the at least one of said potential sites for functionalization in a reaction represented by

S + R(D) -> S(D)

wherein

S represents the substrate molecule having a plurality of potential sites for

functionalization;

R(D) represents the functionalizing reagent comprising a functional group; D represents the functional group; and

S(D) represents a product molecule having the functional group linked to a desired site of the substrate molecule.

In one embodiment, the functionalizing reagent is an electron-rich functionalizing reagent.

In one embodiment, the functionalization is acylation.

In one embodiment, the functionalizing reagent is achiral.

In one embodiment, the functionalizing reagent is a /?ara-substituted benzoyl chloride. An aspect of the invention is a method of C2' -selective acylation of amphotericin B

The method comprises the step of contacting amphotericin B with an electron-rich /?ara-substituted benzoyl chloride.

An aspect of the invention is a method of C2' -selective acylation of amphotericin B. The method comprises the step of contacting a compound of formula I

with an electron-rich /?ara-substituted benzoyl chloride, wherein:

P ¹ and P ² represent hydrogen or together form a para -methoxy phenyl- substituted methine;

P ³ and P ⁴ represent hydrogen or together form a /?ara-methoxy phenyl-substituted methine;

P ⁵ represents hydrogen or methyl;

P ⁶ represents hydrogen or methyl; and

P ⁷ represents hydrogen or -C(0)Bn;

1 2 3 4 5 6 7 ^"

provided that at least one of P ¹ , P , P ^J , F, P ³ , P°, and P' is not hydrogen.

An aspect of the invention is a method of C2' -selective acylation of amphotericin B. The method comprises the step of contacting a compound of formula II

II

with an electron-rich /?ara-substituted benzoyl chloride,

wherein PMP represents /?ara-methoxyphenyl.

In one embodiment, the electron-rich /?ara-substituted benzoyl chloride is achiral.

In one embodiment, the electron-rich /?ara-substituted benzoyl chloride is selected from the group consisting of /?-N,N-dimethylaminobenzoyl chloride and /?-tertbutylbenzoyl chloride.

An aspect of the invention is compound 6

wherein

PMP represents /?ara-methoxyphenyl; and

R represents diethylisopropylsilyl.

An as ect of the invention is compound 10

10 wherein PMP represents /?ara-methoxyphenyl; and R represents diethylisopropylsilyl.

An aspect of the invention is compound 7

wherein PMP represents /?ara-methoxyphenyl; and

R represents diethylisopropylsilyl.

An aspect of the invention is a method of making compound 7. The method comprises the reaction scheme

1 ) DEIPSOTf

2,6-lutidine

wherein PMP represents /?ara-methoxyphenyl; and R represents diethylisopropylsilyl (DEIPS). BRIEF DESCRIPTION OF THE DRAWINGS

Figure la is a graph depicting the Hammond postulate applied to site selectivity. More electron-rich acylpyridinium ions are predicted to react via a more product-like transition state in which the site-discriminating interactions between the acylating reagent and the polyol substrate are magnified. These enhanced interactions increase the difference in activation energies (ΔΔϋ') for the acylation of one hydroxyl group (solid line) versus another (dashed line).

Figure lb depicts Amphotericin B (AmB) and a putative interaction between the hydroxyl group at C2' and ergosterol.

Figure lc is a schematic representation depicting a strategy for site-selective

functionalization of just one of many distinct secondary hydroxyl groups appended to AmB.

Figure 2a is a Hammett study of site-selective acylation. As the electron-withdrawing capacity of the substituent increased, the selectivity decreased and the rate increased. Values for the % C2'-OH selectivity represent the average of three trials.

Figure 2b is a Hammett plot of the log of the ratio of the product monoacylated at C2' to all other products as a function of a _para . Values for the ratio of site isomers represent the average of three trials.

Figure 2c is a Hammett plot of the log of the initial rate as a function of a _para . Values for the initial rate represent the average of three trials.

Figure 2d is a plot of the ratio of site isomers as a function of the initial rate. Values for the ratio of site isomers and initial rate represent the average of three trials.

Figure 3 is a schematic depicting selective functionalizations at the C2' position of AmB. Electronic tuning of the acyl donor enables selective acylation at the C2' hydroxyl of intermediate 1. The acyl group acts as a temporary protecting group that is removed after orthogonal protection of the remaining hydroxyl groups leaving only the C2' hydroxyl exposed. The C2' hydroxyl can then undergo a variety of functionalizations such as

deoxygenation, inversion, or formation of AmB-small molecule conjugates. DETAILED DESCRIPTION

In order for site-selective reactions to occur under kinetic control, information encoded in distinct local chemical environments within a substrate must be effectively translated into substantial differences in the energies of the corresponding transition states for

functionalization. The Hammond postulate predicts that as a reaction becomes less exothermic, the corresponding transition state will become more product-like. As a consequence, any potentially site-discriminating interactions between reagents and substrates should be amplified, thus leading to enhanced site selectivities. Although the Hammond postulate has classically been invoked to explain a variety of different trends in reactivity, a general approach for engaging this phenomenon to maximize site-selective functionalizations of complex small molecules has not, to the best of our knowledge, been previously reported.

We found inspiration for this approach in the context of asymmetric catalysis, where electronic tuning of reagents can have a substantial impact. For example, with the Jacobsen epoxidation, more electron-releasing salen ligands lead to substantially increased

enantioselectivities. Mechanistic studies of this phenomenon are consistent with the Hammond postulate, i.e., electron-releasing ligands create milder manganese-based oxidants which, in turn, react via more product- like transition states. This presumably amplifies the enantiotopic face-discriminating interactions between the chiral oxidant and the prochiral olefin thus yielding higher levels of enantioselectivity. Guided by the logic outlined above and this encouraging precedent, we hypothesized that increasing the electron-richness of an acyl donor would lead to an increase in the product-like nature of the transition state for rate-limiting acyl transfer, resulting in amplified site-discriminating interactions between an acyl donor and a polyol substrate and thus greater site selectivity (Fig. la).

The decahydroxylated natural product amphotericin B (AmB) (Fig. lb) represents an outstanding platform for testing this hypothesis, and recent advances have made the hydroxyl group at C2' a particularly important target for site-selective acylation (Fig. lb). Palacios, DS et al, J. Am. Chem. Soc. 129, 13804-13805 (2007); Palacios, DS et al, Proc. Natl. Acad. Sci. USA 108, 6733-6738 (2011); Gray, KC et al, Proc. Natl. Acad. Sci. U.S.A. 109, 2234-2239 (2012). Specifically, in contrast to the widely accepted channel model, AmB primarily kills yeast via simply binding ergosterol, a lipid that is vital for many aspects of yeast physiology. Gray, KC et al, Proc. Natl. Acad. Sci. U.S.A. 109, 2234-2239 (2012). Competitive binding of cholesterol in human cells likely plays an important role in the substantial toxicity of this clinically vital antifungal agent. Palacios, DS et al, Proc. Natl. Acad. Sci. USA 108, 6733-6738 (2011). Sterol binding is also critical for formation of the AmB-based ion channel, (Palacios, DS et al, Proc. Natl. Acad. Sci. USA 108, 6733-6738 (2011)) a prototype for the development of small molecules that might replicate the function of deficient protein ion channels that underlie human diseases. For all of these reasons, gaining an atomistic understanding of AmB- sterol interactions represents a critical goal.

The mycosamine appendage is required for binding both ergosterol and cholesterol,

(Palacios, DS et al, Proc. Natl. Acad. Sci. USA 108, 6733-6738 (2011)) but the specific role(s) played by the three functional groups at C2', C3', and C4' remain unclear. In a leading model, the C2'-OH is predicted to form a hydrogen bond with the 3β-ΟΗ on the A-ring of the sterol (Fig. lb). If C2'-selective acylation could be achieved, orthogonal protection of the remaining hydroxyl groups and subsequent cleavage of the C2' ester would generate a uniquely exposed hydroxyl group at this position. This site could then be selectively manipulated in a variety of ways to generate derivatives designed to more deeply probe the fundamental underpinnings of AmB function (Fig. lc).

Separating constitutional isomers of highly complex small molecules can be very challenging. This classic problem is magnified substantially in the case of AmB because the physical properties of this natural product, including its amphipathicity and poor solubility in many organic solvents, make the utilization of standard chromatographic techniques ineffective in many cases. Because of this, and the fact that multiple grams of mono-acylated material would be needed to prepare site-selectively modified derivatives of AmB via the sequence outlined in Fig. lc, a highly site-selective acylation of the C2' hydroxyl group was required.

As described below, electronic tuning proved to be an exceptionally effective strategy for solving this challenging problem. This methodology enables the rational transformation of a minimally site-selective reaction into a highly selective one that is preparatively useful.

Importantly, this approach can be applied to increase site selectivities even if the factors underlying site-discrimination in a specific case are unknown. We further report the surprising discovery that concomitant electronic tuning of both the acylpyridinium ion and its counterion can lead to site-divergent functionalization reactions. Collectively, these findings establish electronic tuning as a strategy for discovering and optimizing site-selective functionalizations of complex small molecules.

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 amide (Gray, KC et al., Proc. Natl. Acad. Sci. U.S.A. 109, 2234-2239 (2012)) and methyl ester. With the goal of biasing acylation towards the C2' position, we then selectively masked the hydroxyl groups at CI 3, C3/C5, and C9/C11 as a methyl ketal (Palacios, DS et al, J. Am. Chem. Soc. 129, 13804-13805 (2007)) and /?-methoxybenzylidene acetals, respectively (Example 1). Gray, KC et al., Proc. Natl. Acad. Sci. U.S.A. 109, 2234-2239 (2012). 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 (Table 1, entry 1). Analysis of this product mixture revealed that it contained only 2% of the C2' monoacylated product 3a.

In Table 1, C2' selectivity is the percent of the total product mixture that is monoacylated at C2'. Results represent the average of three trials.

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, 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 or isobutyric anhydride (2b) (Table 1, entry 2), and no conversion was observed for pivalic anhydride. 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, 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 (Table 1, 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 (Table 1, 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 1H NMR analysis and high resolution mass spectrometry established that 3b was mono-acylated at C2'.

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 Fig. la, 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 /?-nitrobenzoyl chloride (2e) provided only a modest 39% site selectivity for formation of the corresponding C2' acylated product 3c (Table 1, entry 5). In contrast, simply switching to the much more electron-rich /?-N,N-dimethylaminobenzoyl chloride (2f) donor provided the desired C2' acylated product 3d with an outstanding site selectivity of 72%

(Table 1, entry 6).

To systematically evaluate whether this effect is primarily attributable to the electronic nature of the acyl donor, we performed a Hammett study with a series of sterically similar /?ara-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 (Example 5). Thus, the ratio of site -isomers (C27other) 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 (ΔΔϋ ).

As predicted by the analysis in Fig. la, C2' site selectivity progressively decreased as the electron- withdrawing capacity of the /?ara-substituent increased (Fig. 2a). A Hammett plot of log[ratio of site-isomers (C27other)] vs. a _para revealed a linear correlation with a negative slope (rho = -0.395) (Fig. 2b). A complementary prediction of the analysis presented in Fig. la is that the reaction rate should also exhibit a linear correlation with a _para , 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 a _para (Fig. 2c). Combining these experiments, a plot of C2' selectivity vs. initial rate (Fig. 2d) also revealed a linear correlation, as collectively predicted by the Hammond analysis presented in Fig. la. 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.

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 (Table 1, entry 4) and the corresponding anhydride 2b (Table 1, entry 2) produced different outcomes. 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.

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, 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 /?-tertbutylbenzoic anhydride 2h (σ _ραΓα t-Bu = -0.20) or its electron-deficient counterpart /?-nitrobenzoic anhydride 2i, a very interesting pair of stereodivergent acylation reactions were observed. Specifically, with the electron-rich anhydride 2h site-selective acylation of the C4' hydroxyl group is the primary pathway yielding 4a as the major product (Table 2, entry 1). In contrast, utilization of the electron-poor anhydride 2i caused a remarkable turnover in site selectivity, with a new major product 5b resulting from selective acylation at C15 (Table 2, entry 2). It has been demonstrated that chiral catalysts can be used to achieve reagent-based site-divergent functionalizations of complex small molecules. 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 (2g) 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 (Table 1, entry 7). 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 Fig. lc. Specifically, as shown in Fig. 3, 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.

Compound 6, 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 macro lide core, efficient deoxygenation at C2' to form 7 was achieved via nucleophilic displacement of the axial C2' -hydroxyl group to generate the equatorial iodide ⁴⁶ followed by a novel AgO Ac-mediated reductive deiodination with NaBH ₄ . 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 conjugates 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 (Fig. lb).

We have thus far demonstrated the electronic tuning strategy only with AmB, and the generality of this approach remains to be determined. However, because our studies are consistent with linking of this effect to the Hammond postulate, and the Hammond postulate is broadly applicable to many reactions, our results suggest that electronic tuning may represent a general strategy for improving site-selective functionalizations of complex small molecules. For example, it may be possible to combine this approach with a range of other nucleophilic catalysts en route to highly optimized site-selective acylations and/or other group transfer processes. Moreover, this same approach should be applicable to a wide range of other substrate-based site-selective transformations as long as the corresponding functionalization reagents can be tuned electronically. In addition, because increasing the product-like nature of a transition state should increase the consequences of site-discriminating interactions regardless of their origins, this strategy should be applicable even in cases where the specific site- discriminating interactions are not understood. Of course, if the specific underpinnings can be elucidated, then a combination of electronic tuning and rational design of the reagent would potentially be synergistic.

Electronic tuning might also prove to be generally useful in the development of reagent- based site-divergent functionalization reactions. For example, if two different acylation catalysts produce modest levels of site-divergency with the same acid chloride, electronic tuning of the acid chloride should lead to parallel optimization of both site-selectivities, thereby yielding highly optimized site-divergency. Moreover, we found that site-divergency can also be achieved via concomitant electronic tuning of both an acylpyridinium ion and its carboxylate counterion, which is accessible by simply modifying the corresponding readily available anhydride donors.

It is also highly notable that all of the site-divergent acylation reactions described herein involve only the use of achiral acylating reagents and catalysts. These findings suggest that physical and/or mechanistic features independent of stereochemistry stand ready to impact site- divergent functionalizations of complex small molecules in ways that exceed the current level of understanding and utilization.

In conclusion, the rate-limiting step in small molecule science is still all too often the synthesis of targeted derivatives. Site-selective functionalization represents a synthetic strategy with an exceptional level of theoretical efficiency, but the challenge in reducing this approach to practice with complex small molecule substrates can be substantial. Electronic tuning can enable the development of highly practical and synthetically useful site-selective

functionalization reactions. This strategy may therefore facilitate advanced understanding and harnessing of the still largely untapped functional potential that complex small molecules possess.

It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the compositions and methods described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

EXAMPLES

General Methods

Materials

Amphotericin B was a gift from the Bristol-Myers Squibb Company. All other commercially available reagents were obtained from Sigma-Aldrich, TCI America, Fischer Scientific, Combi-Blocks Inc., and Oakwood Products. Chemicals were used without further purification unless otherwise specified. Camphorsulfonic acid was purified before use by recrystallization with ethyl acetate. Triethyl amine, diisopropylethyl amine, pyridine, and 2,6- lutidine were freshly distilled over calcium hydride under nitrogen atmosphere. All solvents were obtained from a solvent purification system utilizing packed columns as described by Pangborn and coworkers (Organometallics 1996, 15, 1518-1520).

Reactions

All reactions were performed under argon atmosphere in low light conditions with flame dried glassware unless otherwise indicated. All compounds were stored in the dark under argon atmosphere. Thin layer chromatography or reverse phase HPLC was used to monitor reaction progress. Thin layer chromatography was performed on silica gel 60 F254 plates from Merck with the indicated solvent. Visualization of the compounds was accomplished with a UV lamp (λ ₂ 54) and eerie ammonium molybdate (CAM) stain. HPLC was done on an Agilent 1100 Series HPLC with a CI 8 5 μιη, 4.6 x 150 mm, Symmetry® column from Waters Corp. The detection wavelength was set to 383 nm.

Purification and Analysis

Merck silica gel 60 230-400 mesh was used for flash chromatography with the indicated solvent. 1H NMR spectra were taken at 23 °C on a Varian Unity Inova Narrow Bore spectrometer at a frequency of 500 MHz with a Varian 5 mm 1H { ³ C/ ¹⁵ N} pulsed-field gradient Z probe. Chemical shifts (δ) are reported in parts per million (ppm) downfield from

tetramethylsilane and referenced internally to the residual protium in the NMR solvent

(CHD ₂ OD, δ = 3.30, center line, CD ₃ C(0)CHD ₂ , δ = 2.04, center line). Data is reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, b = broad, app = apparent), coupling constant (J) in Hertz (Hz) and integration. ¹³ C spectra were obtained at 23 °C with a Varian Unity Inova spectrometer at a ¹³ C frequency of 125 MHz with a 5 mm Nalorac gradient { ¹³ C/ ¹⁵ N} 1H quad probe. Chemical shifts (δ) are reported downfield of tetramethylsilane and are referenced to the carbon resonances in the NMR solvent (CD3OD, δ = 49.0, center line, CD ₃ C(0)CD ₃ , δ = 29.8, center line). ESI high resolution mass spectra (FIRMS) and MALDI spectra were obtained at the University of Illinois mass spectrometry facility.

Example 1. Synthesis of AmB derivatives

N-phenylacetyl, methyl ketal, methyl ester SI1

NHC(0)Bn

SI1

Phenyl acetic acid (662 mg, 4.86 mmol, 3 eq) was dissolved in THF (30 mL), Trimethyl acetyl chloride (400 μΐ,, 3.25 mmol, 2 eq) was added followed by triethyl amine (900 μί, 6.46 mmol, 4 eq). The reaction was allowed to stir for 8 hrs at room temperature. The reaction was placed in an ice bath, and DMSO (30 mL) was added over 2 min as it cooled. Once the reaction reached 0 °C, AmB (1.5 g, 1.62 mmol, 1 eq) was added. The yellow-tan suspension slowly became soluble over 90 min stirring at 0 °C. The reaction was poured slowly into rapidly stirring diethyl ether (1.8 L) at 0 °C. After 15 min of stirring, the resulting yellow precipitate was vacuum filtered with a Buchner funnel equipped with Whatman 50 filter paper and washed 3 times with diethyl ether (200 mL). The yellow powder was dried under vacuum for 8 hours.

The powder was then suspended in THF:MeOH 1 : 1 (60 mL) and cooled to 0 °C.

Camphorsulfonic acid (94 mg, 405 μιηοΐ, 0.25 eq) was added, and the yellow-tan suspension slowly became soluble over 45 min of stirring at 0 °C. The reaction was quenched by triethyl amine (57 μί, 405 μιηοΐ, 0.25 eq) at 0 °C. The reaction solution was concentrated by approximately 2/5 by rotary evaporation and poured into diethyl ether:hexane 1 : 1 (1.2 L) while stirring rapidly. After stirring 15 min, the yellow precipitate was collected in a Buchner funnel equipped with Whatman 50 filter paper by vacuum filtration. The precipitate was washed 3 times with diethyl ether (200 mL). The powder was dried under vacuum for 8 hrs.

The powder was suspended in THF (60 mL) and cooled to 0 °C. Freshly distilled diazomethane (8.10 mmol, 5 eq) was added dropwise to the suspension over 20 min at 0 °C. The reaction was allowed to stir for 30 additional min at 0 °C. After quenching with acetic acid (8.10 mmol, 5 eq) at 0 °C, the solution was then concentrated under reduced pressure and purified by flash chromatography (Si0 ₂ ; DCM:MeOH 9: 1) to give SI1 as a yellow solid (971 mg, 907 μιηοΐ, 56 %).

TLC (DCM:MeOH 9: l)

R _f = 0.2, stained by CAM

HPLC

tR = 18.1 min; flow rate = 1 mL/min, gradient = 5→ 95 % MeCN in water over 30 min.

1H NMR (500 MHz, pyridine d-5:CD ₃ OD 10: 1)

δ 9.01 (d, J = 8.5 Hz, 1H), 7.53 (m, 2H),7.25 (m, 3H), 6.58-632 (m, 12H), 6.23 (m, 1H), 5.69 (m, 2H), 4.95 (m, 1H), 4.90 (s, 1H), 4.83 (m, 1H), 4.67 (m, 2H), 4,46 (m, 2H), 4.38 (app d, J = 3 Hz, 1H), 4.17 (m. 1H), 4.01 (m, 2H), 3.86 (m, 2H), 3,74 (m,

5H), 3.56 (m, 1H), 3.26 (s, 3H), 2.94 (m, 1H), 2.84 (t, J = 10.5 Hz, 1H), 2.69 (m, 2H), 2.54 (m, 1H), 2.31-1.81 (M, 13 H), 1.72 (m, 1H), 1.57 (d, J = 6 Hz, 3H), 1.44 (d, J = 6 Hz, 3H), 1.32 (d, J = 6.5 Hz, 3H), 1.24 (d, J = 7 Hz, 3H)

1 ³ C NMR (125 MHz, pyridine d-5:CD ₃ OD 10: 1)

δ 174.4, 174.2, 172.3, 171.9, 137.9, 137.5, 134.8, 134.7, 134.3, 134.2, 133.8, 133.7,

133.6, 133.5, 133.1, 132.9, 132,4, 130.5, 130.1, 129.1, 127.3, 102.3, 99.4, 78.2, 75.8,

75.7, 75.4, 75.0, 72.1, 71.4, 71.0, 68.5, 67.8, 67.7, 67.1, 57.8, 56.8, 52.2, 45.2, 44.1,

43.8, 43.6, 42.1, 36.6, 31.0, 19.2, 18.9, 18.0, 12.8.

HRMS (ESI)

Calculated for C ₅₇ H ₈₃ NOi ₈ (M + Na) ⁺ : 1092.5508

Found: 1092.5515 p-Methoxybenzylidene acetal 1

To a suspension of SI1 (1.50 g, 1.40 mmol, 1 eq) in MeOH:THF 2: 1 (17 mL) was added anisaldehyde dimethyl acetal (2 mL) followed by camphorsulfonic acid (81 mg, 0.35 mmol, 0.25 eq). The solution was stirred for 20 min. The reaction was quenched with triethylamine dropwise until the dark tan solution underwent a color change to light tan. The reaction was poured into saturated sodium bicarbonate and extracted 3 times with ethyl acetate. The organic layers were washed with water followed by a wash with saturated sodium chloride. The organic layers were combined and dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash chromatography (Si0 ₂ ; EtOAc:Hexane:MeOH 77:20:3) purification yielded 1 as a yellow-orange solid (1.10 g, 0.84 mmol, 60%).

TLC (EtOAc:Hexane:MeOH 77:20:3)

R _f = 0.25 , stained by CAM

HPLC

tR = 15.4 min; flow rate = 1 mL/min, gradient = 5% MeCN in water for 2 min then 5→ 54% MeCN in water over 3 min then 54→ 95% MeCN in water over 13 min.

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.42 (m, 2H), 7.35 (m, 4H), 7.29 (m, 2H), 7.21 (m, 2H), 6.86 (m, 4H), 6.43-6.20 (m,

12H), 5.88 (m, 1H), 5.58 (m, 1H), 5.51 (s, 1H), 5.46 (s, 1H), 5.26 (m, 1H), 4,64 (m, 1H), 4.58 (app s, 1H), 4.20-4.10 (in, 2H), 4.02 (m, 1H), 3.95-3.86 (m, 3H), 3.78 (m, 6H), 3.75 (m, 2H), 3.66 (s, 3H), 3.63 (m, 1H), 3.45 (m, 2H), 3.36 (m, 1H), 3.30 (m, 2H), 3.05 (s, 3H), 2.57 (m, 1H), 2.40 (m, 1H), 2.31-2.24 (m, 3H), 1.96 (m, 1H), 1.89- 1.45 (m, 9H), 1.37 (m, 2H), 1.22 (in, 4H), 1.19 (d, J= 6 Hz, 3H), 1.17 (m, 1H), 1.11 (d,

J = 6.5 Hz, 3H), 1.01 (d, J = 7.5 Hz, 3H)

1 ³ C NMR (125 MHz, CD ₃ C(0)CD3) δ 173.6, 172.7, 169.7, 160.6, 160,5, 137.5, 136.9, 136.2, 134.1 , 134.0, 133.9, 133.7, 133.6, 133.5, 132.9, 132.6, 132.5, 132.2, 129.9, 129.8, 128.9, 128.3, 128.2, 127.2, 120.9, 1 17.6, 1 13.8, 101.0, 100.7, 100.6, 98.1 , 81.1 , 77.9, 76.2, 74.7, 74.4, 73.2, 73.1 , 72.9, 72.8, 70.7, 70.5, 67.2, 66.9, 57.3, 56.4, 55.4, 51.8, 48.6, 43.4, 43.3, 42.6, 41.8, 41.5, 37.8, 36.8, 33.8, 33.2, 28.7, 18.7, 18.0, 17.4, 1 1.8.

HRMS (ESI)

Calculated for CysHgsNOzo (M + Na) ⁺ : 1328.6345

Found: 1328.6388

-Tertbutylbenzoic ester 3e

3e

THF (160 mL) was added to a flask containing 1 (6.16 g, 4.72 mmol, 1 eq). DMAP (922 mg, 7.54 mmol, 1.6 eq) was added to a separate flask and dissolved in THF (100 mL). 4- tertbutylbenzoyl chloride (1.29 mL, 6.60 mmol, 1.4 eq) was added dropwise to the DMAP solution creating a white suspension. DIPEA (1.31 mL, 7.54 mmol, 1.6 eq) was added to the solution of 1. A portion of the white suspension was then transferred dropwise to the solution of 1 and DIPEA (over approximately 1 hr) until the majority of 1 had been consumed as evidenced by TLC. The reaction was poured into EtOAc and washed with water followed by saturated sodium bicarbonate. Two more washes with water were performed followed by a wash with saturated sodium chloride. The organic layer was then dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Flexane:MeOH 60:37:3) purification yielded 3e as a yellow-orange solid (3.1 1 g, 2.12 mmol, 45%) as well as unreacted 1.

TLC (EtOAc:Hexane:MeOH 60:37:3)

R _f = 0.22, stained by CAM HPLC

tR = 19.4 min; flow rate = 1 mL/min, gradient = 5% MeCN in water for 2 min then 5→54% MeCN in water over 3 min then 54→95% MeCN in water over 13 min.

1H NMR (500 MHz, CD ₃ C(0)C0 ₃ )

δ 7.99 (d, J= 8.5 Hz, 2H), 7.59 (d, J= 8.5 Hz, 2H), 7.39 (m, 3H), 7.34 (m, 2H), 7.23 (m, 2H), 7.17 (m, 2H), 7.12 (m, 1H), 6.85 (m, 4H), 6.39-6.13 (m, 10H), 6.07 (m, 1H), 5.92 (m, 1H), 5.76 (m, 1H), 5.68 (m, 1H), 5.56 (m, 1H), 5.48 (s, 1H), 5.43 (s, 1H), 5.14 (m, 1H), 4.88 (app s, 1H), 4.65 (m, 1H), 4.24 (m, 1H), 4.15 (m, 2H), 3.97 (m, 1H), 3.91- 3.82 (m, 2H), 3.77 (s, 6H), 3.68 (m, 5H), 3.51 (m, 2H), 3.47 (m, 1H), 3.40 (m, 2H), 2.84 (s, 3H), 2.54 (m, 1H), 2.41 (m, 1H), 2.27 (m, 1H), 2.13 (m, 1H), 1.95 (m, 1H), 1.86 (m, 1H), 1.78-1.42 (m, 10H), 1.40-1.31 (m, 13H), 1.30-1.19 (m, 2H), 1.18 (d, J= 6.5 Hz, 3H), 1.11 (d, J = 6.5 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H)

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 172.7, 171.1, 169.2, 165.3, 160.1, 160.0, 156.7, 137.1, 136.4, 136.3, 133.6, 133.5, 133.4, 133.3, 133.2, 133.1, 132.5, 132.1, 132.0, 131.2, 131.1, 130.0, 129.4, 128.3, 127.7, 126.5, 125.6, 120.7, 113.3, 100.5, 96.1, 80.5, 77.2, 75.7, 74.1, 73.1, 72.6, 72.4, 71.7, 70.6, 66.4, 66.2, 57.2, 54.8, 54.1, 51.3, 47.9, 43.0, 42.7, 41.9, 40.9, 37.2, 36.3, 35.1, 33.4, 32.6, 30.8, 17.8, 17.4, 11.3.

HRMS (EST)

Calculated for C84H107NO21 (M + Na) ⁺ : 1488.7233

Found: 1488.7212

Diethyliso ropyl silyl ether SI2

3e (2.30 g, 1.57 mmol, 1 eq) was azeotropically dried with acetonitrile and left under vacuum overnight. DCM (40 mL) was added followed by hexane (40 mL) slowly while stirring to prevent 3e from crashing out of solution. 2,6-lutidine (2.4 mL, 20.4 mmol, 13 eq) was added and the solution was cooled to 0 °C. Diethylisopropylsilyl trifiate (DEIPSOTf) (2.5 mL, 12.5 mmol, 8 eq) was added dropwise at 0 °C over 20 min. The reaction was stirred for an additional 30 min. The reaction was diluted with diethyl ether and quenched with saturated sodium bicarbonate at 0 °C. The reaction was extracted with diethyl ether and washed with 1 M copper sulfate until no white precipitate was observed. The organic layers were washed twice with water and then once with saturated sodium chloride. The organic layers were then dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Hexane 1 :9— " 1 :4) purification yielded SI2 as a yellow-orange solid (2.24 g, 1.13 mmol, 72%).

TLC (EtOAc:Hexane 1 :3)

R _f = 0.25, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 8.01 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.34 (m, 4H), 7,22 (m, 3H), 7.17 (m, 3H), 6.84 (m, 4H), 6.35-6.13 (m, 9H), 6.04 (m, 1H), 5.91 (m, 1H), 5.74 (m, 2H),

5.49 (m, 1H), 5.41 (s, 1H), 5.39 (s, 1H), 4.92 (app s, 1H), 4.75 (m, 1H), 4.66 (m, 1H), 4.31 (m, 1H), 4.25 (m, 1H), 4.12 (m, 1H), 3.84 (m, 1H), 3.81-3.77 (m, 9H), 3.68 (m, 4H), 3.64 (s, 3H), 3.57-3.45 (m, 3H), 2.74 (s, 3H), 2.45 (m, 2H), 2.26 (m, 1H), 2.17 (m, 1H), 2.09 (m, 1H), 1.90 (m, 2H), 1.73-1.59 (m, 4H), 1.51-1.34 (m, 18H), 1.26-1.1 1 (m, 6H), 1.08-0.76 (m, 54H), 0.73-0.39 (m, 19H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 173.1 , 171.1 , 169.8, 165.9, 160.7, 157.4, 138.2, 136.5, 134.6, 134.4, 134.2, 133.9, 133.8, 133.4, 132.8, 132.7, 132.5, 132.1 , 131.1 , 130.7, 130.6, 130.2, 129.0, 128.9, 128.8, 128.6, 128.2, 127.1 , 126.2, 121.3, 1 17.9, 1 15.1 , 1 13.8, 1 13.7, 101.7, 100.9, 100.5, 96.6, 81.3, 75.6, 74.7, 73.7, 73.2, 72.9, 72.7, 72.6, 68.6, 66.6, 58.1 , 55.4, 54.5,

51.8, 48.3, 43.4, 41.0, 37.9, 36.8, 35.7, 31.4, 19.0, 18.0, 17.9, 17.8, 17.7, 17.6, 17.4, 14.0, 13.8, 13.4, 7.7, 7.6, 7.5, 7.4, 7.1 , 5.1 , 4.8, 4.7, 4.6, 4.4, 4.1.

HRMS (ESI)

Calculated for (M + Na) ⁺ : 2001.1318

Found: 2001.1221 Hydroxyl 6

SI2 (550 mg, 278 μηιοΐ, 1 eq) was dissolved in THF:MeOH 1 :2 (13.5 mL), and KCN (27.0 mg, 417 μιηοΐ, 1.5 eq) was added. The reaction was heated to 40 °C for 2 days. The reaction was diluted with diethyl ether and washed with water three times followed by a wash of saturated sodium chloride. The organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ;

EtOAc:Hexane 1 :4→ 3:7) purification yielded 6 as a yellow-orange solid (329 mg, 181 μιηοΐ, 65%).

TLC (EtOAc:Hexane 3:7)

R _f = 0.22, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.35 (M, 6H), 7.29 (m, 2H), 7.22 (m, 1H), 6.93 (d, J= 9.5 Hz, 1H), 6.85 (m, 4H),

6.40-6.17 (m, 11H), 6.09 (m, 1H), 5.82 (m, 1H), 5.77 (m, 1H), 5.43 (s, 1H), 5.42 (s, 1H), 4.80 (m, 1H), 4.62 (m, 1H), 4.60 (app s, 1H), 4.23 (m, 1H), 4.15 (m, 1H), 3.98 (m, 1H), 3.85 (m, 3H), 3.78 (m, 7H), 3.71 (m, 4H), 3.66 (m, 2H), 3.62 (s, 3H), 3.58-3.50 (m, 2H), 3.32 (m, 1H), 3.02 (s, 3H), 2.48 (m, 1H), 2.42 (m, 1H), 2.29-2.19 (m, 3H), 1.95-1.87 (m, 3H), 1.74 (m, 2H), 1.62-1.28 (m, 7H), 1.24-1.15 (m, 7H), 1.04-0.76 (m,

56H), 0.72-0.50 (m, 13H), 0.44-0.36 (m, 4H)

1 ³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 173.4, 170.8, 169.9, 160.8, 160.6, 137.2, 136.9, 134.6, 134.5, 134.1, 133.9, 133.8, 133.6, 133.2, 132.7, 132.6, 132.2, 130.9, 130.3, 129.5, 129.1, 128.7, 128.3, 127.3, 121.3, 117.9, 113.9, 113.8, 110.6, 101.8, 101.1, 100.8, 98.5, 81.4, 75.9, 75.0, 74.7, 74.6,

73.4, 73.0, 72.8, 71.5, 68.8, 67.1, 57.7, 55.8, 55.5, 52.0, 48.5, 43.8, 43.0, 41.2, 37.9, 36.7, 33.5, 32.7, 28.1, 18.9, 18.0, 17.9, 17.8, 17.4, 14.0, 13.9, 13.8, 13.5, 7.7, 7.6, 7.5, 7.2, 5.1 , 4.9, 4.7, 4.6, 4.4, 4.1.

HRMS (ESI)

Calculated for CioiHi ₅₉ N02oSi4 (M + Na) ⁺ : 1841.0430

Found: 1841.0464

Iodide SI3

6 (350 mg, 192 μηιοΐ, 1 eq), triiodoimidazole (130 mg, 288 μηιοΐ, 1.5 eq), triphenyl phosphine (152 mg, 378 μιηοΐ, 3 eq), and imidazole (60 mg, 866 μιηοΐ, 4.5 eq) were placed in a flask and dissolved in toluene (9.6 mL). The reaction was heated to 70°C for 3 hrs. The reaction was diluted with diethyl ether and washed with saturated sodium bicarbonate followed by water four times. A final wash of saturated sodium chloride was performed, and the organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Hexane 3 : 17) purification yielded SI3 as a yellow-orange solid (296 mg, 154 μιηοΐ, 80 %).

TLC (EtOAc:Hexane 3 :7)

R _f = 0.55, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.38-7.28 (m, 9H), 7.22 (m, 1H), 6.86 (m, 4H), 6.40-6.09 (m, 12H), 5.83 (m, 2H), 5.43 (s, 1H), 5.41 (s, 1H), 4.79 (m, 1H), 4.69 (app d, J = 7.5 Hz, 1H), 4.57 (m, 1H), 4.28 (m, 1H), 4.15 (m, 1H), 4.04 (m, 1H), 3.89 (m, 1H), 3.84 (m, 1H), 3.77 (m, 7H), 3.72 (m, 3H), 3.65 (s, 3H), 3.57 (m, 2H), 3.40 (m, 1H), 3.05 (s, 3H), 2,51-2.40 (m, 2H), 2.25 (m, 3H), 2.00 (m, 1H), 1.88 (m, 2H), 1.74 (m, 2H), 1.62-1.40 (m, 7H), 1.31-1.15 (m, 9H), 1.07-0.76 (m, 58H), 0.72-0.50 (m, 13H), 0.44-0.36 (m, 4H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ ) δ 172.5, 169.3, 160.6, 160.2, 160.0, 158.4, 137.5, 134.2, 134.1, 133.9, 133.6, 133.4, 133.2, 132.9, 132.5, 132.1, 130.0, 131.9, 131.3, 130.2, 130.0, 128.3, 128.1, 127.7, 126.8, 113.3, 113.2, 102.1, 101.3, 100.5. 100,1, 94.2, 80.8, 76.3, 75.4, 74.4, 74.0, 72.5, 72.2, 68.1, 66.7, 57.0, 54.8, 51.4, 47.9, 43.8, 43.4, 42.5, 40.6, 37.3, 36.8, 32.9, 27.5, 18.1, 17.4, 17.3, 17.2, 16.8, 13.4, 13.2, 12.9, 7.1, 7.0, 6.9, 6.6, 4.5, 4.3, 4.2, 4.1, 3.8, 3.6.

HRMS (ESI)

Calculated for CioiHi ₅₈ NOi ₉ Si ₄ I (M + Na) ⁺ : 1950.9448

Found: 1950.9543 Methylene 7

SI3 (320 mg, 166 μηιοΐ, 1 eq) was placed in a vial and azeotropically dried with toluene and placed under vacuum overnight. The vial was backfilled with argon and DMPU (6.6 mL) was added. Sodium borohydride (50 mg, 1.33 mmol, 8 eq) and silver(I) acetate (42 mg, 249 μιηοΐ, 1.5 eq) was added in a glovebox. The reaction was heated in the range of 50-55°C for 3 hrs. After 3 hrs, an aliquot was removed in the glovebox every 30 min to monitor the reaction by TLC. The reaction was allowed to run to approximately 85% conversion until the rate of decomposition exceeded conversion of the starting material. The reaction was cooled to room temperature and then diluted with dry diethyl ether that had been cooled to 0°C. The reaction was quenched with saturated sodium bicarbonate cooled to 0°C. Room temperature diethyl ether was used to extract the aqueous layer. The organic layer was then washed with water twice. A final wash of saturated sodium chloride was performed, and the organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Hexane 3: 17) purification yielded 7 as a yellow-orange solid (89.8 mg, 49.8 μιηοΐ, 30%).

This reaction is quite sensitive to water and air. DMPU was obtained from Aldrich absolute over molecular sieves H ₂ 0 < 0.03%. The product is unstable to the reaction conditions and decomposes over time; the best yields are obtained by stopping the reaction before complete conversion and recovering the starting material and product.

The reaction was found to be dependent upon the identity of the protecting group on the C3 ' amine. Extensive elimination or inactivity was observed for other protecting groups.

TLC (EtOAc:Hexane 1 :3)

R _f = 0.47, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.40-7.33 (m, 9H), 7.26 (m, 1H), 6.87 (m, 4H), 6.42-6.06 (m, 12H), 5.70 (m, 2H),

5.46 (s, 1H), 5.44 (s, 1H), 4.69 (app d, J = 5 Hz, 1H), 4.97 (m, 1H), 4.23 (m, 3H), 3.93 (m, 1H), 3.82-3.70 (m, 10H), 3.66 (m, 4H), 3.58 (m, 2H), 3.39 (m, 1H), 3.17 (m, 1H), 3.04 (s, 3H), 2.63 (m, 2H), 2.42 (m, 1H), 2.30 (m, 3H), 1.98 (m, 1H), 1.88 (m, 1H), 1.76-1.34 (m, 10H), 1.29-1.14 (m, 8H), 1.05-0.76 (m, 59H), 0.72-0.50 (m, 13H), 0.44- 0.36 (m, 4H).

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 173.6, 170.0, 160.8, 160.7, 136.9, 134.6, 134.3, 133.6, 136.4, 133.0, 132.6, 132.5, 130.0, 129.4, 128.7, 128.4, 127.6, 1 13.9, 1 13.8, 102.0, 101.2, 81.7, 80.9, 76.5, 75.2, 73.4, 73.1 , 55.5, 51.7, 44.1 , 43.8, 42.7, 41.5, 37.9, 33.6, 32.6, 28.5, 18.0, 17.9, 17.8, 17.6, 17.4, 13.9, 13.5, 7.7, 7.6, 7.5, 7.4, 7.2, 4.8, 4.7, 4.4, 4.2.

HRMS (ESI)

Calculated for CioiHi ₅₉ NOi9Si4 (M + Na) ⁺ : 1825.0481

Found: 1825.0496

6 (18.2 mg, 10 μηιοΐ, 1 eq), triphenylphosphine (4.0 mg, 15 μηιοΐ, 1.5 eq), and p- nitrobenzoic acid (2.0 mg, 12 μιηοΐ, 1.15 eq) were placed in a flask and azeotroped in toluene to dryness (3x 0.5 mL). The reaction was then dissolved in toluene (0.3 mL) and cooled to 0°C for the dropwise addition of DIAD (3.0 μί, 15 μιηοΐ, 1.5 eq). The reaction was stirred for 20 min at 0°C then heated to 70°C for 2 hrs. The reaction was diluted with diethyl ether (10 mL) and washed with saturated sodium bicarbonate (3.0 mL). The aqueous phase was extracted with diethyl ether (10 mL). A final wash of saturated sodium chloride was performed, and the organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Hexane 5:95 to 1 :3) purification yielded the C2' nitrobenzoate ester as a yellow-orange solid. Two reactions were run: (11.7 mg, 5.9 μιηοΐ, 59%) (13.5 mg, 6.8 μιηοΐ, 68%).

The C2' /^-nitrobenzoate ester was combined (25.2 mg, 12.8 μιηοΐ, 1.0 eq) and taken up in MeOH:THF 2: 1 (435 μί). Potassium cyanide (2.5 mg, 38 μιηοΐ, 3.0 eq) was then added and the reaction was stirred for 72 hrs at 30°C. The reaction was then diluted with diethyl ether (10 mL) and washed with saturated sodium bicarbonate (3.0 mL). The aqueous phase was extracted with diethyl ether (2x 10 mL). A final wash of saturated sodium chloride was performed, and the organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ : EtOAc:Hexane 1 :4→ 3:7) purification yielded 8 as a yellow-orange solid (15.8 mg, 8.7 μιηοΐ, 68%>).

TLC (EtOAc:Hexane 3:7)

R _f = 0.2, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.36 (m, 6H), 7.28 (m, 2H), 7.21 (m, 1H), 7.15 (m, 1H), 6.86 (m, 4H), 6.41-6.18 (m, 11H), 6.10 (m, 1H), 5.86 (m, 1H), 5.77 (m, 1H), 5.44 (s, 1H), 5.43 (s, 1H), 4.81 (m,

1H), 4.56 (m, 1H), 4.36 (app d, J = 7.5, 1H), 4.26 (m, 1H), 4.17 (m, 1H), 3.97-3.85 (m, 4H), 3.79 (m, 7H), 3.73 (m, 4H), 3.66 (s, 3H), 3.61-3.48 (m, 3H), 3.43 (m, 1H), 3.33 (m, 1H), 3.04 (s, 3H), 2.49 (m, 1H), 2.42 (m, 1H), 2.30-2.21 (m, 3H), 1.95-1.87 (m, 3H), 1.80-1.67 (m, 3H), 1.64-1.27 (m, 6H), 1.24-1.16 (m, 7H), 1.04-0.76 (m, 56H), 0.73-0.51 (m, 13H), 0.46-0.37 (m, 4H)

¹³ C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 173.9, 172.1, 170.1, 161.0, 137.9, 137.0, 134.8, 134.7, 134.3, 134.1, 133.9, 133.7,

133.3, 132.9, 132.4, 131.1, 130.5, 129.7, 129.1, 128.9, 128.5, 127.3, 114.1, 114.0,

103.4, 102.0, 101.3, 100.9, 81.6, 76.5, 76.1, 75.2, 74.7, 73.3, 73.0, 69.0, 67.6, 57.8, 55.6, 52.1, 48.6, 44.3, 44.2, 38.1, 33.7, 32.9, 19.1, 18.2, 18.1, 18.0, 17.6, 14.3, 14.2, 14.1, 13.7, 7.8, 7.7, 7.6, 7.3, 5.4, 5.1, 5.0, 4.9, 4.6, 4.4

HRMS (ESI)

Calculated for CioiHi ₅₉ N0 ₂ oSi ₄ (M + Na) ⁺ : 1841.0430

Found: 1841.0464

Conjugate 10

Ergosterol (400 mg, 1.01 mmol, 1 eq) and succinic anhydride (1.01 g, 10.1 mmol, 10.0 eq) were azeotroped with toluene (3x 1.0 mL) in a 40 mL vial. Dry pyridine (20 mL 0.05 M) was then added followed by dimethylaminopyridine (DMAP) (154.2 mg, 1.26 mmol, 1.25 eq). The reaction was sealed with a teflon lined cap and heated to 140°C for 16 hrs. The resulting black solution was extracted with HCl (10% v/v) and EtOAc. The organic phase was dried with sodium sulfate, filtered, and concentrated. Chromatography (Si0 ₂ ; EtOAc:Hexane 1 :5 with 1%) AcOH) purification yielded A as a white solid (282 mg, 0.57 mol, 56%>).

A 9

A (12.5 mg, 25 μηιοΐ, 2.5 eq) was dissolved in toluene (0.3 mL) and oxalyl chloride (10.0 μί, 118 μηιοΐ, 4.75 eq) was added and the reaction was heated to 50°C and stirred for 15 min. The resulting yellow solution was azeotroped with toluene (3x 0.3 mL) to dryness. The resulting off-white solid 9 was then dissolved in THF (0.3 mL) whereupon DMAP (3.1 mg, 25 μιηοΐ, 2.5 eq) was added to generate a cloudy white suspension. In a separate vial 6 (18.2 mg, 10 μιηοΐ, 1.0 eq) was dissolved in THF (0.15 mL) and diethylisopropyl amine (10 μΐ,, 57 μιηοΐ, 5.7 eq) was added. The resulting yellow/orange solution was added dropwise via cannula to the first suspension and stirred for 3 hrs at room temperature. The reaction was diluted with diethyl ether (10 mL) and washed with saturated sodium bicarbonate (3.0 mL). The aqueous phase was extracted with diethyl ether (2x 10 mL). A final wash of saturated sodium chloride was performed, and the organic layers were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and column chromatography (Si0 ₂ ; EtOAc:Hexane 5:95 ^→ 1 :3) purification yielded the 10 as a yellow-orange solid (14.5 mg, 6.3 μιηοΐ, 63%).

TLC (EtOAc:Hexane 1 :3)

Rf = 0.76, stained by CAM

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.36 (m, 4H), 7,28 (m, 4H), 7.23 (m, 1H), 6.93 (m, 1H), 6.86 (m, 4H), 6.39-6.12 (m,

2H), 5.84 (m, 2H), 5.61 (m, 1H), 5.41 (m, 3H), 5.26 (m, 3H), 5.83-4.61 (m, 4H), 4.29- 4.13 (m, 3H), 3.90-3.81 (m, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.72 (m, 2H), 3.67 (s, 3H), 3.56 (m, 2H), 3.47 (m, 2H), 3.00 (s, 3H), 2.71-2.34 (m, 9H), 2.31-2.08 (m, 5H), 2.00- 1.45 (m, 30H), 1.41-0.38 (m, 98H) C NMR (125 MHz, CD ₃ C(0)CD ₃ )

δ 173.4, 173.2, 172.3, 171.2, 170.1 , 160.8, 142.3, 139.6, 137.8, 136.8, 136.7, 134.8, 134.5, 134.1 , 133.7, 133.3, 132.9, 132.6, 132.1 , 131.0, 130.5, 129.2, 128.9, 128.5, 127.5, 126.2, 121.4, 1 17.5, 1 14.1 , 1 14.0, 102.0, 101.3, 100.9, 81.6, 76.2, 75.2, 75.1 ,

74.1 , 73.9, 73.3, 73,1 , 72.3, 68.9, 67.1 , 58.1 , 56.7, 55.7, 55.4, 54.6, 52.1 , 48.8, 47.1 ,

44.2, 43.9, 43.8, 43.7, 43.2, 41.4, 39.9, 38.8, 38.0, 37.6, 34.0, 33.8, 30.9, 29.1 , 29.0, 28.4, 23.9, 21.7, 20.5, 20.2, 19.2, 18.3, 18.2, 18.1 , 18.0, 17.9, 17.6, 16.6, 14.2, 14.1 , 13.7, 12.6, 7.9, 7.8, 7.7, 7.6, 7.3, 5.4, 5.1 , 5.0, 4.9, 4.7, 4.4, 1.5

MS (MALDI)

Calculated for Ci ₃₃ H ₂₀₅ NO ₂ Si ₄ (M + Na) ⁺ : 2319

Found: 2319

Example 2. DMAP-mediated Acylation Studies

General Procedure for Acylation Reactions

1 (5 mg, 3.83 μηιοΐ, 1 eq) was added to a vial and dissolved in THF (450 μί). DIPEA

(0.8 μΕ, 4.60 μηιοΐ, 1.2 eq) and DMAP (0.515 mg, 4.21 μιηοΐ, 1.1 eq) were added to the solution. The acyl chloride or anhydride (1 eq) in THF (50 i ) was added to the reaction. The reaction was allowed to stir at room temperature for 15 hrs. The reaction was filtered and analyzed by reversed-phase HPLC (C18, MeCN/H ₂ 0).

HPLC gradient

flow rate = lmL/min, gradient = 5% MeCN in water for 2 min then 5 ^→ 54% MeCN in water over 3 min then 54 ^→ 95% MeCN in water over 13 min, hold 95% MeCN in water for 7 min followed by gradient to 5% water, 75% MeCN, 20% THF over 3 min then increasing to 5% water, 95% THF in one min and holding 95% THF in water for 3 min.

Determination of Selectivity

The conversion and ratio of products were determined by integration of the HPLC traces. The structure of the mono-acylated products was determined by multi-dimensional NMR and HRMS (Example 3). Conversion was calculated as the sum of the product peak areas over the total area. % C2' selectivity was calculated as the area for the C2' mono-acylated product over the total product area. The ratio of site isomers used in the Hammett analysis is the C2' mono-acylated product over the sum of the other product areas. Product Characterization

p-Nitrobenzoic ester 3c

3c

HPLC

tR= 17.0 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 8.40 (d, J= 8.5 Hz, 2H), 8.29 (d, J= 8.5 Hz, 2H), 7.44 (m, IH), 7.39 (d, J= 9.0 Hz, 2H) 7.34 (d,J= 8.5 Hz, 2H), 7.21 (m, 2H), 7.16 (m, 2H), 7.11 (m, IH), 6.85 (m, 4H), 6.39-6.12 (m, 10H), 6.01 (m, 2H), 5.79 (m, IH), 5.65 (m, IH), 5.61 (m, IH), 5.48 (s, IH), 5.43 (s, IH), 5.18 (m, IH), 4.92 (app s, IH), 4.65 (m, IH), 4.27 (m, IH), 4.14 (m, 2H), 3.98 (m, IH), 3.88 (m, 2H), 3.77 (m, 6H), 3.69 (m, 5H), 3.65-3.38 (m, 5H), 2.86 (s, 3H), 2.55 (m, IH), 2.40 (m, IH), 2.28 (m, 2H), 2.17 (m, IH), 1.94 (m, IH), 1.85 (m, IH), 1.80-1.40 (m, 10H), 1.34 (d,J=5.0Hz, 3H), 1.27 (m, 2H), 1.18 (d,J= 6.5 Hz, 3H), 1.11 (d, J =6.5 Hz, 3H), 1.00 (d,J=7.0Hz, 3H)

HRMS (ESI)

Calculated for (M + Na) ⁺ : 1477.6458

Found: 1477.6404

SI4

HPLC

tR = 18.0 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 8.06 (m, 2H), 7.60 (m, 2H), 7.39 (m, 4H), 7.34 (m, 2H) 7.22-7.11 (m, 4H), 6.85 (m, 4H), 6.41-6.13 (m, 10H), 6.02 (m, 1H), 5.91(m, 1H), 5.77 (m, 1H), 5.67 (m, 1H), 5.57 (m, 1H), 5.48 (s, 1H), 5.43 (s, 1H), 5.14 (m, 1H), 4.89 (app s, 1H), 4.64 (m, 1H), 4.23 (m, 1H), 4.14 (m, 2H), 3.89 (m, 3H), 3.77 (m, 6H), 3.69 (m, 5H), 3.52-3.39 (m, 5H), 2.85 (s, 3H), 2.55 (m, 1H), 2.40 (m, 1H), 2.28 (m, 2H), 2.16 (m, 1H), 1,93 (m, 1H), 1.86 (m, 1H), 1.80-1.40 (m, 10H), 1.32 (d, J= 5.0 Hz, 3H), 1.27 (m, 2H), 1.18 (d, J= 6.5 Hz, 3H), 1.10 (d, J = 6.5 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H)

HRMS (ESI)

Calculated for CsoHgsNClOzi (M + H) ⁺ : 1444.6393

Found: 1444.6330 p-Methylbenzoic ester SI5

SI5 HPLC

tR= 17.8 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.95 (d,J= 8.5 Hz, 2H), 7.36 (m, 8H), 7.22-7.11 (m, 4H), 6.85 (m, 4H), 6.41-6.12 (m, 10H), 6.04 (m, 1H), 5.89 (m, 1H), 5.76 (m, 1H), 5.68 (m, 1H), 5.56 (m, 1H), 5.48 (s, 1H), 5.43 (s, 1H), 5.15 (m, 1H), 4.87 (app s, 1H), 4.64 (m, 1H), 4.22 (m, 1H), 4.13 (m, 2H), 3.88 (m, 3H), 3.77 (m, 6H), 3.68 (m, 5H), 3.51-3.39 (m, 5H), 2.85 (s, 3H), 2.55 (m, 1H), 2.45 (s, 3H), 2.40 (m, 1H), 2.27 (m, 2H), 2.16 (m, 1H), 1.93 (m, 1H), 1.86 (m, 1H), 1.79-1.40 (m, 10H), 1.32 (d,J=5.5Hz, 3H), 1.26 (m, 2H), 1.18 (d,J= 6.5 Hz, 3H), 1.10 (d, J =6.5 Hz, 3H), 1.01 (d,J=7.0Hz, 3H)

HRMS (ESI)

Calculated for C ₈ iHi ₀ iNO ₂ i (M + Na) ⁺ : 1446.6764

Found: 1446.6718 τρ-Methoxybenzoic ester SI6

SI6

HPLC 1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 8.01 (d,J= 8.5 Hz, 2H), 7.37 (m, 6H), 7.23-7.11 (m, 4H), 7.07 (m, 2H), 6.85 (m, 4H), 6.41-6.12 (m, 10H), 6.04 (m, 1H), 5.88 (m, 1H), 5.76 (m, 1H), 5.68 (m, 1H), 5.54 (m, 1H), 5.48 (s, 1H), 5.43 (s, 1H), 5.14 (m, 1H), 4.86 (app s, 1H), 4.64 (m, 1H), 4.21 (m, 1H), 4.13 (m, 2H), 3.90 (m, 6H), 3.77 (m, 6H), 3.67 (m, 5H), 3.51-3.39 (m, 5H), 2.85 (s, 3H), 2.55 (m, 1H), 2.40 (m, 1H), 2.27 (m, 2H), 2.15 (m, 1H), 1.93 (m, 1H), 1.86 (m, 1H), 1.79-1.40 (m, 10H), 1.32 (d,J=5.5Hz, 3H), 1.26 (m, 2H), 1.18 (d,J= 6.5 Hz, 3H), 1.10 (d, J =6.5 Hz, 3H), 1.01 (d,J=7.0Hz, 3H) HRMS (ESI)

Calculated for C ₈ iHi ₀ iNO ₂ 2 (M + Na) 1462.6713

Found: 1462.6680

3d

HPLC

tR = 17.3 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.89 (d, J= 9.0 Hz, 2H), 7.36 (m, 6H), 7.18 (m, 4H), 6.82 (m, 6H), 6.40-6.04 (m, 11H), 5.82 (m, 1H), 5.73 (m, 2H), 5,52 (m, 1H), 5.48 (s, 1H), 5.43 (s, 1H), 5.11 (m, 1H), 4.84 (app s, 1H), 4.65 (m, 1H), 4.16 (m, 3H), 3.87 (m, 3H), 3.77 (s, 6H), 3.68 (m, 5H), 3.51-3.38 (m, 5H), 3.09 (s, 6H), 2.85 (s, 3H), 2.53 (m, 1H), 2.40 (m, 1H), 2.27 (m, 2H), 2.14 (m, 1H), 1.93 (m, 1H), 1.87 (m, 1H), 1.78-1.40 (m, 10H), 1.32 (d, J= 5.5 Hz, 3H), 1.26 (m, 2H), 1.18 (d, J = 6.5 Hz, 3H), 1.10 (d, J = 6.5 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H)

HRMS (ESI)

Calculated for C ₈₂ H ₁₀₄ N ₂ O ₂₁ (M +Na) ⁺ : 1475.7029

Found: 1475.6982 Isobutyric ester 3b

3b

HPLC

tR = 17.3 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.45 (app d, J= 8.5 Hz, 2H), 7.37 (app d, J= 8.5 Hz, 2H), 7.29 (m, 4H), 7.24 (m, IH), 7.09 (app d, J= 7.5 Hz, IH), 6.90 (app d, J= 7.5 Hz, 4H), 6.46-6.21 (m, 12H), 5.90 (m, IH), 5.66 (m, IH), 5.54 (s, IH), 5.48 (s, IH), 5.36 (m, IH), 5.24 (m, IH), 4.77 (app s, IH), 4.65 (m, IH), 4.19 (m, 2H), 4.11 (m, IH), 3.96 (m, 3H), 3.81 (s, 6H), 3.74 (m, 5H), 3.52-3.36 (m, 5H), 3.05 (s, 3H), 2.60 (m, 2H), 2.43 (m, IH), 2.32 (m, 2H), 2.23 (m, IH), 1.99-1.86 (m, 2H), 1.79-1.66 (m, 4H), 1.61-1.48 (m, 4H), 1.36 (m, 2H), 1.30 (d, J= 6.0 Hz, 3H), 1.21 (m, 5H), 1.18-1.13 (m, 9H), 1.04 (d, J= 7.0 Hz, 3H)

HRMS (ESI)

Calculated for C ₇₇ H ₁₀₁ NO ₂₁ (M + Na) ⁺ : 1398.6764

Found: 1398.6758

Isobutyric ester SI7

SI7

HPLC tR= 18.7 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.41 (app d, J= 8.5 Hz, 2H), 7.35-7.26 (m, 6H), 7.21 (m, IH), 6.87 (m, 5H), 6.42-6.18 (m, 12H), 5.88 (m, IH), 5.57 (m, IH), 5.51 (s, IH), 5.45 (s, IH), 5.25 (m, IH), 4.79 (t, J = 10.0, IH), 4.65 (m, 2H), 4.21-4.10 (m, 3H), 4.01-3.91 (m, 3H), 3.77 (s, 6H), 3.71 (m, 3H), 3.65 (s, 3H), 3.54 (m, 2H), 3.44-3.35 (m, 2H), 3.05 (s, 3H), 2.56 (m, IH), 2.41- 2.22 (m, 5H), 1.99-1.45 (m, 10H), 1.31 (m, 2H), 1.18 (m, 4H), 1.10 (m, 7H), 1.02-0.98 (m, 9H)

HRMS (ESI)

Calculated for C ₇₇ H ₁₀₁ NO ₂₁ (M +Na) ⁺ : 1398.6764

Found: 1398.6740

Isobutyric ester SI8

SI8

HPLC

tR = 19.1 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.38-7.18 (m, 10H), 6.85 (m, 4H), 6.43-6.18 (m, 12H), 5.88 (m, IH), 5.58 (m, IH), 5.51 (s, IH), 5.45 (s, IH), 5.29 (m, 2H), 4.67 (m, IH), 4.59 (app s, 114), 4.18 (m, IH), 4.04 (m, IH), 3.93 (m, 2H), 3.86 (m, IH), 3.77 (m, 6H), 3.71 (m, 2H), 3.64 (m, 4H), 3.50 (m, IH), 3.45 (m, IH), 3.34 (m, IH), 3.29 (m, 2H), 3.09 (s, 3H), 2.61-2.28 (m, 5H), 2.00 (m, IH), 1.93-1.25 (M, 12H), 1.19 (m, 6H), 1.12 (m, 5H), 1.05 (m, 6H), 1.01 (d, J= 6.0 Hz, 3H)

HRMS (ESI)

Calculated for C ₇₇ H ₁₀₁ NO ₂₁ (M + Na) ⁺ : 1398.6764 Found: 1398.6755

4a

HPLC

tR = 21.4 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.84 (d, J= 8.0 Hz, 2H), 7.49 (d, J= 8.5 Hz, 2H), 7.42 (d, J= 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.04 (m, 6H), 6.86 (m, 4H), 6.43-6.19 (m, 12H), 5.90 (m, 1H), 5.57 (m, 1H), 5.52 (s, 1H), 5.46 (s, 1H), 5.26 (m, 1H), 5.06 (t, J= 10 Hz, 1H) 4.76 (app s, 1H), 4.69 (m, 1H), 4.40 (m, 1H), 4.16 (m, 2H), 4.04 (m, 1H), 3.95 (m, 2H), 3.85 (m, 1H), 3.79 (s, 6H), 3.72 (m, 3H), 3.69 (s, 3H), 3.52-3.34 (m, 3H), 3.08 (s, 3H), 2.59 (m, 1H), 2.40 (m, 1H), 2.29 (m, 3H), 2.00 (m, 1H), 1.92-1.83 (m, 3H), 1.77-1.62 (m, 3H), 1.58- 1.46 (m, 4H), 1.36 (s, 9H), 1.33(m, 1H), 1.20-1.16 (m, 8H), 1.11 (d, J= 6.0 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H)

HRMS (ESI)

Calculated for C ₈₄ H ₁₀₇ NO ₂₁ (M + Na) ⁺ : 1488.7233

Found: 1488.7230

τρ-Tertbutylbenzoic ester 5a

HPLC

tR = 21.7 min

1H NMR (500 MHz, CD ₃ C(0)CD ₃ )

δ 7.91 (d, J= 8.5 Hz, 2H), 7.56 (d, J= 8.5 Hz, 2H), 7.32 (m, 8H), 7.19 (m, 2H), 6.86 (d, J= 8.5 Hz, 2H), 6.65 (d, J= 8.5 Hz, 2H), 6.41-6.18 (m, 12H), 5.91 (m, 1H), 5.55 (m, 2H), 5.50 (s, 1H), 5.45 (s, 1H), 5.27 (m, 1H), 4.69 (m, 1H), 4.62 (app s, 1H), 4.17 (m, 1H), 4.12 (m, 1H), 3.95 (m, 2H), 3.87 (m, 1H), 3.80 (m, 1H), 3.78 (s, 3H), 3.71 (m,

2H), 3.64 (m, 6H), 3.52-3.43 (m, 2H), 3.35-3.28 (m, 3H), 3.14 (s, 3H), 2.71 (m, 2H), 2.58 (m, 1H), 2.39 (m, 1H), 2.30 (m, 1H), 1.97-1.84 (m, 3H), 1.74-1.38 (m, 9H), 1.34 (s, 9H), 1.22-1.16 (m, 8H), 1.10 (d, J = 6.5 Hz, 3H), 1.01 (d, J= 7.0 Hz, 3H)

HRMS (ESI)

Calculated for C84H107NO21 (M + Na) ⁺ : 1488.7233

Found: 1488.7214

Example 3. Determination of the Site of Acylation

The major products of the acylation reaction were purified by HPLC. Mass

spectrometry was utilized to determine if the products were mono- or bis-acylated. Proton assignments for 1 and its mono-acylated derivatives were made via 1H COSY NMR spectrum analysis. The signal of the proton on the carbon bearing the acylated hydroxyl was shifted downfield approximately 1.5 ppm relative to its non-acylated counterpart in each case.

Included here are expanded sections of the COSY spectrum with the relevant cross-peaks labeled of the continuous segments containing the acylated hydroxyls (C14-C20 for the C15 hydroxyl, Cl '-C6' for C2' and C4' hydroxyls). In the case of the /?-nitrobenzoic anhydride, the C4' and C15 products co-eluted on HPLC as indicated by 1H NMR. Two sets of diagnostic signals that don't overlap with any other signals, the CI '-H and methyl ketal C¾ (A), gave a ratio of 1 to 3. The minor constituent was acylated at C4', and the major constituent was acylated C15*.

Example 4: Determination of the Initial Rates

Acylation reactions were set up according to the general procedure described in Example 2. Aliquots were taken at multiple time points, quenched in a solution of piperidine in DMF, filtered, and analyzed by HPLC as described in the general procedure. The conversion was determined and plotted against time. The initial rate was then determined from the slope of a linear line fitted to the earliest time points.

Example 5: Control Experiments

Determining the Reversibility of Acylation.

Acylation reactions were setup according to the general procedure described in

Example 2. The mono-acylated products of the acylation reactions were purified by preparative HPLC. Each product was then resubmitted to acylating conditions:

Acylated product (1 eq) was added to a vial and dissolved in THF (450 μί). DIPEA (0.8 μί, 4.60 μιηοΐ, 1.2 eq) and DMAP (0.515 mg, 4.21 μιηοΐ, 1.1 eq) were added to the solution. The solution was allowed to stir at room temperature for 15 hrs. The reaction was then analyzed by reversed-phase HPLC (CI 8, MeCN/H ₂ 0).

In all cases the formation of 1 and other acylated products were not observed, confirming the reaction is irreversible.

Assessing the Extent of Background Acylation.

Acylation reactions for the para-substituted benzoyl chloride reagents were set up according to the general procedure described in Example 2 excluding the DMAP reagent:

1 (5 mg, 3.83 μιηοΐ, 1 eq) was added to a vial and dissolved in THF (450 μί). DIPEA (0.8 μί, 4.60 μιηοΐ, 1.2 eq) was added to the solution. The acyl chloride (1 eq) in THF (50 μί) was added to the reaction. The reaction was allowed to stir at room temperature for 15 hrs. The reaction was quenched in a solution of piperidine in DME and filtered. The reactions were then analyzed by reversed-phase HPLC (CI 8, MeCN/H ₂ 0). In all cases the conversion was less than 10%. Table 1. C2' Selectivity of acylation reactions

entry acyi donor (2) hpfc trace C2' selectivity Table 2. Site-divergent acylation enabled by electronic tuning of the acylpyridinium ion and its carboxylate counterion.

The electron-donating feri-butyl group leads to acylation at C4' as the major product, whereas the electron-withdrawing nitro group gives acylation at C15 as the major product. ^l H NMR insert in entry 2 depicts the ratio of 4b to 5b.