WO2023044365A1

WO2023044365A1 - Use of cyclodextrin to enhance solubility of substrates and increase enzymatic glycosylation reaction efficiency

Info

Publication number: WO2023044365A1
Application number: PCT/US2022/076459
Authority: WO
Inventors: Yasmin-Pei Kamal CHAU; Jacob Donald Stanley WIRTH; Sheng Ding; Jing-ke WENG
Original assignee: Doublerainbow Biosciences Inc
Current assignee: Doublerainbow Biosciences Inc
Priority date: 2021-09-17
Filing date: 2022-09-15
Publication date: 2023-03-23
Anticipated expiration: 2024-03-17

Abstract

Methods of glycosylating a substrate by using a uridine diphosphate glycosyltransferases (UGT) in combination with cyclodextrin are disclosed.

Description

USE OF CYCLODEXTRIN TO ENHANCE SOLUBILITY OF SUBSTRATES AND INCREASE ENZYMATIC GLYCOSYLATION REACTION EFFICIENCY

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/245,671, filed on September 17, 2021. The entire teachings of the above application are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: File name: 57671004001. xml; created September 9, 2022, 3,830 Bytes in size.

BACKGROUND

[0003] Many therapeutic molecules have low solubility in aqueous media, which presents challenges for enzymatic reactions involving those molecules.

SUMMARY

[0004] Synthesis of desirable small molecules by enzyme reaction systems have great potential to cheaply and efficiently produce unique small molecules of therapeutic or commercial interest. The low solubility of many enzyme reaction substrates is a barrier to efficient enzyme reactions. Including a solubilizing agent, particularly cyclodextrins, can increase the solubility of hydrophobic substrates in aqueous solutions resulting in increased enzyme reaction efficiency, thereby saving money and time. Cyclodextrins can be used to enhance the glycosylation of small molecules catalyzed by uridine diphosphate glycosyltransferase (UGTs) in purified enzyme reaction systems, immobilized enzyme reaction systems, or in vivo yeast or bacterial transformation reaction systems.

[0005] Described herein is a method of glycosylating a substrate. The method can include a) providing an aqueous reaction mixture; and b) allowing the reaction mixture to convert the substrate to a monosaccharide, a disaccharide, or an oligosaccharide of the substrate. The aqueous reaction mixture can include: i) a substrate; ii) uridine diphosphate glycosyltransferases (UGT); iii) uridine diphosphate-monosaccharide; and iv) a cyclodextrin. [0006] The uridine diphosphate-monosaccharide can be uridine diphosphate-glucose (“UDP -glucose”), uridine diphosphate-galactose (“UDP-galactose”), uridine diphosphatexylose (“UDP -xylose”), or uridine diphosphate-N-acetylglucosamine (“UDP-N- acetylglucosamine”).

[0007] The substrate can be ivacaftor, enasidenib, or etoposide.

[0008] The cyclodextrin can be alpha-cyclodextrin, gamma-cyclodextrin, hydroxypropyl- beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, sulfobutylether-beta-cyclodextrin.

[0009] The concentration of the cyclodextrin can be at least about 1 mM, or from about 1 mM to about 150 mM.

[0010] The UGT can be immobilized in an affinity column in the presence of a cyclodextrin in the reaction media.

[0011] Described herein is a method of glycosylating a substrate. The method can include a) providing an aqueous reaction mixture and b) allowing the reaction mixture to convert the substrate to a monosaccharide, a disaccharide, or an oligosaccharide of the substrate. The aqueous reaction mixture can include i) a substrate; ii) a yeast or bacteria that expresses a uridine diphosphate glycosyltransferases (UGT); iii) uridine diphosphatemonosaccharide; and iv) a cyclodextrin.

[0012] Described herein is an enzyme reaction medium that can include: a) a buffer that maintains the medium at a pH from 6.5 to 8.0; b) from about 40 mM to about 60 mM of a salt selected from KC1 and NaCl; c) from about 200 mM to about 500 mM sucrose; d) from about ImM to about 2 mM UDP-glucose; e) from about 1 pM to about 2 pM of a uridine diphosphate glycosyltransferases (UGT); and f) from about 1 mM to about 150 mM of a cyclodextrin.

[0013] The buffer can be 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) (e.g., approximately 50 mM of the HEPES). The enzyme reaction medium can further include a preservative, such as sodium azide (e.g., up to 0.02% (w/v) sodium azide). The enzyme reaction medium can further include a yeast or bacteria that expresses a uridine diphosphate glycosyltransferases (UGT).

[0014] Described herein is an enzyme reaction medium that can include a) a buffer that maintains the medium at a pH from 6.5 to 8.0; b) from about 40 mM to about 60 mM of a salt selected from KC1 and NaCl; c) from about 200 mM to about 500 mM sucrose; d) from about 1 pM to about2 pM of a uridine diphosphate glycosyltransferases (UGT); e) from about 0.1 pM to about 0.2 pM sucrose synthase; f) from about 0.5 mM to about 1 mM uridine diphosphate (UDP); g) from about 1 mM to about 150 mM of a cyclodextrin.

[0015] The buffer can be 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES)

(e.g., approximately 50 mM of the HEPES). The enzyme reaction medium can further include a preservative, such as sodium azide (e.g., up to 0.02% (w/v) sodium azide).

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0017] FIG. l is a bar graph showing that each drug tested shows different solubilities in different CDs in the enzyme reaction buffer, and these properties can only be determined through experimental screening. Error bars are the standard deviation of 3 replicates performed on separate days. CD: cyclodextrin, HP: hydroxypropyl, SBE: sulfobutylether. [0018] FIG. 2 is a bar graph showing that most drugs show a linear relationship between solubility and increasing CD concentration. Error bars are the range of two replicate assays performed on separate days. CD: cyclodextrin, HP: hydroxypropyl, SBE: sulfobutylether. [0019] FIG. 3 is a bar graph showing that an optimal concentration of hydroxypropyl- beta-cyclodextrin (HPBCD) improves the glycosylation of the small molecule ivacaftor in an in vivo transformation reaction with yeast cultures expressing a UGT. The % glycosylated is calculated by dividing the total sum of the peak areas of all glycosides by the peak area of the starting aglycone concentration. In this experiment, the glycosides quantified are ivacaftor monoglucoside and ivacaftor diglucoside.

[0020] FIG. 4 is a bar graph showing that HPBCD concentration affects the growth of yeast cultures expressing a UGT.

[0021] FIG. 5 is a bar graph showing that the presence of HPBCD promotes the addition of multiple sugar groups on the same substrate. The fold increase in ivacaftor triglucoside was calculated by dividing the triglucoside peak area at the indicated HPBCD concentration by the triglucoside peak area at 0 mM HPBCD. The amount of ivacaftor triglucoside in 0 mM HPBCD is set to 1. This data comes from an experiment similar to the one described above, with the following modifications: only 0-21.9 mM (0-3.2% w/v) HPBCD was tested, and samples were taken after 96 hours of incubation. [0022] FIG. 6 is a bar graph showing that including the cyclodextrin HPBCD in a purified enzyme batch glycosylation reaction results in increased glycosylation of etoposide while not negatively affecting the enzyme activity. Data shows representative results from one out of three replicate experiments. All three experiments showed the same general trends. Each reaction contained 0.1 mg/mL etoposide as the sugar acceptor. At this concentration, etoposide is soluble in 0, 10, and 50 mM HPBCD. The % glycosylated is calculated by dividing the total sum of the peak areas of all glycosides by the peak area of the starting aglycone concentration. In this experiment, the glycoside quantified is a single etoposide monoglucoside.

DETAILED DESCRIPTION

[0023] A description of example embodiments follows.

Cyclodextrins (CDs)

[0024] Cyclodextrins (CDs) are naturally-occurring, water-soluble cyclic oligosaccharides.^1-3 The most common CDs are composed of 6, 7, or 8 monosaccharide units called alpha-, beta-, and gamma-CDs, respectively. Other types of CDs contain chemical modifications on the monosaccharide units, such as addition of a methyl group, hydroxypropyl group, sugar group, sulfate group, or other specialized chemical modification. [0025] CDs form a toroidal three-dimensional structure with an external hydrophilic surface and an internal hydrophobic cavity. The cavity size and aqueous solubility of each CD depends on the composition of the monosaccharide units and any chemical modifications thereof. Hydrophobic molecules can insert into the hydrophobic cyclodextrin cavity to form inclusion complexes. The ability of a hydrophobic molecule to form a complex with a cyclodextrin is a function of compatibility in size, shape, and chemical interactions between the molecule and the cyclodextrin cavity.

[0026] CDs can interact with molecules of various sizes from small molecule therapeutics to peptides, lipids, and even proteins.¹’^{3 4} The most common application of CDs as solubility enhancers is in drug formulation development to enhance the bioavailability of pharmaceutical drugs.⁵

[0027] CDs can also enhance substrate solubility in enzyme reactions. Use of CDs as solubilizers in this context allows the enzyme reaction to proceed in a completely aqueous environment with minimal impact on enzyme activity. For example, glycosylation of lipids to form glycolipids in an aqueous buffer only occurred with the addition of gamma-cyclodextrin into the reaction buffer.⁶ Addition of hydroxypropyl-beta-cyclodextrin (HPBCD) enhanced the solubility of styrene oxide, which enhanced the initial reaction rate of hydrolysis by epoxide hydrolase.⁷ CDs have also been used to complex free fatty acids to enhance enzymatic reactions on those fatty acid substrates. One example of such a reaction is hydrolysis of diolein by Humicola lanuginosa lipase.⁸ Use of CDs to enhance substrate solubility to improve enzyme reactions has also been described in a patent for a droplet actuator device for enzyme assays to detect the cleavage of an umbelliferone substrate (Patent no. US 8,093,062 B2).

[0028] Improving the solubility of substrates with CDs can also enhance in vivo transformation reactions in yeast or bacterial cultures. For example, the addition of CDs to the fermentation broth improved the solubility and thus the availability of cholesterol, sitosterol, and delta4-cholesten-3-one for enzymatic cleavage by Mycobacterium sp. cultures.⁹

[0029] It is not obvious which CD will solubilize a molecule most efficiently. For example, different CDs solubilize different flavonoid molecules to different degrees.¹⁰ As another example, gamma-cyclodextrin enhanced the solubility of lipid substrates and allowed glycosylation of these lipid substrates in an aqueous enzyme reaction. However, alphacyclodextrin failed to solubilize the lipid substrates, and glycosylation in a fully aqueous enzyme reaction was not observed.⁶

[0030] It is difficult to predict how a particular CD will affect the enzyme reaction efficiency. For example, methylated alpha- and beta-CD complexes with L-tyrosine, the substrate for tyrosine phenol-lyase, thereby solubilizing L-tyrosine in an aqueous environment. However, instead of enhancing the enzymatic reaction with tyrosine phenollyase, this complex formation with CD prevents the reaction from occurring.¹¹ Furthermore, cyclodextrins can interact with enzymes and modulate enzyme activity. Methylated gammaCD binds tyrosine phenol-lyase, causing conformational changes in the enzyme and inhibiting its activity.¹¹ In another example, HPBCD inhibits the activity of E. coli methionine aminopeptidase by forming a bridging complex between the substrate and the enzyme active site.^{12 13}

[0031] Many other cyclodextrins are suitable, such as alpha-, beta-, or gammacyclodextrins substituted with one or more hydrophilic groups, such as monosaccharide (e.g., glucosyl, maltosyl), carboxyalkyl (e.g., carboxylmethyl, carboxyethyl), hydroxyalkyl- substituted (e.g., hydroxyethyl, 2-hydroxypropyl (sometimes referred to as simply hydroxypropyl)) and sulfoalkylether-substituted beta-cyclodextrin. Particularly suitable gamma- or beta-cyclodextrins include hydroxypropyl beta-cyclodextrin (HPBCD), hydroxypropyl gamma-cyclodextrin, and sulfobutylether beta-cyclodextrin (SBECD).¹⁸’ ¹⁹ [0032] Alpha-cyclodextrin and the following substituted alpha-cyclodextrins: 6-0- maltosyl-alpha-cyclodextrin, carboxymethyl-alpha-cyclodextrin, carboxyethyl-alpha- cyclodextrin, hydroxypropyl-alpha-cyclodextrin.

[0033] Substituted beta-cyclodextrins including: 6-O-glucosyl-beta-cyclodextrin, 6-O- maltosyl-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxyethyl-beta- cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin.

[0034] Gamma-cyclodextrin and the following substitute gamma-cyclodextrins: carboxymethyl-gamma-cyclodextrin, carboxyethyl-gamma-cyclodextrin, hydroxypropyl- gamma-cyclodextrin.

[0035] Particularly suitable cyclodextrins are hydroxypropyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, and sulfobutylether-beta-cyclodextrin.

[0036] Other cyclodextrins include randomly methylated cyclodextrin, succinyl-alpha- CD, succinyl-beta-CD, succinyl-hydroxypropyl-beta-CD, acetyl-beta-CD, triacetyl-beta-CD. [0037] Preferred cyclodextrins include alpha-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, and sulfobutylether- beta-cyclodextrin.

Glycosylation of Small Molecule-Based Therapeutics

[0038] A potential strategy for improving or modulating the efficacy, safety, and/or PK/PD profile of a small molecule therapeutics is modification by glycosylation. The small molecule, or aglycone, is modified by the addition of one or more sugar groups or chains of two or more sugar groups (called oligosaccharides) to nucleophilic centers of the aglycone. In general, glycosylation of a small molecule can lead to increased aqueous solubility, altered interactions with proteins and membranes, altered absorption and excretion, changes in metabolic stability, and other changes in PK/PD characteristics.¹⁴

[0039] Glycosyltransferases (GTs) are a class of enzyme with the potential to act as the catalyst for the generation of novel glycosylated therapeutic small molecules.¹⁵ GTs catalyze the transfer of a sugar from an activated sugar donor molecule to an acceptor molecule.¹⁵ As a class, GTs can glycosylate a wide variety of acceptor structures, with many GTs exhibiting promiscuity towards the sugar donor and acceptor. Uridine diphosphate GTs (UGTs) utilize uridine diphosphate (UDP) sugar donors, and form the largest group of Leloir GTs in plants.¹⁶

[0040] Examples of glycosylation of small molecule are disclosed in international patent applications PCT/US2021/022416, PCT/US2021/022410, and PCT/US2021/022414, which disclose glycosylation of ivacaftor, enasidenib, and etoposide, respectively.

[0041] While the sugar donor molecule is generally hydrophilic, the acceptor molecule for glycosylation may not be. In particular, many therapeutic small molecules are characterized by limited aqueous solubility. It is estimated that -40% of new chemical entities exhibit low aqueous solubility (solubility <10 pM or 5 pg/mL for a molecule with a molecular weight of 500).¹⁷ A significant limitation to glycosylation reaction efficiency in aqueous environments is the limited aqueous solubility of the acceptor molecule.

[0042] Cyclodextrins present an easy and inexpensive solution to solubilize higher concentrations of acceptor molecules into aqueous enzyme reaction solutions. This will allow for the production of sufficient quantities of glycosylated product for preclinical research and development, clinical trials, and industrial-scale production.

Enzyme Reaction Media (without sucrose synthase)

[0043] Media for conducting the enzymatic reaction typically include a buffer, a salt, a stabilizing agent, UDP -glucose, a uridine diphosphate glycosyltransferases (UGT), and a cyclodextrin.

[0044] The buffer maintains the reaction medium at a pH from about 6.5 to about 8.0. Many suitable buffers are known, including 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), Tris-HCl and phosphate buffer. In preferred embodiments, HEPES is used as a buffer, preferably at a concentration of about 50 mM.

[0045] Typically, the salt is sodium chloride (NaCl) or potassium chloride (KC1). In preferred embodiments, the salt is KC1. The salt can be present at a concentration from about 40 mM to about 60 mM.

[0046] The stabilizing agent can be sucrose, glucose, and/or glycerol. In preferred embodiments, the stabilizing agent is sucrose. The sucrose can be present at a concentration from about 200 mM to about 500 mM. Preferably, the sucrose is present at a concentration of about 300 mM. [0047] UDP-glucose is present at a concentration from about ImM to about 2 mM.

[0048] The UGT is present at a concentration from about 1 pM to about 2 pM.

[0049] The cyclodextrin is present at a concentration from about 1 mM to about 150 mM.

[0050] In some embodiments, enzyme reaction medium includes a preservative. In preferred embodiments, the preservative is sodium azide, preferably at a concentration up to about 0.02% (w/v), even more preferably at a concentration of about 0.01% (w/v).

Enzyme Reaction Media for Purified Enzyme or Immobilized Enzyme Reactions (with sucrose synthase)

[0051] Where the enzymatic reaction includes a sucrose synthase (SuSy), the reaction media includes a sucrose synthase (SuSy) and UDP, but does not need to include UDP- glucose. Typically, the reaction media includes a buffer, a salt, sucrose, a SuSy, UDP, a uridine diphosphate glycosyltransferases (UGT), and a cyclodextrin.

[0052] The buffer maintains the reaction medium at a pH from about 6.5 to about 8.0.

Many suitable buffers are known, including 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), Tris-HCl and phosphate buffer. In preferred embodiments, HEPES is used as a buffer, preferably at a concentration of about 50 mM.

[0053] Typically, the salt is sodium chloride (NaCl) or potassium chloride (KC1). In preferred embodiments, the salt is KC1. The salt can be present at a concentration from about 40 mM to about 60 mM.

[0054] The sucrose can be present at a concentration from about 200 mM to about 500 mM. Preferably, the sucrose is present at a concentration of about 300 mM.

[0055] The UGT is present at a concentration from about 1 pM to about 2 pM.

[0056] Sucrose synthase is present at a concentration from about 0.1 pM to about 0.2 pM.

[0057] UDP is present at a concentration from about 0.5 mM to about 1 mM.

[0058] The cyclodextrin is present at a concentration from about 1 mM to about 150 mM. In some embodiments, enzyme reaction medium includes a preservative. In preferred embodiments, the preservative is sodium azide, preferably at a concentration up to about 0.02%, even more preferably at a concentration of about 0.01%. EXEMPLIFICATION

Example 1

[0059] Determining which CD will solubilize the glycosylation acceptor molecule is not obvious and is determined experimentally. Five drugs (etoposide, enasidenib, ivacaftor, birabresib, paclitaxel) with molecular weights ranging from 392 to 854 g/mol were screened against five CDs (alpha-, gamma-, hydroxypropyl-gamma-, hydroxypropyl-beta-, sulfobutylether-beta-cyclodextrin). The solubility of each drug in the absence of a CD was also determined.

[0060] To a mixture comprised of UGT enzyme reaction buffer (50 mM HEPES pH 7.5, 50 mM KC1, 300 mM sucrose) and the indicated CD (10 mM, or approximately 2% w/v depending on the CD average molecular weight), drug from DMSO stocks was added to a final concentration of 2 mM. In all samples, the drug immediately precipitated. The samples were vortexed for 30 seconds at room temperature, then incubated for 1 hour at room temperature. Then, the samples were vortexed for 30 seconds at room temperature, centrifuged for 5 minutes at 3000 rpm and filtered through a 0.45 pm filter. Methanol was added to a final concentration of 50% v/v. Samples were analyzed by UV-HPLC (FIG. 1 and Table 1).

[0061] Each drug is soluble to different degrees with different CDs. Some drugs (e.g. etoposide) are soluble in all CDs tested. Some drugs (e.g. ivacaftor, paclitaxel) are soluble in only a subset of CDs. In all cases, at least one CD greatly improves the solubility of the drug compared to the absence of a CD.

Table 1: Data of FIG. 1.

Nd = not detected

0.000 = detected, but below the limit of quantitation

Example 2

[0062] After one or two promising CDs were identified for the glycosylation acceptor molecule, the solubility of the molecule in a range of CD concentrations was experimentally determined. Each of the five drugs were screened in a CD that resulted in high solubility according to Example 1. CD concentrations ranged from 1.56 mM to 50 mM (approximately 0.2-8% w/v depending on the CD average molecular weight).

[0063] To a mixture comprised of UGT reaction buffer (50 mM HEPES pH 7.5, 50 mM KC1, 300 mM sucrose) and the indicated CD (0, 1.56, 3.13, 6.25, 12.5, 25, or 50 mM), drug in DMSO stock was added to a final concentration of 2 mM. In all samples, the drug immediately precipitated. The samples were vortexed for 30 seconds at room temperature, then incubated for 1 hour at room temperature. Then, the samples were vortexed for 30 seconds at room temperature, centrifuged for 5 minutes at 3000 rpm and filtered through a 0.45 pm filter. Methanol was added to a final concentration of 50% v/v. Samples were analyzed by UV-HPLC (FIG. 2 and Table 2).

[0064] Most drugs show a linear relationship between increasing solubility and increasing CD concentration. However, this is not always the case. For example, birabresib sees no enhanced solubility at HP-beta-CD concentrations greater than 25 mM.

Table 2: Data FIG. 2.

Nd = not detected

0.000 = detected, but below the limit of quantitation

Example 3

[0065] While higher CD concentrations generally correspond to increased drug solubility, the higher CD concentrations may interfere with glycosylation activity by affecting other aspects of the reaction system, such as cell growth. To experimentally test this, the extent of glycosylation of the sugar acceptor molecule ivacaftor was measured in an in vivo yeast culture expressing the UGT DRB0752 (SEQ ID NO: 1) in the presence of a range of HPBCD concentrations. The OD600 of the yeast culture was measured over the course of the experiment.

[0066] HPBCD (0-55 mM or 0-8% w/v) was added to selective yeast media (lx yeast nitrogen base (BioWorld), 2% w/v glucose). Then, ivacaftor was diluted from a DMSO drug stock to a final concentration of 0.2 mg/mL. The media was briefly vortexed to mix, and incubated at 30°C while shaking at 200 rpm for 30 minutes to allow the CD-drug complex to form. An overnight yeast culture was diluted into the media to a starting OD600 of 0.1 to start the reaction. The reaction was incubated for the indicated lengths of time. At each time point, 100 pL of the reaction was diluted with 100 pL of methanol, vortexed, and filtered with a 0.45 pM filter. Samples were analyzed by UV-HPLC (FIG. 3). To experimentally test if the HPBCD concentration affects the growth of the yeast cultures, the optical density at 600 nm (OD600) of each yeast culture was measured at the 24 hour time point with a spectrophotometer (FIG. 4).

[0067] The reaction mixture was completely clear at HPBCD concentrations greater than or equal to 21.9 mM, indicating complete solubilization of ivacaftor. However, the glycosylation efficiency was not the highest at these high HPBCD concentrations.

Surprisingly, 2.7 mM HPBCD resulted in the highest percentage of glycosylated ivacaftor (FIG. 3). Furthermore, HPBCD concentrations greater than or equal to 21.9 mM begins to inhibit growth of the yeast culture (FIG. 4). Lower concentrations of HPBCD do not seem to affect yeast growth at these time scales. The decreased culture density at higher HPBCD concentrations may have contributed to the decreased glycosylation efficiency of ivacaftor at high HPBCD concentrations.

[0068] Additionally, it was observed that the presence of HPBCD in the yeast culture led to an increased accumulation of triglucosylated ivacaftor (mass confirmed by LC-MS;

[M+H]⁺ = 879.92 m/z) compared to cultures with no HPBCD (FIG. 5). This suggests that the increased substrate solubility or substrate or enzyme stability promoted by CDs may push the glycosylation reaction forward towards the addition of multiple sugars on the same substrate. Example 4

[0069] In order to test if the presence of a CD can increase glycosylation efficiency without negatively affecting UGT activity in a batch purified enzyme reaction, the glycosylation activity of the purified UGT DRB0458 (SEQ ID NO: 2) in the presence of no CD, a low concentration of CD (10 mM or 1.5% w/v), and a high concentration of CD (50 mM or 7.3% w/v) was compared. The sugar acceptor molecule was etoposide and the sugar donor molecule, UDP-glucose, was regenerated using the enzyme sucrose synthase and sucrose.

[0070] UDP (0.5 mM), HPBCD (0, 10, or 50 mM), and then etoposide (0.1 mg/mL) was added in the order stated to the UGT enzyme reaction buffer (50 mM HEPES pH 7.5, 50 mM KC1, 300 mM sucrose). The solution was vortexed until clear. At 0.1 mg/mL, etoposide is soluble in the buffer with no HPBCD and in buffer with 10 and 50 mM HPBCD. Once the solution was clear, the UGT (DRB0458 (SEQ ID NO: 2); 2 pM) and SuSy (0.2 pM) enzymes were added. The mixture was gently inverted to mix, then incubated at 30°C with a gentle, slow rotation to constantly mix the reaction. At each time point, a sample of the reaction was quenched by adding 1 : 1 ice cold methanol. Samples were analyzed by UV-HPLC (FIG. 6). [0071] Glycosylation occurred faster in reactions containing 10 and 50 mM HPBCD compared to reactions with no HPBCD, supporting the conclusion that the presence of a CD can increase glycosylation efficiency. Since the concentration of etoposide used in this experiment is already completely soluble in buffer without HPBCD and in buffer with 10 and 50 mM HPBCD, HPBCD likely promotes glycosylation by further stabilizing the etoposide in solution (precipitation is less likely to occur over time), stabilizing the UGT or SuSy enzymes, or stabilizing the enzyme-substrate complex.

[0072] Furthermore, the presence of 50 mM HPBCD did not reduce glycosylation efficiency compared to 10 mM HPBCD. This suggests that a 5-fold higher HPBCD concentration does not affect the glycosylation activity of the UGT DRB0458 (SEQ ID NO: 2) in a batch purified enzyme reaction. This is different from the results seen in Example 3 where higher CD concentrations led to a reduction in both glycosylation activity and yeast culture growth. REFERENCES

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INCORPORATION BY REFERENCE; EQUIVALENTS

[0092] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0093] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of glycosylating a substrate, the method comprising: a) providing an aqueous reaction mixture comprising: i) a substrate; ii) uridine diphosphate glycosyltransferases (UGT); iii) uridine diphosphate-monosaccharide; and iv) a cyclodextrin; and b) allowing the reaction mixture to convert the substrate to a monosaccharide, a disaccharide, or an oligosaccharide of the substrate.

2. The method of claim 1, wherein the uridine diphosphate-monosaccharide is uridine diphosphate-glucose (“UDP-glucose”) .

3. The method of claim 1, wherein the uridine diphosphate-monosaccharide is uridine diphosphate-galactose (“UDP-galactose”).

4. The method of claim 1, wherein the uridine diphosphate-monosaccharide is uridine diphosphate-xylose (“UDP -xylose”).

5. The method of claim 1, wherein the uridine diphosphate-monosaccharide is uridine diphosphate-N-acetylglucosamine (“UDP-N-acetylglucosamine”).

6. The method of claim 1, wherein the substrate is ivacaftor.

7. The method of claim 1, wherein the substrate is enasidenib.

8. The method of claim 1, wherein the substrate is etoposide.

9. The method of any one of claims 1 through 8, wherein the cyclodextrin is alphacyclodextrin.

10. The method of any one of claims 1 through 8, wherein the cyclodextrin is gammacyclodextrin. The method of any one of claims 1 through 8, wherein the cyclodextrin is hydroxypropyl-beta-cyclodextrin. The method of any one of claims 1 through 8, wherein the cyclodextrin is hydroxypropyl-gamma-cyclodextrin. The method of any one of claims 1 through 8, wherein the cyclodextrin is sulfobutylether-beta-cyclodextrin. The method of any one of claims 1 through 8, wherein the concentration of the cyclodextrin is at least about 1 mM. The method of any one of claims 1 through 8, wherein the concentration of the cyclodextrin is from about 1 mM to about 150 mM. The method of any one of claims 1 through 8, wherein the UGT is immobilized in an affinity column. A method of glycosylating a substrate, the method comprising: a) providing an aqueous reaction mixture comprising: i) a substrate; ii) a yeast or bacteria that expresses a uridine diphosphate glycosyltransferases (UGT); iii) uridine diphosphate-monosaccharide; and iv) a cyclodextrin; and b) allowing the reaction mixture to convert the substrate to a monosaccharide, a disaccharide, or an oligosaccharide of the substrate. An enzyme reaction medium comprising: a) a buffer that maintains the medium at a pH from 6.5 to 8.0; b) from about 40 mM to about 60 mM of a salt selected from KC1 and NaCl; c) from about 200 mM to about 500 mM sucrose; d) from about ImM to about 2 mM UDP-glucose; e) from about 1 pM to about 2 pM of a uridine diphosphate glycosyltransferases (UGT); f) from about 1 mM to about 150 mM of a cyclodextrin. The enzyme reaction medium of claim 18, wherein the buffer is 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES). The enzyme reaction medium of claim 19, wherein the enzyme reaction medium comprise approximately 50 mM of the HEPES. The enzyme reaction medium of any one of claims 18 through 20, further comprising a preservative. The enzyme reaction medium of claim 21, wherein the preservative is sodium azide. The enzyme reaction medium of claim 22, wherein the enzyme reaction buffer comprises up to 0.02% (w/v) sodium azide. An enzyme reaction medium comprising: a) a buffer that maintains the medium at a pH from 6.5 to 8.0; b) from about 40 mM to about 60 mM of a salt selected from KC1 and NaCl; c) from about 200 mM to about 500 mM sucrose; d) from about 1 pM to about2 pM of a uridine diphosphate glycosyltransferases (UGT); e) from about 0.1 pM to about 0.2 pM sucrose synthase; f) from about 0.5 mM to about 1 mM uridine diphosphate (UDP); and g) from about 1 mM to about 150 mM of a cyclodextrin. The enzyme reaction medium of claim 24, wherein the buffer is 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES). The enzyme reaction medium of claim 25, wherein the enzyme reaction medium comprise approximately 50 mM of the HEPES. The enzyme reaction medium of any one of claims 24 through 26, further comprising a preservative. The enzyme reaction medium of claim 27, wherein the preservative is sodium azide. The enzyme reaction medium of claim 28, wherein the enzyme reaction buffer comprises up to 0.02% (w/v) sodium azide.

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