copyright by mireya luna robles 2020

Copyright

by

Mireya Luna Robles

2020

The Thesis Committee for Mireya Luna Robles

Certifies that this is the approved version of the following Thesis:

Ketoreductases as Biocatalysts in the Synthesis of Chiral Diketides

APPROVED BY

SUPERVISING COMMITTEE:

Adrian Keatinge-Clay, Supervisor

Emily Que, Co-Supervisor


by

Mireya Luna Robles

Thesis

Presented to the Faculty of the Graduate School of

The University of Texas at Austin

in Partial Fulfillment

of the Requirements

for the Degree of

Master of Arts

The University of Texas at Austin

May 2020

iv

Acknowledgments

I am thankful for the places I have been, the people I have met, and the people

already in my life. I am lucky to have such wonderful supportive friends and family. My

inspiration has always been my two sisters Erika and Yuleni, who always inspire me to

thrive in uncertainty, to be brave when I am the most afraid and to work hard to

accomplish my goals. To my dear friends Angelica, Jovana, and Rubi, who gifted me

with days of laughter and solidarity I will forever be grateful. VL has never failed to

provide spontaneous and perfect adventures. Asia who has seen me at my worst and best

I can only pray for a thousand years more of love and support. I will always be in debt to

the people I met at UT who made my time here one to remember for years to come.

Abstract


Mireya Luna Robles, MA

The University of Texas at Austin, 2020

Supervisors: Adrian Keatinge-Clay, Emily Que

Ketoreductases (KRs) are capable of setting different combinations of

stereocenters at the α and β position, resulting in complex polyketides with multiple

chiral centers. In order to increase the stereocontrol of KRs a structure–activity

relationship study was performed using four diketides of various lengths. The

stereocontrol of KRs was increased with a longer hydrophobic chain or a pantetheine

handle.

v

vi

Table of Contents

List of Tables ................................................................................................................... viii

List of Figures .................................................................................................................... ix

INTRODUCTION ...................................................................................................................1

Polyketide Synthase ......................................................................................................1

Types of Ketoreductasess ..............................................................................................2

Previous In Vitro reductions with PKS Ketoreductases ................................................4

Results and Discussion ........................................................................................................6

Methods..............................................................................................................................11

Protein preparation ........................................................................................................11

Equipment and settings for characterization .................................................................11

Synthesis of 2 ................................................................................................................12

Characterization of 2 .....................................................................................................12

Synthesis and Characterization of 3 ..............................................................................14

KR reduction of 3 and 5 ................................................................................................17

Characterization of 3a, 3b, 3c, and 3d ..........................................................................17






KR reduction of 7 and 9 ................................................................................................44


vii

Synthesis and Charecterization of 9 .............................................................................52

Overlay of carbon NMRs between 80-70 ppm of the reductions of substrate 7 ..........56



Reference ...........................................................................................................................59

viii

List of Tables

Table 1: Percentage of each 2-Methyl-3-Hydroxy stereoisomer produced by the

reduction of 3, 5, 7, and 9 by KRs. .................................................................7

ix

List of Figures

Figure 1: Chemical structure of Erythromycin A containing 18 chiral centers .............1

Figure 2: Pro-S hydride from NADPH attacking Re or Si face of β-keto group

depending on which side the substrate enters the active sites ........................3

Figure 3: Reactions with KR and NADPH regeneration system to reduce

substrates with varies lengths of alkyl chains such as propionyl (3),

pentanoyl (5), and octanoyl (7) and a substrate linked to pantetheine (9) ......6

Figure 4: Chiral HPLC of KR reductions of substrate 7 ................................................9

Figure 5: Chiral HPLC of KR reductions of substrate 3 (* gem-methylated

substrate confirmed by LCMS). ....................................................................25

Figure 6: Chiral HPLC of KR reductions of substrate 5 ..............................................39

Figure 7: LCMS of 9 with a retention of 5.1 min and low-resolution mass of 391. ....54

Figure 8: LCMS of reduction of 9 with a retention of 5.5 min and low-resolution

mass of 393. ..................................................................................................54

Figure 9: LCMS of thioesterification of 9 with SNAC with a retention of 4.3 min

and low-resolution mass of 234. ...................................................................54

Figure 10: Chiral HPLC of KR reductions of substrate 9 (* gem-methylated product

confirmed by LCMS). ...................................................................................55

Figure 11: Overlay of carbon NMRs between 80-70 ppm of the reductions of

substrate 7. ....................................................................................................56


substrate 5.. ...................................................................................................57


substrate 3. ....................................................................................................58

1

Introduction

Polyketides Synthases

Polyketides are secondary metabolites with bioactivities including

anticancer (doxorubicin), antibacterial (erythromycin A), antifungal (amphotericin B),

antiparasitic (ivermectins), and cholesterol-lowering (mevastatin) properties (Buss and

Waigh 1995; Walsh 2003). These stereochemically diverse compounds are synthesized

by multimodular polyketide synthases (PKSs). PKSs contain several modules that consist

of an acyltransferase (AT) which selects an extender unit, an acyl carrier protein (ACP)

which shuttles the growing polyketide through the assembly line via a

phosphopantetheinyl arm, a ketosynthase (KS) which condenses the extender unit unto

the growing polyketide chain and optionally contains, a ketoreductase that reduces the β-

keto group, a dehydratase (DH) that eliminates the β-hydroxyl group to form an olefin, an

enoyl reductase (ER) which reduces the olefin, and a thioesterase (TE) catalyzes an

intramolecular cyclization of the final polyketide product (Zheng et al., 2013; Piasecki et

al., 2011; Dodge et al., 2018) . The KR sets the stereochemistry at the β and α-positions

resulting in one of four stereochemical combinations with either D- or L- orientations.

These reductions set the majority of stereocenters found in complex polyketide structures

like Erythromycin A (Figure 1).

Figure 1: Chemical structure of Erythromycin A containing 18 chiral centers.

2

Erythromycin A is a macrolide antibiotic consisting of 18 chiral centers, a 14

membered lactone ring, and 2 sugar moieties. Erythromycin is used as treatment against

infections of the skin, soft tissue, respiratory tract, and the urinary tract (Kaneko et al.,

2007). Although erythromycin synthase is one of the most well studied PKS systems,

over 50 years of research has been conducted to improve production and purity at the

fermentation stage (Adrio et al., 2006). Large scale production of erythromycin A in

industry is done by the organism Saccharopolyspora erythraea which converts seven

molecules of propionate into one molecule of 6-deoxyerythronolide B (6-dEB). The first

attempt of the total synthesis of erythromycin resulted in 45 step synthesis which relied

on chiral auxiliaries and metals that are both expensive and environmentally harmful

(Woodward et al., 1981). Polyketides are desirable natural products, but their large-scale

syntheses often are very challenging to synthetic chemists. For example, the shortest total

synthesis of 6-dEB is 14 steps with a yield of 5% (Gao et al., 2013)

Types of Ketoreductases

Understanding how KR control stereospecificity in polyketides will help in

engineering PKSs to produce medically relevant molecules. There are 3 different types of

ketoreductases: type A, type B, and type C. Type A KRs reduce the β-keto group to yield

an L-hydroxyl group, Type B yields D-hydroxyl group, and type C is unable to reduce

the β-keto group (Yin et al., 2001; Moretto et al., 2017). Deuterated NADPH studies have

shown that the pro-S hydride is used by both A- and B-type KRs, signifying that hydride

attacks the re or si face of a substrate by entering the active site from opposite sides

(Reid et al., 2003; Caffrey et al., 2003).Type A KRs contain a conserved tryptophan

motif which guides the β-ketothioester substrate bound to the phosphopantetheinyl arm

3

behind the lid helix into the active site groove from the left side. In contrast, B-type KRs

contain an LDD motif that blocks the phosphopantetheinyl arm from slipping behind the

lid helix causing the substrate to enter the active site from the right side.

Figure 2: Pro-S hydride from NADPH attacking re or si face of β-keto group depending

on which side the substrate enters the active site.

KRs are able to epimerize substrates that have an α-substituent resulting in

subcategories for the A- and B-type KRs adding a 1 if no epimerization occurs and 2 if

the α-group is epimerized. The polyketide in A1-type KRs enter the active site from the

left where a glutamine or leucine interacts with the α-group to yield a 2D, 3L product.

For A2-type KRs a base deprotonates the α-proton which tautomerizes to give the

epimerized polyketide to yield a 2L, 3L product. In B1- type KRs, the unepimerized

polyketide enters the active site from the right to give the 2D, 3D product. For B2-type

KRs it is speculated that a tyrosine residue deprotonates the acidic α-proton (Keatinge-

Clay, 2007). Next, the lid helix along with a leucine or a glutamine selects the epimerized

polyketide to give the 2L, 3D product.

4

Previous In Vitro reductions with PKS Ketoreductases

Typically, in vitro experiments with KRs use N-acetylcysteamine (SNAC)

thioester analogs instead of the acyl carrier protein’s (ACP) phosphopantetheinyl arm

which naturally carries the growing polyketide into the correct side of the KR. Our goal is

to understand how KRs mediate stereocontrol in polyketides. One approach used in

manipulating the stereospecificity of KR is to mutate residues within the domain (Baerga-

Ortiz et al., 2006; Zheng et al., 2010; Kushnir et al., 2012). For example, EryKR2

(redefined modules, Zhang et al., 2017) was converted from a B2 KR into a A2 KR

through double mutations (Baerga-Ortiz et al., 2006), and AmpKR3 and EryKR7 (both

type A1) were converted into A2 KRs (Zheng et al., 2010; Siskos et al., 2005). However,

in vivo when the same alterations are made on the intact polyketide synthase the

stereochemical outcome is not the same signifying that additional factors contribute to the

stereospecificity of KRs (Kwan et al., 2011). Other studies vary the handle region of the

substrate by using ethanethiol or oxoesters and increasing the length of the α-substituents

(Bailey et al., 2016; Häckh et al., 2013; Piasecki et al., 2011). These studies and previous

studies done with (2RS)-methyl-3-oxopentanoyl-SNAC substrates used isolated KR

domains which gave the anticipated reduced product as the major product (Siskos et al.,

2005; Holzbaur et al., 1999). Isolated KRs that naturally reduce smaller intermediates

such as TylKR2, PikKR2, and AmpKR3 are more active towards diketide-SNACs

compare to KRs that reduce larger polyketide intermediates. To solve this problem, a

longer handle might allow for a greater level of stereocontrol than the SNAC-linked

diketides because the KRs gain binding energy from the interactions with a longer

handle.

Only two studies have used pantetheine linked substrates and in these studies

EryKR2 and AmpKR3 were used to reduce 2-methyl-3-oxopentanoyl-S-pantetheine

5

which resulted in identical stereochemical product distributions for the EryKR1

reduction, but for the AmpKR3 reduction the pantetheine linked substrates resulted in an

increase of A1 products compared to the SNAC-linked substrates (Kwan et al., 2011; Liu

et al., 2018). We hypothesize that the alkane chain in native diketides might have

interactions with residues in the KRs active site resulting in the hydrophobic chain

maximizing the van der Waals forces to stabilize the contact. Thus, the activity of KRs

that naturally reduce longer chains should be increased with longer chains. The goal is to

explore the relationship between the α- and γ- region of the substrate and the

stereochemical outcome. To accomplish this goal, we synthesized compounds 3, 5,7 and

9 and reduced them with 6 KRs then characterized the corresponding chiral products

using chiral HPLC, NMR, and mass spectroscopy.

6

Results and Discussion

Six enzymes: PicKR2 (B2-type), MycKR6 (A-type), TylKR2 (B1-type),

AmpKR3 (A1-type), EryKR3 (A1-type), and EryKR7(A1-type) were selected to reduce

3, 5, 7, and 9 in 10% DMSO and analyzed by chiral HPLC chromatography (Table 1).

Figure 3. Reactions with KR and NADPH regeneration system to reduce substrates with

varies lengths of alkyl chains such as propionyl (3), pentanoyl (5), and octanoyl (7) and a

substrate linked to pantetheine (9).

The elution order is assumed to be the same as previous studies in which B1

eludes first followed by A2, then A1 and lastly B2 (Piasecki et al., 2011; Siskos et al.,

2005). The hypothesis was that a pantetheine handle would make more extensive

interactions with the KR active site as seen by the crystal structure of AmpKR3

complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine to guide the

pantetheine-bound polyketide intermediate to enter the active site from the correct side

(Liu et al., 2018). Substrate 9 was synthesized through thioesterification of compound 3

with pantetheine under basic conditions. Flash chromatography with 5%

methanol/chloroform was used to obtain pure compound 9. The next reaction is a KR

biocatalytic reduction powered by a NADPH-regeneration system. The substrate is added

7

to a mixture of the potassium phosphate buffer (pH 7.7), KR, glucose dehydrogenase

(GDH), glucose, sodium chloride, and NADP+. GDH helps drive the reduction of the

diketides by generating NADPH from NADP+ as glucose is oxidized to gluconolactone.

The reduction reaction is driven toward completion by having a large concentration of

glucose to establish a large NADPH:NADP+ ratio (Piasecki et al., 2011). Enzymes are

added last to the buffered aqueous solution and allowed to react with the substrate

overnight. A second thioesterification reaction of 9 with SNAC is completed in order to

compare the elution order and stereochemical outcome to 3. All the reactions were

repeated, resulting in the averages of the stereoisomers percentages to be reported with no

deviation larger than 4%.

Table 1. Percentage of each 2-Methyl-3-Hydroxy stereoisomer produced by the

reduction of 3, 5, 7, and 9 by KRs.

8

PikKR2 generated B2 product exclusively for all substrates. TylKR2 and

AmpKR3 increased their stereocontrol by 4% and 6% and generated less of A2 product

than when reducing 3. MycKR6 and EryKR3 mostly generated A2 product when

reducing 3 and 9, but the longer pantetheine handle resulted in 10% and 18% increase of

A2 product compared to 3. EryKR7 largely retained stereocontrol but generated more B2

and B1 products than when reducing 3. This data suggests the pantetheine handle has

influence on the stereochemical outcome. However, there is still room for improvement

since TylKR2, AmpKR3, and MycKR6 did not exclusively make one product. Also, both

EryKR3 and EryKR7 did not make the expected A1 product and had poor stereocontrol.

This might indicate that some KRs require a longer handle to maintain their native

stereoselectivity thus ACP-bound substrate might be needed (Castonguay et al., 2007).

We decided to extend the interactions on the γ-position to create a hydrophobic pocket.

The alkyl chain at the γ-position was extended to give a five-carbon chain, and an

eight-carbon chain by acylating Meldrum's acid with pentanoyl chloride and octanoyl

chloride. The acidity of carbon 5 of Meldrum’s acid allows for deprotonation and easy

acylation at this position. The acylated product can be manipulated to produce thioester

compounds because Meldrum’s acid undergoes a pericyclic reaction through a

decarboxylation and release of acetone to form a ketene. The resulting ketene is very

electrophilic and can undergo an addition reaction with SNAC to give 4 and 6 (Dumas et

al., 2010). The α-position of 4 and 6 is methylated with iodomethane to give 5 and 7. This

reaction gives the monomethylated substrate as the major product and the gem-

methylated as the minor product which were both observed with LCMS and HPLC. The

gem-methylated product of 5 and 7 eluted before the B1 product which can be purified

easily. However, the gem-methylated product of 3 eluted between B1 and A2 product

(Figure 4 and 10).

9

The reduction of 5 resulted in better stereospecificity with TylKR2, AmpKR3,

and EryKR3 compared to 3 and 9 making 99% of B1 product and A1 product, and 91%

of A2 product, respectively. MycKR6 is a robust, A-type KR and one of the most active

and stereocontrolled KRs in these studies, which is surprising since this enzyme naturally

operates on α-unsubstituted intermediates, however with the pentanoyl chain MycKR6

slightly favors A1 product over A2. Similarly, EryKR7 makes slightly more A1 product

than A2 but it also produces B2 product. KRs from later modules (EryKR3 and EryKR7)

that reduce longer polyketide intermediates are not as stereoselective, which might

indicate a longer chain is required (Siskos et al., 2005; Holzbaur et al., 2001).

Figure 4. Chiral HPLC of KR reductions of substrate 7.

The SNAC-linked substrates and the pantetheine-linked substrate resulted in good

stereospecificity on KRs that reduce small diketides but struggle with KRs that reduce

larger polyketides within their native PKS. However, when the longer substrate 7 was

10

reduced all KRs showed good stereocontrol producing mostly one stereoisomer (Figure

3). TylKR2, AmpKR3, and EryKR7 had 98% ee, MycKR6 had 96% de, and EryKR3 had

90% de. Remarkably, the longer octanoyl chain made 99% of A1 when reduced with

EryKR7, which is 24% better than 3, and 33% better than 9. To further support our

findings, carbon NMRs of the β-hydroxy carbon was taken to show the relative

diastereomer produced. Carbon NMRs between 80-70 ppm show the peak from the

carbon bonded to the β-hydroxy, thus cis vs trans diastereomers can be distinguished

(Figure 10 and 11). The trans enantiomers appear more downfield at 74 ppm while the cis

enantiomers appear upfield at 72 ppm. Interestingly, only AmpKR3 made 99% of A1

product for both 5 and 7 while EryKR3, and EryKR7 favored producing A2 product. It is

hypothesized that the A2 stereoisomer is produced through a default, low-energy

pathway, which can be rationalized through the Felkin–Anh model (Mengel et al., 1999;

Bailey et al., 2016). A2 is favored over the A1 product because the hydride from NADPH

attacks the β-carbonyl to minimize torsional and steric strain.

In summary, we employed four diketides to elucidate the interactions utilized by

KRs to set chiral centers. The hydrophobic pocket created by the longer alkyl chain made

important contacts between the substrate and the KRs which increased stereocontrol. This

result demonstrates that the hydrophobic chain is more important for stereocontrol than

the pantetheine handle.

11

Methods

Protein preparation

The expression plasmids for MycKR6, TylKR2, EryKR3, EryKR7, PikKR2,

AmpKR3 and GDH as well as the purification of GDH have been previously described

(Piasecki et al., 2011; Siskos et al., 2005). After growing 6 L of transformed E. coli

BL21(DE3) in LB supplemented with 50 mg/L kanamycin to OD600=0.6, cells were

harvested through centrifugation (4000 x g, 10 min). They were then resuspended in 100

mL lysis buffer [30 mM HEPES, 500 mM NaCl, 5% (v/v) glycerol, pH 7.0], sonicated,

and centrifuged (30,000 x g, 30 min) to obtain the cell lysate which was stored at -80 °C.

To purify GDH, nickel affinity chromatography was performed using HisPur Ni-NTA

resin (Thermo Scientific, Waltham, MA). For GDH, the column was washed with 10 cv

of 15 mM imidazole in lysis buffer and eluted with 1 cv 150 mM imidazole in lysis

buffer to obtain pure GDH (10mg/mL).

Equipment and settings for characterization

NMR spectra were recorded on Varian DirectDrive 400 MHz spectrometers, or

Bruker AVANCE III 500. High-performance liquid chromatography (HPLC) analysis

was performed with Beckman System Gold 125 Solvent Module with a 166 Detector

equipped with Daicel Chiralcel OC-H column, 4.6 mm x 25 cm, 5 µm, flow rate 0.8

mL/min, 7% ethanol/hexane, detection at 230 nm, 25 °C. Mass spectra were recorded on

Agilent Technologies 6530 Accurate-Mass Q-TOF, Direct Inject, Jet Stream Ion Source

ESI, in positive/negative mode or Agilent 6120 Quadrupole ESI instrument in positive

mode; ZORBAX Eclipse Plus 95 Å C18 column, 2.1 mm × 50 mm, 5μm; column

temperature of 40°C; flow rate of 0.7 mL/min; gradient from 0 to 12 min of 5 to 100%

methanol and from 12 to 15 min of 100% methanol).

12

Synthesis of 2

Meldrum’s acid (4 g, 27.7 mmol) was dissolved in 45 mL dry DCM at 0 °C, and

pyridine (4.4 mL, 56 mmol), and propionyl chloride (2.4 mL, 28mmol) was then added

dropwise. The dark orange reaction was allowed to warm to rt and stirred overnight. The

reaction was washed with 6 x 30 mL 0.1 M HCl to remove pyridine. The organic layer

was dried over Na2SO4 and concentrated under reduced pressure. Crude 1 (4.2 g, 76.0%)

was used directly in the next step.

1 (4.2 g, 50 mmol) was dissolved in 38 mL dry toluene, and N-acetylcysteamine

(NAC, 2.4 g, 30 mmol) was added. The reaction was refluxed at 115 °C. After 5h the

reaction was then cooled down to room temperature, and the solvent was removed

through reduced pressure. Purification of the residue through CuSO4-impregnated silica

gel gave 2 (2.7 g, 61%) as an orange solid. Column conditions used for half of the crude

product: 6 x 11 cm; 200mL (2:1 hexanes: EtOAc), 200 mL (1:1 hexanes: EtOAc),

followed by pure EtOAc until all of the product was eluted.

Characterization of 2

1H NMR of 2 (400 MHz, CDCl3) δ 5.97 (s, 1H), 3.69 (s, 2H), 3.45 (q, J = 6.1 Hz,

2H), 3.08 (td, J = 6.3, 3.2 Hz, 2H), 2.56 (q, J = 7.2 Hz, 2H), 1.96 (d, J = 2.7 Hz, 3H),

1.10 (dt, J = 22.0, 7.4 Hz, 3H).

13

13C NMR of 2 (400 MHz, CDCl3) δ 202.61, 192.40, 170.45, 56.91, 39.17, 36.73,

29.20, 23.19, 7.48.

14

HRESIMS of 2 m/z 218.0849 [M+H]+ (218.0845 calculated for C9H15NO3S).

Synthesis of 3

2 (4.0 g, 18.4 mmol) and tBuOK (1.96 g, 17.4 mmol) was added to 80mL dry

THF at 0 °C for 45 min. Iodomethane (5.74 mL, 92 mmol) was added and then the

solution was allowed to warm to room temperature. The light orange solution was stirred

overnight. The reaction was quenched with 0.1 M HCl (150 mL) and extracted with

ethyl acetate (3 × 150 mL). The organic layer was dried over NaSO4, filtered, and the

solvent was removed through reduced pressure to give the 3 as a light-yellow oil (2.9 g,

68%).

15

1H NMR of 3 (500 MHz, CDCl3) δ 6.3 (s, broad, 1H), 3.8 (q, 1H), 3.4 (m, 2H),

3.1 (m, 2H), 2.5 (m, 2H), 1.9 (s, 3H), 1.4 (d, J = 8.0 Hz, 3H), 1.04 (t, J = 8.0 Hz, 3H).

16

13C NMR of 3 (500 MHz, CDCl3) δ 205.66, 197.10, 171.35, 61.28, 39.44, 35.05, 29.21,

23.35, 14.01,8.02.

HRESIMS of 3 m/z 254.0826 [M+Na]+ (254.0821 calculated for C10H17NO3SNa).

17

KR reductions of 3 and 5

3 (or 5) (200 mg, 0.87 mmol) was combined with 16 mL water, 18 mL 1 M

sodium phosphate buffer (pH 7.7), 1.2 mL 5 M NaCl solution, 10 mL 2 M d-glucose, 40

μL 150 mM NADP+, 200 μL 10 mg/mL GDH, and 3 mL of KR lysate. The reaction was

stirred at 22 °C overnight. To prevent emulsification, heat was applied to denature the

enzymes and separated by centrifugation. After that, the reaction was extracted with

EtOAc, and dried over Na2SO4. The solvent was removed by reduced pressure and

purified by flash column chromatography (10% EtOAc/Hexane) to give a yellow oil 3a,

3b, 3c, 3d, 5a, 5b, 5c, or 5d (~100 mg, 65%).

Characterization of 3a, 3b, 3c, and 3d

18

1H NMR of 3a (500 MHz, CDCl3). δ 5.95 (s, broad, 1H), 3.65 (m, 1H), 3.44 (m, 2H),

3.03 (m, 2H), 2.76 (m, 2H), 1.95 (s, 3H), 1.51, (m, 2H),1.21 (d, J = 8.0 Hz, 3H), 0.98 (t, J

= 7.5 Hz, 3H).

19

13C NMR of 3a (500 MHz, CDCl3) δ 204.26, 170.61, 75.07, 53.93, 39.44, 28.71, 27.76,

23.33, 15.20, 9.86.

20

1H NMR of 3b (500 MHz, CDCl3). δ 5.92 (s, broad, 1H), 3.83 (m, 1H, minor), 3.65 (m,

1H, major), 3.45 (m, 2H), 3.03 (m, 2H), 2.75 (m, 2H), 1.96 (s, 3H), 1.51 (m, 2H), 1.21 (d,

J = 8.0 Hz, 3H), 0.97 (t, J = 7.5 Hz, 3H).

21

13C NMR of 3b (500 MHz, CDCl3) δ 204.27, 170.58, 75.24 (major), 73.76 (minor),

53.11, 39.55, 28.71, 27.25, 23.33, 15.21, 9.87.

22

1H NMR of 3c (500 MHz, CDCl3). δ 5.95 (s, broad, 1H), 3.83 (m, 1H), 3.43 (m, 2H),


= 7.5 Hz, 3H).

23

13C NMR of 3c (500 MHz, CDCl3) δ 204.28, 170.63, 75.22 (minor), 73.76 (major),

53.11, 39.51, 28.69, 27.61, 23.30, 15.19, 9.85.

24

1H NMR of 3d (500 MHz, CDCl3). δ 56.17 (s, broad, 1H), 3.81 (m, 1H), 3.41 (m, 2H),


= 7.5 Hz, 3H).

13C NMR of 3d (500 MHz, CDCl3) δ 204.09, 170.70, 73.75, 53.15, 39.43, 28.88, 27.26,

23.23, 14.27, 10.51.

25

Figure 5. Chiral HPLC of KR reductions of substrate 3 (* gem-methylated substrate

confirmed by LCMS).

26

Synthesis of 4

The synthesis for 2 was followed with the exception that pentanoyl chloride (3.3

ml, 27.8 mmol, 1 eq.) was substituted for propionyl chloride. Flash chromatography was

performed in the same manner, yielding 5 g (73%) of the desired product.

1H NMR of 4 (500 MHz, CDCl3) δ 5.97 (s, broad, 1H), 3.7 (s, 2H), 3.45 (t, J= 6.1 Hz,

2H), 3.1 (m, 2H), 2.5 (t, J= 7.4 Hz 2H), 1.97 (s, 3H), 1.57 (m, 2H), 1.3 (m, 2H), 0.91 (t,

J=6.5 Hz, 3H).

27

13C NMR of 4 (500 MHz, CDCl3) δ 200.22, 196.32, 170.22, 57.06, 43.06, 39.86, 29.10,

25.37, 23.09, 23.03, 13.67.


28

Synthesis of 5

The synthesis for 3 was followed with the exception that 4 (2 g, 8.15 mmol, 1 eq.)

was substituted for 2. Flash chromatography was performed using gradient 33%-100

ethyl acetate hexane, yielding 1.3 g (62%) of the desired product 5.

1H NMR of 5 (500 MHz, CDCl3) δ 6.15 (s, broad, 1H), 3.76 (q, 1H), 3.4 (m, 2H), 3.04

(m, 2H), 2.5 (m, 2H), 1.95 (s, 3H), 1.52 (m, 2H), 1.4 (m, 2H), 1.37 (d, J= 7.1 Hz, 3H),

0.87 (t, J= 7.0 Hz, 3H).

29

13C NMR of 5 (500 MHz, CDCl3) δ 205.16, 196.98, 170.60, 61.07, 41.34, 39.30, 28.80,

25.56, 23.10, 22.13, 13.80, 13.60.

30


Characterization of 5a, 5b, 5c, & 5d

HRESIMS of reduced compound 5 m/z 284.1296 [M+Na] + (284.1291 calculated for

C12H23NO3SNa).

31

1H NMR of 5a (500 MHz, CDCl3) δ 6.00 (s, broad, 1H), 3.7 (m, 1H), 3.43 (m, 2H), 3.04


0.88 (t, J= 7.0 Hz, 3H).

32


28.70, 23.29, 22.71, 15.18, 14.14.

33

1H NMR of 5b (500 MHz, CDCl3) δ 5.85 (s, broad, 1H), 3.92 (m, 1H), 3.69 (m, 1H), 3.45

(m, 2H), 3.02 (m, 2H), 2.42 (m, 2H), 1.98 (s, 3H), 1.43 (m, 2H), 1.30 (m, 2H), 1.21 (d, J=

7.1 Hz, 3H), 0.93 (t, J= 7.0 Hz, 3H).

34

13C NMR of 5b (500 MHz, CDCl3) δ 204.41, 170.58, 74.02, 72.97, 53.40, 39.58, 34.65,

28.72, 28.29, 23.46, 22.7, 14.16, 11.20.

35

1H NMR of 5c (500 MHz, CDCl3) δ 5.95 (s, broad, 1H), 3.91(m, 1H), 3.44 (m, 2H), 3.02


0.89 (t, J= 7.0 Hz, 3H).

36

13C NMR of 5c (500 MHz, CDCl3) δ 204.33, 170.64, 72.26, 53.42, 41.45, 39.51, 34.11,

28.68, 23.28, 22.38,14.14, 11.19.

37

1H NMR of 5d (500 MHz, CDCl3) δ 5.87 (s, broad, 1H), 3.92(m, 1H), 3.45 (m, 2H), 3.03


0.9 (t, J= 7.0 Hz, 3H).

38


28.29, 23.35, 22.72, 14.16, 11.20.

39

Figure 6. Chiral HPLC of KR reductions of substrate 5.

40

Synthesis of 6

The synthesis for 3 was followed with the exception that Octanoyl chloride (7.5

ml, 43.8 mmol, 1 eq.) was substituted for propionyl chloride. Flash chromatography was

performed in the same manner, yielding 6 g (60%) of the desired product.

1H NMR of 6 (500 MHz, CDCl3) δ 5.9 (s, broad, 1H), 3.7 (s, 2H), 3.47 (q, J = 6.4 Hz,

2H), 3.09 (t, J = 5.9 Hz, 2H), 2.52 (q, J = 7.4 Hz, 2H), 1.97 (s, 3H), 1.58 (t, J = 6.9 Hz,

2H), 1.34 – 1.21 (m, 8H), 0.88 (t, J = 6.9 Hz, 3H).

41

13C NMR of 7 (500 MHz, CDCl3) δ 202.24, 194.20, 177.62, 57.06, 43.31, 39.12, 34.80,

31.53, 29.13, 28.87, 27.73, 23.34, 22.53, 13.94.

HRESIMS of 6 m/z 310.1450 [M+Na]+ (310.1447 calculated for C14H25O3SNa).

42

Syntheses of 7

The synthesis for 3 was followed with the exception that 6 (4 g, 13.9 mmol, 1 eq.)

was substituted for 2. Flash chromatography was performed using gradient 25%-100

ethyl acetate hexane, yielding 3.03 g (72%) of the desired product.

1H NMR of 7 (500 MHz, CDCl3) δ 6.07 (s, broad, 1H), 3.78 (q, 1H), 3.45 (m, 2H), 3.06

(m, 2H), 2.48 (m, 2H), 1.98 (s, 3H), 1.57 (dd, 2H), 1.4 (q, 3H), 1.27 (m, 8H), 0.86 (t, J =

7.0 Hz, 3H).

43

13C NMR of 7 (500 MHz, CDCl3) δ 205.53, 197.40, 171.04, 61.48, 42.06, 39.76, 38.43,

32.03, 32.01, 29.49, 29.40, 23.90, 22.98, 22.63, 14.44.

HRESIMS of 7 m/z 324.1609 [M+Na]+ (324.1604 calculated for C15H27O3SNa).

44

KR reductions of 7 and 9

7 (or 9) (200 mg, 0.66 mmol) was mixed in 1 mL DMSO (2 %v/v) and combined

with 16 mL water, 18 mL 1 M sodium phosphate buffer (pH 7.7), 1.2 mL 5 M NaCl

solution, 10 mL 2 M d-glucose, 40 μL 150 mM NADP+, 200 μL 10 mg/mL GDH, and 3

mL of KR lysate. The reaction was stirred at 22 °C overnight. To prevent emulsification,

heat was applied to denature the enzymes and separated by centrifugation. For 9a, 9b, 9c,

or 9d the solution pH was increased to 8.5 and SNAC (200 mg) was added dropwise and

stirred for 2 hrs. After that, the reaction was extracted with EtOAc, and dried over

Na2SO4. The solvent was removed by reduced pressure and purified by flash column

chromatography (10-50% EtOAc/Hexane) to give a yellow oil 7a, 7b, 7c, 7d, 9a, 9b, 9c,

or 9d (~70 mg, 35%).

Characterization of 7a, 7b, 7c, and 7d

HRESIMS of reduced compound 7 m/z 326.1772 [M+Na]+ (326.1760 calculated for

C15H29NO3SNa).

45

1H NMR of 7a (500 MHz, CDCl3) δ 5.83 (s, broad, 1H), 3.71 (m, J= 8.0 Hz, 1H), 3.46

(m, 2H), 3.04 (m, 2H), 2.75 (q, J= 7.0 Hz, 1H), 1.98 (m, 2H), 1.96 (s, 3H), 1.50 (m, 2H),

1.27 (m, 8H), 1.22 ( d = 7.0 Hz, 3H) 0.88 (t, J = 6.8 Hz, 3H).

46


31.68, 29.38, 28.47, 25.31, 23.10, 22.48, 15.03, 14.79.

47

1H NMR of 7b (500 MHz, CDCl3) δ 5.83 (s, broad, 1H), 3.70 (m, 1H), 3.50 (m, 2H), 3.05

(m, 2H), 2.79 (q, J= 7.0 Hz, 1H), 2.01 (m, 2H), 1.96 (s, 3H), 1.53 (m, 2H), 1.29 (m, 8H),

1.25 ( d = 7.0 Hz, 3H) 0.87 (t, J = 6.8 Hz, 3H).

13C NMR of 7b (500 MHz, CDCl3) δ 204.36, 170.87, 74.05, 54.31, 39.57, 34.01, 31.94,

29.84, 29.64, 29.38, 25.57, 23.38, 22.78, 15.04, 14.24.

48

1H NMR of 7c (500 MHz, CDCl3) δ 5.92 (s, broad, 1H), 3.91 (m, 1H), 3.44 (m, 2H), 3.02


1.20 ( d = 7.0 Hz, 3H) 0.87 (t, J = 6.8 Hz, 3H).

49

13C NMR of 7c (500 MHz, CDCl3) δ 204.32, 170.59, 72.28, 53.43, 41.10, 34.32, 31.91,

29.60, 29.36, 28.71, 26.13, 23.33, 22.72, 14.22, 11.19.

50

1H NMR of 7d (500 MHz, CDCl3) δ 5.81 (s, broad, 1H), 3.91 (m, 1H), 3.46 (m, 2H), 3.05


1.25 (d = 7.0 Hz, 3H) 0.88 (t, J = 6.8 Hz, 3H).

51


29.85, 29.64, 29.62, 26.14, 23.37, 22.78, 14.22, 11.21.

52

Synthesis of 8

Pantetheine was synthesized by following the procedure in literature (Tran et al., 2008).

Synthesis of 9

Pantetheine (7.5 g) was added dropwise into a saturated solution of 100 mL

NaHCO3 (pH 8.5). Then 3 (1 g) was added and stirred for 2h and extracted with ethyl

acetate. The solvent was removed by reduced pressure and purified by flash column

chromatography (1-5% methanol/chloroform) to give a 9 (750 mg, 45%).

53

1H NMR of 9 (500 MHz, CDCl3) δ 6.99 (s, broad, 1H), 6.81 (s, broad, 1H), 3.96 (s, 1H),

3.77 (m, 2H), 3.51 (m, 2H), 3.39 (m, 4H), 3.01 (m, 2H), 2.79 (t, J = 6.5 Hz, 2H), 2.40 (t,

J = 6.0 Hz, 2H), 1.34 (d, J= 8.0, 3H), 1.02 (t, J = Hz, 3H), 0.92 (s, 3H), 0.88 (s, 3H).

13C NMR of 9 (500 MHz, CDCl3) δ 2056.14, 196.92, 174.14, 171.97, 67.80, 60.98,

53.54, 52.67, 39.36, 38.53, 37.79, 35.08, 28.78, 21.35, 20.60, 13.11, 8.21.

54

Figure 7. LCMS of 9 with a retention of 5.1 min and low-resolution mass of 391.

Figure 8. LCMS of reduction of 9 with a retention of 5.5 min and low-resolution mass of

393.

Figure 9. LCMS of thioesterification of 9 with SNAC with a retention of 4.3 min and

low-resolution mass of 234.

55

Figure 10. Chiral HPLC of KR reductions of substrate 9 (* gem-methylated product

confirmed by LCMS).

56

Figure 11. Overlay of carbon NMRs between 80-70 ppm of the reductions of substrate 7

57

Figure 12. Overlay of carbon NMRs between 79-73 ppm of the reductions of substrate 5.

58

Figure 13. Overlay of carbon NMRs between 80-70 ppm of the reductions of substrate 3.

59

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