Towards Organoboron-mediated Functionalization of Erythromycin A and Synthesis of its Aglycon

by

Christopher D. Adair

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Christopher D. Adair 2014

  ii

Towards Organoboron-mediated Functionalization of

Erythromycin A and Synthesis of its Aglycon

Christopher D. Adair

Master of Science

Department of Chemistry University of Toronto

2014

Abstract

Many natural products, including antibiotics, are structurally complex and contain a wide

variety of functional groups. As a consequence, the selective functionalization of these

molecules often requires the use of inefficient protecting group strategies. Inspired by this

obstacle, our group recently developed a borinic acid-catalyzed method to

regioselectively functionalize the equatorial position of cis-vicinal diols in carbohydrates

with limited use of protecting groups.

The work presented in this thesis describes progress made towards selective

functionalization of the cis-vicinal diol present in the macrolide antibiotic erythromycin

A. This was attempted using the boronic and borinic acid-mediated methodologies

developed previously in our group. Finally, a semisynthesis of erythronolide A was

carried out with the goal of using our methodology to prepare novel analogues for

biological evaluation.  

  iii

Acknowledgements

There was a time when I believed that personal success was driven solely by hard work

and perseverance. While the definition of success is dependent on whom you ask, I think

that many will agree that it is very difficult to be successful without the love and support

from others.

Firstly, I would like to acknowledge my parents. They continue to serve as my primary

inspiration and always will. Perhaps unknowingly, they’ve instilled within me a sense of

ambition, pride and humbleness that I will always cherish. My mother has always been

there to support me through the toughest times of my academic career and, for that, I am

forever grateful. My father has served a complementary role, pushing me to realize that I

have the potential to accomplish anything that I desire.

I should note that my choice to pursue synthetic organic chemistry wasn’t made until the

fourth year of my undergraduate career. As such, I have to thank to Professor France-

Isabelle Auzanneau for taking a chance on a student with limited synthesis experience.

She provided me with a wonderful introduction to carbohydrate chemistry and catalyzed

my passion for a very interesting branch of synthesis. I would also like to thank Professor

Mark S. Taylor. He taught me how to think like a scientist and suggested a project that

challenged me to go above and beyond what I thought possible.

A big thank you to the Taylor group! Being a part of such a smart and talented group of

people was truly a pleasure. A special thank you to Kyan D’Angelo and Kashif Tanveer

for sharing their vast knowledge of chemistry and contributing to many insightful

conversations about my work over the year.

And of course, I’m thankful for my brother and friends. There were times when I had to

make sacrifices to succeed academically and they were always supportive. Lastly, thank

you to Craig McDougall for being the best  friend  anyone  could  ask  for.  

 

  iv

Table of Contents

Abstract .................................................................................................................................... ii

Acknowledgements .................................................................................................................. iii

Table of Contents ..................................................................................................................... iv

List of Tables ........................................................................................................................... vi

List of Figures .......................................................................................................................... vii

List of Schemes ........................................................................................................................ viii

Abbreviations ........................................................................................................................... xi

Chapter 1: Boron-Diol Interactions

1.0 Introduction ...................................................................................................... 1

1.1 Organoboron methodology in carbohydrate synthesis .................................... 3

1.2 Application of organoboron methodology to natural products ........................ 5

1.3 Conclusions ...................................................................................................... 8

Chapter 2: The Evolution of Antibiotics

2.0 Introduction ...................................................................................................... 10

2.1 Historical overview .......................................................................................... 10

2.2 Antibiotic resistance ......................................................................................... 13

2.3 Exploring new antibiotic landscape with chemical synthesis .......................... 14

2.4 Biosynthesis of novel antibiotic analogues ...................................................... 17

2.5 Conclusions and outlook .................................................................................. 19

Chapter 3: Application of Organoboron-mediated Transformations to Erythromycin A 3.0 Introduction ...................................................................................................... 20

3.1 Biosynthesis of erythromycin A ...................................................................... 21

3.2 Total synthesis of the erythromycins ............................................................... 25

3.3 Acid-catalyzed rearrangements of erythromycin A ......................................... 28

3.4 Semisynthetic analogues of erythromycin A ................................................... 30

3.5 Regioselective functionalization of erythromycin A ....................................... 31

3.6 Research goals ................................................................................................. 33

3.7 Results and discussion ..................................................................................... 34

  v

3.7.1 Organoboron-mediated glycosylation of erythromycin A ................... 34

3.7.2 Organoboron-mediated benzoylation of erythromycin A .................... 37

3.7.3 NMR experiments with erythromycin A ............................................. 44

3.8 Conclusions and outlook .................................................................................. 48

3.9 Experimental details ......................................................................................... 49

3.10 Characterization data ....................................................................................... 50

Chapter 4: Semisynthesis of Erythronolide A 4.0 Introduction ...................................................................................................... 57

4.1 Semisynthesis of erythronolide A .................................................................... 57

4.2 Research goals ................................................................................................. 60

4.3 Results and discussion ..................................................................................... 60

4.4 Conclusions and outlook .................................................................................. 65

4.5 Experimental details ......................................................................................... 67

4.6 Characterization data ....................................................................................... 68

Appendix A: NMR spectra ..................................................................................................... 75

  vi

List of Tables Table 3.1 – Borinic acid-mediated glycosylationa .................................................................. 35

Table 3.2 – Boronic acid-mediated glycosylationa ................................................................. 36

Table 3.3 – Organoboron-mediated benzoylation at 23 °Ca ................................................... 39

Table 3.4 – Organoboron-mediated benzoylation at 80 °Ca ................................................... 41

  vii

List of Figures Figure 1.1 – Deprotected pentasaccharide target of our synthesis (1.1) and pentasaccharide derived target of the Du synthesis (1.2) ........................................................ 7 Figure 2.1 – Dimer, trimer and pentamer forms of arsphenamine (Salvarsan) effective for treating syphilis ........................................................................................................................ 11

Figure 2.2 – Selected antibiotics discovered in the 20th century of historical importance ..... 12

Figure 2.3 – Overview of the cephalosporin scaffold and examples of modern adaptations ............................................................................................................................... 15

Figure 3.1 – Components of the macrolide antibiotic erythromycin A .................................. 20

Figure 3.2 – Polyketide synthase-mediated chain elongation process to form 6-deoxyerythronolide B [adopted from (47)] .............................................................................. 22

Figure 3.3 – Select examples of 6-deoxyerythronolide B analogues generated by site-directed mutagenesis of polyketide synthase domains (McDaniel, 1999) ............................... 24

Figure 3.4 – Total syntheses of erythromycin derivatives ...................................................... 25

Figure 3.5 – Seco acid derivative for erythromycin A synthesis (Woodward, 1981) ............. 26

Figure 3.6 – Erythromycin A enol ether and anhydroerythromycin A ................................... 28

Figure 3.7 – Inherent reactivity of the hydroxyl groups in erythromycin A ........................... 32

Figure 3.8 – (a) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of Ph2BOH (3.37) (b) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of erythromycin A (3.1) upon addition of Ph2BOH (3.37) .......................................................... 45

  viii

List of Schemes

Scheme 1.1 – Boronic acid-diol complexation equilibria in aqueous media .......................... 2

Scheme 1.2 – Boronic acid-mediated monoalkylation of methyl α-L-fucopyranoside with Lewis base activation ............................................................................................................... 3

Scheme 1.3 – Borinic acid-catalyzed regioselective monoacylation of carbohydrate derivatives ................................................................................................................................ 4

Scheme 1.4 – Borinic acid-catalyzed regioselective glycosylation of carbohydrate derivatives ................................................................................................................................ 4

Scheme 1.5 – Organoboron-catalyzed regio- and stereoselective formation of β-2-deoxyglycosidic linkages ......................................................................................................... 5

Scheme 1.6 – Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin ................................................................................. 6

Scheme 1.7 – Preparation of disaccharide fragment 1.4 using the borinic acid-catalyzed methodology ............................................................................................................................ 7

Scheme 1.8 – Preparation of disaccharide fragment 1.6 using the catalytic borinic acid and stoichiometric boronic acid methods ................................................................................ 8

Scheme 2.1 – Reductive removal of the C6-hydroxy group in 6-demethyltetracycline to give sancycline (Pfizer, 1958) .................................................................................................. 16

Scheme 2.2 – Semisynthesis of minocycline from sancycline (Lederle, 1967) ...................... 17

Scheme 2.3 – Semisynthesis of tigecycline from minocycline (Wyeth, 1994) ....................... 17

Scheme 2.4 – Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues by genetically engineered polyketide synthase (Khosla, 1996) .................................................... 18

Scheme 2.5 – Biosynthesis of unnatural erythromycin A derivatives ..................................... 19

Scheme 3.1 – Formation of 6-deoxyerythronolide B from propionyl CoA and methyl malonyl CoA ............................................................................................................................ 21

Scheme 3.2 – Post-PKS enzyme cascade to give erythromycin A .......................................... 23

  ix

Scheme 3.3 – Key steps in Woodward’s total synthesis of erythromycin A .......................... 27

Scheme 3.4 – Acid degradation mechanism of erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C ........................................................................................................ 29

Scheme 3.5 – Semisynthesis of clarithromycin (Taisho, 1980) .............................................. 30

Scheme 3.6 – Semisynthesis of azithromycin (Pliva, 1980) ................................................... 31

Scheme 3.7 – Site-selective acylation of erythromycin A using a peptide catalyst (Miller, 2006) ........................................................................................................................................ 33

Scheme 3.8 – Proposed regioselective monofunctionalization of erythromycin A catalyzed by a diarylborinic acid ............................................................................................. 33

Scheme 3.9 – Monobenzoylation of erythromycin A using acetic anhydride in pyridine ...... 38

Scheme 3.10 – Monobenzoylation of erythromycin A enol ether under boron-free conditions ................................................................................................................................. 42

Scheme 3.11 – Erythromycin A acid-catalyzed rearrangement products and their molecular masses ..................................................................................................................... 43

Scheme 4.1 – Semisynthesis of erythronolide A (LeMahieu, 1974) ....................................... 58

Scheme 4.2 – Cope elimination procedure employed by Celmer for removal of the tertiary amine from D-desosamine in oleandomycin ............................................................... 59

Scheme 4.3 – Synthesis of erythromycin A 9-oxime N-oxide (4.2) ....................................... 61

Scheme 4.4 – Synthesis of 3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) via Cope elimination ....................................................................................................... 61

Scheme 4.5 – Synthesis of erythronolide A 9-oxime (4.4) under acidic conditions ............... 62

Scheme 4.6 – Nitrous acid-mediated oxime cleavage to give erythronolide A 5,9-enol ether (4.6) ................................................................................................................................. 63

Scheme 4.7 – Final steps of the erythronolide A total synthesis (Carreira, 2009) .................. 64

Scheme 4.8 – Oxime cleavage with Raney Nickel in the semisynthesis of erythronolide A (4.5) .......................................................................................................................................... 65

  x

Abbreviations 1H proton (NMR spectroscopy)

13C carbon (NMR spectroscopy)

°C degrees Celsius

Å Ångstrom(s)

aq. aqueous

Ac acetyl

ACP acyl carrier protein

AT acyl transferase

Bn benzyl

Bz benzoyl

cat. catalytic or catalyst

d doublet

DCM dichloromethane

DEBS deoxyerythronolide B synthase

DIPEA N,N-diisopropylethylamine (Hünig’s base)

DMSO dimethylsulfoxide

equiv. equivalent(s)

ESI electrospray ionization

Et ethyl

EtOAc ethyl acetate

FTIR Fourier transform infrared spectroscopy

g gram(s)

  xi

hr hour(s)

HMBC heteronuclear multiple bond correlation (NMR spectroscopy)

HPLC high-performance liquid chromatography

HRMS high-resolution mass spectrometry

i-Pr isopropyl

J coupling constant (NMR spectroscopy)

KS β-ketoacyl synthase

LC-MS liquid chromatography-mass spectrometry

M molar

m multiplet

m/z mass over charge

Me methyl

MeCN acetonitrile

MHz megahertz

mg milligram(s)

min minute(s)

mL milliliter(s)

mmol millimole(s)

mol mole(s)

MS molecular sieves

NBS N-bromosuccinimide

NMR nuclear magnetic resonance

PBP penicillin binding protein

  xii

Ph phenyl

PKS polyketide synthase

PMP para-methoxyphenyl

ppm parts per million

q quartet

Ra-Ni Raney nickel

rpm revolutions per minute

RT room temperature

s singlet

sat. saturated

t triplet

TBDPS tert-butyldiphenylsilyl

TLC thin-layer chromatography

UDP uridine diphosphate

µL microliter(s)

  1

1

Boron-Diol Interactions

1.0 Introduction

The scientific discipline known as organic synthesis has a rich history that has been

documented for nearly two hundred years. Remarkable advances in this field have been

observed during the 20th century and have significantly increased our understanding of

life on the atomic and molecular level.1 Despite these advances, organic chemistry

continues to be an ever-evolving field of study.

Recent efforts in organic synthesis have focused on asymmetric catalysis, driven

particularly by the pharmaceutical industry’s demand for chiral compounds. While a wide

variety of methods have been developed for this purpose, the functional group tolerance

of these methods is highly variable.2 As a result, strategic use of protective groups has

become commonplace when carrying out asymmetric synthesis.

Regioselective functionalization of hydroxyl groups in complex molecules represents a

significant challenge for synthetic chemists.3 This is especially true for polyol natural

products such as carbohydrates. Development of protecting group-free strategies to

selectively functionalize polyols would be of considerable value and have the potential to

revolutionize carbohydrate synthesis. In this regard, progress has been made using

                                                                                                                 

1 Seebach, D. Angew. Chem. Int. Ed. 2003, 29, 1320–1367. 2 Johansson Seechurn, C. C. C.; Kitching, M. O.; Colocat, T. J.; Snieckus, V. Angew. Chem. Int. Ed. 2012, 51, 5062–5085. 3 Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007.    

  2

approaches such as organocatalytic processes4, Lewis acid-promoted methods5 and

enzyme-catalyzed methods.6

Most recently, organoboron reagents have emerged as an attractive approach to

selectively functionalize carbohydrates. Their ability to form reversible covalent

interactions with diols has been studied extensively in aqueous media, with initial reports

made by Lorand and Edwards in 1959 using phenylboronic acid (Scheme 1.1).7 It was

observed that boronate ester formation is favourable in solutions of high pH. This effect

was attributed to the lower angle strain present in the tetracoordinate boronate complex

relative to the tricoordinate conjugate acid. Subsequent study of this equilibrium has

revealed that structure and stereochemistry of the diol are important. It was found that

1,2-diols complex to boronic acids preferentially over 1,3-diols8 and that cis diols bind

preferentially to trans or simple acyclic diols.9

Scheme 1.1 – Boronic acid-diol complexation equilibria in aqueous media

                                                                                                                 4 (a) Griswold, K. S.; Miller, S. J. Tetrahedron. 2003, 59, 8869–8875. (b) Kawabata, T.; Muramatsu, W.; Nishio, T.; Shibata, T.; Schedel, H. J. Am. Chem. Soc. 2007, 129, 12890–12895. 5 Sn(IV) derivatives: (a) Iwasaki, F.; Maki, T.; Onomura, O.; Nakashima, W.; Matsumura, Y. J. Org. Chem. 2000, 65, 996–1002. (b) Martinelli, M. J.; Vaidyanathan, R.; Pawlak, J. M.; Nayyar, N. K.; Dhokte, U. P.; Doecke, C. W.; Zollars, L. M. H.; Moher, E.D.; Van Khau, V.; Kosmrjl, B. J. Am. Chem. Soc. 2002, 124, 3578–3585. (c) Demizu, Y.; Kubo, Y.; Miyoshi, H.; Maki, T.; Matsumura, Y.; Moriyama, N.; Onomura, O. Org. Lett. 2008, 10, 5075–5077. La(III) salts: Dhiman, R. S.; Kluger, R. Org. Biomol. Chem. 2010, 8, 2006–2008. 6 (a) Therisod, M.; Klibanov, A. M. J. Am. Chem. Soc. 1987, 109, 3977–3981. (b) Wang, Y.-F.; Lalonde, J. J.; Momongan, M.; Bergbreiter, D. E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200–7205. 7 Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769–774. 8 Pizer, R.; Tihal, C. Inorg. Chem. 1992, 31, 3243–3247. 9 James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem. Int. Ed. 1996, 35, 1910–1922.  

BOH

OH HO

HOB

O

O2H2O

OH

BO

O2H2O

HOB

OH

OH HO

HOHO

OH

pH 7.5

pH >10

  3

1.1 Organoboron Methodology in Carbohydrate Synthesis

Although the oxygen atoms involved in tricoordinate boronic ester formation are

deactivated, complexation with organoboron compounds can also be used as an activation

method. The group of Aoyama was the first to exploit this type of activation strategy

using a phenylboronate derived from methyl α-fucopyranoside.10 In the presence of

triethylamine, the phenylboronate underwent regioselective alkylation at O-3 (Scheme

1.2). It was proposed that coordination of the Lewis base to the boron atom resulted in

activation of the boronic ester towards reaction with iodobutane. This methodology was

later expanded to glycosylations of peracetylated glucosyl bromide donors with

deprotected carbohydrates containing cis-1,2-diol or 1,3-diol moieties.11

Scheme 1.2 – Boronic acid-mediated monoalkylation of methyl α-L-fucopyranoside with Lewis base activation

Inspired by the work of Aoyama, our group set out to develop organoboron-catalyzed

methods for regioselective carbohydrate activation. In 2011, our group reported a method

for catalytic acylation of carbohydrates using 2-aminoethyl diphenylborinate as a

precatalyst (Scheme 1.3).12 This work displayed general regioselectivity for the

equatorial hydroxyl group of the cis-diol in pyranoside derivatives of galactose, mannose,

fucose, and rhamnose. Of note, acylation of carbohydrates containing a free primary

hydroxyl group, such as β-galactopyranoside, resulted in competitive functionalization at

the primary hydroxyl group and desired secondary hydroxyl group.

                                                                                                                 10 Oshima, K.; Kitazono, E.-i.; Aoyama, Y. Tetrahedron Lett. 1997, 38, 5001–5004. 11 Oshima, K.; Aoyama, Y. J. Am. Chem. Soc. 1999, 121, 2315–2316. 12 Lee, D.; Taylor, M. S. J. Am. Chem. Soc. 2011, 133, 3724–3727.  

O

OCH3

OHOOB

O

OCH3

OHOn-BuHO

Ph NEt3

n-BuI, Ag2O, NEt3

PhH, reflux, 22 hr

O

OCH3

OHOOB

Ph

O

OCH3

OHOHHO

PhB(OH)2

CH2Cl250%

  4

Scheme 1.3 – Borinic acid-catalyzed regioselective monoacylation of carbohydrate derivatives

Another recent development in our group arose from adaptation of the borinic acid-

catalyzed acylation procedure to glycosylation of various carbohydrate derivatives.13 This

work served as the first reported example of a regioselective glycosylation procedure

using a nonenzymatic catalyst.14 Koenigs-Knorr glycosylations of several armed and

disarmed glycosyl halides with minimally or unprotected glycosyl acceptors gave good to

excellent yields with silver(I) oxide as a promoter (Scheme 1.4).

Scheme 1.4 – Borinic acid-catalyzed regioselective glycosylation of carbohydrate derivatives

Most recently, a strategy was developed in our group that enables regio- and

stereoselective glycosylations of pyranoside-derived cis-1,2- and 1,3-diols using both 2-

deoxy and 2,6-dideoxyglycosyl chloride donors with 2-aminoethyl diphenylborinate as a

precatalyst (Scheme 1.5).15 The stereoselective synthesis of these linkages is quite

challenging due to the anomeric effect and absence of participating protective groups at

                                                                                                               

13 Gouliaras, C.; Lee, D.; Chan, L.; Taylor, M. S. J. Am. Chem. Soc. 2011, 133, 13926–13929. 14 Mensah, E. A.; Nguyen, H. M. J. Am. Chem. Soc. 2009, 131, 8778–8780. 15 Beale, T. M.; Moon, P. J.; Taylor, M. S.; Org. Lett. 2014, 16, 3604–3607.  

R1

HO

HO R2

OB

NH2

PhPh

(5–10 mol%)

RCOCl (1.2–2.0 equiv.)i-Pr2NEt (1.2–2.0 equiv.)

MeCN, RT

R1

HO

ROCO R2

69–95% (14 examples)

R3R4HO

OH

1.1 equiv

R3R4O

OH

R2

OR1

(10 mol%)

Ag2O (1 equiv.)MeCN, 23–60 oC

OB

NH2

Ph

Ph

R2

OR1

X

X = Br, Cl 68–99% (13 examples)

  5

the C-2 position, resulting in bias toward α-configured 2-deoxy glycosides.16 Despite this

bias, the borinic acid catalyst favoured an SN2-type pathway to give β:α ratios ranging

from 4:1 to >19:1 with good yields of the desired regioisomer in 16 examples.

Scheme 1.5 – Organoboron-catalyzed regio- and stereoselective formation of β-2-deoxyglycosidic linkages

1.2 Application of organoboron methodology to natural

products

The carbohydrate scope of the borinic acid-catalyzed methodology developed in our

group was initially limited to simple mono- and disaccharide acceptors containing cis-

1,2-diols. To expand upon this work, it was of great interest to apply our methodology

towards the functionalization of complex polyol natural products.

With this goal in mind, the cardiac glycoside digitoxin was chosen as a target for

regioselective glycosylations using our methodology. Consistent with previous studies,

the equatorial position of the cis-1,2-diol of digitoxin was selectively glycosylated out of

the possible five free hydroxyl groups and gave good to excellent yields for all six

glycosyl donors (Scheme 1.6).17 A variety of peracetylated glycosyl bromides were

successfully employed and resulted in β-configuration of the newly formed glycosidic

bond. Cleavage of the acetyl groups from the newly linked sugars with lithium hydroxide

in methanol/water furnished the deprotected products, which could serve as new analogs

                                                                                                                 16 (a) Hou, D.; Lowary, T. L. Carbohydr. Res. 2009, 344, 1911−1940. (b) Crich, D. J. Org. Chem. 2011, 76, 9193−9209. 17 Beale, T. M.; Taylor, M. S. Org. Lett. 2013, 15, 1358–1361.  

OAcO

OAc

AcOCl

O

O

HOOCH3

OHO

TBDPSH

OB

NH2

PhPh

(10 mol%)

Ag2O (2 equiv.)CH2Cl2, RT

OAcO

OAc

AcOO

O

OOCH3

OHO

TBDPSH

Catalyzed reaction: 72% yield, 7.3:1 β:α Uncatalyzed reaction: 18% yield, 2:1 β:α

  6

for biological evaluation. Notably, the levels of regiocontrol for the 4”-O-glycosylated

isomer were excellent, with the major byproduct being unreacted digitoxin rather than

regioisomers.

Scheme 1.6 – Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin

While late stage glycosylation of complex natural products presents a useful strategy to

prepare novel semisynthetic analogues, it would also be advantageous to apply our

O

OH

HOH3C

O O

OH

H3C

O O

OH

H3C

O

OB

NH2

Ph

Ph

(25 mol%)

Ag2O (2 equiv.)

CH2Cl2, 23 ˚C(2 equiv.)

O

OH

OH3C

O O

OH

H3C

O O

OH

H3C

O

R2

OR1

Br

R2

OR1

OAcO

OAc

AcOBrAcO

OAcO

AcOBr

OAcOAcO

AcO

OAc

77%

O

OAc

74%

O OAcOAcAcO

Br

63%

O OAcOAcAcO

Br

51%

OAcO

AcOBr

OAcO

AcO

OAc

O

OAc

64%

OAc

O

OAc

AcOBrAcO

63%

OAc

OH

OOCH3CH3

OH

OOCH3CH3

  7

borinic acid-catalyzed methodology to oligosaccharide total synthesis. In particular, we

envisioned using borinic acid catalysts for two regioselective glycosylation reactions in

the synthesis of a pentasaccharide derivative (1.1) isolated from Spergularia ramosa

(Figure 1.1). The first synthesis of this oligosaccharide was completed by Du and co-

workers and involved 14 steps to reach target 1.2.18 Although each step in the synthesis is

relatively efficient, nine of the fourteen steps are protective group manipulations.

Figure 1.1 – Deprotected pentasaccharide target of our synthesis (1.1) and pentasaccharide derived target of the Du synthesis (1.2)

To improve upon this synthesis, our group used the catalytic borinic acid-methodology to

facilitate glycosylation of a peracetylated glucosyl bromide donor (1.3) and unprotected

pentenyl rhamnose acceptor (1.4), which proceeded in 80% yield (Scheme 1.7).

Scheme 1.7 – Preparation of disaccharide fragment 1.4 using the borinic acid-catalyzed methodology

                                                                                                                 18 Gu, G.; Du, Y. J. Chem. Soc., Perkin Trans., 1. 2002, 2075–2079.

O

O

O

OOH

O

HOHOHOHO

O

HOHOHO

O

HO

HO

HOO

HO

OOR

O

O

O

OOAc

O

AcOAcOAcOAcO

O

AcOAcOAcO

O

BzO

BzO

BzOO

BzO

O

O

1.21.1

O

O

HOHO

OHO

AcOAcO

AcO

Br

OAc

O

O

O

HOOHO

AcOAcO

AcO

OAc

80%Ag2O (1 equiv.)

MeCN

OB

NH2

PhPh

(10 mol%)

(1.1 equiv.)

1.3 1.4

  8

Unfortunately, attempts at glycosylating a peracetylated fucosyl bromide donor (1.5) with

unprotected PMP arabinose acceptor (1.6) using the catalytic procedure resulted in poor

yields. This prompted the development of a stoichiometric boronic acid-mediated

approach, which led to significant improvement in yield (Scheme 1.8).19 Further

optimization of this synthesis is currently underway.

Scheme 1.8 – Preparation of disaccharide fragment 1.6 using the catalytic borinic acid and stoichiometric boronic acid methods

1.3 Conclusions

Our group’s development of regioselective functionalization reactions for carbohydrates

using borinic acid-derived catalysts provides several advantages over traditional

                                                                                                                 19 McClary, C. A. 2013. Exploring Noncovalent and Reversible Covalent Interactions as Tools for Developing New Reactions. (Doctor of Philosophy Dissertation).

O

AcO

AcO

AcO

O

OH

HO

HO OPMP

Br

O

AcO

AcO

AcOO

OH

HO

O OPMP

Ag2O (1 equiv.)MeCN, RT

(1.1 equiv.) 20%

O

AcO

AcO

AcO

O

OH

HO

HO OPMP

Br

O

AcO

AcO

AcOO

OH

HO

O OPMP

Ag2O (1 equiv.)NEt3 (3 equiv.)

MeCN, RT

(1.1 equiv.)

(1 equiv.)

77%

B(OH)2F F

FF

F

OB

NH2

PhPh

(10 mol%)

1.5 1.6

1.5 1.6

Catalytic (borinic)

Stoichiometric (boronic)

  9

oligosaccharide synthesis, organotin protocols and enzymatic methods. Highlights of our

methodology include the use of a relatively benign, inexpensive catalyst and a simplistic

reaction setup that does not require high temperatures, long reaction times or exclusion of

air. Furthermore, the regiochemical outcome of these reactions is predictable and

reproducible for substrates bearing a cis-1,2-diol motif. Current efforts are focused on

developing new borinic acid-catalyzed glycosylation protocols that avoid using

stoichiometric quantities of heterogeneous silver(I) salts and adopting our current

methodology to regio- and stereocontrolled functionalization of complex natural

products.

  10

2

The Evolution of Antibiotics

2.0 Introduction

During the 19th century, infections such as pneumonia, diphtheria and diarrhea

represented the principle causes of death in children and adults.20 As a consequence of

the industrial revolution and subsequent urbanization, incidence rates of these ailments

and others, such as syphilis and tuberculosis, increased significantly. The introduction of

aseptic technique in 1867 served as a starting point for minimizing the risk of bacterial

infection but many of these diseases remained incurable.21 It wasn’t until the early 20th

century that the first modern chemotherapeutic agents were discovered and implemented

in treatment of common bacterial infections.

2.1 Historical overview

One of the first antibacterial agents used to treat infections was arsphenamine. Soon after

its discovery in 1910, arsphenamine was marketed under the trade name Salvarsan and

referred to as the “magic bullet” for treatment of syphilis.22 This synthetic organoarsenic

compound was a significant improvement to inorganic mercury compounds used

previously to treat syphilis but was relatively difficult to administer due to its

hygroscopic nature and remarkable sensitivity to atmospheric conditions. Interestingly,

its chemical composition was recently shown to be that of two different organoarsenic

structures (2.2, 2.3) rather than the previously described dimer 2.1 (Figure 2.1).23

                                                                                                                20 Christoffersen, R. E. Nat. Biotechnol. 2006, 24, 1512–1514. 21 Wallace, W. C.; Cinat, M. E.; Nastanski, F. Am. Surg. 2000, 66, 874–878. 22 Riethmiller, S. Chemotherapy. 2005, 51, 234–242. 23 Lloyd, N.C.; Morgan, H.W.; Nicholson, B.K.; Ronimus, R. S. Angew. Chem. Int. Ed. 2005, 44, 941–944.  

  11

Figure 2.1 – Dimer, trimer and pentamer forms of arsphenamine (Salvarsan) effective for treating syphilis

The first general-purpose antibiotic to gain widespread use was prontosil (2.4), developed

in the 1930s by Bayer Laboratories.24 Prontosil is a synthetic diazo dye containing a

sulfonamide functionality. The discovery of this compound marked the beginning of a

new class of antibiotics known as the sulfa drugs. These sulfonamide containing

compounds act as analogues of para-aminobenzoic acid and ultimately inhibit folate

synthesis.25 This induces the inhibition of DNA, RNA and protein synthesis in a broad

range of both Gram-positive and Gram-negative bacteria.

Though antibiotics of synthetic origin are important, they account for only a small

fraction of antibiotics in use today. In fact, most antibacterial agents used commonly in

hospitals originated from natural products.26 Perhaps the most revolutionary example of a

naturally occurring antibiotic is penicillin, discovered by Alexander Fleming in 1928.

The penicillins [see penicillin G (2.5)] belong to a large family of β-lactam antibiotics

that also include the cephalosporins and are responsible for saving the lives of countless

soldiers during World War II. The β-lactam ring structure is essential for antimicrobial

activity and has been shown to inhibit formation of the peptidoglycan crosslink in the

bacterial cell wall, thereby activating cell wall autolysis in Gram-positive bacteria.

                                                                                                               

24 Owa, T.; Nagasu, T.; Expert Opin. Ther. Pat. 2000, 10, 1725–1740. 25 Kalkut, G.; Cancer Invest. 1998, 16, 612–615. 26 Singh, S. B.; Barrett, J. F. Biochem. Pharmacol. 2006, 71, 1006–1015.  

As AsAs

OHNH2

OHNH2

HO

H2NAs As

AsAs

As

OHNH2

NH2

OH

OHH2NHO

H2N

HO

H2N

AsAs

H2N

HO

OH

NH2

2.1 2.2 2.3

  12

Figure 2.2 – Selected antibiotics discovered in the 20th century of historical importance

In the decades following, several new classes of naturally occurring antibiotics were

discovered and implemented in routine medical practice. Among them are the

tetracyclines [see tetracycline (2.6)], the macrolides [see erythromycin A (2.7)] and the

glycopeptides [see vancomycin (2.8)]. While all having varying modes of action, the

usual targets of these antibacterial agents are cell wall synthesis, protein synthesis,

nucleic acid synthesis, or important biosynthetic pathways.27

The significant growth experienced in the mid 20th century in the development of

antibiotics was not sustained in the following decades, resulting in nearly 40 years before

the introduction of a new class of antibiotics. This has, in part, been attributed to the

belief that bacterial infections were becoming an issue of the past.28 However, the

discovery of antibiotic resistant bacteria proved this hypothesis false. Indeed, resistance

                                                                                                               

27 Hartmann, G.; Behr, W.; Beissner, K.-A.; Honikel, K.; Sippel, A. Angew. Chem. Int. Ed. 1968, 7, 693–701. 28 Overbye, K. M.; Barrett, J. F. Drug Discovery Today. 2005, 10, 45–52.  

H2N

NH2

NN

SO2NH2

HN

O N

S MeMe

CO2H

H

OOH O O

OH

CONH2O

NMe2H

O

Me OHH

HH

O

NHHN

Cl

NHO

O

HNNH

HOO H

N

O

O

O

HO

Cl

OO NH2

OH

OH

ONH

HO OH

OO

OO

OH

HH

O

O

N

Me

O

Me

H2H

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

2.4: prontosil (sulfonamide) 2.5: penicillin G (β-lactam) 2.6: tetracycline (tetracycline)

2.7: erythromycin A (macrolide)

2.8: vancomycin (glycopeptide)

  13

to last resort antibiotics such as vancomycin has become a significant problem that only

recently gained widespread attention.

2.2 Antibiotic resistance

From a biological perspective, antibacterial drug resistance is an intriguing aspect of

evolution. Under the selective pressure of antibiotics, bacteria evolve to spread resistance

mechanisms that eventually become prevalent among other pathogenic and

nonpathogenic bacteria. Alternatively, bacteria may also become resistant to a class of

antibiotics through random spontaneous mutation of their genetic material. In general,

resistance is exhibited through the following mechanisms:29

1) upregulation of enzymes that inactivate the antibiotic (e.g., β-lactamases) or

modify of the cellular target (e.g., ribosomal methylase in Staphylococci

preventing erythromycin binding);

2) modification or loss of the target with which the antibiotic interacts (e.g.,

alteration of penicillin-binding protein in Pneumococci);

3) upregulation of pumps that expel the antimicrobial agent from the cell (e.g.,

efflux of fluoroquinolones in Staphylococcus aureus);

4) downregulation or inactivation of outer membrane protein channels required

for entry of the antibiotic into the cell (e.g., resistance to β-lactams by OmpF

porin downregulation in Escherichia coli).

Even with judicious use of antibiotics, the onset of bacterial resistance is inevitable. Thus,

the development of new antibiotics is a significant priority. Fortunately, developments in

chemistry and biology have improved our ability to discover new antibiotics. This has

been accomplished through exploration of new natural product chemical space,

modification of previously existing structures and genetic engineering of antibiotic-

producing biosynthetic pathways.30 Collectively, these advances have facilitated the

                                                                                                               

29 Gallo, G.; Puglia, A. M. Antibiotics: Targets, Mechanisms and Resistance.; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; pp 73–80. 30 Nicolaou, K. C.; Chen, J. S.; Edmonds, D. J.; Estrada, A. A. Angew. Chem. Int. Ed. 2009, 48, 660–719.  

  14

development of antimicrobial agents that avoid resistance and have novel mechanisms of

action.

2.3 Exploring new antibiotic landscape with chemical

synthesis

Since the discovery of the sulfonamide drugs, chemical synthesis has played a critical

role in the development of new antibiotics.31 Even though fermentation is the preferred

method to manufacture large quantities of clinically used antibiotics, chemical synthesis

has served an important and complementary purpose. For example, structural

modification of naturally occurring antibiotics has yielded compounds with improved

biological properties. Furthermore, the de novo synthesis of natural product antibiotics

and their analogues has contributed to our understanding of their mode of action through

structure-activity relationships (SARs). These studies assist scientists in designing

improved antibiotic analogues that are effective against resistant bacterial strains.

One class of antibiotics that has been subjected to thorough medicinal chemistry efforts is

the cephalosporin class of β-lactam antibacterials. There are now at least four recognized

generations of the cephalosporins, which are differentiated by their activity spectrum and

efficacy rather than by structural diversity. While each generation of these β-lactams is

different, this is not to say that compounds of earlier generations are obsolete. In fact,

there are several antibiotics in each generation that are still in clinical use today.32

The cephalosporins bind to enzymes known as penicillin-binding proteins (PBPs) through

acylation of the β-lactam amide bond, which is mediated by a nucleophilic serine residue

in the active site. Though β-lactamase enzymes are largely responsible for antibiotic

resistance to the cephalosporins, the presence of penicillin-binding protein 2a (PBP2a) in

                                                                                                                 31 Nussbaum, F. V.; Brands, B. M.; Hinzen, S.; Weigand, D.; Habich, C. Angew. Chem. 2006, 118, 5194–5254. 32 Page, M. G. Expert Opin. Invest. Drugs. 2004, 13, 973–985.  

  15

certain strains of Staphylococcus aureus has led to resistance to many β-lactams.33 This is

because PBP2a has a very low affinity for traditional β-lactam antibiotics. Consequently,

even when other PBPs are inhibited, PBP2a can continue to mediate cell wall

biosynthesis, thus leading to β-lactam resistance.

The discovery of ceftobiprole (2.9) provided extensive insight into the inhibition

mechanism of many cephalosporin antibiotics. Strynadka and co-workers obtained a

crystal structure of ceftobiprole bound to the PBP2a active site, which led to the

discovery that the hydrophobic nature and planarity of the R2 group was essential for

effective binding to the active site (Figure 2.3).34 With this knowledge, synthetic

modifications were made to increase the hydrophobicity of the R2 group in ceftobiprole

that resulted in the development of ceftaroline (2.10). Ceftaroline displays greater affinity

for the PBP2a active site, which increases rate of acylation of the β-lactam amide bond

and, thus, improves antibacterial activity.35

Figure 2.3 – Overview of the cephalosporin scaffold and examples of modern adaptations

                                                                                                                33 Saravolatz, L. D.; Stein, G. E.; Johnson, L. B. Clin. Infect. Dis. 2011, 52, 1156–1163. 34 Lovering, A. L.; Gretes, M. C.; Safadi, S. S.; Danel, F.; Castro, L.; Page, M. G. P.; Strynadka, N. C. J. J. Biol. Chem. 2012, 287, 32096–32102. 35 Llarrull, L. I.; Fisher, J. F.; Mobashery, S. Antimicrob. Agents Chemother. 2009, 53, 4051–4063.  

N

S

CO2HR2O

HNR1

O

Cephalosporin Scaffold

R1 – essential for stabilityto hydrolysis by β-lactamases

R2 – essential for achieving higherbinding affinity for PBP2a

β-lactam ring – essential for inhibition oftranspeptidase activity in cell wall biosynthesis

N

S

CO2HO

HN

O N NH

N OMeN

S N

H2N

O

2.9: ceftobiprole

N

S

CO2HSO

HN

O

N OEtN

S N

HN

S

N

NMe

POHO

HO

2.10: ceftaroline

  16

The tetracyclines, discovered in 1945, were the first broad-spectrum antibiotics

incorporated into routine medical practice.36 Effective against Gram-positive and Gram-

negative bacteria, the tetracyclines have been used extensively in human and veterinary

medicine for treatment of bacterial infections and as feed additives.37 As a consequence

of their widespread use, high levels of bacterial resistance have been reported. However,

in light of their broad-spectrum activity, good safety profile and abundant supply,

tetracyclines remain first-line antibiotics for ailments such as pneumonia, Lyme disease,

cholera, and acne vulgaris.

Beginning with the semisynthesis of tetracycline from chlorotetracycline, the

development of semisynthetic tetracycline analogues has been instrumental in tackling

complications associated with bacterial resistance. Approximately 10 years after the

discovery of tetracycline, Pfizer demonstrated that the C6-hydroxy group of tetracycline,

oxytetracycline and 6-demethyltetracycline could be removed reductively (Scheme

2.1).38 The resulting 6-deoxytetracyclines were found to be more stable than their

predecessors, while retaining similar broad-spectrum antibacterial activity.

Scheme 2.1 – Reductive removal of the C6-hydroxy group in 6-demethyltetracycline to give sancycline (Pfizer, 1958)

Additionally, the improved chemical stability of the 6-deoxytetracyclines enabled acid-

and base-mediated structural modifications that had not been previously possible, leading

                                                                                                               

36 Duggar, B. M. Ann. N. Y. Acad. Sci. 1948, 51, 177–181. 37 Stockstad, E. L. R.; Jukes, T. H.; Pierce, J.; Page, A. C.; Franklin, A. L. J. Biol. Chem. 1949, 180, 647–654. 38 McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P. J. Am. Chem. Soc. 1960, 82, 3381–3388.  

OH O O

OH

CONH2O

NMe2H

O

H

HH

6-demethyltetracycline(natural product)

HO H

6Pd, H2, HCl

MeOHOH O O

OH

CONH2O

NMe2H

O

H

HH

sancycline

H H

6

  17

to the discovery of minocycline in 1967.39 Minocycline was synthesized from 6-deoxy-6-

demethyltetracycline (sancycline) by an electrophilic aromatic substitution at C7

(Scheme 2.2) and exhibited a broader spectrum of antimicrobial activity than previous

tetracyclines.

Scheme 2.2 – Semisynthesis of minocycline from sancycline (Lederle, 1967)

Aiming to overcome tetracycline resistance in the late 1990s, the group of Tally and co-

workers synthesized 7,9-disubstituted tetracycline analogues, which led to the discovery

of tigecycline in 1994 (Scheme 2.3).40 These new derivatives greatly extended the

antimicrobial spectrum of tetracyclines, especially towards tetracycline-resistant bacteria.

After its FDA approval in 2005, tigecycline quickly became the antibiotic of choice for

last-line of defense against multidrug-resistant bacteria.54

Scheme 2.3 – Semisynthesis of tigecycline from minocycline (Wyeth, 1994)

2.4 Biosynthesis of novel antibiotic analogues

Many antibiotic-producing biosynthetic pathways have been studied extensively over the

past several decades.41 This has helped scientists develop a better understanding of the

                                                                                                                 

39 Church, R. F. R.; Schaub, R. E.; Weiss, M. J. J. Org. Chem. 1971, 36, 723–725. 40 Sum, P. E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–188.

41 Moellering, R. C. N. Engl. J. Med. 2010, 363, 2377–2379.

OH O O

OH

CONH2O

NMe2H

OHH

sancycline

H7

9

KNO3, H2SO4

OH O O

OH

CONH2O

NMe2H

OHH

HNO2

Pd, H2, CH2O

MeOH OH O O

OH

CONH2O

NMe2H

OHH

HNMe2

minocyclineMixture of 7- and 9-nitro isomers

OH O O

OH

CONH2O

NMe2H

OHH

HNMe2

minocycline

1) KNO3, H2SO4

2) Pd/C, H2 OH O O

OH

CONH2O

NMe2H

OHH

HNMe2

H2N

O

ClHN

t-BuHCl

OH O O

OH

CONH2O

NMe2H

OHH

HNMe2

NH

OHN

t-Bu

tigecycline

  18

antibacterial agent’s mechanism of action and, more recently, to develop novel antibiotic

analogues. Despite the biological complexity of these pathways and enzymes involved,

biosynthesis presents a unique alternative to chemical synthesis and can be particularly

advantageous for synthesizing structurally complex antibiotics.

In 1997, Khosla and co-workers genetically modified the enzyme polyketide synthase

(PKS) to synthesize new derivatives of the macrolide antibiotic erythromycin A.42 In this

work, a genetic block was introduced to deoxyerythronolide B synthase (DEBS), which

disrupted the first condensation step in erythromycin A biosynthesis. Expressing this

mutation in a strain of Streptomyces coelicolor with inactive ketosynthase KS1 allowed

for introduction of unnatural synthetic building blocks into the 6-deoxyerythronolide B

scaffold (Scheme 2.4). This strategy furnished several new analogues of 6-

deoxyerythronolide B not previously accessible by chemical synthesis.

Scheme 2.4 – Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues by genetically engineered polyketide synthase (Khosla, 1996)

The successful synthesis of these unnatural intermediates prompted investigation into

whether the post-PKS enzymes in the erythromycin biosynthetic pathway might also

accept unnatural substrates. Positive results were obtained for substrates containing

                                                                                                               

42 Jacobsen, J. R.; Hutchinson, R. C.; Cane, D. E.; Khosla, C. Science. 1997, 277, 367–369.

O

O

OH

O OH

OHSCoA

OOH DEBS

O

O

OH

O OH

OHSNAC

OOH DEBS KS1°

in vivo

O

O

OH

O OH

OHSNAC

OOH DEBS KS1°

in vivo

O

O

OH

OH

OHSNAC

O DEBS KS1°

in vivo

OH

O

Typical propionyl CoA substrate to give 6-deoxyerythronolide B

  19

methyl, n-propyl and phenyl R groups when subjected to S. erythraea mutants unable to

synthesize 6-deoxyerythronolide B (Scheme 2.5).

Scheme 2.5 – Biosynthesis of unnatural erythromycin A derivatives

2.5 Conclusions

The history of antibiotics describes a fascinating scientific journey through the 20th

century. From the beginning of the antibiotic era to present day, the role of chemical

synthesis remains of critical importance. Total- and semisynthesis, in combination with

medicinal chemistry efforts, continue to yield next-generation antibacterial agents with

improved biological activity that aid in deterring bacterial resistance. Although

biosynthesis remains a relatively underdeveloped strategy for antibiotic development, the

ability to generate novel antibiotic analogues through genetic engineering represents an

intriguing approach worth further exploration. Though recent developments in biology

and chemistry have improved our ability to discover new antibiotics and manipulate

privileged structures, the inevitable onset of bacterial resistance will demand the

continued search for new antimicrobial agents in the years ahead.

O

O

OH

O OH

OHR

O

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

R

R = Methyl, n-Propyl, Phenyl

  20

3

Application of Organoboron-mediated Transformations to Erythromycin A

3.0 Introduction

In 1952, the pharmaceutical company Eli Lily commercialized the first macrolide

antibiotic, erythromycin A (3.1, Figure 3.1). This marked the discovery of an important

subclass of polyketide antibiotics that are used extensively in the treatment of bacterial

infections and remain one of the most widely studied antibiotic classes in modern

medicine.43 Erythromycin A was discovered in 1949 when researchers from Eli Lily

isolated the metabolic products of Saccharopolyspora erythraea in a soil sample from the

Philippines. It was found that erythromycin A is effective against many Gram-positive

bacteria, mediated by ribosomal binding and subsequent inhibition of protein synthesis.

Advantageously, its antimicrobial spectrum has been reported to be wider than that of

penicillin and is often prescribed to individuals allergic to the penicillins.44 In terms of

Figure 3.1 – Components of the macrolide antibiotic erythromycin A

                                                                                                               

43 Pal, S. Tetrahedron. 2006, 14, 3171–3200. 44 Washington, J. A.; Wilson, W. R. Mayo Clin. Proc. 1985, 60, 189–203.  

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

erythromycin A (3.1)

Aglycone(erythronolide A)

D-desosamine

L-cladinose

  21

structure, erythromycin A is described as a macrolide. This term was introduced by R. B.

Woodward to denote a class of substances produced by Streptomyces bacteria that

contain a macrocyclic lactone to which one or more carbohydrates are attached.45 The

aglycon of erythromycin A, referred to as erythronolide A, is linked to two unusual

sugars, D-desosamine and L-cladinose.

3.1 Biosynthesis of erythromycin A

With its plethora of stereocenters and 14-membered cyclic backbone, erythromycin A

represents a relatively complex natural product. Thus, it is of interest to discuss the

underlying mechanism of its biosynthesis. Macrolides, such as erythromycin A, contain a

macrocyclic lactone scaffold that is synthesized by polyketide synthase (PKS) in a multi-

enzyme process. Using one unit of propionyl CoA (3.2) and six units of methylmalonyl

CoA (3.3), PKS mediates a sequential chain elongation process. This is followed by a

termination event that results in separation of the newly formed chain from PKS and

cyclization to yield 6-deoxyerythronolide B (3.4, Scheme 3.1).46

Scheme 3.1 – Formation of 6-deoxyerythronolide B from propionyl CoA and methyl malonyl CoA

Figure 3.2 provides an excellent representation of the chain elongation process and the

steps involved in between each condensation. The three essential domains – β-ketoacyl

synthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) – co-operate to

catalyze carbon-carbon bond formation by Claisen condensation, which results in a β-                                                                                                                

45 Woodward, R. B. Angew. Chem. 1957, 69, 50. 46 Corcoran, J. W.; Vygantas, A. M. Biochemistry. 1982, 21, 263.  

SCoA

O

SCoA

O

CO2H

Chain assembly on PKS

Cyclization and release from enzyme

O

O

OH

O

Et

OH

OH

6-deoxyerythronolide B (3.4)

(3.2) (3.3)

  22

keto ester intermediate. The variable set of domains positioned between the AT and ACP

then carry out reductive modification of the keto group before the next round of chain

extension. After the sixth unit of methylmalonyl CoA is added, thioesterase catalyzes

chain cleavage and cyclization to give 6-deoxyerythronolide B.47

Figure 3.2 – Polyketide synthase-mediated chain elongation process to form 6-deoxyerythronolide B [adopted from (47)]

                                                                                                               

47 Staunton, J.; Wilkinson, B. Chem. Rev. 1997, 97, 2611–2629.

 

  23

6-deoxyerythronolide B then undergoes a series of site-selective functionalization

reactions to yield erythromycin A (Scheme 3.2).

Scheme 3.2 – Post-PKS enzyme cascade to give erythromycin A

Firstly, C-6 hydroxylation of 6-deoxyerythronolide B (3.4) is accomplished by a

cytochrome P450 enzyme and occurs with retention of configuration to give

erythronolide B (3.5).48 In the next step, L-mycarose is linked to the C-3 hydroxyl group

by TDP-mycarose glycosyltransferase to yield 3-O-mycarosylerythronolide B (3.6).49

Then, the amino carbohydrate D-desosamine is linked to the C-5 hydroxyl group by

                                                                                                               

48 Corcoran, J. W. In Antibiotics, Volume IV: Biosynthesis; Corcoran, J. W., Ed.; Springer-Verlag: New York; 1981, pp 132. 49 Martin, J. R.; Perun, T. J.; Girolami, R. L. Biochemistry. 1966, 5, 2852.  

O

O

OH

O

Et

OH

OH

6-deoxyerythronolide B (3.4)

O

O

OH

O

Et

OH

OH

OH b

erythronolide B (3.5)

O

O

OH

O

Et

O

OH

OH

3-O-Mycarosylerythronolide B (3.6)

OCH3

OHCH3

OH

a

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

erythromycin A (3.1)

O

O

OH

O

Et

O

O

OH

OCH3

OHCH3

OMe

O

N(CH3)2

CH3HO

erythromycin B (3.8)

c

O

O

OH

O

Et

O

O

OH

OCH3

OHCH3

OH

O

N(CH3)2

CH3HO

erythromycin D (3.7)

O

O

OH

O

Et

O

O

OH

OCH3

OHCH3

OH

O

N(CH3)2

CH3HO

erythromycin C (3.9)

HO

erythromycin D (3.7)

d e

de

a – C-6 erythronolide hydroxylaseb – TDP-mycarose glycosyltransferasec – TDP-desosamine glycosyltransferased – (O)-methyltransferasee – C-12 hydroxylase

  24

TDP-desosamine glycosyltransferase. The resulting intermediate, erythromycin D (3.7),

is the first to show antibacterial activity and occurs at a branch in the synthetic pathway.50

Either O-methylation of the C-3” hydroxyl on the mycarose sugar follows, to produce

erythromycin B (3.8), or C-12 hydroxylation takes place with retention of configuration

to furnish erythromycin C (3.9).51 Finally, erythromycin A (3.1) is generated either by C-

12 hydroxylation of 3.8 or O-methylation of 3.9.

As shown in chapter 2, Scheme 2.4, genetic manipulation of PKS allows for production

of novel 6-deoxyerythronolide B analogues. In the example presented by Khosla and co-

workers, genetically modified PKS enabled the use of substrates other than propionyl

CoA for the chain elongation process in preparation of 6-deoxyerythronolide B. More

recently, McDaniel and co-workers manipulated several genetic modules within

polyketide synthase and generated a library of more than 50 macrocycles that would be

impractical to produce by chemical synthesis (select examples, Figure 3.3).52

Figure 3.3 – Select examples of 6-deoxyerythronolide B analogues generated by site-directed mutagenesis of polyketide synthase domains (McDaniel, 1999)

In this work, the authors systematically engineered single and multiple enzymatic domain

substitutions in deoxyerythronolide B synthase (DEBS) to demonstrate the utility of PKS

mutagenesis techniques. Firstly, substitutions were made to the acyl transferase (AT)

domain that resulted in mutants incorporating acetate rather than propionate units to

generate analogues lacking a methyl substituent at the engineered position (see 3.10,

                                                                                                               

50 Weber, J. M.; Leung, J. O.; Maine, G. T.; Potenz, R. H. B.; Paulus, T. J.; DeWitt, J. P. J. Bacteriol. 1990, 172, 2372. 51 Corcoran, J. W.; Vygantas, A. M. Fed. Proc. 1977, 36, 663. 52 McDaniel, R.; Thamchaipenet, A.; Gustaffson, C.; Fu, H.; Betlach, M.; Betlach, M.; Ashley, G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1846–1851.  

O

O

OH

O

Et

OH

OH

O

O

OH

O

Et

OH

OH

O

O

O

Et

OH

OH

O

O

OH

O

Et

OH

O

O

OH

O

Et

O

OH

O

O

OH

O

Et

OH

O

(3.10) (3.11) (3.12) (3.13) (3.14) (3.15)

  25

3.11). Similarly, mutagenesis allowed for replacement of β-ketoacyl- and enoyl

reductases with domains from the rapamycin PKS that resulted in the corresponding

alcohol moieties being replaced with alkene and alkane carbons (see 3.12, 3.13). Lastly,

deletion mutagenesis of the reductase domains converted hydroxyl groups to ketones in

several examples (see 3.14, 3.15). Combining these genetic alterations in varying orders

allowed for rapid access to a large library of 6-deoxyerythronolide derivatives. These

novel compounds could in themselves provide the basis for new pharmaceuticals or could

serve as scaffolds for new semisynthetic analogues.

3.2 Total synthesis of the erythromycins

Beyond its impact on human medicine, erythromycin A has been closely tied to the

evolution of synthetic organic chemistry. Its discovery has prompted numerous total

syntheses of erythromycin biosynthetic precursors over the past 35 years (Figure 3.4).53

Figure 3.4 – Total syntheses of erythromycin derivatives                                                                                                                  

53 Gao, X.; Woo, S. K.; Krische, M. J. J. Am. Chem. Soc. 2013, 135, 4223–4226. 54 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3215. 55 Martin, S. F.; Hida, T.; Kym, P. R.; Loft, M.; Hodgson, A. J. Am. Chem. Soc. 1997, 119, 3193. 56 Corey, E. J.; et al. J. Am. Chem. Soc. 1979, 101, 713. 57 Nakata, M.; Arai, M.; Tomooka, K.; Ohsawa, N.; Kinoshita. M. Bull. Chem. Soc. Jpn. 1989, 62, 2618. 58 Muri, D.; Lohse-Fraefel, N.; Carreira, E. M. Angew. Chem., Int. Ed. 2005, 117, 4036. 59 Corey, E. J.; et al. J. Am. Chem. Soc. 1978, 100, 4620. 60 Sviridov, A. F.; et al. Tetrahedron Lett. 1987, 28, 3839. 61 Mulzer, J.; Kirstein, H. M.; Buschmann, J.; Lehmann, C.; Luger, P. J. Am. Chem. Soc. 1991, 113, 910. 62 Masamune, S.; Hirama, M.; Mori, S.; Ali, S. A.; Garvey, D. S. J. Am. Chem. Soc. 1981, 103, 1568. 63 Myles, D. C.; Danishefsky, S. J. J. Org. Chem. 1990, 55, 1636. 64 Evans, D. A.; Kim, A. S. Tetrahedron Lett. 1997, 38, 53. 65 Stang, E. M.; White, M. C. Nat. Chem. 2009, 1, 547.  

Erythromycin A – R1 = OH, R2 = D-desosamine, R3 = L-cladinose, R4 = OH Woodward (1981): 55 steps (LLS), 77 steps (TS)54

Erythromycin B – R1 = OH, R2 = D-desosamine, R3 = L-cladinose, R4 = H Martin (1997): 28 steps (LLS), 33 steps (TS)55

Erythronolide A – R1 = OH, R2 = H, R3 = H, R4 = OH Corey (1979): 39 steps (LLS), 50 steps (TS)56

Kinoshita (1989): 50 steps (LLS), 74 steps (TS)57

Carreira (2005): 26 steps (LLS), 36 steps (TS)58

Erythronolide B – R1 = OH, R2 = H, R3 = H, R4 = H Corey (1978): 33 steps (LLS), 47 steps (TS)59

Kochetkov (1987): 36 steps (LLS), 51 steps (TS)60

Mulzer (1991): 27 steps (LLS), 41 steps (TS)61

6-deoxyerythronolide B – R1 = H, R2 = H, R3 = H, R4 = H Masamune (1981): 26 steps (LLS), 39 steps (TS)62

Danishefsky (1990): 42 steps (LLS), 42 steps (TS)63

Evans (1997): 23 steps (LLS), 28 steps (TS)64

White (2009): 23 steps (LLS), 25 steps (TS)65

O

O

OH

O

Et

OR3

OR2

R1R4

  26

Notably, the only reported total synthesis of erythromycin A is that of Woodward in 1981

(55 steps, LLS). This is likely due to the inherent complexity of the erythromycin A

aglycon, with its 10 stereocenters (five of which are consecutive) and five free hydroxyl

groups. Furthermore, regio- and stereoselective glycosidation of the aglycon presented a

significant challenge.

The vast majority of erythromycin and erythronolide total syntheses follow the same

strategy.66 The protected aglycons are formed by lactonization of a seco acid backbone.

These seco acids are constructed by coupling smaller chiral fragments that are obtained

by chiral resolution or enantioselective synthesis. Indeed, the evolution of enantio- and

diastereomeric control has assisted in decreasing the step count of aglycon synthesis and

eliminated the requirement of chiral resolution.67

The seco acid target of Woodward’s erythromycin A synthesis is shown in Figure 3.5.

Protection of the C-9 ketone and the C-3, C-5 and C-11 hydroxyl groups proved

necessary for the lactonization step in order to prevent polymerization and undesired

cyclizations. Additionally, their protective group strategy was instrumental for inducing

conformations favourable for cyclization. For example, the 3,5-acetal unit in 3.16 locks

the C2-C6 fragment of the molecule into a rigid, linear structure due to the diequatorial

nature of the 1,3-dioxane chair. This allows for the 6-OH group to remain unprotected

because it can only participate in lactonization after flipping the acetal to the diaxial

conformation.68

Figure 3.5 – Seco acid derivative for erythromycin A synthesis (Woodward, 1981)

                                                                                                               

66 Paterson, I.; Mansuri, M. M. Tetrahedron, 1985, 41, 3569. 67 Bartlett, P. A. Tetrahedron. 1980, 36, 1. 68 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3210.  

Me Me Me

O

MeOH

Me Me

OOHOH

Me

OO O

HH OHH

Me Me Me

NH

MeOH

Me Me

OOO

Me

OO O

HH OH

35691112

RO

erythronolide A seco acid Woodward's seco acid target (3.16)

  27

Following preparation of seco acid 3.16, Woodward and co-workers used the Corey-

Nicolaou double activation method for macrolactonization of 3.16 (Scheme 3.3).69 After

deprotection of 3.17, the next task was to glycosylate the aglycon. Previous efforts with

an erythronolide A derivative revealed that glycosylation of the C-5 hydroxyl group was

more favourable than the C-3 and C-11 hydroxyls.70 Thus, glycosylation of 3.18 with an

O-2’ protected D-desosamine thioglycoside was attempted, which furnished the desired

O-5 functionalized product (3.19) in 36% yield. The use of O-2’ protected D-desosamine

was crucial for the subsequent glycosylation with L-cladinose because, if left

unprotected, functionalization of the 2’-OH is preferred over the 3-OH group of the

aglycon. Glycosidation of L-cladinal with 3.19 and methanolysis of the O-2’ ester group

gave 3.20 in 55% yield. Finally, deprotection of the macrolide and regeneration of the C-

9 ketone afforded erythromycin A (3.1).

Scheme 3.3 – Key steps in Woodward’s total synthesis of erythromycin A

The synthesis of erythromycin A by Woodward and co-workers involved the

collaboration of 49 scientists and took nearly ten years to complete. This work serves as a

testimony for the sheer difficulty of its synthesis. Although erythromycin A can be                                                                                                                

69 Corey, E. J.; Nicolaou, K. C. J. Am. Chem. Soc. 1974, 96, 5614–5616. 70 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3216.  

Me Me Me

NH

MeOH

Me Me

OOO

Me

OO O

HH OH

35691112

O

1) N

SS N

Me Me

Me

Ph3P

2) toluene, 110 oC O

OOH

O

Et

HO

O

O

HNO

Me

Me

Me70%

(3.16) (3.17)

O

OHOH

O

Et

HO

OH

OH

HN

(3.18)

BPCO

S O

N(CH3)2

CH3O

OMe

ON

N AgOTf

36%

O

OHOH

O

Et

HO

OH

O

HNBPCO

O

N(CH3)2

CH3O

O OMe

OCH3

OAcCH3

OCH3

Pb(ClO4)2, MeCN

S

N

55%

1)

2) MeOH

HN

O

OHOH

O

Et

HO

O

O

OCH3

OAcCH3

OCH3

O

N(CH3)2

CH3HO

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

(3.19)(3.20)(3.1)

BPCO

3

5

911

9

5

3

11

911

5

3

2'

911

5

3

2'

  28

obtained in large quantities by fermentation, the total syntheses of its derivatives have led

to the development of new synthetic methodology that can be applied to other complex

natural products that may not be accessible by alternative means.

3.3 Acid-catalyzed rearrangements of erythromycin A

One of the major drawbacks of erythromycin A is its remarkable acid sensitivity, leading

to degradation in the stomach following oral administration.71 Outside of clinical use, the

groups of Corey and Carreira also observed its susceptibility to acidic degradation in their

total syntheses of erythronolide A.72,73

It has long been known that erythromycin A converts rapidly under acidic conditions to

erythromycin A enol ether (3.21) and anhydroerythromycin A (3.22), eliminating its

antibiotic activity. Indeed, this rapid inactivation necessitates the administration of large

doses in humans.

Figure 3.6 – Erythromycin A enol ether and anhydroerythromycin A

Barber and co-workers have completed extensive kinetic studies over the past 20 years to

determine the degradation mechanism of erythromycin A.74,75,76 In their work, they

showed that erythromycin A enol ether and anhydroerythromycin A are in equilibrium

                                                                                                               

71 Mordi, M. N.; Pelta, M. D.; Boote, V; Morris, G. A.; Barber, J. J. Med. Chem. 2000, 43, 467–474. 72 Schomburg, D.; Hopkins, P. B.; Lipscomb, W. N.; Corey, E. J. J. Org. Chem. 1980, 45, 1544–1546. 73 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712. 74 Alam, P.; Buxton, P.C.; Embrey K. J.; Parkinson, J. A.; Barber, J. Magn. Reson. Chem. 1996, 559–561. 75 Awan, A.; Brennan, R. J.; Regan, A. C.; Barber, J. J. Chem. Soc., Perkin Trans. 2. 2000, 2, 1645–1652. 76 Hassanzadeh, A.; Barber, J.; Morris, G. A.; Gorry, P. A. J. Phys. Chem. A. 2007, 111, 10098–10104.  

O

O

Et

O

O

OOH

HO

O

N(CH3)2CH3

HO

OCH3

OHCH3

OCH3

CH3

O

O

Et

O

OCH3

OHCH3

OCH3

O O

HO

O O

N(CH3)2CH3

HO

erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)

  29

with erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C. It was also

noted that erythromycin A exists as both 6,9- and 9,12-cyclic hemiketal tautomers (3.23,

3.24) under neutral aqueous conditions, albeit in relatively small quantities with the 9,12-

hemiketal preferred. These hemiketal intermediates were rapidly converted to their

respective enol ether and anhydro forms when exposed to acidic conditions. (Scheme

3.4).

Scheme 3.4 – Acid degradation mechanism of erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C

An alternative pathway is the hydrolysis of L-cladinose from the aglycon of erythromycin

A to give 5-desosaminylerythronolide A (3.25). This process was found to be

irreversible and significantly slower than the tautomerization pathways.

Prediction of conditions that lead to the selective formation of either erythromycin A enol

ether or anhydroerythromycin A is not trivial because the tautomerization and

dehydration steps in the erythromycin degradation pathway are reversible.77 Through

                                                                                                                77 Hassanzadeh, A.; Barber, J.; Morris, G. A.; Gorry, P. A. J. Phys. Chem. A. 2007, 111, 10098–10104.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

HO

O

O

Et

O

O

OOH

HO

O

N(CH3)2CH3

HO

OCH3

OHCH3

OCH3

CH3

O

O

Et

O

O O

N(CH3)2CH3

HO

OCH3

OHCH3

OCH3

O

OHHO OH

O

O

Et

O

OCH3

OHCH3

OCH3

O O

HO

O O

N(CH3)2CH3

HO

O

O

OHOH

O

Et

HO

OH

O O

N(CH3)2CH3

HO

erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)

erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)

5-desosaminylerythronolide A (3.25)

1

12 6

9

1

612

9

9

612

11

12

9

6

O

O

Et

O

O

OOH

HO

O

N(CH3)2CH3

HO

OCH3

OHCH3

OCH3

6,9-hemiketal of erythromycin (3.23)

1

12

9

6

HO

  30

experimentation, standard conditions have been developed to form each as the major

product. Erythromycin A enol ether (3.21) can be synthesized by subjecting erythromycin

A (3.1) to glacial acetic acid at room temperature.78 Alternatively, anhydroerythromycin

A (3.22) can be formed by exposing erythromycin A enol ether (3.21) to methanolic

hydrochloric acid at room temperature.79

3.4 Semisynthetic analogues of erythromycin A

Knowledge of the chemical basis for erythromycin A’s acid instability prompted the

development of semisynthetic macrolides that lacked this significant limitation. As

shown in Scheme 3.4, nucleophilic attack at the C-9 ketone is the cause of erythromycin

A enol ether and anhydroerythromycin A formation. To discourage acid-catalyzed

rearrangements, Taisho Pharmaceutical Co. developed a 6-step sequence to selectively

methylate the C-6 hydroxyl substituent, affording the antibiotic clarithromycin (3.26,

Scheme 3.5).80 By functionalizing O-6, the possibility of enol ether formation is

eliminated. Although formation of the 9,12-hemiketal is still possible, the 6-OH group is

no longer available to participate in forming anhydroerythromycin A. In addition to being

both acid-stable and orally active, clarithromycin displays a slightly expanded

antimicrobial spectrum relative to erythromycin A.

Scheme 3.5 – Semisynthesis of clarithromycin (Taisho, 1980)

                                                                                                               

78 Alam, P.; Buxton, C.; Parkinson, J. A.; Barber, J. J. Chem. Soc. Perkin Trans. 2. 1995, 1163–1168. 79 Kurath, P.; Jones, P. H.; Egan, R. S.; Perun, T. J. Experientia. 1971, 27, 362. 80 Morimoto, S.; Takahashi, Y.; Watanabe, Y.; Omura, S. J. Antibiot. 1984, 37, 187–189.  

N

O

OHOH

O

Et

HO

O

O

OCH3

OTMSCH3

OCH3

O

N(CH3)2

CH3O

OOi-Pr

TMS

N

O

OHOMe

O

Et

HO

O

O

OCH3

OTMSCH3

OCH3

O

N(CH3)2

CH3O

OOi-Pr

TMSKOH

MeI

O

O

OHOMe

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

1) HCO2H

2) NaHSO3

oxime intermediate(3 steps from erythromycin A)

clarithromycin (3.26)

  31

Another innovative semisynthetic strategy to reduce the chemical instability of

erythromycin A was developed by Pliva in 1980. In this case, the C-9 ketone was

completely removed from the erythromycin scaffold in a 4-step sequence to give

azithromycin (3.28, Scheme 3.6).81 The first step in the synthesis involved formation of

an oxime to protect the C-9 ketone. Then, the aglycon underwent ring expansion through

a Beckmann rearrangement to give an iminoether (3.27). Hydrogenolysis of 3.27 and

subsequent N-methylation led to the discovery of an “azalide” structure that became

known as azithromycin. Azithromycin was found to have excellent acid stability, oral

bioavailability, and an expanded antimicrobial spectrum relative to erythromycin A. In

1991, azithromycin gained FDA approval and rose to the 7th most prescribed drug in the

U.S. in 2010.

Scheme 3.6 – Semisynthesis of azithromycin (Pliva, 1980)

3.5 Regioselective functionalization of erythromycin A

In 2006, the group of Miller was the first to report a site-selective, catalytic method for

acylation of erythromycin A. With three secondary hydroxyl groups and two tertiary

hydroxyl groups, erythromycin A presents a challenge for regioselective catalysis. A

seminal report from Abbott Laboratories revealed that the C-2’ hydroxyl group on the

desosamine sugar of erythromycin A was the most reactive towards acetylation using

acetic anhydride in pyridine (Figure 3.7)82 The next most reactive position was the C-4”

                                                                                                               

81 Kobrehel, G.; Radobolja, G.; Tamburasev, Z.; Djokic, S. 11-Aza-4-0-cladinosyl-6-0-desosaminyl-15-ethyl-7,13,14-trihydroxy-3,5,7,9,12,14-hexamethyloxacyclopentadecan-2-one derivatives as well as process for their production, DE3012533A1, 1980. 82 Jones, P. H.; Baker, E. J.; Rowley, E. K.; Perun, T. J. J. Med. Chem. 1972, 15, 631–634.  

N

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

OH

erythromycin A oxime

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

N

PhSO2Cl

NaHCO3O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

NH3C

1) H2, Pt

2) CH2O, HCO2H

azithromycin (3.28)(3.27)

  32

hydroxyl on the cladinose sugar, as evidenced by preferential formation of a C2’,C4”-

diacetate when additional Ac2O is used. Finally, the least reactive secondary site was the

C-11 hydroxyl group on the aglycon, which acetylates to form a C2’,C4”,C11-triacetate

after prolonged reaction time. The tertiary alcohols are significantly less reactive under

these conditions and acetylation was not observed at these sites. Interestingly, the C2’-

actetate can be cleaved when the reaction is quenched with methanol. This phenomenon

has been attributed to the autocatalytic nature of the tertiary amine-containing

desosamine sugar.

Figure 3.7 – Inherent reactivity of the hydroxyl groups in erythromycin A

The goal of the Miller group was to identify a small molecule catalyst that would reverse

the inherent reactivity such that the 11-OH group would be modified preferentially over

the more reactive 2’-OH and 4”-OH groups. They examined 137 peptide catalysts chosen

at random from their catalyst libraries. Notably, most of the peptides displayed pyridine-

like behavior, favouring the C2’,C4”-diacetate. However, when peptides containing β-

turn-like structures were employed, a reversal in selectivity was observed. Overall, their

approach created a bias towards formation of the C2’,C11-diacetate as opposed to the

C2’,C4”-diacetate.83 It should be noted that preferential acetylation of the 2’-OH group

was unavoidable under their reaction conditions. However, methanolysis of the C2’,C11-

diacetate revealed the C11-monoacetate as the major product (Scheme 3.7). The product

distribution after methanolysis was as follows: C11-monoacetate (37%), recovered

erythromycin A (37%), C4”-monoacetate (8%), and C4”,C11-diacetate (9%).

Interestingly, the C11-monoacetate exists almost exclusively as its hemiketal tautomer.

                                                                                                               

83 Lewis, C. A.; Miller, S. J. Angew. Chem. Int. Ed. 2006, 188, 5744–5747.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

- 2'-OH, most reactive- Biologically inactive upon functionalization

- 4"-OH, 2nd most reactive

- 11-OH, 3rd most reactive- Desired selectivity

  33

Everett and co-workers have rationalized the hemiketalization of C11-monoacylated

erythromycin A derivatives as a consequence of the loss of a macrolide-stabilizing

hydrogen bond across the C11-OH and C9 ketone in native erythromycin.84

Scheme 3.7 – Site-selective acylation of erythromycin A using a peptide catalyst (Miller, 2006)

3.6 Research goals

Our goal was to selectively functionalize erythromycin A using the organoboron-

mediated methodology previously developed in our group. The presence of the cis-vicinal

diol on the aglycon at C11-C12 served as the target for activation with diarylborinic acids

(Ar2BOH) and aryl boronic acids [ArB(OH)2]. As shown in the work of Miller, the C-11

hydroxyl group is the least reactive of the secondary alcohols present in erythromycin A.

Therefore, our methodology would have to bias selectivity towards the C11-OH as

opposed to the C2’-OH and C4”-OH groups.

Scheme 3.8 – Proposed regioselective monofunctionalization of erythromycin A catalyzed by a diarylborinic acid

                                                                                                               

84 Everett, J. R.; Hunt, E.; Tyler, J. W. J. Chem. Soc. Perkin Trans. 2. 1991, 1481–1487.

O

O

Et

O

O O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

O

OHO OH

1

612

9

O

O

OOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

1

12 6

9

N

N

NH

Me N

O

Boc

HNH

O Me MeO

HN

NBocNHO

Ph OMe

O(5 mol%)

Ac2O (2 equiv.), NEt3 (5 equiv.), CHCl3, RT 24 hr

OO

37%, major product

1)

2) MeOH, RT 72 hrC11-monoacetate (3.29)

3.1

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

HO

O

O

OOH

O

Et

O

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

HO

BPh Ph

Ar2BOH (cat.) electrophile E+

O

O

OOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

E

  34

3.7 Results and discussion

3.7.1 Glycosylation of erythromycin A

The work of Scott Miller and co-workers showed that regioselective acylation of

erythromycin A was possible using a small molecule catalyst. Therefore, we decided to

attempt glycosylations using the borinic acid-catalyzed methodology previously

developed in our group. Similar conditions to that of the digitoxin work from our group

were used as a starting point. Erythromycin A was subjected to peracetylated glucosyl

bromide donor 3.31) (2 equiv.), Ag2O (2 equiv.) and 25 mol% of 2-aminoethyl

diphenylborinate (3.30) in acetonitrile for 24 hours at 23 °C. Following silica gel

chromatography, <5% of the O-2’ glucosylated product (3.32) was observed, with

recovered erythromycin A accounting for 87%. The control reaction (without catalyst)

provided equivalent results in terms of regioselectivity and yield. Increasing the loading

of 3.30 had no observed effect in terms of selectivity and yield. Additionally, increasing

reaction time to 48 hours and electrophile loading to 5 equivalents had little to no effect

on the outcome of the reaction.

  35

Table 3.1 – Borinic acid-mediated glycosylationa

Entry Catalyst loading (mol%) Yieldb (%)

1 0 <5

2 25 <5

3 100 <5

a Reaction conditions: erythromycin A (0.068 mmol), catalyst (0–100 mol%), peracetylated glucosyl bromide donor (0.136 mmol), Ag2O (0.136 mmol), MeCN (6 mL). b Isolated yield.

Based on our work with pentasaccharide target 1.1 (see Scheme 1.10), the stoichiometric

boronic acid-mediated glycosylation method appeared to be a suitable alternative in cases

when the catalytic borinic acid conditions failed to produce favourable results.

Differences from the catalytic method include solvent choice (CH2Cl2), addition of a

Lewis base (NEt3) and stoichiometric use of a boronic acid instead of borinic acid

precatalyst 3.30. Furthermore, the presence of molecular sieves has been noted to affect

results in some cases. (Pentafluorophenyl)boronic acid (3.33) was chosen as the boron

source because it gave favourable results in glycosylations previously attempted in our

group.85 The results from the stoichiometric boronic acid-mediated glycosylations are

summarized in Table 3.2.

                                                                                                               

85 McClary, C. A. 2013. Exploring Noncovalent and Reversible Covalent Interactions as Tools for Developing New Reactions. (Doctor of Philosophy Dissertation).

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

OB

NH2

Ph

Ph

(x mol%)

Ag2O MeCN, 23 oC

24 hr

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3O

O

OOAc

OAc

AcOAc

OAcOAcO

BrAcO

OAc

(3.30)

(3.1) (3.32)

(3.31)

  36

Table 3.2 – Boronic acid-mediated glycosylationa

Entry Boronic acid 4Å MS Yieldb (%)

1 none yes 6

2 none no 8

3 3.32 yes <5

4 3.32 no <5

a Reaction conditions: erythromycin A (0.068 mmol), (pentafluorophenyl)boronic acid (0.068 mmol), peracetylated glucosyl bromide donor (0.136 mmol), Ag2O (0.136 mmol), NEt3 (0.204 mmol), DCM (6 mL). b Isolated yield.

The stoichiometric method is carried out using either a one-pot reaction setup or a two-

step procedure. The former involves complexation of the boronic acid with the diol in

CH2Cl2 for 6 hours at room temperature, followed by addition of Lewis base, glycosyl

donor and Ag2O. The latter is accomplished by complexing the boronic acid and diol in

toluene for 3 hours at 110 °C, followed by removing the solvent in vacuo. To the

resulting solid are added DCM, Lewis base, glycosyl donor, and Ag2O. While both

methods were attempted, the results displayed in Table 3.2 are from the two-step

procedure.

Identical selectivity and similar yields were observed for the control and boronic acid-

mediated reactions. The presence of molecular sieves did not have a significant effect in

terms of yield. Notably, the one-pot and two-step complexation methods gave trace yields

of 3.32. As with the borinic acid-mediated method, increasing reaction time to 48 hours

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

Ag2O, NEt3DCM, 23 oC

24 hr

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3O

O

OOAc

OAc

AcOAc

OAcOAcO

BrAcO

OAcFB(OH)2

F

FF

F

(3.33)

(3.1) (3.32)

(3.31)

  37

and electrophile loading to 5 equivalents had no observable effect on the outcome of the

reaction.

At this point, we had not observed any differences in selectivity between the control and

organoboron-mediated reactions. The inherent bias towards functionalization of the C-2’

hydroxyl group on the desosamine sugar could not be modified under the reaction

conditions employed. However, it is difficult to draw meaningful conclusions from these

results because the extent of starting material conversion was nearly negligible. Thus, it

was clear that the reaction conditions and/or choice of electrophile would need to be

modified. Increasing the reaction temperature was thought be a suitable option.

Alternatively, a more “armed” glycosyl donor, such as perbenzylated glucosyl bromide,

could be used. Ultimately, the decision was made to switch the electrophile to benzoyl

chloride. It was envisioned that our previously developed benzoylation methodology

would result in a greater extent of erythromycin functionalization, such that differences in

regioselectivity may be observed.

3.7.2 Benzoylation of erythromycin A

The first step towards developing a procedure for selective benzoylation of erythromycin

A was to synthesize and characterize any products that could form under boron-free

conditions. This would make the screening process more efficient by enabling quick

comparison of pure compounds to those present in crude reaction mixtures. When using

acetic anhydride with pyridine as the solvent, Scott Miller and co-workers reported the

formation of the C2’-monoacetate, C2’,C4”-diacetate and C2’,C4”,C11-triacetate when

the reaction was at room temperature for 72 hours. Expecting similar results, we

subjected erythromycin A to benzoic anhydride (3 equiv.) in pyridine at 23 °C for 72

hours. Interestingly, the only product observed was C2’-monobenzoylated erythromycin

A (3.34) in 92% isolated yield (Scheme 3.9).

  38

Scheme 3.9 – Monobenzoylation of erythromycin A using benzoic anhydride in pyridine

Despite observing only monofunctionalization, we did not conclude that

difunctionalization would be required to see differences in regioselectivity between the

control and catalyzed reactions under our conditions. Therefore, benzoylation was

attempted using conditions similar to those previously described in our carbohydrate

acylation work. When 3.1 was subjected to benzoyl chloride (3 equiv.), DIPEA (3 equiv.)

and boronic/borinic acid at 23 °C for 24 hours, C2’-monobenzoylated erythromycin A

(3.34) was the major product in all cases. In the control reaction, a yield of 87% was

obtained for the C2’-monobenzoylated product, with 5% recovered erythromycin A. The

organoboron-mediated reactions provided the same regioselectivity as the control

reaction but a new product was observed. After purification by silica gel chromatography,

C2’-monobenzoylated erythromycin A enol ether (3.35) was recovered as a minor

product in the reactions with boronic and borinic acids. Notably, the yield of 3.35

increased from 15% to 26% when the amount of 2-aminoethyl diphenylborinate was

increased from 0.25 equivalents to 1 equivalent. The yield of 3.35 increased further when

the 2-step stoichiometric boronic acid procedure was employed.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO (3 equiv.)

pyridine23 oC, 72 hr

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3O

(3.1) (3.34)

BzPh O

O

Ph

O

92%

  39

Table 3.3 – Organoboron-mediated benzoylation at 23 °Ca

Entry Boron source Yield (%)c [3.34] Yield (%)c [3.35]

1 none 87 0

2 2-aminoethyl diphenylborinate (0.25

equiv.)

72 15

3 2-aminoethyl diphenylborinate (1 equiv.)

63 26

4 (pentafluorophenyl)boronic acid (1 equiv.)b

55 34

a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt (0.204 mmol), MeCN (6 mL). b 2-step procedure with NEt3 (0.204 mmol) as the Lewis base and DCM as the solvent. c Determined by 1H NMR of the crude reaction mixture after elution through a silica gel plug.

These results suggest that the organoboron species is participating in the reaction and is

promoting intramolecular rearrangement of erythromycin A to its enol ether form. Based

on this observation, it was difficult to say whether benzoylation or enol ether formation

occurred first. Regardless, the organoboron reagents did not alter the regiochemical

outcome of the reaction, nor increase the yield of C2’-monobenzoylated erythromycin A

relative to the control reaction. Another interesting result was that

(pentafluorophenyl)boronic acid reaction yielded less of 3.34 and nearly 10% more C2’-

monobenzoylated erythromycin A enol ether (3.35) compared to the borinic acid reaction

when used in equivalent stoichiometric amounts. Perhaps the thermally promoted

condensation of 3.33 with erythromycin A in the two-step stoichiometric method

promoted enol ether formation even before benzoyl chloride was introduced to the

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3O

(3.1) (3.34)

BzBoron source (x equiv.)BzCl, i-Pr2NEt

MeCN, 23 oC24 hr

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3O

OCH3

OHCH3

OCH3

CH3

(3.35)

Bz

  40

reaction. This could explain the decreased ratio of 3.34:3.35 in the boronic acid-mediated

reaction relative to the borinic acid reaction.

Although interesting results were obtained from the organoboron-mediated reactions, the

desired O-11 selectivity was not achieved. The extent of erythromycin A

functionalization increased significantly for benzoylation compared to glycosylation but

the regiochemical outcome remained the same. Perhaps, like the work of Miller, we

would require difunctionalization to observe differences in regioselectivity between the

control and organoboron-mediated reactions. In attempt to accomplish this, the reaction

temperature was increased to 80 °C, with the remaining parameters unchanged (Table

3.4).

At 80 °C, the extent of C2’-monobenzoylated enol ether formation increased significantly

for the control and organoboron-mediated reactions. Moreover, when stoichiometric

boronic/borinic acid was used, C2’-monobenzoylated erythromycin A (3.34) was not

observed. Furthermore, a new product was observed when boronic or borinic acids were

employed. In the cases where stoichiometric organoboron reagent was used, nearly 50%

of the reaction mixture contained unfunctionalized erythromycin A enol ether (3.21).

This was an interesting observation because the presence of organoboron reagent resulted

in a significant decrease in benzoylation compared to the control reaction. This result

provided insight to the question of whether benzoylation or enol ether formation occurs

first. Perhaps the organoboron reagent promoted formation of erythromycin A enol ether

(3.21), which discouraged functionalization of O-2’ relative to native erythromycin A.

  41

Table 3.4 – Organoboron-mediated benzoylation at 80 °Ca

Entry Boron source Yield (%)c [3.34]

Yield (%)c [3.35]

Yield (%)c [3.21]

1 none 57 35 0

2 2-aminoethyl diphenylborinate (0.25

equiv)

28 54 9

3 2-aminoethyl diphenylborinate (1 equiv.)

0 52 44

4 (pentafluorophenyl)boronic acid (1 equiv.)b

0 49 47

a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt (0.204 mmol), MeCN (6 mL). b 2-step procedure with NEt3 (0.204 mmol) as the Lewis base and MeCN as the solvent. c Determined by 1H NMR of the crude reaction mixture after elution through a silica gel plug.

To test this hypothesis, erythromycin A enol ether (3.21) was prepared according to a

literature procedure.86 Then, it was subjected to benzoyl chloride (3 equiv.) and DIPEA

(3 equiv.) in MeCN for 24 hours at 23 °C (Scheme 3.10). After purification by silica gel

chromatography, C2’-monobenzoylated enol ether (3.35) was isolated in 32% yield, with

65% recovered starting material. Under equivalent conditions, erythromycin A formed

the C2’-monobenzoylated product (3.4) in 87% yield, with 5% recovered starting

material (Table 3.3). Comparing these results illustrates that the formation of

erythromycin A enol ether discourages benzoylation. Furthermore, the presence of

                                                                                                                 86 Alam, P.; Buxton, C. P.; Parkinson, J. A.; Barber, J. J. Chem. Soc. Perkin Trans. 2. 1995, 1163–1167.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3O

(3.34)

BzBoron source (x equiv.)BzCl, i-Pr2NEt

MeCN, 80 oC24 hr

3.1O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3O

OCH3

OHCH3

OCH3

CH3

(3.35)

Bz

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

(3.21)

  42

organoboron reagent, whether catalytic or stoichiometric, appears to accelerate

erythromycin A enol ether formation at both 23 °C and 80 °C.

Scheme 3.10 – Monobenzoylation of erythromycin A enol ether under boron-free conditions

Evidently, when subjected to benzoyl chloride under thermal conditions, erythromycin A

was significantly less stable than at room temperature. Addition of organoboron-reagent

further complicated the reaction by promoting the formation of undesired by products,

which discouraged benzoylation. From here, the goal was to reduce enol ether formation,

while attempting to increase the extent of benzoylation.

To accomplish this goal, erythromycin A was subjected to the same organoboron reagent

screen as described in Table 3.3 at 23 °C for 72 hours. Though only preliminary results

were obtained, it was noted that C2’-monobenzoylated erythromycin A (3.34) was the

major product in the control and boronic acid-mediated reactions. Formation of

dibenzoylated products was not observed. Interestingly, the reaction with stoichiometric

2-aminoethyl diphenylborinate (3.30) gave a complex mixture of products that were

inseparable by silica gel chromatography. In pursuit of purifying this reaction mixture,

semi-preparative reversed-phase HPLC and LC-MS were employed. Preliminary

screening of the crude reaction mixture with LC-MS showed m/z peaks corresponding to

3.35 and 3.21 but not C2’-monobenzoylated erythromycin A (3.34). Though this wasn’t

an entirely unexpected result, it seemed unusual that 3.34 was not observed – especially

considering that the boronic acid-mediated reaction gave 3.34 as the major product under

the same conditions. In attempt to troubleshoot this problem, pure erythromycin A was

subjected to semi-preparative reversed-phase HPLC and LC-MS. Information from mass

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3O

OCH3

OHCH3

OCH3

CH3

(3.35)

Bz

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

(3.21)

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

(3.36)

BzCl, i-Pr2NEt

MeCN, 23 oC24 hr

32% 65%

  43

spectrometry alone was not sufficient to identify the isolated compound. This is because

the acid-catalyzed rearrangement products of erythromycin A have identical molecular

masses in certain instances (Scheme 3.11). After characterization by 1H and 13C NMR, it

was revealed that erythromycin A (3.1) was converted quantitatively to

anhydroerythromycin A (3.22), which gave a signal of (M-18)+. This result prompted

investigation of the conditions used for separation. A gradient of 80 → 5% H2O in MeCN

was employed with 0.1% formic acid as the buffer. The presence of buffer in the mobile

phase is imperative to obtain effective separation of ionizable compounds and its identity

should be based on the compounds being separated.87 Given the acid-sensitivity of

erythromycin A, we hypothesized that formic acid was likely the cause of

anhydroerythromycin A formation and, thus, was an incompatible buffer choice for the

separation.

Scheme 3.11 – Erythromycin A acid-catalyzed rearrangement products and their molecular masses

                                                                                                                 

87 Unger, K. K.; Ditz, R.; Machtejevas, E.; Skudas, R. Angew. Chem. Int. Ed. 2010, 49, 2300–2312.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

O

O

Et

O

O O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

O

OHHO OH

O

O

Et

O

OCH3

OHCH3

OCH3

O O

HO

O O

N(CH3)2

CH3HO

erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)

erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)

O

O

Et

O

O

OOH

HO

O

N(CH3)2

CH3HO

OCH3

OHCH3

OCH3

6,9-hemiketal of erythromycin (3.23)

HO

m/z = 733m/z = 733 m/z = 733

m/z = 715 m/z = 715

  44

Notably, Miller and co-workers relied extensively on semi-preparative reversed-phase

HPLC for purification of acetylated erythromycin A regioisomers. Their choice of buffer

for separation of the regioisomers was potassium phosphate dibasic (K2HPO4), which has

an optimal buffering range of pH = 6.2–8.2. Future efforts in our group are directed

towards optimizing this separation by using similar HPLC conditions to those employed

by Miller and co-workers.

3.7.3 NMR experiments with erythromycin A

Although regioselective functionalization was not accomplished using our methodology,

there was interest in gaining a better understanding of the interaction between boronic

and borinic acids with erythromycin A. Foremost, we wanted to establish whether the

organoboron reagents were capable of binding to the cis-vicinal diol in erythromycin A.

In light of the results obtained for benzoylation, we also wished to study the effect of

boron reagents 3.30 and 3.33 on enol ether formation in absence of electrophile.

NMR spectroscopy has been instrumental in our group for observing boron-diol

complexation in organic solvents. In terms of boronic ester formation, monitoring

chemical shift changes in 1H NMR is a useful method for determining which site(s) of the

substrate the boronic acid complexes. Furthermore, analyzing chemical shift changes in 19F NMR can be beneficial when using boronic acids containing fluorine groups. To

begin, erythromycin A was complexed with (pentafluorophenyl)boronic acid (3.33) in

toluene at 110 °C. One of the first challenges was finding a suitable deuterated solvent

that would dissolve the reaction mixture after complexation. The use of protic solvents

would interfere with the boron-diol equilibria, thus polar protic solvents such as methanol

and water were not suitable. Additionally, acetonitrile, acetone, chloroform,

dichloromethane, and toluene were unable to solubilize the reaction mixture at room

temperature. Deuterated DMSO was effective in this regard but led to inconclusive

results by 1H and 19F NMR. When comparing 19F NMR spectra of 3.33 in CDCl3 to that

of DMSO, we noted that the expected 3 signals were now more than 10 separate signals,

suggesting interaction of DMSO with the boronic acid or that trace water in the solvent

  45

could have caused protodeborylation. Despite being the only solvent to dissolve the

reaction mixture, DMSO was unsuitable for this experiment.

Next, we turned our attention to studying the interaction between borinic acids and

erythromycin A. Previous studies in our group revealed that simple diol substrates such

as cis-1,2-cyclohexanediol were incapable of displacing the ethanolamine ligand from

precatalyst 3.30. In contrast, the free base of diphenylborinic acid (3.37) was effective in

complexing with cis-1,2-cyclohexanediol in the presence of DIPEA as shown by 1H and 11B NMR. Although 11B NMR does not give relevant information in terms of

integrations, it is useful for distinguishing between tricoordinate and tetracoordinate

boron species. Peaks corresponding to tetracoordinate boron exhibit an upfield shift

relative to those of tricoordinate boron and appear sharper.88

                                                                                                               

88 Solovyev, A.; Chu, Q.; Geib, S. J.; Fensterbank, L.; Malacria, M.; Lacôte, E.; Curran, D. P. J. Am. Chem. Soc. 2010, 132, 15072–15080.

  46

Figure 3.8 – (a) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of Ph2BOH (3.37) (b) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of erythromycin A (3.1) upon addition of Ph2BOH (3.37)

Figure 3.8 shows the 11B NMR spectra of free base 3.37 (Figure 3.8a) and erythromycin

A with free base 3.37 (3 equiv.) and DIPEA (5 equiv.) (Figure 3.8b). The 11B NMR

spectrum of free diphenylborinic acid (3.37) showed a sharp peak at 45.16 ppm (top).

Upon addition of one equivalent of erythromycin A, we observed no signal at 45.16 ppm

and appearance of two sharp peaks at 7.24 ppm and 3.34 ppm (bottom). Brown and co-

workers have reported the 11B NMR signal corresponding to the “ate” complex of

diphenylborinic acid and 2-propanol at 6.41 ppm.89 Therefore, these results suggest that

erythromycin A was indeed complexing with diphenylborinic acid to form a

tetracoordinate borinate complex. 1H NMR of this interaction revealed spectra that were

not interpretable.

                                                                                                               

89 Brown, H.C.; Srebnik, M.; Cole, T. E. Organometallics. 1986, 5, 2300–2303.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

HO

(3.1)

i-Pr2NEt

CD3CNPh2BOH

(3.37)

O

O

OOH

O

Et

O

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

HO

BPh Ph

(3.1–3.37)

H2O

i-Pr2NEtH+

(a) 3.37

(b) 3.1–3.37

45.16 ppm

7.24 ppm 3.34 ppm

  47

Evidently, studying the presence of reversible covalent interactions between

erythromycin A and boronic/borinic acids was challenging. Though, this was not entirely

surprising given the structural complexity of erythromycin A. Next, our goal was to

observe the effect of organoboron reagents on erythromycin A in the absence of an

electrophile. To do so, we subjected erythromycin A to (pentafluorophenyl) boronic acid

(3.33) (1 equiv.) and DIPEA (3 equiv.) in acetonitrile for 24 hours at 80 °C. After eluting

through a silica plug to remove the boronic acid, the crude sample was analyzed by TLC, 1H NMR and 13C NMR. Several products were observed by TLC and 1H NMR, which

made identification of the products a difficult task without further purification. However, 13C NMR was useful for identifying individual products in the crude reaction mixture.

In 13C NMR, the C-1 carbonyl signal of erythromycin A appears at 178 ppm. This region

of the spectrum is useful for determining how many erythromycin-related products are

present in the reaction mixture. In the case of the reaction with

(pentafluorophenyl)boronic acid (3.33), three peaks were observed in this region. Further

analysis of the 13C NMR spectrum showed the presence of the C-9 ketone peak for

erythromycin A (3.1) at 220 ppm, the C-9 signal for 6,9-hemiketal 3.23 at 111 ppm and

the C-9 peak for 9,12-hemiketal 3.24 at 108 ppm. This was also completed for

erythromycin A and free diphenylborinic acid (3.37) under the same conditions. In this

case, three peaks were observed between 172–178 ppm. Further analysis revealed the

absence of the C-9 ketone peak for erythromycin A at 220 ppm, which was replaced by

three signals corresponding to C-9 of erythromycin A enol ether (3.21) at 150 ppm, the

6,9-hemiketal (3.23) at 111 ppm and the 9,12-hemiketal (3.24) at 108 ppm. Although

relative ratios of these products were not obtained due to overlapping signals in the 1H

NMR, these results indicate that erythromycin A enol ether is more likely to form in the

presence of diphenylborinic acid than (pentafluorophenyl)boronic acid when reacted

under the same conditions.

Although the two-step stoichiometric boronic acid procedure produced comparable

results to the stoichiometric borinic acid conditions for benzoylation of erythromycin A,

these NMR studies suggest that (pentafluorophenyl)boronic acid could be a more suitable

  48

reagent to discourage enol ether formation. With that said, both of the organoboron

reagents employed in this work promoted formation of enol ether 3.35 in the presence of

benzoyl chloride at 23 °C and 80 °C. This suggests that we may be putting ourselves at a

disadvantage by using Lewis acidic organoboron reagents to selectively functionalize

erythromycin A. Attempts made to study the boron-diol equilibria of boronic and borinic

acids with erythromycin A proved difficult. As a result, it is hard to conclude whether the

acid-promoted rearrangements are influenced by complexation of boron with the cis-

vicinal diol at C11–C12 or are a result of the organoboron reagents’ Lewis acidity.

Preference for enol ether formation could suggest that the C11–C12 diol is participating

in complexation, which would eliminate the stabilizing hydrogen bond between the 11-

OH group and the C-9 ketone. This could result in bias towards formation of the 6,9-

hemiketal and, subsequently, the enol ether.

3.8 Conclusions and outlook

As described herein, efficient and selective functionalization of complex natural products

can be a very difficult task. The regioselective functionalization of erythromycin A

presented the challenge of competing reaction pathways with unequal activation barriers.

This was further complicated because the desired C-11 hydroxyl group was the least

reactive secondary hydroxyl group in erythromycin A. Furthermore, the presence of

borinic and boronic acids promoted rearrangement to the enol ether form of erythromycin

A in the presence and absence of an electrophile. The source of this issue arises from the

C-9 ketone of erythromycin A. Previous efforts to avoid acid-catalyzed intramolecular

rearrangements have focused on selectively capping the hydroxyl groups that participate

in the rearrangements [see clarithromycin (3.26)] or removing the C-9 ketone

functionality all together [see azithromycin (3.28)]. Perhaps our methodology may be

better suited for acid-stable erythromycin A derivatives such as these.

  49

3.9 Experimental details

General Procedures: All reactions were carried out in oven-dried glassware fitted with

rubber septa. Stainless steel syringes were used to transfer air- and moisture-sensitive

liquids. Analytical TLC was performed using EMD aluminum-backed silica gel 60 F254

plates and visualized using UV light and/or KMnO4 stain with heat. Flash

chromatography was performed using silica gel 60 (230–400 mesh) from Silicycle.

Materials: HPLC grade acetonitrile, dichloromethane and toluene were dried and

purified using a solvent purification system (Innovative Technology, Inc.). Distilled

water was obtained from an in-house supply. Nuclear magnetic resonance (NMR)

solvents were purchased from Cambridge Isotope Laboratories. The remaining reagents

were purchased from Sigma-Aldrich or ACROS Organics and were used without further

modification.

Instrumentation: 1H and 13C NMR spectra were recorded in CDCl3, CD3OD and

(CD3)2SO using Agilent DD2-500 (500 MHz) and DD2-700 (700 MHz) spectrometers

equipped with a XSens cryogenic probe or using a Varian Mercury 400 MHz

spectrometer. Chemical shifts are reported in parts per million (ppm) relative to

tetramethylsilane and are referenced to residual protium in the solvent. For 1H NMR:

CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for 13C NMR: CDCl3 -

77.16 ppm, CD3OD - 49.00 ppm, (CD3)2SO - 39.52 ppm. Spectral information is

tabulated in the following order: chemical shift (δ, ppm); multiplicity (s-singlet, d-

doublet, t-triplet, q-quartet, m-complex multiplet); coupling constant (J, Hz); number of

protons; assignment. Assignments for proton and carbon resonances were based on two-

dimensional 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC correlation experiments.

High-resolution mass spectra (HRMS) were obtained on a VS 70-250S (double focusing)

mass spectrometer at 70 eV. Fourier transform infrared (FTIR) spectra were obtained on

a Perkin-Elmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe

ATR accessory in a solid or liquid state as indicated. Data are tabulated as follows:

wavenumber (cm-1); intensity (s-strong, m-medium, w-weak, br-broad).

  50

3.10 Characterization data 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31)

Compound 3.31 was synthesized according to a modified literature procedure.90

1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose (2.50 g, 6.41 mmol) was dissolved in

dichloromethane (1.5 M) and added to a round-bottom flask under an argon atmosphere

containing a stir bar. The solution was cooled to 0 °C in an ice bath followed by drop

wise addition of HBr (33 wt.%) in acetic acid (5.10 mL, 28.82 mmol, 4.5 equiv.). The

reaction was slowly warmed to 23 °C and then stirred at this temperature for 4 hours. The

reaction mixture was diluted with dichloromethane and poured into ice-cold water. The

aqueous layer was extracted with dichloromethane three times. The combined organic

layers were washed with water, saturated NaHCO3 (aq) and brine. The organic layers were

dried over MgSO4, filtered and concentrated under vacuum. The resulting crude product

was recrystallized from ethanol to give a white solid (2.21 g, 5.38 mmol, 84% yield). Rƒ

= 0.36 (EtOAc/pentane; 20/80). Spectral data are in agreement with previous reports.91

1H NMR (400 MHz, Chloroform-d): δ 6.60 (d, J = 4.0 Hz, 1H, H-1), 5.59–5.51 (m, 1H,

H-3), 5.15 (dd, J = 10.3, 9.4 Hz, 1H, H-4), 4.83 (dd, J = 10.0, 4.0 Hz, 1H, H-2), 4.37–

4.25 (m, 2H, H-6, H-5), 4.16–4.09 (m, 1H, H-6’), 2.10 (s, 3H, -OCOCH3), 2.09 (s, 3H, -

OCOCH3), 2.05 (s, 3H, -OCOCH3), 2.03 (s, 3H, -OCOCH3). 13C NMR (101 MHz Chloroform-d): δ 170.6, 170.0, 169.9, 169.6, 86.7, 72.3, 70.7,

70.3, 67.3, 61.1, 20.8, 20.8, 20.8, 20.7.

                                                                                                               

90,91 Brown, H.C.; Srebnik, M.; Cole, T. E. Organometallics. 1986, 5, 2300–2303.

O

Br

OAc

AcOAcO

OAc

  51

2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32)

To a 20 mL scintillation vial equipped with a stir bar were added erythromycin A (100

mg, 0.136 mmol), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl bromide (62 mg, 0.149

mmol, 1.1 equiv.), silver(I) oxide (63 mg, 0.272 mmol, 2 equiv.), 4Å molecular sieves

(500 mg), and dichloromethane (6 mL). The resulting suspension was stirred at 23 °C for

30 hours. The reaction was then filtered through Celite® and eluted with

dichloromethane. The filtrate was concentrated in vacuo and the resulting crude solid was

purified by silica gel chromatography (0 → 20% methanol in dichloromethane) to give a

white solid. Rƒ = 0.60 (DCM/MeOH; 75/25).

1H NMR (700 MHz, Chloroform-d): δ 6.48 (d, J = 8.9 Hz, 1H), 5.51–5.43 (m, 1H),

5.34–5.26 (m, 1H), 5.20–5.16 (m, 1H), 5.02 (dd, J = 11.1, 2.3 Hz, 1H), 4.88–4.82 (m,

1H), 4.63 (d, J = 6.8 Hz, 1H), 4.53–4.48 (m, 1H), 4.40–4.29 (m, 1H), 4.27–4.18 (m, 2H),

4.06–3.89 (m, 3H), 3.84 (s, 1H), 3.75–3.61 (m, 1H), 3.55 (d, J = 7.4 Hz, 1H), 3.29 (s,

3H), 3.22 (s, 3H), 3.15–3.08 (m, 1H), 3.07–2.97 (m, 4H), 2.89–2.79 (m, 1H), 2.73–2.61

(m, 1H), 2.36–2.28 (m, 1H), 2.18 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.98–

1.81 (m, 4H), 1.78–1.66 (m, 1H), 1.66–1.47 (m, 3H), 1.43 (s, 3H), 1.40–1.35 (m, 1H),

1.35–1.11 (m, 22H), 1.08 (d, J = 7.3 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H).

HRMS (ESI, m/z): Calculated for [C51H85NO22] (M+H)+ 1064.5636; found 1064.5648.

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2CH3

O

O

OOAc

OAc

AcOAc

  52

2’-(O-benzoyl)erythromycin A (3.34)

Erythromycin A (25 mg, 0.034 mmol) was added to a 30 mL screw cap test tube

containing a magnetic stir bar. The tube was sealed with a rubber septum and purged with

a balloon of argon. Anhydrous acetonitrile (2 mL) was added to the tube, followed by

DIPEA (6 µL, 0.034 mmol, 1 equiv.) and benzoyl chloride (4 µL, 0.034 mmol, 1 equiv.).

The resulting mixture was capped and stirred at 23 °C for 24 hours. The solvent was then

removed in vacuo and the crude mixture was purified by silica gel chromatography (0 →

15% methanol in chloroform with 1% NH4OH) to give a white solid. Rƒ = 0.50

(CHCl3/MeOH; 90/10). [𝛼]!!"= -66.7 (c0.53, CHCl3).

1H NMR (700 MHz, Chloroform-d): δ 8.06–7.99 (m, 2H, ortho), 7.59–7.52 (m, 1H,

para), 7.47–7.39 (m, 2H, meta), 5.04 (dd, J = 10.6, 7.5 Hz, 1H, H-2’), 4.98 (dd, J = 10.9,

2.3 Hz, 1H, H-13), 4.86 (d, J = 4.9 Hz, 1H, H-1”), 4.68 (d, J = 7.5 Hz, 1H, H-1’), 3.99

(dq, J = 9.3, 6.2 Hz, 1H, H-5”), 3.93 (dd, J = 9.3, 1.4 Hz, 1H, H-3), 3.74 (d, J = 1.5 Hz,

1H, H-11), 3.60–3.53 (m, 1H, H-5’), 3.51 (d, J = 7.6 Hz, 1H, H-5), 3.40 (s, 3H, -OCH3),

3.07–2.99 (m, 2H, H-4”, H-10), 2.85–2.76 (m, 1H, H-3’), 2.73–2.68 (m, 1H, H-2), 2.38–

2.32 (m, 1H, H-2”eq), 2.28 (s, 6H, -N(CH3)2), 1.90–1.68 (m, 4H, H-14eq, H-4’eq, H-4,

H-7eq), 1.67–1.62 (m, 1H, H-7ax), 1.61–1.56 (m, 1H, H-2”ax), 1.47 (s, 3H, H-18), 1.44–

1.36 (m, 2H, H-4’ax, H-14ax), 1.31–1.23 (m, 9H, H-19, H-21, H-6’), 1.17 (d, J = 7.0 Hz,

3H, H-6”), 1.14–1.09 (m, 6H, H-20, H-16), 1.02 (s, 3H, H-7”), 0.79 (t, J = 7.4 Hz, 3H, H-

15), 0.71 (d, J = 7.5 Hz, 3H, H-17).

13C NMR (126 MHz Chloroform-d): δ 222.3 (C-9), 175.6 (C-1), 165.3 (C=O Benzoyl),

132.6 (para), 130.7 (ipso), 129.7 (ortho), 128.2 (meta), 101.0 (C-1’), 96.0 (C-1”), 83.2

(C-5), 79.6 (C-3), 77.9 (C-4”), 76.8 (C-13), 75.0 (C-6), 74.5 (C-12), 72.8 (C-3”), 72.2 (C-

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

  53

2’), 68.9 (C-11), 68.5 (C-5’), 65.7 (C-5”), 63.7 (C-3’), 49.5 (-OCH3), 44.7 (C-2), 40.8 (-

N(CH3)2), 39.3 (C-4), 38.0 (C-7), 37.7 (C-10), 35.0 (C-2”), 31.1 (C-4’), 27.1 (C-18), 21.5

(C-21), 21.2 (C-6”), 21.0 (C-14), 18.6 (C-19), 18.1 (C-16), 16.2 (C-7”), 15.8 (C-20), 12.0

(C-6’), 10.6 (C-15), 9.3 (C-17).

FTIR (powder, cm-1): 3414 (w, br), 2971 (w), 2940 (w), 1733 (m), 1718 (w), 1695 (w),

1456 (w), 1271 (s), 1052 (s), 995 (s), 734 (s), 711 (s).

HRMS (ESI, m/z): Calculated for [C44H71NO14] (M+H)+ 838.4948; found 838.4942.

2’-(O-benzoyl)erythromycin A 6,9-enol ether (3.35)

 

Compound 3.35 was synthesized according to a modified literature procedure.92

Erythromycin A (25 mg, 0.034 mmol) was added to a 30 mL screw cap test tube

containing a magnetic stir bar. The tube was sealed with a rubber septum and purged with

a balloon of argon. Anhydrous acetonitrile (2 mL) was added to the tube, followed by

boronic or borinic acid (0.034 mmol, 1 equiv.), DIPEA (6 µL, 0.034 mmol, 1 equiv.) and

benzoyl chloride (4 µL, 0.034 mmol, 1 equiv.). The resulting mixture was capped and

stirred at 80 °C for 24 hours. The solvent was then removed in vacuo and the crude

mixture was purified by silica gel chromatography (0 → 15% methanol in chloroform

with 1% NH4OH) to give a white solid. Rƒ = 0.55 (CHCl3/MeOH; 90/10). [𝛼]!!"= -43.3

(c0.55, CHCl3).

                                                                                                               

92 Lee, D.; Williamson, C.L.; Chan, L.; Taylor, M. J. Am. Chem. Soc. 2012, 134, 8260–8267.

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

  54

1H NMR (500 MHz, Chloroform-d): δ 8.06–7.95 (m, 2H, ortho), 7.57–7.49 (m, 1H,

para), 7.45–7.35 (m, 2H, meta), 5.12–5.03 (m, 2H, H-2’, H-1”), 4.82 (dd, J = 10.5, 2.4

Hz, 1H, H-13), 4.66 (d, J = 7.5 Hz, 1H, H-1’), 4.14–4.05 (m, 1H, H-5”), 4.05–3.98 (m,

1H, H-3), 3.84 (d, J = 7.5 Hz, 1H, H-5), 3.67–3.52 (m, 1H, H-5’), 3.46 (s, 3H, -OCH3),

3.37 (d, J = 7.9 Hz, 1H, H-11), 3.12–3.00 (m, 1H, H-4”), 2.92–2.80 (m, 1H, H-3’), 2.80–

2.69 (m, 1H, H-10), 2.60–2.50 (m, 1H, H-2), 2.49–2.37 (m, 2H, H-7eq, H-2”eq), 2.30 (s,

6H, -N(CH3)2), 1.97–1.92 (m, 1H, H-7ax), 1.89–1.77 (m, 2H, H-14eq, H-4’eq), 1.75–1.65

(m, 1H, H-4), 1.66–1.55 (m, 1H, H-2”ax), 1.52 (s, 3H, H-19), 1.49–1.37 (m, 2H, H-4’ax,

H-14ax), 1.37–1.21 (m, 12H, H-18, H-6”, H-21, H-6’), 1.08 (d, J = 7.4 Hz, 3H, H-16),

1.02 (d, J = 7.1 Hz, 3H, H-20), 0.96 (s, 3H, H-7”), 0.83 (t, J = 7.4 Hz, 3H, H-15), 0.71 (d,

J = 7.4 Hz, 3H, H-17).

13C NMR (126 MHz Chloroform-d): δ 178.4 (C-1), 165.4 (C=O Benzoyl), 151.7 (C-9),

132.8 (para), 130.8 (ipso), 129.9 (ortho), 128.3 (meta), 101.8 (C-8), 101.1 (C-1’), 94.7

(C-1”), 85.6 (C-6), 79.8 (C-5), 78.4 (C-13), 78.2 (C-4”), 76.3 (C-3), 75.4 (C-12), 73.3 (C-

3”), 72.4 (C-2’), 70.0 (C-11), 68.7 (C-5’), 65.9 (C-5”), 63.9 (C-3’), 49.7 (-OCH3), 44.7

(C-2), 43.3 (C-4), 42.4 (C-7), 41.0 (-N(CH3)2), 34.8 (C-2”), 31.8 (C-4’), 30.6 (C-10), 26.4

(C-18), 21.8 (C-21), 21.4 (C-6’), 21.1 (C-14), 18.4 (C-6”), 16.1 (C-7”), 15.2 (C-20), 13.6

(C-16), 12.1 (C-19), 11.0 (C-15), 9.0 (C-17).

FTIR (powder, cm-1): 3425 (w, br), 2970 (w), 1728 (m), 1703 (w), 1451 (w), 1267 (m),

1062 (s), 999 (s), 737 (s), 710 (s).

HRMS (ESI, m/z): Calculated for [C44H69NO13] (M+H)+ 820.4842; found 820.4858.

  55

Erythromycin A 6,9-enol ether (3.21)

Compound 3.21 was synthesized according to a literature procedure.93 Erythromycin A

(200 mg, 0.273 mmol) was dissolved in glacial acetic acid (5 mL) and allowed to stir in a

round-bottom flask at 23 °C for 4 hours. The reaction was then quenched with saturated

NaHCO3 (aq), followed by addition of dichloromethane. The two layers were separated

and the aqueous layer was further extracted twice with dichloromethane. The combined

organic layers were washed with saturated NaHCO3 (aq) to remove any trace acetic acid.

The organic layers were combined, dried over Na2SO4, filtered, and concentrated under

vacuum. The resulting crude product was recrystallized from hexane-ethanol to give a

white powder (95 mg, 0.133 mmol, 49% yield). Rƒ = 0.42 (CHCl3/MeOH; 85/15).

Spectral data are in agreement with previous reports.94

1H NMR (500 MHz, Chloroform-d): δ 5.02–4.95 (m, 2H), 4.48 (d, J = 7.3 Hz, 1H),

4.27–4.16 (m, 1H), 4.06–4.02 (m, 1H), 3.92 (d, J = 7.4 Hz, 1H), 3.79–3.69 (m, 1H), 3.41

(d, J = 8.9 Hz, 1H), 3.38 (s, 3H), 3.25 (dd, J = 10.3, 7.3 Hz, 1H), 3.05 (d, J = 9.5 Hz, 1H),

2.78–2.62 (m, 4H), 2.51–2.44 (m, 1H), 2.34 (s, 6H), 1.97–1.81 (m, 3H), 1.78–1.71 (m,

1H), 1.59 (s, 3H), 1.52–1.41 (m, 1H), 1.34 (s, 3H), 1.28–1.23 (m, 7H), 1.18 (d, J = 6.0

Hz, 3H), 1.15 (d, J = 7.5 Hz, 3H), 1.10 (d, J = 7.6 Hz, 3H), 1.08–1.04 (m, 6H), 0.93–0.87

(m, 3H).

13C NMR (126 MHz Chloroform-d): δ 177.8, 152.2, 102.9, 100.9, 95.1, 85.4, 79.9,

78.0, 77.6, 76.8, 75.2, 73.0, 71.2, 69.7, 67.7, 65.3, 64.2, 48.7, 44.6, 43.5, 39.4, 34.4, 31.3,

31.0, 30.6, 25.5, 22.3, 20.5, 20.2, 17.5, 15.8, 14.7, 13.0, 12.6, 10.8, 9.8.

                                                                                                               

93,94 Alam, P.; Buxton, C.; Parkinson, J. A.; Barber, J. J. Chem. Soc., Perkin Trans. 2. 1995, 6, 1163–1167.

O

O O

O

OOH

HO

ON(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

  56

Diphenylborinic acid (3.37)

 

Compound 3.37 was synthesized according to a modified literature procedure.95 In a 2-

dram vial equipped with a stir bar were added 2-aminoethyl diphenylborinate (200 mg,

0.889 mmol), acetone (0.5 mL) and methanol (0.5 mL). 1M HCl (aq) (1 mL) was added to

the solution and the reaction was stirred at 23 °C for 1 hour. The mixture was then diluted

in diethyl ether, washed with water and extracted three times with diethyl ether. The

combined organic layers were dried over MgSO4, filtered and concentrated under vacuum

to give a white solid (125 mg, 0.687 mmol, 77% yield). Spectral data are in agreement

with previous reports.96

1H NMR (400 MHz, DMSO-d6): δ 9.97 (s, 1H, OH), 7.74–7.66 (m, 4H, ArH), 7.54–7.45

(m, 2H, ArH), 7.45–7.37 (m, 4H, ArH).

13C NMR (101 MHz DMSO-d6): δ 134.5, 130.2, 127.5.

                                                                                                               

95 Hosoya, T.; Uekusa, H.; Ohashi, Y.; Ohhara, T.; Kuroki, R. Bull. Chem. Soc. Jpn. 2006, 79, 692–701. 96 Chen, X.; Ke, H.; Chen, Y.; Guan, C.; Zou, G. J Org. Chem. 2012, 77, 7572–7578.  

B OHPh

Ph

  57

4

Semisynthesis of Erythronolide A

4.0 Introduction

Several total syntheses of the erythromycin aglycons have been reported over the past 35

years (see Figure 3.4).97 Although extensive efforts have been made to decrease the step

count of these syntheses, accessing large quantities of the aglycons through total

synthesis remains an inefficient process. 6-deoxyerythronolide B (3.4) and erythronolide

B (3.5) can be obtained by fermentation because they are intermediates in the

erythromycin biosynthetic pathway (see Scheme 3.2) but the aglycon of erythromycin A,

known as erythronolide A, has only been attainable by total- or semisynthesis.98,99

4.1 Semisynthesis of erythronolide A

In 1974, LeMahieu and co-workers from Hoffmann-La Roche reported a 4-step

procedure to synthesize erythronolide A from erythromycin A 9-oxime (Scheme 4.1).100

The purpose of their work was to illustrate that selective cleavage of the cladinose sugar

was possible, in addition to removal of both sugars to furnish erythronolide A.

Furthermore, the biological activity of erythronolide A was compared to that of

erythromycin A. Despite obtaining disappointing results in terms of biological activity,

this semisynthesis was the first, and remains the only, known practical method to obtain

erythronolide A in sufficient quantities.

                                                                                                               

97 Gao, X.; Woo, S. K.; Krische, M. J. J. Am. Chem. Soc. 2013, 135, 4223–4226. 98 Muri, D.; Carreira, E. J. Org. Chem. 2009, 74, 8695–8712. 99 Staunton, J.; Wilkinson, B. Chem. Rev. 1997, 97, 2611–2629. 100 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.  

  58

Scheme 4.1 – Semisynthesis of erythronolide A (LeMahieu, 1974)

Prior to this synthesis, the acid sensitivity of erythromycin A had been well documented,

including characterization of erythromycin A enol ether and anhydroerythromycin A.

Thus, the first step in LeMahieu and co-workers’ semisynthesis of erythronolide A

involved the protection of the C-9 ketone, which was accomplished with an oxime. The

use of oximes for carbonyl protection has become quite rare in recent times because they

contain an acidic hydrogen and a somewhat reactive C=N functionality.101 With that said,

carbonyl protection is often limited to acetals and ketals, which would likely be cleaved

under the conditions necessary to hydrolyze L-cladinose and D-desosamine. Surprisingly,

LeMahieu did not report the conditions employed for oxime formation in their report. As

a result, the conditions described in Scheme 4.1 for oxime protection were adopted from

a more recent literature procedure by Ma and co-workers.102

Next, LeMahieu and co-workers attempted to cleave both sugars with 1% hydrochloric

acid in methanol. They noticed that L-cladinose had been hydrolyzed but the desosamine

moiety was left intact. Treatment of 4.1 under more vigorous acidic conditions failed to

cleave desosamine from the aglycon. The group of Celmer had previously completed

work with the macrolide antibiotic oleandomycin, which also has a desosamine sugar,                                                                                                                

101 Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007; pp 515. 102 Zhang, L.; Jiao, B.; Yang, X.; Liu, L.; Ma, S. J. Antibiot. 2011, 64, 243–247.  

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

N

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

OHN

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N

CH3HO

OH

CH3H3C O

N

O

OHOH

O

Et

HO

OH

OH

OHO

O

OHOH

O

Et

HO

OH

OH

NH2OH⋅HClNaOAc, AcOH

MeOH, 55 oC, 24 hr

98%

3% H2O2

MeOH, 23 oC, 19 hr

81%

155 oC (0.1 mm Hg)

3 hr

56%

3% HCl

MeOH, 23 oC, 21 hr

69%

NaNO2, 1M HCl

MeOH, 0 oC, 6 hr

40%

(3.1) (4.1) (4.2)

(4.5)(4.4)(4.3)

N

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

HO

OHO CH3

  59

and experienced similar problems when attempting to cleave the sugar moieties from the

aglycon. Celmer later employed a Cope elimination procedure,103 whereby the tertiary

amine of desosamine was converted to an N-oxide, followed by thermally induced syn

elimination to form an alkene-containing neutral sugar and N,N-dimethyl hydroxylamine

(Scheme 4.2).104 This newly formed neutral sugar was cleaved under much milder acidic

conditions than those needed to cleave a basic sugar such as desosamine.

Scheme 4.2 – Cope elimination procedure employed by Celmer for removal of the tertiary amine from D-desosamine in oleandomycin

LeMahieu and co-workers adopted this procedure for the 9-oxime protected erythromycin

A substrate (4.1), which furnished 4.3 in moderate yield. Cleavage of 4.3 with 3%

hydrochloric acid in methanol smoothly removed both sugars and yielded erythronolide

A 9-oxime (4.4) in good yield.

Typically, carbonyl compounds are regenerated from oximes by oxidation, reduction, or

hydrolysis. The hydrolytic methods often involve a strong Lewis- or Brønsted acid.

Alternatively, the oxidative and reductive procedures are generally unsuitable for highly

functionalized molecules. Aware of the possibility of acid-promoted intramolecular

rearrangements, LeMahieu and co-workers opted for a milder hydrolytic approach

wherein nitrous acid was generated in situ with sodium nitrite and 1M hydrochloric acid.

The nitrous acid that is formed then decomposes and results in nitrosonium ion

formation, which promotes nucleophilic attack upon the carbon-nitrogen double bond

such that hydrolytic cleavage can occur.105 Adopting this protocol resulted in cleavage of

the oxime from 4.4 to afford erythronolide A (4.5), albeit in a 40% yield.

                                                                                                               

103 Cope, A. C.; Ciganek, E.; Howell, C. F.; Schweizer, E. E. J. Am. Chem. Soc. 1960, 82, 4663–4669. 104 W. D. Celmer.; Biogenesis of Antibiotic Substances.; Z.Vanek and Z. Hostalek, Ed., Academic Press.: New York, NY, 1965; pp 103-105. 105 Balaban, T. S.; et al. Science of Synthesis: Houben-Weyl Methods of Molecular Transformations, Vol. 26: Ketones., Georg Thieme Verlag.: Göttingen, Germany, 2009. pp 317.  

RO O

N

CH3HO

RO O

N

CH3HO

H3CCH3

HH3CH3C

O

(CH3)2NOHH2O2 150 °C

RO OHO CH3

  60

4.2 Research goals

Our goal was to reproduce LeMahieu and co-worker’s literature procedure for

preparation of erythronolide A. In doing so, we hoped to access a new substrate to

showcase our group’s organoboron-mediated methodology and to synthesize novel

antibiotic analogues for biological evaluation. Erythronolide A presented the opportunity

for a unique intramolecular competition experiment because it contains a 1,2-cis diol, a

1,2-trans diol and a 1,3-cis diol. Like our work with erythromycin A, our goal was to

selectively functionalize the 11-OH group on the aglycon, which is present within the

only cis-vicinal diol of erythronolide A.

4.3 Results and discussion

The semisynthesis reported by LeMahieu and co-workers was completed on a relatively

large scale when compared to modern syntheses. For example, synthesis of erythromycin

A 9-oxime N-oxide (4.2) was accomplished using 50 grams of starting material. The only

step in their synthesis that was not completed on multi-gram scale was that of the oxime

deprotection, which was optimized using 300 milligrams of erythronolide A 9-oxime

(4.4). The purification strategies employed in their work were also notable: all of the

reported steps involved several two-solvent recrystallizations. Ultimately, our goal was to

obtain a significant amount of erythronolide A but on a smaller scale than LeMahieu and

co-workers. This presented the opportunity to simplify difficult purifications using silica

gel chromatography as opposed to using recrystallization.

To begin, erythromycin A 9-oxime (4.1) was synthesized according to the protocol

described in Scheme 4.1, using 5 grams of erythromycin A. The only purification

described in the procedure by Ma and co-workers was an aqueous workup with 2M

sodium hydroxide. Although a 98% yield was reported for 4.1 in the literature procedure,

we found our product to be impure by 1H NMR. Subsequent reaction of the crude

material with a 3% solution of H2O2 in methanol furnished the desired N-oxide 4.2 in

67% yield over two steps (Scheme 4.3).

  61

Scheme 4.3 – Synthesis of erythromycin A 9-oxime N-oxide (4.2)

The next step was the pyrolysis of 4.2 to obtain 3’-de(dimethylamino)-3’,4’-

dehydroerythromycin A 9-oxime (4.3) (Scheme 4.4). Using a Kugelrohr glass oven, N-

oxide 4.2 was heated under high vacuum at 150 °C in solvent-free conditions for 3 hours.

Upon investigation by 1H NMR, we noticed that the crude material still contained a

significant quantity of starting material. This could have been a consequence of the

difference in pressure within the reaction vessel between our method and the literature

procedure. To solve this problem, the temperature was increased to 170 °C, which

resulted in full conversion of 4.2.

Scheme 4.4 – Synthesis of 3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) via Cope elimination

The resulting brown solid was purified by recrystallization from acetone-hexanes but

several impurities remained in the recovered product. Thus, silica gel chromatography

was attempted and proved to be very effective for isolation of the desired product.

Optimal yields were obtained when the reaction was performed using 800 mg of starting

O

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N(CH3)2

CH3HO

N

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N

CH3HO

OH

CH3H3C O

(3.1) (4.2)67%

1) NH2OH•HCl, NaOAc, AcOH MeOH, 55 oC, 24 hr

2) 3% H2O2 (aq), MeOH 23 oC, 19 hr

N

O

OHOH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

O

N

CH3HO

OH

CH3H3C O

(4.2)

59%

170 °C, high vacuum

2 hr

(4.3)

N

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

HO

OHO CH3

  62

material. Notably, our 59% yield was comparable to the 57% yield obtained by

LeMahieu and co-workers.

The step involving hydrolysis of cladinose and desosamine from the aglycon was the first

challenge we experienced in our synthesis of erythronolide A. Using a 37% (w/w) source

of hydrochloric acid, we prepared a 3% solution of methanolic hydrochloric acid. Upon

reaction with 4.3, a complex mixture of products was observed. Attempts at purifying

individual products by recrystallization and silica gel chromatography were unsuccessful

and none of the desired erythronolide A 9-oxime was observed. Notably, 1H NMR

spectra of the partially purified reaction mixture showed signals corresponding to alkene

protons (5.8–5.6 ppm) that were of a different chemical shift than those of the starting

material. Since we did not observe any of the desired product by NMR or mass

spectrometry, it was possible that the cladinose sugar was cleaved while the desosamine

sugar remained linked to the aglycon. We then decided to prepare anhydrous

hydrochloric acid in situ through reaction of acetyl chloride in methanol. After allowing

this mixture to stir at room temperature for 15 minutes, the solution was transferred via

cannula to a new flask containing 4.3. This procedure proved effective and gave

erythronolide A 9-oxime (4.4) in 63% yield after purification by silica gel

chromatography when performed with 1.3 g of starting material (Scheme 4.5).

Scheme 4.5 – Synthesis of erythronolide A 9-oxime (4.4) under acidic conditions

The last step in the synthesis involved regeneration of the C-9 ketone through hydrolytic

cleavage of the oxime using sodium nitrite and 1M hydrochloric acid. Our first attempt at

this procedure yielded none of the desired erythronolide A (4.5). Instead, we observed a

complex mixture of compounds with erythronolide A enol ether (4.6) as the major

63%

0.78 M AcCl

MeOH, 23 o C, 4 hr

N

O

OHOH

O

Et

HO

OH

OH

OH

(4.4)(4.3)

N

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

HO

OHO CH3

  63

product in 32% yield (Scheme 4.6). Unlike erythromycin A, the preferred cyclization

product of erythronolide A is the 5,9-enol ether as opposed to the 6,9-enol ether. This has

been attributed to the increased stability of the resulting 6-membered ring in the 5,9-enol

ether.106 Additionally, the C5 secondary hydroxyl group was found to be more prone to

acetylation than the C6 tertiary hydroxyl group, suggesting increased reactivity of the 5-

OH group.107

Scheme 4.6 – Nitrous acid-mediated oxime cleavage to give erythronolide A 5,9-enol ether (4.6)

Slow addition of the 1M hydrochloric acid solution by syringe pump was attempted but

resulted in a similar product distribution with none of the desired product observed.

During this stage of troubleshooting, we questioned whether erythronolide A was

decomposing during purification by column chromatography. To ensure this was not the

case, crude reaction mixtures were screened by 1H and 13C NMR. Additionally, HMBC

experiments were instrumental for this task and provided increased sensitivity relative to 13C NMR. Since the 1H NMR of oxime protected erythronolide A (4.4) and deprotected

erythronolide A (4.5) were known to be relatively similar, HMBC NMR experiments

were used to determine if the characteristic C-9 ketone signal (220 ppm) of erythronolide

A was present. Still, erythronolide A was not observed when nitrous acid was used to

cleave the oxime.

At this point, it became evident that a new strategy would have to be taken to cleave the

oxime. Many of the literature protocols for oxime removal have only been tested on

simple substrates and their functional group tolerance is relatively unexplored. Thus, we                                                                                                                

106 Schomburg, D.; Hopkins, P. B.; Lipscomb, W. N.; Corey, E. J. J. Org. Chem. 1980, 45, 1544–1546. 107 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3213–3215.  

N

O

OHOH

O

Et

HO

OH

OH

OH

(4.4)

32%

O

H3C

OH

OHOEtOH

HO

O

5

9

NaNO2, 1M HCl(aq)

MeOH, 0 oC, 6 hr

(4.6)

  64

made hypotheses as to which methods would be tolerant of a polyol such as erythronolide

A.

The first set of conditions employed involved reaction of 4.4 with sodium bisulfite

(NaHSO3) in a 1:1 mixture of ethanol and water at reflux. This procedure was used for

cleavage of the oxime in the semisynthesis of clarithromycin (see Scheme 3.5), and

therefore, appeared to be a suitable starting point. Similar to the nitrous acid protocol,

only enol ether 4.6 was observed. Lowering the temperature to both 50 °C and room

temperature resulted in sluggish reactivity, with none of the desired product obtained

after silica gel chromatography. We then tried an oxidative procedure with NBS in a 10:1

mixture of acetone and water, which was performed at room temperature. Analysis of the

crude and partially purified reaction mixtures by 1H NMR and HMBC experiments

revealed a complex mixture of products with none of the desired product formed.

Another interesting set of conditions were those used in Carreira and co-workers’ total

synthesis of erythronolide A for the cleavage of an isoxazoline. In this step, the

isoxazoline was reductively opened using Raney Nickel, hydrogen gas and acetic acid in

methanol (Scheme 4.7). In addition to isoxazoline cleavage, Scheme 4.6 shows the last

step of Carreira and co-workers’ total synthesis of erythronolide A. This is provided to

illustrate the point that the final deprotection of erythronolide A is challenging, regardless

of the protecting group strategy used.

Scheme 4.7 – Final steps of the erythronolide A total synthesis (Carreira, 2009)

Typically, Raney Nickel and H2(g) will result in reduction of an oxime to the

corresponding amine but the presence of acetic acid promotes hydrolysis of the imine

N

O

OH

O

Et

HO

O

O

Ph

ORa-Ni, AcOH, H2

MeOH, 23 oC, 20 min

O

O

OH

O

Et

HO

O

O

Ph

OH

93%

Pd(OAc)2, H2O, H2

MeOH, 23 oC, 6 hr

40%

O

O

OH

O

Et

HO

OH

OH

OH

  65

formed in situ to regenerate the carbonyl compound.108 Thus, we adopted these reductive

conditions for deprotection of oxime 4.4, which gave erythronolide A in 38% yield

(Scheme 4.8).

Scheme 4.8 – Oxime cleavage with Raney Nickel in the semisynthesis of erythronolide A (4.5)

Although a relatively poor yield was obtained for oxime deprotection, we were not overly

surprised given that LeMahieu and co-workers achieved a 40% yield in this step. Of note,

this was the only step of our semisynthesis that was sensitive to reaction scale. Optimal

yields were observed when using 80 mg of starting material. Increasing the amount of 4.4

to 200 mg resulted in a complex reaction mixture with none of the desired product

observed.

4.4 Conclusions and outlook

In summary, a semisynthesis of erythronolide A is reported herein, which was modified

from the synthesis developed previously by LeMahieu and co-workers. Aside from the

oxime removal, we found their synthesis to be entirely reproducible. Like erythromycin

A, acid-promoted intramolecular rearrangements pose a problem for erythronolide A.

Thus, care must be taken when handling these substrates. Based on the results from

chapter 3 and those obtained from our attempts at cleaving the oxime, using Lewis acidic

organoboron reagents may cause problems when attempting to selectively functionalize

erythronolide A. Perhaps an erythronolide A 9-oxime derivative with the hydroxyl group

                                                                                                               

108 Trost, B. M.; Fleming, I. Comprehensive Organic Synthesis – Selectivity, Strategy & Efficiency in Modern Organic Chemistry, Vol. 8: Reduction., Elsevier Ltd.: Kidlington, Oxford, 1991. pp 143.

N

O

OHOH

O

Et

HO

OH

OH

OHO

O

OHOH

O

Et

HO

OH

OH

Ra-Ni, AcOH, H2

MeOH, 23 oC, 10 hr

38%

(4.4) (4.5)

  66

of the oxime protected would be more suitable for the organoboron-mediated

methodology developed in our group. Alternatively, the aglycon of azithromycin (3.28)

would also be an interesting substrate to apply our methodology.

  67

4.5 Experimental details

General Procedures: All reactions were carried out in oven-dried glassware fitted with

rubber septa. Stainless steel syringes were used to transfer air- and moisture-sensitive

liquids. Analytical TLC was performed using EMD aluminum-backed silica gel 60 F254

plates and visualized using UV light and/or KMnO4 stain with heat. Flash

chromatography was performed using silica gel 60 (230–400 mesh) from Silicycle.

Materials: HPLC grade acetonitrile, dichloromethane and toluene were dried and

purified using a solvent purification system (Innovative Technology, Inc.). Distilled

water was obtained from an in-house supply. Nuclear magnetic resonance (NMR)

solvents were purchased from Cambridge Isotope Laboratories. The remaining reagents

were purchased from Sigma-Aldrich or ACROS Organics and were used without further

modification.

Instrumentation: 1H and 13C NMR spectra were recorded in CDCl3, CD3OD and

(CD3)2SO using Agilent DD2-500 (500 MHz) and DD2-700 (700 MHz) spectrometers

equipped with a XSens cryogenic probe or using a Varian Mercury 400 MHz

spectrometer. Chemical shifts are reported in parts per million (ppm) relative to

tetramethylsilane and are referenced to residual protium in the solvent. For 1H NMR:

CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for 13C NMR: CDCl3 -

77.16 ppm, CD3OD - 49.00 ppm, (CD3)2SO - 39.52 ppm. Spectral information is

tabulated in the following order: chemical shift (δ, ppm); multiplicity (s-singlet, d-

doublet, t-triplet, q-quartet, m-complex multiplet); coupling constant (J, Hz); number of

protons; assignment. Assignments for proton and carbon resonances were based on two-

dimensional 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC correlation experiments.

High-resolution mass spectra (HRMS) were obtained on a VS 70-250S (double focusing)

mass spectrometer at 70 eV. Fourier transform infrared (FTIR) spectra were obtained on

a Perkin-Elmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe

ATR accessory in a solid or liquid state as indicated. Data are tabulated as follows:

wavenumber (cm-1); intensity (s-strong, m-medium, w-weak, br-broad).

  68

4.6 Characterization data Erythromycin A 9-oxime N-oxide (4.2)

Compound 4.2 was synthesized according to modified literature procedures.108 ,109 To a

solution of erythromycin A (5.00 g, 6.81 mmol) in methanol (80 mL) were added

hydroxylamine hydrochloride (2.20 g, 34.06 mmol, 5 equiv.), sodium acetate (3.91 g,

47.69 mmol, 7 equiv.) and acetic acid (351 µL, 6.13 mmol, 0.9 equiv.). The mixture was

heated to 55 °C and stirred for 24 hours. After solvent was removed under vacuum, the

residue was taken up in ethyl acetate and water and was adjusted to pH 11–12 with 2M

sodium hydroxide. The resulting solution was extracted three times with ethyl acetate.

The organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated

in vacuo to give a white solid. The crude product was added to methanol (150 mL) and

H2O2 (3% [v/v]) in water (150 mL) and stirred for 20 hours. Most of the methanol was

removed in vacuo and the precipitate that separated was filtered, rinsed with 10 mL of

cold deionized water and air dried to give a pure white solid (3.49 g, 4.56 mmol, 67%

yield). Rƒ = 0.44 (DCM/MeOH/NH4OH; 75/25/1 v/v/v).

1H NMR (500 MHz, Methanol-d4): δ 5.20 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 4.92 (d, J =

5.0 Hz, 1H, H-1”), 4.63 (d, J = 7.0 Hz, 1H, H-1’), 4.13 (dq, J = 9.4, 6.2 Hz, 1H, H-5”),

3.98–3.92 (m, 1H, H-3), 3.83 (dqd, J = 12.2, 6.1, 1.4 Hz, 1H, H-8), 3.72–3.66 (m, 2H, H-

2’, H-5’), 3.59 (d, J = 6.9 Hz, 1H, H-5), 3.52 (ddd, J = 12.4, 10.2, 4.1 Hz, 1H, H-3’), 3.38

                                                                                                               

108 Zhang, L.; Jiao, B.; Yang, X.; Liu, L.; Ma, S. J. Antibiot. 2011, 64, 243-247. 109 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953– 956.  

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O CH3HO

OH

N+ CH3H3C O-

  69

(s, 3H, -OCH3), 3.22 (d, J = 8.3 Hz, 6H, -N(CH3)2), 3.04 (d, J = 9.4 Hz, 1H, H-4”), 2.99–

2.90 (m, 1H, H-2), 2.74 (q, J = 7.4 Hz, 1H, H-10), 2.45 (d, J = 15.1 Hz, 1H, H-2”eq),

2.14 (ddd, J = 12.4, 4.1, 2.1 Hz, 1H, H-4’eq), 2.08–1.97 (m, 1H, H-4), 1.90 (m, 1H, H-

14eq), 1.67–1.56 (m, 2H, H-7eq, H-2”ax), 1.55–1.47 (m, 1H, H-14ax), 1.45 (s, 3H, H-

18), 1.43–1.39 (m, 1H, H-4’ax), 1.30–1.25 (m, 9H), 1.23 (d, J = 6.0 Hz, 3H), 1.20 (d, J =

7.1 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (s, 3H), 1.12 (d, J = 7.6 Hz, 3H), 0.85 (t, J =

7.4 Hz, 3H, H-15).

13C NMR (126 MHz, Methanol-d4): δ 177.4 (C-1), 171.6 (C-9), 103.0 (C-1’), 97.8 (C-

1”), 84.6 (C-5), 81.0 (C-3), 79.2 (C-4”), 78.3 (C-13), 77.9 (C-6), 77.5 (C-3’), 76.6 (C-12),

76.1 (C-11), 74.1 (C-3”), 73.6 (C-2’), 72.2 (C-5’), 67.8 (C-8), 66.6 (C-5”), 58.2 (-

N(CH3)2), 54.5 (-N(CH3)2), 50.1 (-OCH3), 49.3, 46.2 (C-2), 39.0 (C-4), 36.2 (C-2”), 35.3

(C-4’), 27.4 (C-18), 26.6, 22.3, 21.8, 21.6, 19.2, 19.1, 17.2, 16.7, 14.8, 11.1 (C-15), 10.1.

HRMS (ESI, m/z): Calculated for [C37H68N2O14] (M+H)+ 765.4744; found 765.4742.

3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3)

Compound 4.3 was synthesized according to a modified literature procedure.110 N-oxide

4.2 (778 mg, 1.02 mmol) was added to a round bottom flask and placed in a Büchi® Glass

Oven B-585 Kugelrohr under high vacuum. The substrate was pyrolyzed at 170 °C for 2

hours at 35 rpm. The resulting dark brown solid was purified by silica gel

                                                                                                               

110 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

OH

OHO CH3

  70

chromatography (0 → 10% methanol in diethyl ether) to give a light brown foam (422

mg, 0.599 mmol, 59% yield). Rƒ = 0.66 (Et2O/MeOH; 95/5).

1H NMR (500 MHz, Methanol-d4): δ 5.69 (ddd, J = 10.1, 2.2, 1.4 Hz, 1H, H-3’), 5.56–

5.51 (m, 1H, H-4’), 5.20 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 4.87 (d, J = 5.0 Hz, 1H, H-1”),

4.57 (d, J = 6.6 Hz, 1H, H-1’), 4.49–4.43 (m, 1H, H-5’), 4.22 (dq, J = 9.5, 6.2 Hz, 1H, H-

5”), 4.12–4.07 (m, 1H, H-3), 3.96–3.92 (m, 1H, H-2’), 3.71–3.65 (m, 2H, H-5, H-11),

3.30 (s, 3H, -OCH3), 3.03 (d, J = 9.5 Hz, 1H, H-4”), 2.96–2.88 (m, 1H, H-2), 2.77–2.70

(m, 1H, H-10), 2.41 (d, J = 15.1 Hz 1H, H-2”eq), 2.07–1.98 (m, 1H, H-4), 1.95–1.85 (m,

1H, H-14eq), 1.68–1.54 (m, 2H, H-7eq, H-2”ax), 1.53–1.44 (m, 4H, , H-14ax, H-18),

1.28 (d, J = 6.2 Hz, 3H), 1.25 (s, 1H), 1.23 (s, 3H), 1.21–1.16 (m, 12H), 1.15 (s, 3H),

1.13 (d, J = 7.5 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H, H-15).

13C NMR (126 MHz, Methanol-d4): δ 177.6 (C-1), 171.7 (C-9), 133.9 (C-3’), 127.8 (C-

4’), 102.9 (C-1’), 98.4 (C-1”), 82.7 (C-5), 81.5 (C-3), 79.3 (C-4”), 78.3 (C-13), 76.4 (C-

6), 74.1 (C-12), 72.2 (C-11), 70.8 (C-5’), 69.6 (C-2’), 66.6 (C-5”), 50.1 (-OCH3), 46.2

(C-2), 40.7 (C-4), 39.3 (C-7), 36.3 (C-2”), 32.1, 29.5, 27.5 (C-18), 26.6, 22.2, 21.6, 21.5,

19.2, 18.9, 17.2, 16.6, 14.8, 11.2 (C-15), 9.6.

HRMS (ESI, m/z): Calculated for [C35H61NO13] (M+H)+ 704.4216; found 704.4214.

  71

Erythronolide A 9-oxime (4.4)

Compound 4.4 was synthesized according to a modified literature procedure.111 A

solution of 0.78 M acetyl chloride (4.20 mL, 59.10 mmol, 32 equiv.) in methanol (75

mL) was stirred in a round-bottom flask for 15 minutes. The solution was then transferred

via cannula to a round bottom flask containing 4.3 (1.30 g, 1.85 mmol) dissolved in

methanol (5 mL) and was stirred at 23 °C for 4 hours. After solvent was removed in

vacuo, the residue was taken up in ethyl acetate and washed with 1M NaHCO3 (aq). The

organic layers were combined, dried over Na2SO4, filtered, and concentrated under

vacuum. The resulting crude product was purified by silica gel chromatography (0 →

10% methanol in diethyl ether) to give a light brown solid (0.504 g, 1.16 mmol, 63%

yield). Rƒ = 0.60 (Et2O/MeOH; 95/5).

1H NMR (500 MHz, Methanol-d4): δ 5.29 (dd, J = 11.3, 2.4 Hz, 1H, H-13), 3.80–3.71

(m, 1H, H-8), 3.67 (d, J = 1.3 Hz, 1H, H-11), 3.47 (dd, J = 10.5, 1.6 Hz, 1H, H-3), 3.42

(d, J = 3.6 Hz, 1H, H-5), 2.75 (qd, J = 7.0, 1.3 Hz, 1H, H-10), 2.67 (dq, J = 10.5, 6.6 Hz,

1H, H-2), 2.06–1.97 (m, 1H, H-4), 1.96–1.86 (m, 1H, H-14eq), 1.66–1.57 (m, 1H, H-

7eq), 1.55–1.44 (m, 1H, H-14ax), 1.35 (s, 3H, H-18), 1.34–1.30 (m, 1H, H-7ax), 1.22–

1.15 (m, 9H, H-16, H-20, H-21), 1.06 (d, J = 7.0 Hz, 3H, H-19), 0.96 (d, J = 7.4 Hz, 3H,

H-17), 0.84 (t, J = 7.4 Hz, 3H, H-15). 13C NMR (126 MHz, Methanol-d4): δ 177.0 (C-1), 171.9 (C-9), 81.3 (C-5), 79.3 (C-3),

78.3 (C-13), 76.4 (C-6), 75.7 (C-12), 72.7 (C-11), 45.2 (C-2), 37.9 (C-4), 37.8 (C-7), 34.1

                                                                                                               

111 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.

N

O

OHOH

O

HO

OH

OH

OH

  72

(C-10), 30.9 (C-14), 26.8 (C-18), 26.7 (C-8), 19.0 (C-19), 17.3 (C-21), 15.9 (C-20), 14.9

(C-16), 11.0 (C-15), 8.4 (C-17).

HRMS (ESI, m/z): Calculated for [C21H39NO8] (M+H)+ 434.2749; found 434.2748.

Erythronolide A (4.5)

To a solution of Oxime 4.5 (80 mg, 0.184 mmol) in methanol (6 mL) were added acetic

acid (21 µL, 0.369 mmol, 2 equiv.) and Raney®-Nickel (100 mg, 2800 mesh). The round-

bottom flask was purged twice with H2 and the resulting black suspension was stirred

rapidly at 23 °C under an atmosphere of H2 for 10 hours. The reaction mixture was

filtered through Celite® and eluted with methanol. The filtrate was concentrated in vacuo

and the resulting brown residue was purified by silica gel chromatography (20 → 0%

pentanes in diethyl ether, 0 → 10% methanol in diethyl ether) to give a white solid (29

mg, 0.070 mmol, 38% yield). Rƒ = 0.51 (Et2O/MeOH; 95/5). Spectral data are in

agreement with previous reports.112

1H NMR (500 MHz, Methanol-d4): δ 5.19 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 3.87 (d, J =

1.7 Hz, 1H, H-11), 3.56 (dd, J = 10.5, 1.3 Hz, 1H, H-3), 3.51 (d, J = 3.3 Hz, 1H, H-5),

3.15 (qd, J = 6.8, 1.7 Hz, 1H, H-10), 2.76–2.65 (m, 2H, H-8, H-2), 2.06–1.99 (m, 1H, H-

4), 1.95–1.86 (m, 2H, H-14eq, H-7eq), 1.55–1.47 (m, 1H, H-14ax), 1.44–1.41 (m, 1H, H-

7ax), 1.29 (s, 3H, H-18), 1.22–1.12 (m, 12H, H-16, H-21, H-19, H-20), 0.99 (d, J = 7.3

Hz, 3H, H-17), 0.85 (t, J = 7.4 Hz, 3H, H-15).                                                                                                                

112 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712.

O

OHOH

O

HO

OH

OH

O

  73

13C NMR (126 MHz, Methanol-d4): δ 221.3 (C-9), 177.3 (C-1), 82.3 (C-5), 79.9 (C-3),

78.2 (C-13), 76.3 (C-6), 75.5 (C-12), 70.7 (C-11), 45.2 (C-2), 45.1 (C-8), 41.0 (C-10),

39.3 (C-7), 37.6 (C-4), 26.3 (C-18), 22.5 (C-14), 18.3 (C-19), 17.4 (C-21), 15.7 (C-16),

12.1 (C-20), 11.1 (C-15), 8.1 (C-17).

HRMS (ESI, m/z): Calculated for [C21H38O8] (M+Na)+ 441.2459; found 441.2453.

Erythronolide A 5,9-enol ether (4.6)

Compound 4.6 was synthesized according to a modified literature procedure.113 A

solution of sodium nitrite (382 mg, 5.55 mmol, 50 equiv.) and water (2 mL) was stirred in

a round-bottom flask for 10 min and transferred via cannula to a round bottom-flask

containing Oxime 4.4 (48 mg, 0.111 mmol) dissolved in methanol (3 mL). After cooling

the mixture in an ice bath, 1M hydrochloric acid (5.6 mL, 5.55 mmol, 50 equiv.) was

added over 3 hours using a syringe pump while keeping the reaction at 0 °C. The reaction

was then quenched with saturated NaHCO3 (aq) and the methanol was removed in vacuo.

The product was extracted three times with ethyl acetate. The organic layers were

combined, dried over Na2SO4, filtered, and concentrated under vacuum. The resulting

crude product was purified by silica gel chromatography (20 → 0% pentanes in diethyl

ether, 0 → 10% methanol in diethyl ether) to give a yellow glass (16 mg, 0.038 mmol,

32% yield). Rƒ = 0.65 (Et2O/MeOH; 95/5). Spectral data are in agreement with previous

reports.114

                                                                                                               

113 Corey, E. J.; Hopkins, P. B.; Kim, S.; Yoo, S.; Nambiar, K. P.; Falck, J. R. J. Am. Chem. Soc. 1979. 101, 7131–7134. 114 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712.  

O

H3C

OH

OHOOH

HO

O

  74

1H NMR (500 MHz, Methanol-d4): δ 5.18 (dd, J = 11.2, 2.4 Hz, 1H), 3.59–3.53 (m,

2H), 3.50 (dd, J = 10.4, 1.3 Hz, 1H), 2.84–2.73 (m, 2H), 2.67 (dq, J = 10.4, 6.7 Hz, 1H),

2.09 (dd, J = 15.7, 1.3 Hz, 1H), 2.00–1.77 (m, 2H), 1.58–1.46 (m, 4H), 1.35 (s, 3H), 1.17

(d, J = 6.7 Hz, 3H), 1.09 (s, 3H), 1.04 (d, J = 7.2 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H), 0.84

(t, J = 7.4 Hz, 3H).

13C NMR (126 MHz, Methanol-d4): δ 176.9, 152.7, 102.5, 84.7, 82.8, 82.3, 79.2, 76.5,

71.2, 44.7, 43.0, 36.0, 31.7, 28.8, 22.0, 17.2, 16.0, 14.7, 12.5, 10.8, 7.0.

HRMS (ESI, m/z): Calculated for [C21H36O7] (M+K)+ 439.2093; found 439.2089.

  75

2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31) 1H NMR (400 MHz, Chloroform-d)

13C NMR (101 MHz, Chloroform-d)

O

Br

OAc

AcOAcO

OAc

O

Br

OAc

AcOAcO

OAc

  76

2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32) 1H NMR (700 MHz, Chloroform-d)

1H–1H COSY (700 MHz, Chloroform-d)

O

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3O

O

(OAc)4

O

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3O

O

(OAc)4

  77

1H–13C HSQC (700 MHz, Chloroform-d)

1H–13C HMBC (700 MHz, Chloroform-d)

O

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3O

O

(OAc)4

O

O

OH OH

O

Et

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3O

O

(OAc)4

  78

2’-(O-benzoyl)erythromycin A (3.34) 1H NMR (700 MHz, Chloroform-d)

13C NMR (126 MHz, Chloroform-d)

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

  79

1H–1H COSY (700 MHz, Chloroform-d)

1H–13C HSQC (700 MHz, Chloroform-d)

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

  80

1H–13C HMBC (700 MHz, Chloroform-d)

2’-(O-benzoyl)erythromycin A 6,9-enol ether (3.35) 1H NMR (500 MHz, Chloroform-d)

O

O

OH OH

O

HO

O

O

OCH3

OHCH3

OCH3

ON(CH3)2

CH3OBz

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

  81

13C NMR (126 MHz, Chloroform-d)

1H–1H COSY (700 MHz, Chloroform-d)

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

  82

1H–13C HSQC (700 MHz, Chloroform-d)

1H–13C HMBC (700 MHz, Chloroform-d)

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

O

O O

O

OOH

HO

ON(CH3)2

CH3O

OCH3

OHCH3

OCH3

Bz

CH3

  83

Erythromycin A 6,9-enol ether (3.21) 1H NMR (500 MHz, Chloroform-d)

13C NMR (126 MHz, Chloroform-d)

O

O O

O

OOH

HO

ON(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

O

O O

O

OOH

HO

ON(CH3)2

CH3HO

OCH3

OHCH3

OCH3

CH3

  84

Diphenylborinic acid (3.37) 1H NMR (400 MHz, DMSO-d6)

13C NMR (101 MHz, DMSO-d6)

B OHPh

Ph

B OHPh

Ph

  85

Erythromycin A 9-oxime N-oxide (4.2)

1H NMR (500 MHz, Methanol-d4)

13C NMR (126 MHz, Methanol-d4)

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O CH3HO

OH

N+ CH3H3C O-

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O CH3HO

OH

N+ CH3H3C O-

  86

1H–1H COSY (500 MHz, Methanol-d4)

1H–13C HSQC (500 MHz, Methanol-d4)

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O CH3HO

OH

N+ CH3H3C O-

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

O CH3HO

OH

N+ CH3H3C O-

  87

3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) 1H NMR (500 MHz, Methanol-d4)

13C NMR (126 MHz, Methanol-d4)

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

OH

OHO CH3

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

OH

OHO CH3

  88

1H–1H COSY (500 MHz, Methanol-d4)

1H–13C HSQC (500 MHz, Methanol-d4)

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

OH

OHO CH3

N

O

OHOH

O

HO

O

O

OCH3

OHCH3

OCH3

OH

OHO CH3

  89

Erythronolide A 9-oxime (4.4) 1H NMR (500 MHz, Methanol-d4)

13C NMR (126 MHz, Methanol-d4)

N

O

OHOH

O

HO

OH

OH

OH

N

O

OHOH

O

HO

OH

OH

OH

  90

1H–1H COSY (500 MHz, Methanol-d4)

1H–13C HSQC (500 MHz, Methanol-d4)

N

O

OHOH

O

HO

OH

OH

OH

N

O

OHOH

O

HO

OH

OH

OH

  91

Erythronolide A (4.5) 1H NMR (500 MHz, Methanol-d4)

13C NMR (126 MHz, Methanol-d4)

O

OHOH

O

HO

OH

OH

O

O

OHOH

O

HO

OH

OH

O

  92

1H–1H COSY (500 MHz, Methanol-d4)

1H–13C HSQC (500 MHz, Methanol-d4)

O

OHOH

O

HO

OH

OH

O

O

OHOH

O

HO

OH

OH

O

  93

Erythronolide A 5,9-enol ether (4.6) 1H NMR (500 MHz, Methanol-d4)

13C NMR (126 MHz, Methanol-d4)

O

H3C

OH

OHOOH

HO

O

O

H3C

OH

OHOOH

HO

O