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 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.
ii 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.
iii 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
iv 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
v 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
vi 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
11 1 Figure 3.8 – (a) B NMR (128 MHz, decouple H 400 MHz, CD3CN, 295 K) of 11 1 Ph2BOH (3.37) (b) B NMR (128 MHz, decouple H 400 MHz, CD3CN, 295 K) of erythromycin A (3.1) upon addition of Ph2BOH (3.37) ...... 45
vii 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
viii 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
ix 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)
x 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
xi 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)
xii 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.
1 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
OH HO pH 7.5 O B B 2H2O OH HO O
OH OH
OH HO pH >10 O B B 2H2O HO OH HO HO O
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.
2 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
OCH3 OCH3 OCH3 O O OCH3 PhB(OH)2 OH n-BuI, Ag2O, NEt3 OH O O O O OH OH O O OH On-Bu HO CH2Cl2 B PhH, reflux, 22 hr B HO Ph NEt3 Ph 50%
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.
3 Ph O B Ph N H2 HO (5–10 mol%) HO R1 R1 HO R ROCO R 2 RCOCl (1.2–2.0 equiv.) 2 i-Pr NEt (1.2–2.0 equiv.) 2 69–95% (14 examples) MeCN, RT
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).
Ph O B Ph N H2 OH (10 mol%) OH R1 O R4 R1 O R4 R2 O HO R3 R2 R3 X Ag2O (1 equiv.) 1.1 equiv MeCN, 23–60 oC X = Br, Cl 68–99% (13 examples)
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.
4 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.
Ph O B Ph N OAc TBDPSO H OAc TBDPSO OH 2 OH AcO O HO O (10 mol%) AcO O HO O AcO HO AcO O
Cl OCH3 Ag2O (2 equiv.) OCH3 CH2Cl2, RT Catalyzed reaction: 72% yield, 7.3:1 β:α Uncatalyzed reaction: 18% yield, 2:1 β:α
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.
5 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.
O CH3 O CH3 H3C OH H3C HO O O H3C O O O O OH OH OH
O R1 O Ph R2 B Ag2O (2 equiv.) Ph N Br H2 CH2Cl2, 23 ˚C (2 equiv.) (25 mol%)
O CH3 O CH3 H C OH O 3 H C R1 O 3 H C R O O 3 2 O O O O OH OH OH
OAc OAc AcO Br Br O AcO O O O AcO AcO OAc OAc AcO AcO OAc OAc Br Br AcO AcO 77% 63% 63% 51%
OAc OAc OAc AcO OAc O O AcO O O AcO O AcO O AcO AcO AcO AcO AcO AcO Br Br 74% 64%
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
6 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.
HO HO BzO BzO O O O O HO O BzO O HO O OR BzO O O HO O O AcO O O HO O AcO O HO OH AcO OAc HO AcO O O HO O AcO O HO AcO HO AcO
1.1 1.2
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).
Ph O B Ph N H O O 2 (10 mol%) OAc O O HO HO AcO O HO OAc O OH OH AcO AcO AcO O 1.3 AcO 1.4 AcO Br (1.1 equiv.) 80% Ag2O (1 equiv.) MeCN
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.
7 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.
Catalytic (borinic) Ph O B Ph N H2 HO (10 mol%) AcO HO O O O HO OPMP AcO O OPMP AcO OH AcO OH O 1.5 AcO 1.6 AcO Br (1.1 equiv.) 20%
Ag2O (1 equiv.) MeCN, RT
Stoichiometric (boronic)
B(OH)2 F F
F F F HO (1 equiv.) AcO HO O O O HO OPMP AcO O OPMP AcO OH AcO OH O 1.5 AcO 1.6 AcO Br (1.1 equiv.) 77%
Ag2O (1 equiv.) NEt3 (3 equiv.) MeCN, RT
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).
8 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.
9 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.
10 OH
OH NH2
NH2 H2N OH HO OH As As As H2N As As As NH2 As As NH2 H2N As As HO H2N HO OH HO OH NH2 H2N
2.1 2.2 2.3
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.
11 H H Me OH NMe2 H2N N H H S Me OH N Me N O N O CONH2 NH2 CO2H O SO2NH2 OH O HO H O
2.4: prontosil (sulfonamide) 2.5: penicillin G (β-lactam) 2.6: tetracycline (tetracycline)
O HO O NH2 H O H Me O Me OH O HO OH O OH N(CH3)2 O HO Cl Et O O O CH3 O O HO O O OCH Cl OH 3 O O H H CH N 3 N N NH NH O OH H NH HN O CH3 O O O O 2.7: erythromycin A (macrolide) O NH HO 2
HO OH OH
2.8: vancomycin (glycopeptide)
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.
12 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.
13 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.
14 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
Cephalosporin Scaffold
R1 H N S R2 – essential for achieving higher O binding affinity for PBP2a N 2 R1 – essential for stability O R to hydrolysis by β-lactamases CO2H
β-lactam ring – essential for inhibition of transpeptidase activity in cell wall biosynthesis
Me N OMe HO O N HO P OEt H2N N H N N N S HN H S N N S O N NH S N N N O O N S S CO2H O O CO2H 2.9: ceftobiprole 2.10: ceftaroline
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.
15
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.
HO H NMe2 H H NMe2 H H H OH Pd, H , HCl H OH 6 2 6
CONH CONH O 2 MeOH O 2 OH O HO H O OH O HO H O
6-demethyltetracycline sancycline (natural product)
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.
16 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.
NMe NO2 NMe2 NMe2 NMe2 7 H H 2 H H H H OH OH Pd, H , CH O OH KNO3, H2SO4 2 2 9 CONH CONH2 CONH2 O 2 O MeOH O OH O HO H O OH O HO H O OH O HO H O sancycline Mixture of 7- and 9-nitro isomers minocycline
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
O NMe NMe NMe NMe H NMe NMe 2 H H 2 2 H H 2 N 2 H H 2 OH OH t-Bu Cl OH 1) KNO3, H2SO4 O HCl H N CONH2 H2N CONH2 t-Bu N CONH2 O 2) Pd/C, H2 O H O OH O HO H O OH O HO H O OH O HO H O minocycline tigecycline
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.
17 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.
O O
OH O DEBS OH O DEBS KS1° OH OH SCoA SNAC in vivo O OH O OH
O OH O OH
Typical propionyl CoA substrate to give 6-deoxyerythronolide B
O O
OH O DEBS KS1° OH O DEBS KS1° OH OH SNAC SNAC in vivo O OH in vivo O OH
O OH O OH
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.
18 methyl, n-propyl and phenyl R groups when subjected to S. erythraea mutants unable to synthesize 6-deoxyerythronolide B (Scheme 2.5).
O O
HO OH OH OH N(CH3)2 HO O OH O O O CH3 R R O O O OH OCH3
CH3 O OH CH R = Methyl, n-Propyl, Phenyl 3
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.
19 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
O
HO OH D-desosamine OH N(CH3)2 HO Et O O O CH3 O O OCH3 Aglycone (erythronolide A) CH3 OH O L-cladinose CH3
erythromycin A (3.1)
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.
20 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
O
O O Chain assembly on PKS OH SCoA SCoA Et O OH CO2H Cyclization and release from enzyme O OH (3.2) (3.3) 6-deoxyerythronolide B (3.4)
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.
21 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.
22 6-deoxyerythronolide B then undergoes a series of site-selective functionalization reactions to yield erythromycin A (Scheme 3.2).
O O O O
OH OH OH OH OH N(CH3)2 OH a OH b c HO Et O OH Et O O O CH3 Et O OH Et O OH O O OH O O OH O OH O OH CH3 CH3 O OH O OH CH CH 6-deoxyerythronolide B (3.4) erythronolide B (3.5) 3 3 3-O-Mycarosylerythronolide B (3.6) erythromycin D (3.7)
erythromycin D (3.7)
d e O O
OH HO OH OH OH N(CH3)2 N(CH3)2 HO HO Et O O O CH3 Et O O O CH3
O O OMe O O OH
CH3 CH3 O OH O OH CH3 CH3
erythromycin B (3.8) erythromycin C (3.9)
e d
O
a – C-6 erythronolide hydroxylase b – TDP-mycarose glycosyltransferase HO OH OH c – TDP-desosamine glycosyltransferase N(CH3)2 d – (O)-methyltransferase HO e – C-12 hydroxylase Et O O O CH3 O O OCH3
CH3 O OH CH3 erythromycin A (3.1)
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.
23 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
O O O O O O
OH OH OH OH OH
Et O OH Et O OH Et O OH Et O Et O OH Et O O
O OH O OH O OH O OH O O O OH
(3.10) (3.11) (3.12) (3.13) (3.14) (3.15)
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.
24 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
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 O Erythronolide A – R1 = OH, R2 = H, R3 = H, R4 = OH Corey (1979): 39 steps (LLS), 50 steps (TS)56 R4 R1 Kinoshita (1989): 50 steps (LLS), 74 steps (TS)57 OH Carreira (2005): 26 steps (LLS), 36 steps (TS)58 Et O OR2 Erythronolide B – R1 = OH, R2 = H, R3 = H, R4 = H 3 O OR 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
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.
25 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
Me Me Me Me Me Me Me Me Me Me Me Me Me Me 12 6 OH 11 9 5 3 OH O O O O HO H OH O H OH OH O HO H O NH H O O O
O R erythronolide A seco acid Woodward's seco acid target (3.16)
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.
26 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).
BPCO 1) N HN HN Me Me Me Me Me Me Me O 12 6 S N 9 9 11 9 5 3 OH S 11 11 O O Ph P HO OH HO OH HO H O NH H O O O 3 O OH 5 5
O Me Me o Et O O Me Et O OH 2) toluene, 110 C 3 3 O O O OH 70% Me Me Me
(3.16) (3.17) (3.18)
OMe O N(CH3)2 N O S O CH3 36% N AgOTf
N BPCO 1) O HN S OCH3 BPCO 9 CH3 HN 11 O OAc HO OH HO OH CH 9 OH OH 2' 3 N(CH3)2 N(CH3)2 11 O HO 5 HO HO OH OMe Pb(ClO4)2, MeCN OH 2' N(CH ) Et O O O CH3 Et O O O CH3 3 2 3 5 O Et O O O CH3 O O OCH O O OCH 3 3 2) MeOH 3 O OH CH3 CH3 O OH O OAc CH CH 3 3 55% (3.1) (3.20) (3.19)
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.
27 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.
CH3 HO HO O OH N(CH3)2 O O N(CH3)2 HO HO Et O O O CH3 Et O O O CH3
O O O O OCH3 OCH3
CH3 CH3 O OH O OH CH3 CH3
erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)
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.
28 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).
O HO OH 9 HO 9 HO 9 OH O HO OH 12 OH O OH 6 N(CH3)2 12 6 N(CH3)2 12 6 N(CH3)2 HO HO HO Et O O O CH3 Et O O O CH3 Et O O O CH3 1 1 1 O O O O O O OCH3 OCH3 OCH3
CH3 CH3 CH3 O OH O OH O OH CH3 CH3 CH3
6,9-hemiketal of erythromycin (3.23) erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)
CH3 O 9 HO 9 HO O 6 12 6 12 OH N(CH3)2 HO OH O O N(CH3)2 HO OH N(CH ) HO 3 2 O Et O O O CH3 HO Et O O CH3 1 Et O O O CH3 1 O O O O OCH OCH3 3 O OH CH3 CH3 OH O OH O CH CH3 5-desosaminylerythronolide A (3.25) 3 erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)
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.
29 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.
Oi-Pr Oi-Pr O O N N O
HO OH KOH HO OMe 1) HCO H HO OMe OH TMS OH TMS 2 OH N(CH3)2 N(CH3)2 N(CH3)2 O O HO Et O O O CH3 MeI Et O O O CH3 Et O O O CH3 2) NaHSO3 O O O O O O OCH3 OCH3 OCH3
CH3 CH3 CH3 O OTMS O OTMS O OH CH3 CH3 CH3
oxime intermediate clarithromycin (3.26) (3 steps from 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.
30 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.
OH N H3C N N HO HO OH O HO OH OH PhSO2Cl N(CH ) 1) H2, Pt OH N(CH3)2 OH 3 2 N(CH3)2 HO HO HO Et O O O CH Et O O O CH3 3 Et O O O CH3 NaHCO3 2) CH2O, O O O O OCH O O OCH3 3 HCO2H OCH3 CH CH3 CH 3 OH 3 OH O OH O CH O CH3 3 CH3
erythromycin A oxime (3.27) azithromycin (3.28)
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.
31 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.
O - 2'-OH, most reactive - Biologically inactive upon functionalization HO OH OH N(CH3)2 - 11-OH, 3rd most reactive HO - Desired selectivity Et O O O CH3 O O OCH3 - 4"-OH, 2nd most reactive
CH3 O OH CH3
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.
32 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
O Me Me O 1) N O Me N H H O O HN OH N O 9 O 9 NBoc OH N NH O NH HO OH O Boc O 12 6 N(CH3)2 O 12 6 N(CH ) (5 mol%) 3 2 HO HO Et O O O CH Et O O O 3 CH3 1 Ph OMe 1 O O 3.1 O O OCH3 OCH3 Ac O (2 equiv.), NEt (5 equiv.), CHCl , RT 24 hr CH3 2 3 3 CH3 OH OH O O CH3 CH3 2) MeOH, RT 72 hr C11-monoacetate (3.29) 37%, major product
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.
O Ph O Ph O B E HO OH HO OH OH O OH O N(CH3)2 O + N(CH3)2 Ar2BOH (cat.) N(CH3)2 electrophile E HO HO HO Et O O O CH3 Et O O O CH3 Et O O O CH3 O O O O OCH3 O O OCH3 OCH3 CH3 CH CH3 OH 3 OH O O OH O CH3 CH3 CH3
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.
33 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.
34 Table 3.1 – Borinic acid-mediated glycosylationa
Ph O AcO O B OAc O AcO OAc Ph N H2 O (3.30) AcO HO OH AcO HO OH O OAc OH AcO OH N(CH3)2 (x mol%) (3.31) Br N(CH3)2 HO O Et O O O CH3 Et O O O CH3
O O Ag2O O O OCH3 OCH3 MeCN, 23 oC CH3 CH3 O OH 24 hr O OH CH3 CH3 (3.1) (3.32)
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).
35 Table 3.2 – Boronic acid-mediated glycosylationa
B(OH)2 AcO O O AcO OAc F F OAc
O HO OH F F AcO HO OH O OAc OH AcO OH N(CH3)2 F AcO N(CH3)2 Br HO (3.33) (3.31) O Et O O O CH3 Et O O O CH3 O O O O OCH3 OCH3 Ag2O, NEt3 CH CH 3 DCM, 23 oC 3 O OH O OH CH3 24 hr CH3 (3.1) (3.32)
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), b NEt3 (0.204 mmol), DCM (6 mL). 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
36 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).
37 O O
O O HO OH HO OH OH OH Bz N(CH3)2 Ph O Ph N(CH3)2 HO O Et O O O CH3 (3 equiv.) Et O O O CH3 O O O O OCH3 pyridine OCH3 CH3 23 oC, 72 hr CH3 O OH O OH CH3 CH3
(3.1) (3.34) 92%
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.
38 Table 3.3 – Organoboron-mediated benzoylation at 23 °Ca
O O CH3
HO OH HO OH HO OH OH Bz O Bz N(CH3)2 Boron source (x equiv.) N(CH3)2 OH N(CH3)2 HO O O BzCl, i-Pr NEt Et O O O CH3 2 Et O O O CH3 Et O O O CH3
O O O O O O OCH3 MeCN, 23 oC OCH3 OCH3 CH3 24 hr CH3 CH3 O OH O OH O OH CH3 CH3 CH3 (3.1) (3.34) (3.35)
Entry Boron source Yield (%)c [3.34] Yield (%)c [3.35]
1 none 87 0
2 2-aminoethyl 72 15 diphenylborinate (0.25 equiv.)
3 2-aminoethyl 63 26 diphenylborinate (1 equiv.)
4 (pentafluorophenyl)boronic 55 34 acid (1 equiv.)b
a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt b (0.204 mmol), MeCN (6 mL). 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
39 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.
40 Table 3.4 – Organoboron-mediated benzoylation at 80 °Ca
O CH3 CH3
HO OH HO HO OH Bz O Bz O Boron source (x equiv.) N(CH3)2 OH N(CH3)2 OH N(CH3)2 O O HO BzCl, i-Pr NEt 2 Et O O O CH3 Et O O O CH3 Et O O O CH3 3.1 O O O O O O MeCN, 80 oC OCH3 OCH3 OCH3 24 hr CH3 CH3 CH3 O OH O OH O OH CH3 CH3 CH3 (3.34) (3.35) (3.21)
Entry Boron source Yield (%)c Yield (%)c Yield (%)c [3.34] [3.35] [3.21]
1 none 57 35 0
2 2-aminoethyl 28 54 9 diphenylborinate (0.25 equiv)
3 2-aminoethyl 0 52 44 diphenylborinate (1 equiv.)
4 (pentafluorophenyl)boronic 0 49 47 acid (1 equiv.)b
a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt b (0.204 mmol), MeCN (6 mL). 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.
41 organoboron reagent, whether catalytic or stoichiometric, appears to accelerate erythromycin A enol ether formation at both 23 °C and 80 °C.
CH3 CH3 CH3
HO HO HO O O Bz O OH N(CH3)2 OH N(CH3)2 OH N(CH3)2 HO O HO Et O O O CH3 BzCl, i-Pr2NEt Et O O O CH3 Et O O O CH3
O O OCH O O OCH O O OCH 3 MeCN, 23 oC 3 3 CH3 CH3 CH3 O OH 24 hr O OH O OH CH3 CH3 CH3
(3.36) (3.35) (3.21)
32% 65%
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
42 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.
O HO OH HO HO HO OH OH O OH O OH N(CH3)2 N(CH3)2 N(CH3)2 HO HO HO Et O O O CH3 Et O O O CH3 Et O O O CH3
O O O O O O OCH3 OCH3 OCH3
CH3 CH3 CH3 O OH O OH O OH CH3 CH3 CH3
6,9-hemiketal of erythromycin (3.23) erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)
m/z = 733 m/z = 733 m/z = 733
CH3 HO
HO O O O N(CH3)2 OH N(CH3)2 HO HO Et O O O CH3 Et O O O CH3 O O OCH O O 3 OCH3 CH3 CH3 OH OH O O CH3 CH3
erythromycin A enol ether (3.21) anhydroerythromycin A (3.22) m/z = 715 m/z = 715
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.
43 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 1 19 19 results by H and F NMR. When comparing F 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
44 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.
45 Ph O Ph O B + i-Pr2NEtH HO OH O OH OH O N(CH ) N(CH3)2 i-Pr NEt 3 2 HO 2 HO Et O O O CH Et O O O CH3 Ph BOH 3 2 H O CD CN 2 O O 3 O O OCH OCH3 (3.37) 3 CH3 CH3 OH O OH O CH3 CH3 (3.1) (3.1–3.37)
45.16 ppm (a) 3.37
(b) 3.1–3.37 3.34 ppm 7.24 ppm
11 1 Figure 3.8 – (a) B NMR (128 MHz, decouple H 400 MHz, CD3CN, 295 K) of 11 1 Ph2BOH (3.37) (b) B NMR (128 MHz, decouple H 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.
46 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
47 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.
48 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.
1 13 Instrumentation: H and C 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: 13 CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for C 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).
49 3.10 Characterization data
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31)
OAc O AcO AcO AcO Br
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.
50 2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32)
AcO O AcO OAc
HO OH O OAc OH N(CH3)2 O Et O O O CH3 O O OCH3
CH3 O OH CH3
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.
51 2’-(O-benzoyl)erythromycin A (3.34)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
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). [�]! = -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-
52 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)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
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). [�]! = -43.3
(c0.55, CHCl3).
92 Lee, D.; Williamson, C.L.; Chan, L.; Taylor, M. J. Am. Chem. Soc. 2012, 134, 8260–8267.
53 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.
54 Erythromycin A 6,9-enol ether (3.21)
CH3
HO O OH N(CH3)2 HO O O O CH3
O O OCH3
CH3 O OH CH3
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.
55 Diphenylborinic acid (3.37)
Ph B OH Ph
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
1 H 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).
13 C 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.
56 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.
57 OH OH O N N
NH2OH⋅HCl HO OH HO OH HO OH H3C O OH NaOAc, AcOH OH 3% H O OH N(CH3)2 N(CH3)2 2 2 N CH3 HO HO HO Et O O O CH3 Et O O O CH3 Et O O O CH3 MeOH, 55 oC, 24 hr MeOH, 23 oC, 19 hr O O O O O O OCH3 OCH3 OCH3 98% 81% CH3 CH3 CH3 O OH O OH O OH CH3 CH3 CH3
(3.1) (4.1) (4.2)
HO N OH N O o 155 C (0.1 mm Hg) HO OH 3% HCl NaNO2, 1M HCl OH HO HO OH HO OH O CH OH OH 3 hr Et O O 3 MeOH, 23 oC, 21 hr MeOH, 0 oC, 6 hr Et O OH Et O OH O O 56% OCH3 69% 40% O OH O OH CH3 O OH CH3 (4.3) (4.4) (4.5)
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.
58 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.
O H3C H3C H N CH H3C N 3 H2O2 150 °C HO HO HO O CH (CH3)2NOH RO RO O CH RO 3 O CH3 3
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.
59 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).
60
OH O N
HO OH 1) NH OH•HCl, NaOAc, AcOH HO OH H3C O OH N(CH ) 2 OH 3 2 o N CH3 HO MeOH, 55 C, 24 hr HO Et O O O CH3 Et O O O CH3 O O 2) 3% H O MeOH O O OCH3 2 2 (aq), OCH3 23 oC, 19 hr CH3 CH3 O OH O OH CH3 CH3 67% (3.1) (4.2)
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.
OH HO N N
HO OH H3C O HO OH OH OH N CH3 170 C, high vacuum HO ° HO O CH3 Et O O O CH3 Et O O 2 hr O O O O OCH3 OCH3 CH3 59% CH3 O OH O OH CH3 CH3 (4.3) (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
61 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).
HO N OH N
HO OH OH 0.78 M AcCl HO OH HO CH OH Et O O O 3 MeOH, 23 o C, 4 hr Et O OH O O OCH3 O OH CH3 O OH 63% CH3 (4.3) (4.4)
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
62 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
OH N H3C 9 OH HO OH NaNO , 1M HCl HO 5 OH 2 (aq) O OH Et O OH o Et HO MeOH, 0 C, 6 hr O O OH O 32% (4.4) (4.6)
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.
63 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.
N O O
O HO OH Ra-Ni, AcOH, H HO OH Pd(OAc) , H O, H HO OH 2 OH 2 2 2 OH
Et O O MeOH, 23 oC, 20 min Et O O MeOH, 23 oC, 6 hr Et O OH
O O Ph O O Ph O OH 93% 40% 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
64 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).
OH N O
Ra-Ni, AcOH, H2 HO OH HO OH OH OH MeOH, 23 oC, 10 hr Et O OH Et O OH
O OH O OH 38%
(4.4) (4.5)
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.
65 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.
66 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.
1 13 Instrumentation: H and C 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: 13 CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for C 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).
67 4.6 Characterization data
Erythromycin A 9-oxime N-oxide (4.2)
OH N
O- HO OH H3C OH N+ CH3 HO O O O CH3
O O OCH3
CH3 O OH CH3
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).
1 H 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.
68 (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).
13 C 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)
OH N
HO OH OH HO O O O CH3
O O OCH3
CH3 O OH CH3
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.
69 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).
1 H 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).
13 C 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.
70 Erythronolide A 9-oxime (4.4)
OH N
HO OH OH
O OH
O OH
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).
1 H 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).
13 C 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.
71 (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)
O
HO OH OH
O OH
O OH
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
1 H 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.
72 13 C 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)
H3C
OH HO O OH HO O
O
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.
73 1 H 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).
13 C 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.
74 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31) 1H NMR (400 MHz, Chloroform-d)
OAc O AcO AcO AcO Br
13C NMR (101 MHz, Chloroform-d)
OAc O AcO AcO AcO Br
75 2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32) 1H NMR (700 MHz, Chloroform-d)
(OAc)4 O
O HO OH OH N(CH3)2 O Et O O O CH3 O O OCH3
CH3 O OH CH3
1H–1H COSY (700 MHz, Chloroform-d)
(OAc)4 O
O HO OH OH N(CH3)2 O Et O O O CH3 O O OCH3
CH3 O OH CH3
76 1H–13C HSQC (700 MHz, Chloroform-d)
(OAc)4 O
O HO OH OH N(CH3)2 O Et O O O CH3 O O OCH3
CH3 O OH CH3
1H–13C HMBC (700 MHz, Chloroform-d)
(OAc)4 O
O HO OH OH N(CH3)2 O Et O O O CH3 O O OCH3
CH3 O OH CH3
77 2’-(O-benzoyl)erythromycin A (3.34) 1H NMR (700 MHz, Chloroform-d)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
13C NMR (126 MHz, Chloroform-d)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
78 1H–1H COSY (700 MHz, Chloroform-d)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
1H–13C HSQC (700 MHz, Chloroform-d)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
79 1H–13C HMBC (700 MHz, Chloroform-d)
O
HO OH OH Bz N(CH3)2 O O O O CH3 O O OCH3
CH3 O OH CH3
2’-(O-benzoyl)erythromycin A 6,9-enol ether (3.35) 1H NMR (500 MHz, Chloroform-d)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
80 13C NMR (126 MHz, Chloroform-d)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
1H–1H COSY (700 MHz, Chloroform-d)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
81 1H–13C HSQC (700 MHz, Chloroform-d)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
1H–13C HMBC (700 MHz, Chloroform-d)
CH3
HO O Bz OH N(CH3)2 O O O O CH3
O O OCH3
CH3 O OH CH3
82 Erythromycin A 6,9-enol ether (3.21) 1H NMR (500 MHz, Chloroform-d)
CH3
HO O OH N(CH3)2 HO O O O CH3
O O OCH3
CH3 O OH CH3
13C NMR (126 MHz, Chloroform-d)
CH3
HO O OH N(CH3)2 HO O O O CH3
O O OCH3
CH3 O OH CH3
83 Diphenylborinic acid (3.37) 1 H NMR (400 MHz, DMSO-d6)
Ph B OH Ph
13 C NMR (101 MHz, DMSO-d6)
Ph B OH Ph
84 Erythromycin A 9-oxime N-oxide (4.2)
1 H NMR (500 MHz, Methanol-d4)
OH N
O- HO OH H3C OH N+ CH3 HO O O O CH3
O O OCH3
CH3 O OH CH3
13 C NMR (126 MHz, Methanol-d4)
OH N
O- HO OH H3C OH N+ CH3 HO O O O CH3
O O OCH3
CH3 O OH CH3
85 1 1 H– H COSY (500 MHz, Methanol-d4)
OH N
O- HO OH H3C OH N+ CH3 HO O O O CH3
O O OCH3
CH3 O OH CH3
1 13 H– C HSQC (500 MHz, Methanol-d4)
OH N
O- HO OH H3C OH N+ CH3 HO O O O CH3
O O OCH3
CH3 O OH CH3
86 3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) 1 H NMR (500 MHz, Methanol-d4)
OH N
HO OH OH HO O O O CH3
O O OCH3
CH3 O OH CH3
13 C NMR (126 MHz, Methanol-d4)
OH N
HO OH OH HO O O O CH3
O O OCH3
CH3 O OH CH3
87 1 1 H– H COSY (500 MHz, Methanol-d4)
OH N
HO OH OH HO O O O CH3
O O OCH3
CH3 O OH CH3
1 13 H– C HSQC (500 MHz, Methanol-d4)
OH N
HO OH OH HO O O O CH3
O O OCH3
CH3 O OH CH3
88 Erythronolide A 9-oxime (4.4) 1 H NMR (500 MHz, Methanol-d4)
OH N
HO OH OH
O OH
O OH
13 C NMR (126 MHz, Methanol-d4)
OH N
HO OH OH
O OH
O OH
89 1 1 H– H COSY (500 MHz, Methanol-d4)
OH N
HO OH OH
O OH
O OH
1 13 H– C HSQC (500 MHz, Methanol-d4)
OH N
HO OH OH
O OH
O OH
90 Erythronolide A (4.5) 1 H NMR (500 MHz, Methanol-d4)
O
HO OH OH
O OH
O OH
13 C NMR (126 MHz, Methanol-d4)
O
HO OH OH
O OH
O OH
91 1 1 H– H COSY (500 MHz, Methanol-d4)
O
HO OH OH
O OH
O OH
1 13 H– C HSQC (500 MHz, Methanol-d4)
O
HO OH OH
O OH
O OH
92 Erythronolide A 5,9-enol ether (4.6) 1 H NMR (500 MHz, Methanol-d4)
H3C
OH HO O OH HO O
O
13 C NMR (126 MHz, Methanol-d4)
H3C
OH HO O OH HO O
O
93