Towards Organoboron-mediated Functionalization of Erythromycin A and Synthesis of its Aglycon
by
Christopher D. Adair
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Chemistry University of Toronto
© Copyright by Christopher D. Adair 2014
ii
Towards Organoboron-mediated Functionalization of
Erythromycin A and Synthesis of its Aglycon
Christopher D. Adair
Master of Science
Department of Chemistry University of Toronto
2014
Abstract
Many natural products, including antibiotics, are structurally complex and contain a wide
variety of functional groups. As a consequence, the selective functionalization of these
molecules often requires the use of inefficient protecting group strategies. Inspired by this
obstacle, our group recently developed a borinic acid-catalyzed method to
regioselectively functionalize the equatorial position of cis-vicinal diols in carbohydrates
with limited use of protecting groups.
The work presented in this thesis describes progress made towards selective
functionalization of the cis-vicinal diol present in the macrolide antibiotic erythromycin
A. This was attempted using the boronic and borinic acid-mediated methodologies
developed previously in our group. Finally, a semisynthesis of erythronolide A was
carried out with the goal of using our methodology to prepare novel analogues for
biological evaluation.
iii
Acknowledgements
There was a time when I believed that personal success was driven solely by hard work
and perseverance. While the definition of success is dependent on whom you ask, I think
that many will agree that it is very difficult to be successful without the love and support
from others.
Firstly, I would like to acknowledge my parents. They continue to serve as my primary
inspiration and always will. Perhaps unknowingly, they’ve instilled within me a sense of
ambition, pride and humbleness that I will always cherish. My mother has always been
there to support me through the toughest times of my academic career and, for that, I am
forever grateful. My father has served a complementary role, pushing me to realize that I
have the potential to accomplish anything that I desire.
I should note that my choice to pursue synthetic organic chemistry wasn’t made until the
fourth year of my undergraduate career. As such, I have to thank to Professor France-
Isabelle Auzanneau for taking a chance on a student with limited synthesis experience.
She provided me with a wonderful introduction to carbohydrate chemistry and catalyzed
my passion for a very interesting branch of synthesis. I would also like to thank Professor
Mark S. Taylor. He taught me how to think like a scientist and suggested a project that
challenged me to go above and beyond what I thought possible.
A big thank you to the Taylor group! Being a part of such a smart and talented group of
people was truly a pleasure. A special thank you to Kyan D’Angelo and Kashif Tanveer
for sharing their vast knowledge of chemistry and contributing to many insightful
conversations about my work over the year.
And of course, I’m thankful for my brother and friends. There were times when I had to
make sacrifices to succeed academically and they were always supportive. Lastly, thank
you to Craig McDougall for being the best friend anyone could ask for.
iv
Table of Contents
Abstract .................................................................................................................................... ii
Acknowledgements .................................................................................................................. iii
Table of Contents ..................................................................................................................... iv
List of Tables ........................................................................................................................... vi
List of Figures .......................................................................................................................... vii
List of Schemes ........................................................................................................................ viii
Abbreviations ........................................................................................................................... xi
Chapter 1: Boron-Diol Interactions
1.0 Introduction ...................................................................................................... 1
1.1 Organoboron methodology in carbohydrate synthesis .................................... 3
1.2 Application of organoboron methodology to natural products ........................ 5
1.3 Conclusions ...................................................................................................... 8
Chapter 2: The Evolution of Antibiotics
2.0 Introduction ...................................................................................................... 10
2.1 Historical overview .......................................................................................... 10
2.2 Antibiotic resistance ......................................................................................... 13
2.3 Exploring new antibiotic landscape with chemical synthesis .......................... 14
2.4 Biosynthesis of novel antibiotic analogues ...................................................... 17
2.5 Conclusions and outlook .................................................................................. 19
Chapter 3: Application of Organoboron-mediated Transformations to Erythromycin A 3.0 Introduction ...................................................................................................... 20
3.1 Biosynthesis of erythromycin A ...................................................................... 21
3.2 Total synthesis of the erythromycins ............................................................... 25
3.3 Acid-catalyzed rearrangements of erythromycin A ......................................... 28
3.4 Semisynthetic analogues of erythromycin A ................................................... 30
3.5 Regioselective functionalization of erythromycin A ....................................... 31
3.6 Research goals ................................................................................................. 33
3.7 Results and discussion ..................................................................................... 34
v
3.7.1 Organoboron-mediated glycosylation of erythromycin A ................... 34
3.7.2 Organoboron-mediated benzoylation of erythromycin A .................... 37
3.7.3 NMR experiments with erythromycin A ............................................. 44
3.8 Conclusions and outlook .................................................................................. 48
3.9 Experimental details ......................................................................................... 49
3.10 Characterization data ....................................................................................... 50
Chapter 4: Semisynthesis of Erythronolide A 4.0 Introduction ...................................................................................................... 57
4.1 Semisynthesis of erythronolide A .................................................................... 57
4.2 Research goals ................................................................................................. 60
4.3 Results and discussion ..................................................................................... 60
4.4 Conclusions and outlook .................................................................................. 65
4.5 Experimental details ......................................................................................... 67
4.6 Characterization data ....................................................................................... 68
Appendix A: NMR spectra ..................................................................................................... 75
vi
List of Tables Table 3.1 – Borinic acid-mediated glycosylationa .................................................................. 35
Table 3.2 – Boronic acid-mediated glycosylationa ................................................................. 36
Table 3.3 – Organoboron-mediated benzoylation at 23 °Ca ................................................... 39
Table 3.4 – Organoboron-mediated benzoylation at 80 °Ca ................................................... 41
vii
List of Figures Figure 1.1 – Deprotected pentasaccharide target of our synthesis (1.1) and pentasaccharide derived target of the Du synthesis (1.2) ........................................................ 7 Figure 2.1 – Dimer, trimer and pentamer forms of arsphenamine (Salvarsan) effective for treating syphilis ........................................................................................................................ 11
Figure 2.2 – Selected antibiotics discovered in the 20th century of historical importance ..... 12
Figure 2.3 – Overview of the cephalosporin scaffold and examples of modern adaptations ............................................................................................................................... 15
Figure 3.1 – Components of the macrolide antibiotic erythromycin A .................................. 20
Figure 3.2 – Polyketide synthase-mediated chain elongation process to form 6-deoxyerythronolide B [adopted from (47)] .............................................................................. 22
Figure 3.3 – Select examples of 6-deoxyerythronolide B analogues generated by site-directed mutagenesis of polyketide synthase domains (McDaniel, 1999) ............................... 24
Figure 3.4 – Total syntheses of erythromycin derivatives ...................................................... 25
Figure 3.5 – Seco acid derivative for erythromycin A synthesis (Woodward, 1981) ............. 26
Figure 3.6 – Erythromycin A enol ether and anhydroerythromycin A ................................... 28
Figure 3.7 – Inherent reactivity of the hydroxyl groups in erythromycin A ........................... 32
Figure 3.8 – (a) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of Ph2BOH (3.37) (b) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of erythromycin A (3.1) upon addition of Ph2BOH (3.37) .......................................................... 45
viii
List of Schemes
Scheme 1.1 – Boronic acid-diol complexation equilibria in aqueous media .......................... 2
Scheme 1.2 – Boronic acid-mediated monoalkylation of methyl α-L-fucopyranoside with Lewis base activation ............................................................................................................... 3
Scheme 1.3 – Borinic acid-catalyzed regioselective monoacylation of carbohydrate derivatives ................................................................................................................................ 4
Scheme 1.4 – Borinic acid-catalyzed regioselective glycosylation of carbohydrate derivatives ................................................................................................................................ 4
Scheme 1.5 – Organoboron-catalyzed regio- and stereoselective formation of β-2-deoxyglycosidic linkages ......................................................................................................... 5
Scheme 1.6 – Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin ................................................................................. 6
Scheme 1.7 – Preparation of disaccharide fragment 1.4 using the borinic acid-catalyzed methodology ............................................................................................................................ 7
Scheme 1.8 – Preparation of disaccharide fragment 1.6 using the catalytic borinic acid and stoichiometric boronic acid methods ................................................................................ 8
Scheme 2.1 – Reductive removal of the C6-hydroxy group in 6-demethyltetracycline to give sancycline (Pfizer, 1958) .................................................................................................. 16
Scheme 2.2 – Semisynthesis of minocycline from sancycline (Lederle, 1967) ...................... 17
Scheme 2.3 – Semisynthesis of tigecycline from minocycline (Wyeth, 1994) ....................... 17
Scheme 2.4 – Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues by genetically engineered polyketide synthase (Khosla, 1996) .................................................... 18
Scheme 2.5 – Biosynthesis of unnatural erythromycin A derivatives ..................................... 19
Scheme 3.1 – Formation of 6-deoxyerythronolide B from propionyl CoA and methyl malonyl CoA ............................................................................................................................ 21
Scheme 3.2 – Post-PKS enzyme cascade to give erythromycin A .......................................... 23
ix
Scheme 3.3 – Key steps in Woodward’s total synthesis of erythromycin A .......................... 27
Scheme 3.4 – Acid degradation mechanism of erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C ........................................................................................................ 29
Scheme 3.5 – Semisynthesis of clarithromycin (Taisho, 1980) .............................................. 30
Scheme 3.6 – Semisynthesis of azithromycin (Pliva, 1980) ................................................... 31
Scheme 3.7 – Site-selective acylation of erythromycin A using a peptide catalyst (Miller, 2006) ........................................................................................................................................ 33
Scheme 3.8 – Proposed regioselective monofunctionalization of erythromycin A catalyzed by a diarylborinic acid ............................................................................................. 33
Scheme 3.9 – Monobenzoylation of erythromycin A using acetic anhydride in pyridine ...... 38
Scheme 3.10 – Monobenzoylation of erythromycin A enol ether under boron-free conditions ................................................................................................................................. 42
Scheme 3.11 – Erythromycin A acid-catalyzed rearrangement products and their molecular masses ..................................................................................................................... 43
Scheme 4.1 – Semisynthesis of erythronolide A (LeMahieu, 1974) ....................................... 58
Scheme 4.2 – Cope elimination procedure employed by Celmer for removal of the tertiary amine from D-desosamine in oleandomycin ............................................................... 59
Scheme 4.3 – Synthesis of erythromycin A 9-oxime N-oxide (4.2) ....................................... 61
Scheme 4.4 – Synthesis of 3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) via Cope elimination ....................................................................................................... 61
Scheme 4.5 – Synthesis of erythronolide A 9-oxime (4.4) under acidic conditions ............... 62
Scheme 4.6 – Nitrous acid-mediated oxime cleavage to give erythronolide A 5,9-enol ether (4.6) ................................................................................................................................. 63
Scheme 4.7 – Final steps of the erythronolide A total synthesis (Carreira, 2009) .................. 64
Scheme 4.8 – Oxime cleavage with Raney Nickel in the semisynthesis of erythronolide A (4.5) .......................................................................................................................................... 65
x
Abbreviations 1H proton (NMR spectroscopy)
13C carbon (NMR spectroscopy)
°C degrees Celsius
Å Ångstrom(s)
aq. aqueous
Ac acetyl
ACP acyl carrier protein
AT acyl transferase
Bn benzyl
Bz benzoyl
cat. catalytic or catalyst
d doublet
DCM dichloromethane
DEBS deoxyerythronolide B synthase
DIPEA N,N-diisopropylethylamine (Hünig’s base)
DMSO dimethylsulfoxide
equiv. equivalent(s)
ESI electrospray ionization
Et ethyl
EtOAc ethyl acetate
FTIR Fourier transform infrared spectroscopy
g gram(s)
xi
hr hour(s)
HMBC heteronuclear multiple bond correlation (NMR spectroscopy)
HPLC high-performance liquid chromatography
HRMS high-resolution mass spectrometry
i-Pr isopropyl
J coupling constant (NMR spectroscopy)
KS β-ketoacyl synthase
LC-MS liquid chromatography-mass spectrometry
M molar
m multiplet
m/z mass over charge
Me methyl
MeCN acetonitrile
MHz megahertz
mg milligram(s)
min minute(s)
mL milliliter(s)
mmol millimole(s)
mol mole(s)
MS molecular sieves
NBS N-bromosuccinimide
NMR nuclear magnetic resonance
PBP penicillin binding protein
xii
Ph phenyl
PKS polyketide synthase
PMP para-methoxyphenyl
ppm parts per million
q quartet
Ra-Ni Raney nickel
rpm revolutions per minute
RT room temperature
s singlet
sat. saturated
t triplet
TBDPS tert-butyldiphenylsilyl
TLC thin-layer chromatography
UDP uridine diphosphate
µL microliter(s)
1
1
Boron-Diol Interactions
1.0 Introduction
The scientific discipline known as organic synthesis has a rich history that has been
documented for nearly two hundred years. Remarkable advances in this field have been
observed during the 20th century and have significantly increased our understanding of
life on the atomic and molecular level.1 Despite these advances, organic chemistry
continues to be an ever-evolving field of study.
Recent efforts in organic synthesis have focused on asymmetric catalysis, driven
particularly by the pharmaceutical industry’s demand for chiral compounds. While a wide
variety of methods have been developed for this purpose, the functional group tolerance
of these methods is highly variable.2 As a result, strategic use of protective groups has
become commonplace when carrying out asymmetric synthesis.
Regioselective functionalization of hydroxyl groups in complex molecules represents a
significant challenge for synthetic chemists.3 This is especially true for polyol natural
products such as carbohydrates. Development of protecting group-free strategies to
selectively functionalize polyols would be of considerable value and have the potential to
revolutionize carbohydrate synthesis. In this regard, progress has been made using
1 Seebach, D. Angew. Chem. Int. Ed. 2003, 29, 1320–1367. 2 Johansson Seechurn, C. C. C.; Kitching, M. O.; Colocat, T. J.; Snieckus, V. Angew. Chem. Int. Ed. 2012, 51, 5062–5085. 3 Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007.
2
approaches such as organocatalytic processes4, Lewis acid-promoted methods5 and
enzyme-catalyzed methods.6
Most recently, organoboron reagents have emerged as an attractive approach to
selectively functionalize carbohydrates. Their ability to form reversible covalent
interactions with diols has been studied extensively in aqueous media, with initial reports
made by Lorand and Edwards in 1959 using phenylboronic acid (Scheme 1.1).7 It was
observed that boronate ester formation is favourable in solutions of high pH. This effect
was attributed to the lower angle strain present in the tetracoordinate boronate complex
relative to the tricoordinate conjugate acid. Subsequent study of this equilibrium has
revealed that structure and stereochemistry of the diol are important. It was found that
1,2-diols complex to boronic acids preferentially over 1,3-diols8 and that cis diols bind
preferentially to trans or simple acyclic diols.9
Scheme 1.1 – Boronic acid-diol complexation equilibria in aqueous media
4 (a) Griswold, K. S.; Miller, S. J. Tetrahedron. 2003, 59, 8869–8875. (b) Kawabata, T.; Muramatsu, W.; Nishio, T.; Shibata, T.; Schedel, H. J. Am. Chem. Soc. 2007, 129, 12890–12895. 5 Sn(IV) derivatives: (a) Iwasaki, F.; Maki, T.; Onomura, O.; Nakashima, W.; Matsumura, Y. J. Org. Chem. 2000, 65, 996–1002. (b) Martinelli, M. J.; Vaidyanathan, R.; Pawlak, J. M.; Nayyar, N. K.; Dhokte, U. P.; Doecke, C. W.; Zollars, L. M. H.; Moher, E.D.; Van Khau, V.; Kosmrjl, B. J. Am. Chem. Soc. 2002, 124, 3578–3585. (c) Demizu, Y.; Kubo, Y.; Miyoshi, H.; Maki, T.; Matsumura, Y.; Moriyama, N.; Onomura, O. Org. Lett. 2008, 10, 5075–5077. La(III) salts: Dhiman, R. S.; Kluger, R. Org. Biomol. Chem. 2010, 8, 2006–2008. 6 (a) Therisod, M.; Klibanov, A. M. J. Am. Chem. Soc. 1987, 109, 3977–3981. (b) Wang, Y.-F.; Lalonde, J. J.; Momongan, M.; Bergbreiter, D. E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200–7205. 7 Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769–774. 8 Pizer, R.; Tihal, C. Inorg. Chem. 1992, 31, 3243–3247. 9 James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem. Int. Ed. 1996, 35, 1910–1922.
BOH
OH HO
HOB
O
O2H2O
OH
BO
O2H2O
HOB
OH
OH HO
HOHO
OH
pH 7.5
pH >10
3
1.1 Organoboron Methodology in Carbohydrate Synthesis
Although the oxygen atoms involved in tricoordinate boronic ester formation are
deactivated, complexation with organoboron compounds can also be used as an activation
method. The group of Aoyama was the first to exploit this type of activation strategy
using a phenylboronate derived from methyl α-fucopyranoside.10 In the presence of
triethylamine, the phenylboronate underwent regioselective alkylation at O-3 (Scheme
1.2). It was proposed that coordination of the Lewis base to the boron atom resulted in
activation of the boronic ester towards reaction with iodobutane. This methodology was
later expanded to glycosylations of peracetylated glucosyl bromide donors with
deprotected carbohydrates containing cis-1,2-diol or 1,3-diol moieties.11
Scheme 1.2 – Boronic acid-mediated monoalkylation of methyl α-L-fucopyranoside with Lewis base activation
Inspired by the work of Aoyama, our group set out to develop organoboron-catalyzed
methods for regioselective carbohydrate activation. In 2011, our group reported a method
for catalytic acylation of carbohydrates using 2-aminoethyl diphenylborinate as a
precatalyst (Scheme 1.3).12 This work displayed general regioselectivity for the
equatorial hydroxyl group of the cis-diol in pyranoside derivatives of galactose, mannose,
fucose, and rhamnose. Of note, acylation of carbohydrates containing a free primary
hydroxyl group, such as β-galactopyranoside, resulted in competitive functionalization at
the primary hydroxyl group and desired secondary hydroxyl group.
10 Oshima, K.; Kitazono, E.-i.; Aoyama, Y. Tetrahedron Lett. 1997, 38, 5001–5004. 11 Oshima, K.; Aoyama, Y. J. Am. Chem. Soc. 1999, 121, 2315–2316. 12 Lee, D.; Taylor, M. S. J. Am. Chem. Soc. 2011, 133, 3724–3727.
O
OCH3
OHOOB
O
OCH3
OHOn-BuHO
Ph NEt3
n-BuI, Ag2O, NEt3
PhH, reflux, 22 hr
O
OCH3
OHOOB
Ph
O
OCH3
OHOHHO
PhB(OH)2
CH2Cl250%
4
Scheme 1.3 – Borinic acid-catalyzed regioselective monoacylation of carbohydrate derivatives
Another recent development in our group arose from adaptation of the borinic acid-
catalyzed acylation procedure to glycosylation of various carbohydrate derivatives.13 This
work served as the first reported example of a regioselective glycosylation procedure
using a nonenzymatic catalyst.14 Koenigs-Knorr glycosylations of several armed and
disarmed glycosyl halides with minimally or unprotected glycosyl acceptors gave good to
excellent yields with silver(I) oxide as a promoter (Scheme 1.4).
Scheme 1.4 – Borinic acid-catalyzed regioselective glycosylation of carbohydrate derivatives
Most recently, a strategy was developed in our group that enables regio- and
stereoselective glycosylations of pyranoside-derived cis-1,2- and 1,3-diols using both 2-
deoxy and 2,6-dideoxyglycosyl chloride donors with 2-aminoethyl diphenylborinate as a
precatalyst (Scheme 1.5).15 The stereoselective synthesis of these linkages is quite
challenging due to the anomeric effect and absence of participating protective groups at
13 Gouliaras, C.; Lee, D.; Chan, L.; Taylor, M. S. J. Am. Chem. Soc. 2011, 133, 13926–13929. 14 Mensah, E. A.; Nguyen, H. M. J. Am. Chem. Soc. 2009, 131, 8778–8780. 15 Beale, T. M.; Moon, P. J.; Taylor, M. S.; Org. Lett. 2014, 16, 3604–3607.
R1
HO
HO R2
OB
NH2
PhPh
(5–10 mol%)
RCOCl (1.2–2.0 equiv.)i-Pr2NEt (1.2–2.0 equiv.)
MeCN, RT
R1
HO
ROCO R2
69–95% (14 examples)
R3R4HO
OH
1.1 equiv
R3R4O
OH
R2
OR1
(10 mol%)
Ag2O (1 equiv.)MeCN, 23–60 oC
OB
NH2
Ph
Ph
R2
OR1
X
X = Br, Cl 68–99% (13 examples)
5
the C-2 position, resulting in bias toward α-configured 2-deoxy glycosides.16 Despite this
bias, the borinic acid catalyst favoured an SN2-type pathway to give β:α ratios ranging
from 4:1 to >19:1 with good yields of the desired regioisomer in 16 examples.
Scheme 1.5 – Organoboron-catalyzed regio- and stereoselective formation of β-2-deoxyglycosidic linkages
1.2 Application of organoboron methodology to natural
products
The carbohydrate scope of the borinic acid-catalyzed methodology developed in our
group was initially limited to simple mono- and disaccharide acceptors containing cis-
1,2-diols. To expand upon this work, it was of great interest to apply our methodology
towards the functionalization of complex polyol natural products.
With this goal in mind, the cardiac glycoside digitoxin was chosen as a target for
regioselective glycosylations using our methodology. Consistent with previous studies,
the equatorial position of the cis-1,2-diol of digitoxin was selectively glycosylated out of
the possible five free hydroxyl groups and gave good to excellent yields for all six
glycosyl donors (Scheme 1.6).17 A variety of peracetylated glycosyl bromides were
successfully employed and resulted in β-configuration of the newly formed glycosidic
bond. Cleavage of the acetyl groups from the newly linked sugars with lithium hydroxide
in methanol/water furnished the deprotected products, which could serve as new analogs
16 (a) Hou, D.; Lowary, T. L. Carbohydr. Res. 2009, 344, 1911−1940. (b) Crich, D. J. Org. Chem. 2011, 76, 9193−9209. 17 Beale, T. M.; Taylor, M. S. Org. Lett. 2013, 15, 1358–1361.
OAcO
OAc
AcOCl
O
O
HOOCH3
OHO
TBDPSH
OB
NH2
PhPh
(10 mol%)
Ag2O (2 equiv.)CH2Cl2, RT
OAcO
OAc
AcOO
O
OOCH3
OHO
TBDPSH
Catalyzed reaction: 72% yield, 7.3:1 β:α Uncatalyzed reaction: 18% yield, 2:1 β:α
6
for biological evaluation. Notably, the levels of regiocontrol for the 4”-O-glycosylated
isomer were excellent, with the major byproduct being unreacted digitoxin rather than
regioisomers.
Scheme 1.6 – Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin
While late stage glycosylation of complex natural products presents a useful strategy to
prepare novel semisynthetic analogues, it would also be advantageous to apply our
O
OH
HOH3C
O O
OH
H3C
O O
OH
H3C
O
OB
NH2
Ph
Ph
(25 mol%)
Ag2O (2 equiv.)
CH2Cl2, 23 ˚C(2 equiv.)
O
OH
OH3C
O O
OH
H3C
O O
OH
H3C
O
R2
OR1
Br
R2
OR1
OAcO
OAc
AcOBrAcO
OAcO
AcOBr
OAcOAcO
AcO
OAc
77%
O
OAc
74%
O OAcOAcAcO
Br
63%
O OAcOAcAcO
Br
51%
OAcO
AcOBr
OAcO
AcO
OAc
O
OAc
64%
OAc
O
OAc
AcOBrAcO
63%
OAc
OH
OOCH3CH3
OH
OOCH3CH3
7
borinic acid-catalyzed methodology to oligosaccharide total synthesis. In particular, we
envisioned using borinic acid catalysts for two regioselective glycosylation reactions in
the synthesis of a pentasaccharide derivative (1.1) isolated from Spergularia ramosa
(Figure 1.1). The first synthesis of this oligosaccharide was completed by Du and co-
workers and involved 14 steps to reach target 1.2.18 Although each step in the synthesis is
relatively efficient, nine of the fourteen steps are protective group manipulations.
Figure 1.1 – Deprotected pentasaccharide target of our synthesis (1.1) and pentasaccharide derived target of the Du synthesis (1.2)
To improve upon this synthesis, our group used the catalytic borinic acid-methodology to
facilitate glycosylation of a peracetylated glucosyl bromide donor (1.3) and unprotected
pentenyl rhamnose acceptor (1.4), which proceeded in 80% yield (Scheme 1.7).
Scheme 1.7 – Preparation of disaccharide fragment 1.4 using the borinic acid-catalyzed methodology
18 Gu, G.; Du, Y. J. Chem. Soc., Perkin Trans., 1. 2002, 2075–2079.
O
O
O
OOH
O
HOHOHOHO
O
HOHOHO
O
HO
HO
HOO
HO
OOR
O
O
O
OOAc
O
AcOAcOAcOAcO
O
AcOAcOAcO
O
BzO
BzO
BzOO
BzO
O
O
1.21.1
O
O
HOHO
OHO
AcOAcO
AcO
Br
OAc
O
O
O
HOOHO
AcOAcO
AcO
OAc
80%Ag2O (1 equiv.)
MeCN
OB
NH2
PhPh
(10 mol%)
(1.1 equiv.)
1.3 1.4
8
Unfortunately, attempts at glycosylating a peracetylated fucosyl bromide donor (1.5) with
unprotected PMP arabinose acceptor (1.6) using the catalytic procedure resulted in poor
yields. This prompted the development of a stoichiometric boronic acid-mediated
approach, which led to significant improvement in yield (Scheme 1.8).19 Further
optimization of this synthesis is currently underway.
Scheme 1.8 – Preparation of disaccharide fragment 1.6 using the catalytic borinic acid and stoichiometric boronic acid methods
1.3 Conclusions
Our group’s development of regioselective functionalization reactions for carbohydrates
using borinic acid-derived catalysts provides several advantages over traditional
19 McClary, C. A. 2013. Exploring Noncovalent and Reversible Covalent Interactions as Tools for Developing New Reactions. (Doctor of Philosophy Dissertation).
O
AcO
AcO
AcO
O
OH
HO
HO OPMP
Br
O
AcO
AcO
AcOO
OH
HO
O OPMP
Ag2O (1 equiv.)MeCN, RT
(1.1 equiv.) 20%
O
AcO
AcO
AcO
O
OH
HO
HO OPMP
Br
O
AcO
AcO
AcOO
OH
HO
O OPMP
Ag2O (1 equiv.)NEt3 (3 equiv.)
MeCN, RT
(1.1 equiv.)
(1 equiv.)
77%
B(OH)2F F
FF
F
OB
NH2
PhPh
(10 mol%)
1.5 1.6
1.5 1.6
Catalytic (borinic)
Stoichiometric (boronic)
9
oligosaccharide synthesis, organotin protocols and enzymatic methods. Highlights of our
methodology include the use of a relatively benign, inexpensive catalyst and a simplistic
reaction setup that does not require high temperatures, long reaction times or exclusion of
air. Furthermore, the regiochemical outcome of these reactions is predictable and
reproducible for substrates bearing a cis-1,2-diol motif. Current efforts are focused on
developing new borinic acid-catalyzed glycosylation protocols that avoid using
stoichiometric quantities of heterogeneous silver(I) salts and adopting our current
methodology to regio- and stereocontrolled functionalization of complex natural
products.
10
2
The Evolution of Antibiotics
2.0 Introduction
During the 19th century, infections such as pneumonia, diphtheria and diarrhea
represented the principle causes of death in children and adults.20 As a consequence of
the industrial revolution and subsequent urbanization, incidence rates of these ailments
and others, such as syphilis and tuberculosis, increased significantly. The introduction of
aseptic technique in 1867 served as a starting point for minimizing the risk of bacterial
infection but many of these diseases remained incurable.21 It wasn’t until the early 20th
century that the first modern chemotherapeutic agents were discovered and implemented
in treatment of common bacterial infections.
2.1 Historical overview
One of the first antibacterial agents used to treat infections was arsphenamine. Soon after
its discovery in 1910, arsphenamine was marketed under the trade name Salvarsan and
referred to as the “magic bullet” for treatment of syphilis.22 This synthetic organoarsenic
compound was a significant improvement to inorganic mercury compounds used
previously to treat syphilis but was relatively difficult to administer due to its
hygroscopic nature and remarkable sensitivity to atmospheric conditions. Interestingly,
its chemical composition was recently shown to be that of two different organoarsenic
structures (2.2, 2.3) rather than the previously described dimer 2.1 (Figure 2.1).23
20 Christoffersen, R. E. Nat. Biotechnol. 2006, 24, 1512–1514. 21 Wallace, W. C.; Cinat, M. E.; Nastanski, F. Am. Surg. 2000, 66, 874–878. 22 Riethmiller, S. Chemotherapy. 2005, 51, 234–242. 23 Lloyd, N.C.; Morgan, H.W.; Nicholson, B.K.; Ronimus, R. S. Angew. Chem. Int. Ed. 2005, 44, 941–944.
11
Figure 2.1 – Dimer, trimer and pentamer forms of arsphenamine (Salvarsan) effective for treating syphilis
The first general-purpose antibiotic to gain widespread use was prontosil (2.4), developed
in the 1930s by Bayer Laboratories.24 Prontosil is a synthetic diazo dye containing a
sulfonamide functionality. The discovery of this compound marked the beginning of a
new class of antibiotics known as the sulfa drugs. These sulfonamide containing
compounds act as analogues of para-aminobenzoic acid and ultimately inhibit folate
synthesis.25 This induces the inhibition of DNA, RNA and protein synthesis in a broad
range of both Gram-positive and Gram-negative bacteria.
Though antibiotics of synthetic origin are important, they account for only a small
fraction of antibiotics in use today. In fact, most antibacterial agents used commonly in
hospitals originated from natural products.26 Perhaps the most revolutionary example of a
naturally occurring antibiotic is penicillin, discovered by Alexander Fleming in 1928.
The penicillins [see penicillin G (2.5)] belong to a large family of β-lactam antibiotics
that also include the cephalosporins and are responsible for saving the lives of countless
soldiers during World War II. The β-lactam ring structure is essential for antimicrobial
activity and has been shown to inhibit formation of the peptidoglycan crosslink in the
bacterial cell wall, thereby activating cell wall autolysis in Gram-positive bacteria.
24 Owa, T.; Nagasu, T.; Expert Opin. Ther. Pat. 2000, 10, 1725–1740. 25 Kalkut, G.; Cancer Invest. 1998, 16, 612–615. 26 Singh, S. B.; Barrett, J. F. Biochem. Pharmacol. 2006, 71, 1006–1015.
As AsAs
OHNH2
OHNH2
HO
H2NAs As
AsAs
As
OHNH2
NH2
OH
OHH2NHO
H2N
HO
H2N
AsAs
H2N
HO
OH
NH2
2.1 2.2 2.3
12
Figure 2.2 – Selected antibiotics discovered in the 20th century of historical importance
In the decades following, several new classes of naturally occurring antibiotics were
discovered and implemented in routine medical practice. Among them are the
tetracyclines [see tetracycline (2.6)], the macrolides [see erythromycin A (2.7)] and the
glycopeptides [see vancomycin (2.8)]. While all having varying modes of action, the
usual targets of these antibacterial agents are cell wall synthesis, protein synthesis,
nucleic acid synthesis, or important biosynthetic pathways.27
The significant growth experienced in the mid 20th century in the development of
antibiotics was not sustained in the following decades, resulting in nearly 40 years before
the introduction of a new class of antibiotics. This has, in part, been attributed to the
belief that bacterial infections were becoming an issue of the past.28 However, the
discovery of antibiotic resistant bacteria proved this hypothesis false. Indeed, resistance
27 Hartmann, G.; Behr, W.; Beissner, K.-A.; Honikel, K.; Sippel, A. Angew. Chem. Int. Ed. 1968, 7, 693–701. 28 Overbye, K. M.; Barrett, J. F. Drug Discovery Today. 2005, 10, 45–52.
H2N
NH2
NN
SO2NH2
HN
O N
S MeMe
CO2H
H
OOH O O
OH
CONH2O
NMe2H
O
Me OHH
HH
O
NHHN
Cl
NHO
O
HNNH
HOO H
N
O
O
O
HO
Cl
OO NH2
OH
OH
ONH
HO OH
OO
OO
OH
HH
O
O
N
Me
O
Me
H2H
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
2.4: prontosil (sulfonamide) 2.5: penicillin G (β-lactam) 2.6: tetracycline (tetracycline)
2.7: erythromycin A (macrolide)
2.8: vancomycin (glycopeptide)
13
to last resort antibiotics such as vancomycin has become a significant problem that only
recently gained widespread attention.
2.2 Antibiotic resistance
From a biological perspective, antibacterial drug resistance is an intriguing aspect of
evolution. Under the selective pressure of antibiotics, bacteria evolve to spread resistance
mechanisms that eventually become prevalent among other pathogenic and
nonpathogenic bacteria. Alternatively, bacteria may also become resistant to a class of
antibiotics through random spontaneous mutation of their genetic material. In general,
resistance is exhibited through the following mechanisms:29
1) upregulation of enzymes that inactivate the antibiotic (e.g., β-lactamases) or
modify of the cellular target (e.g., ribosomal methylase in Staphylococci
preventing erythromycin binding);
2) modification or loss of the target with which the antibiotic interacts (e.g.,
alteration of penicillin-binding protein in Pneumococci);
3) upregulation of pumps that expel the antimicrobial agent from the cell (e.g.,
efflux of fluoroquinolones in Staphylococcus aureus);
4) downregulation or inactivation of outer membrane protein channels required
for entry of the antibiotic into the cell (e.g., resistance to β-lactams by OmpF
porin downregulation in Escherichia coli).
Even with judicious use of antibiotics, the onset of bacterial resistance is inevitable. Thus,
the development of new antibiotics is a significant priority. Fortunately, developments in
chemistry and biology have improved our ability to discover new antibiotics. This has
been accomplished through exploration of new natural product chemical space,
modification of previously existing structures and genetic engineering of antibiotic-
producing biosynthetic pathways.30 Collectively, these advances have facilitated the
29 Gallo, G.; Puglia, A. M. Antibiotics: Targets, Mechanisms and Resistance.; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; pp 73–80. 30 Nicolaou, K. C.; Chen, J. S.; Edmonds, D. J.; Estrada, A. A. Angew. Chem. Int. Ed. 2009, 48, 660–719.
14
development of antimicrobial agents that avoid resistance and have novel mechanisms of
action.
2.3 Exploring new antibiotic landscape with chemical
synthesis
Since the discovery of the sulfonamide drugs, chemical synthesis has played a critical
role in the development of new antibiotics.31 Even though fermentation is the preferred
method to manufacture large quantities of clinically used antibiotics, chemical synthesis
has served an important and complementary purpose. For example, structural
modification of naturally occurring antibiotics has yielded compounds with improved
biological properties. Furthermore, the de novo synthesis of natural product antibiotics
and their analogues has contributed to our understanding of their mode of action through
structure-activity relationships (SARs). These studies assist scientists in designing
improved antibiotic analogues that are effective against resistant bacterial strains.
One class of antibiotics that has been subjected to thorough medicinal chemistry efforts is
the cephalosporin class of β-lactam antibacterials. There are now at least four recognized
generations of the cephalosporins, which are differentiated by their activity spectrum and
efficacy rather than by structural diversity. While each generation of these β-lactams is
different, this is not to say that compounds of earlier generations are obsolete. In fact,
there are several antibiotics in each generation that are still in clinical use today.32
The cephalosporins bind to enzymes known as penicillin-binding proteins (PBPs) through
acylation of the β-lactam amide bond, which is mediated by a nucleophilic serine residue
in the active site. Though β-lactamase enzymes are largely responsible for antibiotic
resistance to the cephalosporins, the presence of penicillin-binding protein 2a (PBP2a) in
31 Nussbaum, F. V.; Brands, B. M.; Hinzen, S.; Weigand, D.; Habich, C. Angew. Chem. 2006, 118, 5194–5254. 32 Page, M. G. Expert Opin. Invest. Drugs. 2004, 13, 973–985.
15
certain strains of Staphylococcus aureus has led to resistance to many β-lactams.33 This is
because PBP2a has a very low affinity for traditional β-lactam antibiotics. Consequently,
even when other PBPs are inhibited, PBP2a can continue to mediate cell wall
biosynthesis, thus leading to β-lactam resistance.
The discovery of ceftobiprole (2.9) provided extensive insight into the inhibition
mechanism of many cephalosporin antibiotics. Strynadka and co-workers obtained a
crystal structure of ceftobiprole bound to the PBP2a active site, which led to the
discovery that the hydrophobic nature and planarity of the R2 group was essential for
effective binding to the active site (Figure 2.3).34 With this knowledge, synthetic
modifications were made to increase the hydrophobicity of the R2 group in ceftobiprole
that resulted in the development of ceftaroline (2.10). Ceftaroline displays greater affinity
for the PBP2a active site, which increases rate of acylation of the β-lactam amide bond
and, thus, improves antibacterial activity.35
Figure 2.3 – Overview of the cephalosporin scaffold and examples of modern adaptations
33 Saravolatz, L. D.; Stein, G. E.; Johnson, L. B. Clin. Infect. Dis. 2011, 52, 1156–1163. 34 Lovering, A. L.; Gretes, M. C.; Safadi, S. S.; Danel, F.; Castro, L.; Page, M. G. P.; Strynadka, N. C. J. J. Biol. Chem. 2012, 287, 32096–32102. 35 Llarrull, L. I.; Fisher, J. F.; Mobashery, S. Antimicrob. Agents Chemother. 2009, 53, 4051–4063.
N
S
CO2HR2O
HNR1
O
Cephalosporin Scaffold
R1 – essential for stabilityto hydrolysis by β-lactamases
R2 – essential for achieving higherbinding affinity for PBP2a
β-lactam ring – essential for inhibition oftranspeptidase activity in cell wall biosynthesis
N
S
CO2HO
HN
O N NH
N OMeN
S N
H2N
O
2.9: ceftobiprole
N
S
CO2HSO
HN
O
N OEtN
S N
HN
S
N
NMe
POHO
HO
2.10: ceftaroline
16
The tetracyclines, discovered in 1945, were the first broad-spectrum antibiotics
incorporated into routine medical practice.36 Effective against Gram-positive and Gram-
negative bacteria, the tetracyclines have been used extensively in human and veterinary
medicine for treatment of bacterial infections and as feed additives.37 As a consequence
of their widespread use, high levels of bacterial resistance have been reported. However,
in light of their broad-spectrum activity, good safety profile and abundant supply,
tetracyclines remain first-line antibiotics for ailments such as pneumonia, Lyme disease,
cholera, and acne vulgaris.
Beginning with the semisynthesis of tetracycline from chlorotetracycline, the
development of semisynthetic tetracycline analogues has been instrumental in tackling
complications associated with bacterial resistance. Approximately 10 years after the
discovery of tetracycline, Pfizer demonstrated that the C6-hydroxy group of tetracycline,
oxytetracycline and 6-demethyltetracycline could be removed reductively (Scheme
2.1).38 The resulting 6-deoxytetracyclines were found to be more stable than their
predecessors, while retaining similar broad-spectrum antibacterial activity.
Scheme 2.1 – Reductive removal of the C6-hydroxy group in 6-demethyltetracycline to give sancycline (Pfizer, 1958)
Additionally, the improved chemical stability of the 6-deoxytetracyclines enabled acid-
and base-mediated structural modifications that had not been previously possible, leading
36 Duggar, B. M. Ann. N. Y. Acad. Sci. 1948, 51, 177–181. 37 Stockstad, E. L. R.; Jukes, T. H.; Pierce, J.; Page, A. C.; Franklin, A. L. J. Biol. Chem. 1949, 180, 647–654. 38 McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P. J. Am. Chem. Soc. 1960, 82, 3381–3388.
OH O O
OH
CONH2O
NMe2H
O
H
HH
6-demethyltetracycline(natural product)
HO H
6Pd, H2, HCl
MeOHOH O O
OH
CONH2O
NMe2H
O
H
HH
sancycline
H H
6
17
to the discovery of minocycline in 1967.39 Minocycline was synthesized from 6-deoxy-6-
demethyltetracycline (sancycline) by an electrophilic aromatic substitution at C7
(Scheme 2.2) and exhibited a broader spectrum of antimicrobial activity than previous
tetracyclines.
Scheme 2.2 – Semisynthesis of minocycline from sancycline (Lederle, 1967)
Aiming to overcome tetracycline resistance in the late 1990s, the group of Tally and co-
workers synthesized 7,9-disubstituted tetracycline analogues, which led to the discovery
of tigecycline in 1994 (Scheme 2.3).40 These new derivatives greatly extended the
antimicrobial spectrum of tetracyclines, especially towards tetracycline-resistant bacteria.
After its FDA approval in 2005, tigecycline quickly became the antibiotic of choice for
last-line of defense against multidrug-resistant bacteria.54
Scheme 2.3 – Semisynthesis of tigecycline from minocycline (Wyeth, 1994)
2.4 Biosynthesis of novel antibiotic analogues
Many antibiotic-producing biosynthetic pathways have been studied extensively over the
past several decades.41 This has helped scientists develop a better understanding of the
39 Church, R. F. R.; Schaub, R. E.; Weiss, M. J. J. Org. Chem. 1971, 36, 723–725. 40 Sum, P. E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–188.
41 Moellering, R. C. N. Engl. J. Med. 2010, 363, 2377–2379.
OH O O
OH
CONH2O
NMe2H
OHH
sancycline
H7
9
KNO3, H2SO4
OH O O
OH
CONH2O
NMe2H
OHH
HNO2
Pd, H2, CH2O
MeOH OH O O
OH
CONH2O
NMe2H
OHH
HNMe2
minocyclineMixture of 7- and 9-nitro isomers
OH O O
OH
CONH2O
NMe2H
OHH
HNMe2
minocycline
1) KNO3, H2SO4
2) Pd/C, H2 OH O O
OH
CONH2O
NMe2H
OHH
HNMe2
H2N
O
ClHN
t-BuHCl
OH O O
OH
CONH2O
NMe2H
OHH
HNMe2
NH
OHN
t-Bu
tigecycline
18
antibacterial agent’s mechanism of action and, more recently, to develop novel antibiotic
analogues. Despite the biological complexity of these pathways and enzymes involved,
biosynthesis presents a unique alternative to chemical synthesis and can be particularly
advantageous for synthesizing structurally complex antibiotics.
In 1997, Khosla and co-workers genetically modified the enzyme polyketide synthase
(PKS) to synthesize new derivatives of the macrolide antibiotic erythromycin A.42 In this
work, a genetic block was introduced to deoxyerythronolide B synthase (DEBS), which
disrupted the first condensation step in erythromycin A biosynthesis. Expressing this
mutation in a strain of Streptomyces coelicolor with inactive ketosynthase KS1 allowed
for introduction of unnatural synthetic building blocks into the 6-deoxyerythronolide B
scaffold (Scheme 2.4). This strategy furnished several new analogues of 6-
deoxyerythronolide B not previously accessible by chemical synthesis.
Scheme 2.4 – Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues by genetically engineered polyketide synthase (Khosla, 1996)
The successful synthesis of these unnatural intermediates prompted investigation into
whether the post-PKS enzymes in the erythromycin biosynthetic pathway might also
accept unnatural substrates. Positive results were obtained for substrates containing
42 Jacobsen, J. R.; Hutchinson, R. C.; Cane, D. E.; Khosla, C. Science. 1997, 277, 367–369.
O
O
OH
O OH
OHSCoA
OOH DEBS
O
O
OH
O OH
OHSNAC
OOH DEBS KS1°
in vivo
O
O
OH
O OH
OHSNAC
OOH DEBS KS1°
in vivo
O
O
OH
OH
OHSNAC
O DEBS KS1°
in vivo
OH
O
Typical propionyl CoA substrate to give 6-deoxyerythronolide B
19
methyl, n-propyl and phenyl R groups when subjected to S. erythraea mutants unable to
synthesize 6-deoxyerythronolide B (Scheme 2.5).
Scheme 2.5 – Biosynthesis of unnatural erythromycin A derivatives
2.5 Conclusions
The history of antibiotics describes a fascinating scientific journey through the 20th
century. From the beginning of the antibiotic era to present day, the role of chemical
synthesis remains of critical importance. Total- and semisynthesis, in combination with
medicinal chemistry efforts, continue to yield next-generation antibacterial agents with
improved biological activity that aid in deterring bacterial resistance. Although
biosynthesis remains a relatively underdeveloped strategy for antibiotic development, the
ability to generate novel antibiotic analogues through genetic engineering represents an
intriguing approach worth further exploration. Though recent developments in biology
and chemistry have improved our ability to discover new antibiotics and manipulate
privileged structures, the inevitable onset of bacterial resistance will demand the
continued search for new antimicrobial agents in the years ahead.
O
O
OH
O OH
OHR
O
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
R
R = Methyl, n-Propyl, Phenyl
20
3
Application of Organoboron-mediated Transformations to Erythromycin A
3.0 Introduction
In 1952, the pharmaceutical company Eli Lily commercialized the first macrolide
antibiotic, erythromycin A (3.1, Figure 3.1). This marked the discovery of an important
subclass of polyketide antibiotics that are used extensively in the treatment of bacterial
infections and remain one of the most widely studied antibiotic classes in modern
medicine.43 Erythromycin A was discovered in 1949 when researchers from Eli Lily
isolated the metabolic products of Saccharopolyspora erythraea in a soil sample from the
Philippines. It was found that erythromycin A is effective against many Gram-positive
bacteria, mediated by ribosomal binding and subsequent inhibition of protein synthesis.
Advantageously, its antimicrobial spectrum has been reported to be wider than that of
penicillin and is often prescribed to individuals allergic to the penicillins.44 In terms of
Figure 3.1 – Components of the macrolide antibiotic erythromycin A
43 Pal, S. Tetrahedron. 2006, 14, 3171–3200. 44 Washington, J. A.; Wilson, W. R. Mayo Clin. Proc. 1985, 60, 189–203.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
erythromycin A (3.1)
Aglycone(erythronolide A)
D-desosamine
L-cladinose
21
structure, erythromycin A is described as a macrolide. This term was introduced by R. B.
Woodward to denote a class of substances produced by Streptomyces bacteria that
contain a macrocyclic lactone to which one or more carbohydrates are attached.45 The
aglycon of erythromycin A, referred to as erythronolide A, is linked to two unusual
sugars, D-desosamine and L-cladinose.
3.1 Biosynthesis of erythromycin A
With its plethora of stereocenters and 14-membered cyclic backbone, erythromycin A
represents a relatively complex natural product. Thus, it is of interest to discuss the
underlying mechanism of its biosynthesis. Macrolides, such as erythromycin A, contain a
macrocyclic lactone scaffold that is synthesized by polyketide synthase (PKS) in a multi-
enzyme process. Using one unit of propionyl CoA (3.2) and six units of methylmalonyl
CoA (3.3), PKS mediates a sequential chain elongation process. This is followed by a
termination event that results in separation of the newly formed chain from PKS and
cyclization to yield 6-deoxyerythronolide B (3.4, Scheme 3.1).46
Scheme 3.1 – Formation of 6-deoxyerythronolide B from propionyl CoA and methyl malonyl CoA
Figure 3.2 provides an excellent representation of the chain elongation process and the
steps involved in between each condensation. The three essential domains – β-ketoacyl
synthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) – co-operate to
catalyze carbon-carbon bond formation by Claisen condensation, which results in a β-
45 Woodward, R. B. Angew. Chem. 1957, 69, 50. 46 Corcoran, J. W.; Vygantas, A. M. Biochemistry. 1982, 21, 263.
SCoA
O
SCoA
O
CO2H
Chain assembly on PKS
Cyclization and release from enzyme
O
O
OH
O
Et
OH
OH
6-deoxyerythronolide B (3.4)
(3.2) (3.3)
22
keto ester intermediate. The variable set of domains positioned between the AT and ACP
then carry out reductive modification of the keto group before the next round of chain
extension. After the sixth unit of methylmalonyl CoA is added, thioesterase catalyzes
chain cleavage and cyclization to give 6-deoxyerythronolide B.47
Figure 3.2 – Polyketide synthase-mediated chain elongation process to form 6-deoxyerythronolide B [adopted from (47)]
47 Staunton, J.; Wilkinson, B. Chem. Rev. 1997, 97, 2611–2629.
23
6-deoxyerythronolide B then undergoes a series of site-selective functionalization
reactions to yield erythromycin A (Scheme 3.2).
Scheme 3.2 – Post-PKS enzyme cascade to give erythromycin A
Firstly, C-6 hydroxylation of 6-deoxyerythronolide B (3.4) is accomplished by a
cytochrome P450 enzyme and occurs with retention of configuration to give
erythronolide B (3.5).48 In the next step, L-mycarose is linked to the C-3 hydroxyl group
by TDP-mycarose glycosyltransferase to yield 3-O-mycarosylerythronolide B (3.6).49
Then, the amino carbohydrate D-desosamine is linked to the C-5 hydroxyl group by
48 Corcoran, J. W. In Antibiotics, Volume IV: Biosynthesis; Corcoran, J. W., Ed.; Springer-Verlag: New York; 1981, pp 132. 49 Martin, J. R.; Perun, T. J.; Girolami, R. L. Biochemistry. 1966, 5, 2852.
O
O
OH
O
Et
OH
OH
6-deoxyerythronolide B (3.4)
O
O
OH
O
Et
OH
OH
OH b
erythronolide B (3.5)
O
O
OH
O
Et
O
OH
OH
3-O-Mycarosylerythronolide B (3.6)
OCH3
OHCH3
OH
a
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
erythromycin A (3.1)
O
O
OH
O
Et
O
O
OH
OCH3
OHCH3
OMe
O
N(CH3)2
CH3HO
erythromycin B (3.8)
c
O
O
OH
O
Et
O
O
OH
OCH3
OHCH3
OH
O
N(CH3)2
CH3HO
erythromycin D (3.7)
O
O
OH
O
Et
O
O
OH
OCH3
OHCH3
OH
O
N(CH3)2
CH3HO
erythromycin C (3.9)
HO
erythromycin D (3.7)
d e
de
a – C-6 erythronolide hydroxylaseb – TDP-mycarose glycosyltransferasec – TDP-desosamine glycosyltransferased – (O)-methyltransferasee – C-12 hydroxylase
24
TDP-desosamine glycosyltransferase. The resulting intermediate, erythromycin D (3.7),
is the first to show antibacterial activity and occurs at a branch in the synthetic pathway.50
Either O-methylation of the C-3” hydroxyl on the mycarose sugar follows, to produce
erythromycin B (3.8), or C-12 hydroxylation takes place with retention of configuration
to furnish erythromycin C (3.9).51 Finally, erythromycin A (3.1) is generated either by C-
12 hydroxylation of 3.8 or O-methylation of 3.9.
As shown in chapter 2, Scheme 2.4, genetic manipulation of PKS allows for production
of novel 6-deoxyerythronolide B analogues. In the example presented by Khosla and co-
workers, genetically modified PKS enabled the use of substrates other than propionyl
CoA for the chain elongation process in preparation of 6-deoxyerythronolide B. More
recently, McDaniel and co-workers manipulated several genetic modules within
polyketide synthase and generated a library of more than 50 macrocycles that would be
impractical to produce by chemical synthesis (select examples, Figure 3.3).52
Figure 3.3 – Select examples of 6-deoxyerythronolide B analogues generated by site-directed mutagenesis of polyketide synthase domains (McDaniel, 1999)
In this work, the authors systematically engineered single and multiple enzymatic domain
substitutions in deoxyerythronolide B synthase (DEBS) to demonstrate the utility of PKS
mutagenesis techniques. Firstly, substitutions were made to the acyl transferase (AT)
domain that resulted in mutants incorporating acetate rather than propionate units to
generate analogues lacking a methyl substituent at the engineered position (see 3.10,
50 Weber, J. M.; Leung, J. O.; Maine, G. T.; Potenz, R. H. B.; Paulus, T. J.; DeWitt, J. P. J. Bacteriol. 1990, 172, 2372. 51 Corcoran, J. W.; Vygantas, A. M. Fed. Proc. 1977, 36, 663. 52 McDaniel, R.; Thamchaipenet, A.; Gustaffson, C.; Fu, H.; Betlach, M.; Betlach, M.; Ashley, G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1846–1851.
O
O
OH
O
Et
OH
OH
O
O
OH
O
Et
OH
OH
O
O
O
Et
OH
OH
O
O
OH
O
Et
OH
O
O
OH
O
Et
O
OH
O
O
OH
O
Et
OH
O
(3.10) (3.11) (3.12) (3.13) (3.14) (3.15)
25
3.11). Similarly, mutagenesis allowed for replacement of β-ketoacyl- and enoyl
reductases with domains from the rapamycin PKS that resulted in the corresponding
alcohol moieties being replaced with alkene and alkane carbons (see 3.12, 3.13). Lastly,
deletion mutagenesis of the reductase domains converted hydroxyl groups to ketones in
several examples (see 3.14, 3.15). Combining these genetic alterations in varying orders
allowed for rapid access to a large library of 6-deoxyerythronolide derivatives. These
novel compounds could in themselves provide the basis for new pharmaceuticals or could
serve as scaffolds for new semisynthetic analogues.
3.2 Total synthesis of the erythromycins
Beyond its impact on human medicine, erythromycin A has been closely tied to the
evolution of synthetic organic chemistry. Its discovery has prompted numerous total
syntheses of erythromycin biosynthetic precursors over the past 35 years (Figure 3.4).53
Figure 3.4 – Total syntheses of erythromycin derivatives
53 Gao, X.; Woo, S. K.; Krische, M. J. J. Am. Chem. Soc. 2013, 135, 4223–4226. 54 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3215. 55 Martin, S. F.; Hida, T.; Kym, P. R.; Loft, M.; Hodgson, A. J. Am. Chem. Soc. 1997, 119, 3193. 56 Corey, E. J.; et al. J. Am. Chem. Soc. 1979, 101, 713. 57 Nakata, M.; Arai, M.; Tomooka, K.; Ohsawa, N.; Kinoshita. M. Bull. Chem. Soc. Jpn. 1989, 62, 2618. 58 Muri, D.; Lohse-Fraefel, N.; Carreira, E. M. Angew. Chem., Int. Ed. 2005, 117, 4036. 59 Corey, E. J.; et al. J. Am. Chem. Soc. 1978, 100, 4620. 60 Sviridov, A. F.; et al. Tetrahedron Lett. 1987, 28, 3839. 61 Mulzer, J.; Kirstein, H. M.; Buschmann, J.; Lehmann, C.; Luger, P. J. Am. Chem. Soc. 1991, 113, 910. 62 Masamune, S.; Hirama, M.; Mori, S.; Ali, S. A.; Garvey, D. S. J. Am. Chem. Soc. 1981, 103, 1568. 63 Myles, D. C.; Danishefsky, S. J. J. Org. Chem. 1990, 55, 1636. 64 Evans, D. A.; Kim, A. S. Tetrahedron Lett. 1997, 38, 53. 65 Stang, E. M.; White, M. C. Nat. Chem. 2009, 1, 547.
Erythromycin A – R1 = OH, R2 = D-desosamine, R3 = L-cladinose, R4 = OH Woodward (1981): 55 steps (LLS), 77 steps (TS)54
Erythromycin B – R1 = OH, R2 = D-desosamine, R3 = L-cladinose, R4 = H Martin (1997): 28 steps (LLS), 33 steps (TS)55
Erythronolide A – R1 = OH, R2 = H, R3 = H, R4 = OH Corey (1979): 39 steps (LLS), 50 steps (TS)56
Kinoshita (1989): 50 steps (LLS), 74 steps (TS)57
Carreira (2005): 26 steps (LLS), 36 steps (TS)58
Erythronolide B – R1 = OH, R2 = H, R3 = H, R4 = H Corey (1978): 33 steps (LLS), 47 steps (TS)59
Kochetkov (1987): 36 steps (LLS), 51 steps (TS)60
Mulzer (1991): 27 steps (LLS), 41 steps (TS)61
6-deoxyerythronolide B – R1 = H, R2 = H, R3 = H, R4 = H Masamune (1981): 26 steps (LLS), 39 steps (TS)62
Danishefsky (1990): 42 steps (LLS), 42 steps (TS)63
Evans (1997): 23 steps (LLS), 28 steps (TS)64
White (2009): 23 steps (LLS), 25 steps (TS)65
O
O
OH
O
Et
OR3
OR2
R1R4
26
Notably, the only reported total synthesis of erythromycin A is that of Woodward in 1981
(55 steps, LLS). This is likely due to the inherent complexity of the erythromycin A
aglycon, with its 10 stereocenters (five of which are consecutive) and five free hydroxyl
groups. Furthermore, regio- and stereoselective glycosidation of the aglycon presented a
significant challenge.
The vast majority of erythromycin and erythronolide total syntheses follow the same
strategy.66 The protected aglycons are formed by lactonization of a seco acid backbone.
These seco acids are constructed by coupling smaller chiral fragments that are obtained
by chiral resolution or enantioselective synthesis. Indeed, the evolution of enantio- and
diastereomeric control has assisted in decreasing the step count of aglycon synthesis and
eliminated the requirement of chiral resolution.67
The seco acid target of Woodward’s erythromycin A synthesis is shown in Figure 3.5.
Protection of the C-9 ketone and the C-3, C-5 and C-11 hydroxyl groups proved
necessary for the lactonization step in order to prevent polymerization and undesired
cyclizations. Additionally, their protective group strategy was instrumental for inducing
conformations favourable for cyclization. For example, the 3,5-acetal unit in 3.16 locks
the C2-C6 fragment of the molecule into a rigid, linear structure due to the diequatorial
nature of the 1,3-dioxane chair. This allows for the 6-OH group to remain unprotected
because it can only participate in lactonization after flipping the acetal to the diaxial
conformation.68
Figure 3.5 – Seco acid derivative for erythromycin A synthesis (Woodward, 1981)
66 Paterson, I.; Mansuri, M. M. Tetrahedron, 1985, 41, 3569. 67 Bartlett, P. A. Tetrahedron. 1980, 36, 1. 68 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3210.
Me Me Me
O
MeOH
Me Me
OOHOH
Me
OO O
HH OHH
Me Me Me
NH
MeOH
Me Me
OOO
Me
OO O
HH OH
35691112
RO
erythronolide A seco acid Woodward's seco acid target (3.16)
27
Following preparation of seco acid 3.16, Woodward and co-workers used the Corey-
Nicolaou double activation method for macrolactonization of 3.16 (Scheme 3.3).69 After
deprotection of 3.17, the next task was to glycosylate the aglycon. Previous efforts with
an erythronolide A derivative revealed that glycosylation of the C-5 hydroxyl group was
more favourable than the C-3 and C-11 hydroxyls.70 Thus, glycosylation of 3.18 with an
O-2’ protected D-desosamine thioglycoside was attempted, which furnished the desired
O-5 functionalized product (3.19) in 36% yield. The use of O-2’ protected D-desosamine
was crucial for the subsequent glycosylation with L-cladinose because, if left
unprotected, functionalization of the 2’-OH is preferred over the 3-OH group of the
aglycon. Glycosidation of L-cladinal with 3.19 and methanolysis of the O-2’ ester group
gave 3.20 in 55% yield. Finally, deprotection of the macrolide and regeneration of the C-
9 ketone afforded erythromycin A (3.1).
Scheme 3.3 – Key steps in Woodward’s total synthesis of erythromycin A
The synthesis of erythromycin A by Woodward and co-workers involved the
collaboration of 49 scientists and took nearly ten years to complete. This work serves as a
testimony for the sheer difficulty of its synthesis. Although erythromycin A can be
69 Corey, E. J.; Nicolaou, K. C. J. Am. Chem. Soc. 1974, 96, 5614–5616. 70 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3216.
Me Me Me
NH
MeOH
Me Me
OOO
Me
OO O
HH OH
35691112
O
1) N
SS N
Me Me
Me
Ph3P
2) toluene, 110 oC O
OOH
O
Et
HO
O
O
HNO
Me
Me
Me70%
(3.16) (3.17)
O
OHOH
O
Et
HO
OH
OH
HN
(3.18)
BPCO
S O
N(CH3)2
CH3O
OMe
ON
N AgOTf
36%
O
OHOH
O
Et
HO
OH
O
HNBPCO
O
N(CH3)2
CH3O
O OMe
OCH3
OAcCH3
OCH3
Pb(ClO4)2, MeCN
S
N
55%
1)
2) MeOH
HN
O
OHOH
O
Et
HO
O
O
OCH3
OAcCH3
OCH3
O
N(CH3)2
CH3HO
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
(3.19)(3.20)(3.1)
BPCO
3
5
911
9
5
3
11
911
5
3
2'
911
5
3
2'
28
obtained in large quantities by fermentation, the total syntheses of its derivatives have led
to the development of new synthetic methodology that can be applied to other complex
natural products that may not be accessible by alternative means.
3.3 Acid-catalyzed rearrangements of erythromycin A
One of the major drawbacks of erythromycin A is its remarkable acid sensitivity, leading
to degradation in the stomach following oral administration.71 Outside of clinical use, the
groups of Corey and Carreira also observed its susceptibility to acidic degradation in their
total syntheses of erythronolide A.72,73
It has long been known that erythromycin A converts rapidly under acidic conditions to
erythromycin A enol ether (3.21) and anhydroerythromycin A (3.22), eliminating its
antibiotic activity. Indeed, this rapid inactivation necessitates the administration of large
doses in humans.
Figure 3.6 – Erythromycin A enol ether and anhydroerythromycin A
Barber and co-workers have completed extensive kinetic studies over the past 20 years to
determine the degradation mechanism of erythromycin A.74,75,76 In their work, they
showed that erythromycin A enol ether and anhydroerythromycin A are in equilibrium
71 Mordi, M. N.; Pelta, M. D.; Boote, V; Morris, G. A.; Barber, J. J. Med. Chem. 2000, 43, 467–474. 72 Schomburg, D.; Hopkins, P. B.; Lipscomb, W. N.; Corey, E. J. J. Org. Chem. 1980, 45, 1544–1546. 73 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712. 74 Alam, P.; Buxton, P.C.; Embrey K. J.; Parkinson, J. A.; Barber, J. Magn. Reson. Chem. 1996, 559–561. 75 Awan, A.; Brennan, R. J.; Regan, A. C.; Barber, J. J. Chem. Soc., Perkin Trans. 2. 2000, 2, 1645–1652. 76 Hassanzadeh, A.; Barber, J.; Morris, G. A.; Gorry, P. A. J. Phys. Chem. A. 2007, 111, 10098–10104.
O
O
Et
O
O
OOH
HO
O
N(CH3)2CH3
HO
OCH3
OHCH3
OCH3
CH3
O
O
Et
O
OCH3
OHCH3
OCH3
O O
HO
O O
N(CH3)2CH3
HO
erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)
29
with erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C. It was also
noted that erythromycin A exists as both 6,9- and 9,12-cyclic hemiketal tautomers (3.23,
3.24) under neutral aqueous conditions, albeit in relatively small quantities with the 9,12-
hemiketal preferred. These hemiketal intermediates were rapidly converted to their
respective enol ether and anhydro forms when exposed to acidic conditions. (Scheme
3.4).
Scheme 3.4 – Acid degradation mechanism of erythromycin A in deuterated phosphate buffer (pH = 3.0) at 37 °C
An alternative pathway is the hydrolysis of L-cladinose from the aglycon of erythromycin
A to give 5-desosaminylerythronolide A (3.25). This process was found to be
irreversible and significantly slower than the tautomerization pathways.
Prediction of conditions that lead to the selective formation of either erythromycin A enol
ether or anhydroerythromycin A is not trivial because the tautomerization and
dehydration steps in the erythromycin degradation pathway are reversible.77 Through
77 Hassanzadeh, A.; Barber, J.; Morris, G. A.; Gorry, P. A. J. Phys. Chem. A. 2007, 111, 10098–10104.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
HO
O
O
Et
O
O
OOH
HO
O
N(CH3)2CH3
HO
OCH3
OHCH3
OCH3
CH3
O
O
Et
O
O O
N(CH3)2CH3
HO
OCH3
OHCH3
OCH3
O
OHHO OH
O
O
Et
O
OCH3
OHCH3
OCH3
O O
HO
O O
N(CH3)2CH3
HO
O
O
OHOH
O
Et
HO
OH
O O
N(CH3)2CH3
HO
erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)
erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)
5-desosaminylerythronolide A (3.25)
1
12 6
9
1
612
9
9
612
11
12
9
6
O
O
Et
O
O
OOH
HO
O
N(CH3)2CH3
HO
OCH3
OHCH3
OCH3
6,9-hemiketal of erythromycin (3.23)
1
12
9
6
HO
30
experimentation, standard conditions have been developed to form each as the major
product. Erythromycin A enol ether (3.21) can be synthesized by subjecting erythromycin
A (3.1) to glacial acetic acid at room temperature.78 Alternatively, anhydroerythromycin
A (3.22) can be formed by exposing erythromycin A enol ether (3.21) to methanolic
hydrochloric acid at room temperature.79
3.4 Semisynthetic analogues of erythromycin A
Knowledge of the chemical basis for erythromycin A’s acid instability prompted the
development of semisynthetic macrolides that lacked this significant limitation. As
shown in Scheme 3.4, nucleophilic attack at the C-9 ketone is the cause of erythromycin
A enol ether and anhydroerythromycin A formation. To discourage acid-catalyzed
rearrangements, Taisho Pharmaceutical Co. developed a 6-step sequence to selectively
methylate the C-6 hydroxyl substituent, affording the antibiotic clarithromycin (3.26,
Scheme 3.5).80 By functionalizing O-6, the possibility of enol ether formation is
eliminated. Although formation of the 9,12-hemiketal is still possible, the 6-OH group is
no longer available to participate in forming anhydroerythromycin A. In addition to being
both acid-stable and orally active, clarithromycin displays a slightly expanded
antimicrobial spectrum relative to erythromycin A.
Scheme 3.5 – Semisynthesis of clarithromycin (Taisho, 1980)
78 Alam, P.; Buxton, C.; Parkinson, J. A.; Barber, J. J. Chem. Soc. Perkin Trans. 2. 1995, 1163–1168. 79 Kurath, P.; Jones, P. H.; Egan, R. S.; Perun, T. J. Experientia. 1971, 27, 362. 80 Morimoto, S.; Takahashi, Y.; Watanabe, Y.; Omura, S. J. Antibiot. 1984, 37, 187–189.
N
O
OHOH
O
Et
HO
O
O
OCH3
OTMSCH3
OCH3
O
N(CH3)2
CH3O
OOi-Pr
TMS
N
O
OHOMe
O
Et
HO
O
O
OCH3
OTMSCH3
OCH3
O
N(CH3)2
CH3O
OOi-Pr
TMSKOH
MeI
O
O
OHOMe
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
1) HCO2H
2) NaHSO3
oxime intermediate(3 steps from erythromycin A)
clarithromycin (3.26)
31
Another innovative semisynthetic strategy to reduce the chemical instability of
erythromycin A was developed by Pliva in 1980. In this case, the C-9 ketone was
completely removed from the erythromycin scaffold in a 4-step sequence to give
azithromycin (3.28, Scheme 3.6).81 The first step in the synthesis involved formation of
an oxime to protect the C-9 ketone. Then, the aglycon underwent ring expansion through
a Beckmann rearrangement to give an iminoether (3.27). Hydrogenolysis of 3.27 and
subsequent N-methylation led to the discovery of an “azalide” structure that became
known as azithromycin. Azithromycin was found to have excellent acid stability, oral
bioavailability, and an expanded antimicrobial spectrum relative to erythromycin A. In
1991, azithromycin gained FDA approval and rose to the 7th most prescribed drug in the
U.S. in 2010.
Scheme 3.6 – Semisynthesis of azithromycin (Pliva, 1980)
3.5 Regioselective functionalization of erythromycin A
In 2006, the group of Miller was the first to report a site-selective, catalytic method for
acylation of erythromycin A. With three secondary hydroxyl groups and two tertiary
hydroxyl groups, erythromycin A presents a challenge for regioselective catalysis. A
seminal report from Abbott Laboratories revealed that the C-2’ hydroxyl group on the
desosamine sugar of erythromycin A was the most reactive towards acetylation using
acetic anhydride in pyridine (Figure 3.7)82 The next most reactive position was the C-4”
81 Kobrehel, G.; Radobolja, G.; Tamburasev, Z.; Djokic, S. 11-Aza-4-0-cladinosyl-6-0-desosaminyl-15-ethyl-7,13,14-trihydroxy-3,5,7,9,12,14-hexamethyloxacyclopentadecan-2-one derivatives as well as process for their production, DE3012533A1, 1980. 82 Jones, P. H.; Baker, E. J.; Rowley, E. K.; Perun, T. J. J. Med. Chem. 1972, 15, 631–634.
N
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
OH
erythromycin A oxime
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
N
PhSO2Cl
NaHCO3O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
NH3C
1) H2, Pt
2) CH2O, HCO2H
azithromycin (3.28)(3.27)
32
hydroxyl on the cladinose sugar, as evidenced by preferential formation of a C2’,C4”-
diacetate when additional Ac2O is used. Finally, the least reactive secondary site was the
C-11 hydroxyl group on the aglycon, which acetylates to form a C2’,C4”,C11-triacetate
after prolonged reaction time. The tertiary alcohols are significantly less reactive under
these conditions and acetylation was not observed at these sites. Interestingly, the C2’-
actetate can be cleaved when the reaction is quenched with methanol. This phenomenon
has been attributed to the autocatalytic nature of the tertiary amine-containing
desosamine sugar.
Figure 3.7 – Inherent reactivity of the hydroxyl groups in erythromycin A
The goal of the Miller group was to identify a small molecule catalyst that would reverse
the inherent reactivity such that the 11-OH group would be modified preferentially over
the more reactive 2’-OH and 4”-OH groups. They examined 137 peptide catalysts chosen
at random from their catalyst libraries. Notably, most of the peptides displayed pyridine-
like behavior, favouring the C2’,C4”-diacetate. However, when peptides containing β-
turn-like structures were employed, a reversal in selectivity was observed. Overall, their
approach created a bias towards formation of the C2’,C11-diacetate as opposed to the
C2’,C4”-diacetate.83 It should be noted that preferential acetylation of the 2’-OH group
was unavoidable under their reaction conditions. However, methanolysis of the C2’,C11-
diacetate revealed the C11-monoacetate as the major product (Scheme 3.7). The product
distribution after methanolysis was as follows: C11-monoacetate (37%), recovered
erythromycin A (37%), C4”-monoacetate (8%), and C4”,C11-diacetate (9%).
Interestingly, the C11-monoacetate exists almost exclusively as its hemiketal tautomer.
83 Lewis, C. A.; Miller, S. J. Angew. Chem. Int. Ed. 2006, 188, 5744–5747.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
- 2'-OH, most reactive- Biologically inactive upon functionalization
- 4"-OH, 2nd most reactive
- 11-OH, 3rd most reactive- Desired selectivity
33
Everett and co-workers have rationalized the hemiketalization of C11-monoacylated
erythromycin A derivatives as a consequence of the loss of a macrolide-stabilizing
hydrogen bond across the C11-OH and C9 ketone in native erythromycin.84
Scheme 3.7 – Site-selective acylation of erythromycin A using a peptide catalyst (Miller, 2006)
3.6 Research goals
Our goal was to selectively functionalize erythromycin A using the organoboron-
mediated methodology previously developed in our group. The presence of the cis-vicinal
diol on the aglycon at C11-C12 served as the target for activation with diarylborinic acids
(Ar2BOH) and aryl boronic acids [ArB(OH)2]. As shown in the work of Miller, the C-11
hydroxyl group is the least reactive of the secondary alcohols present in erythromycin A.
Therefore, our methodology would have to bias selectivity towards the C11-OH as
opposed to the C2’-OH and C4”-OH groups.
Scheme 3.8 – Proposed regioselective monofunctionalization of erythromycin A catalyzed by a diarylborinic acid
84 Everett, J. R.; Hunt, E.; Tyler, J. W. J. Chem. Soc. Perkin Trans. 2. 1991, 1481–1487.
O
O
Et
O
O O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
O
OHO OH
1
612
9
O
O
OOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
1
12 6
9
N
N
NH
Me N
O
Boc
HNH
O Me MeO
HN
NBocNHO
Ph OMe
O(5 mol%)
Ac2O (2 equiv.), NEt3 (5 equiv.), CHCl3, RT 24 hr
OO
37%, major product
1)
2) MeOH, RT 72 hrC11-monoacetate (3.29)
3.1
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
HO
O
O
OOH
O
Et
O
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
HO
BPh Ph
Ar2BOH (cat.) electrophile E+
O
O
OOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
E
34
3.7 Results and discussion
3.7.1 Glycosylation of erythromycin A
The work of Scott Miller and co-workers showed that regioselective acylation of
erythromycin A was possible using a small molecule catalyst. Therefore, we decided to
attempt glycosylations using the borinic acid-catalyzed methodology previously
developed in our group. Similar conditions to that of the digitoxin work from our group
were used as a starting point. Erythromycin A was subjected to peracetylated glucosyl
bromide donor 3.31) (2 equiv.), Ag2O (2 equiv.) and 25 mol% of 2-aminoethyl
diphenylborinate (3.30) in acetonitrile for 24 hours at 23 °C. Following silica gel
chromatography, <5% of the O-2’ glucosylated product (3.32) was observed, with
recovered erythromycin A accounting for 87%. The control reaction (without catalyst)
provided equivalent results in terms of regioselectivity and yield. Increasing the loading
of 3.30 had no observed effect in terms of selectivity and yield. Additionally, increasing
reaction time to 48 hours and electrophile loading to 5 equivalents had little to no effect
on the outcome of the reaction.
35
Table 3.1 – Borinic acid-mediated glycosylationa
Entry Catalyst loading (mol%) Yieldb (%)
1 0 <5
2 25 <5
3 100 <5
a Reaction conditions: erythromycin A (0.068 mmol), catalyst (0–100 mol%), peracetylated glucosyl bromide donor (0.136 mmol), Ag2O (0.136 mmol), MeCN (6 mL). b Isolated yield.
Based on our work with pentasaccharide target 1.1 (see Scheme 1.10), the stoichiometric
boronic acid-mediated glycosylation method appeared to be a suitable alternative in cases
when the catalytic borinic acid conditions failed to produce favourable results.
Differences from the catalytic method include solvent choice (CH2Cl2), addition of a
Lewis base (NEt3) and stoichiometric use of a boronic acid instead of borinic acid
precatalyst 3.30. Furthermore, the presence of molecular sieves has been noted to affect
results in some cases. (Pentafluorophenyl)boronic acid (3.33) was chosen as the boron
source because it gave favourable results in glycosylations previously attempted in our
group.85 The results from the stoichiometric boronic acid-mediated glycosylations are
summarized in Table 3.2.
85 McClary, C. A. 2013. Exploring Noncovalent and Reversible Covalent Interactions as Tools for Developing New Reactions. (Doctor of Philosophy Dissertation).
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
OB
NH2
Ph
Ph
(x mol%)
Ag2O MeCN, 23 oC
24 hr
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3O
O
OOAc
OAc
AcOAc
OAcOAcO
BrAcO
OAc
(3.30)
(3.1) (3.32)
(3.31)
36
Table 3.2 – Boronic acid-mediated glycosylationa
Entry Boronic acid 4Å MS Yieldb (%)
1 none yes 6
2 none no 8
3 3.32 yes <5
4 3.32 no <5
a Reaction conditions: erythromycin A (0.068 mmol), (pentafluorophenyl)boronic acid (0.068 mmol), peracetylated glucosyl bromide donor (0.136 mmol), Ag2O (0.136 mmol), NEt3 (0.204 mmol), DCM (6 mL). b Isolated yield.
The stoichiometric method is carried out using either a one-pot reaction setup or a two-
step procedure. The former involves complexation of the boronic acid with the diol in
CH2Cl2 for 6 hours at room temperature, followed by addition of Lewis base, glycosyl
donor and Ag2O. The latter is accomplished by complexing the boronic acid and diol in
toluene for 3 hours at 110 °C, followed by removing the solvent in vacuo. To the
resulting solid are added DCM, Lewis base, glycosyl donor, and Ag2O. While both
methods were attempted, the results displayed in Table 3.2 are from the two-step
procedure.
Identical selectivity and similar yields were observed for the control and boronic acid-
mediated reactions. The presence of molecular sieves did not have a significant effect in
terms of yield. Notably, the one-pot and two-step complexation methods gave trace yields
of 3.32. As with the borinic acid-mediated method, increasing reaction time to 48 hours
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
Ag2O, NEt3DCM, 23 oC
24 hr
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3O
O
OOAc
OAc
AcOAc
OAcOAcO
BrAcO
OAcFB(OH)2
F
FF
F
(3.33)
(3.1) (3.32)
(3.31)
37
and electrophile loading to 5 equivalents had no observable effect on the outcome of the
reaction.
At this point, we had not observed any differences in selectivity between the control and
organoboron-mediated reactions. The inherent bias towards functionalization of the C-2’
hydroxyl group on the desosamine sugar could not be modified under the reaction
conditions employed. However, it is difficult to draw meaningful conclusions from these
results because the extent of starting material conversion was nearly negligible. Thus, it
was clear that the reaction conditions and/or choice of electrophile would need to be
modified. Increasing the reaction temperature was thought be a suitable option.
Alternatively, a more “armed” glycosyl donor, such as perbenzylated glucosyl bromide,
could be used. Ultimately, the decision was made to switch the electrophile to benzoyl
chloride. It was envisioned that our previously developed benzoylation methodology
would result in a greater extent of erythromycin functionalization, such that differences in
regioselectivity may be observed.
3.7.2 Benzoylation of erythromycin A
The first step towards developing a procedure for selective benzoylation of erythromycin
A was to synthesize and characterize any products that could form under boron-free
conditions. This would make the screening process more efficient by enabling quick
comparison of pure compounds to those present in crude reaction mixtures. When using
acetic anhydride with pyridine as the solvent, Scott Miller and co-workers reported the
formation of the C2’-monoacetate, C2’,C4”-diacetate and C2’,C4”,C11-triacetate when
the reaction was at room temperature for 72 hours. Expecting similar results, we
subjected erythromycin A to benzoic anhydride (3 equiv.) in pyridine at 23 °C for 72
hours. Interestingly, the only product observed was C2’-monobenzoylated erythromycin
A (3.34) in 92% isolated yield (Scheme 3.9).
38
Scheme 3.9 – Monobenzoylation of erythromycin A using benzoic anhydride in pyridine
Despite observing only monofunctionalization, we did not conclude that
difunctionalization would be required to see differences in regioselectivity between the
control and catalyzed reactions under our conditions. Therefore, benzoylation was
attempted using conditions similar to those previously described in our carbohydrate
acylation work. When 3.1 was subjected to benzoyl chloride (3 equiv.), DIPEA (3 equiv.)
and boronic/borinic acid at 23 °C for 24 hours, C2’-monobenzoylated erythromycin A
(3.34) was the major product in all cases. In the control reaction, a yield of 87% was
obtained for the C2’-monobenzoylated product, with 5% recovered erythromycin A. The
organoboron-mediated reactions provided the same regioselectivity as the control
reaction but a new product was observed. After purification by silica gel chromatography,
C2’-monobenzoylated erythromycin A enol ether (3.35) was recovered as a minor
product in the reactions with boronic and borinic acids. Notably, the yield of 3.35
increased from 15% to 26% when the amount of 2-aminoethyl diphenylborinate was
increased from 0.25 equivalents to 1 equivalent. The yield of 3.35 increased further when
the 2-step stoichiometric boronic acid procedure was employed.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO (3 equiv.)
pyridine23 oC, 72 hr
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3O
(3.1) (3.34)
BzPh O
O
Ph
O
92%
39
Table 3.3 – Organoboron-mediated benzoylation at 23 °Ca
Entry Boron source Yield (%)c [3.34] Yield (%)c [3.35]
1 none 87 0
2 2-aminoethyl diphenylborinate (0.25
equiv.)
72 15
3 2-aminoethyl diphenylborinate (1 equiv.)
63 26
4 (pentafluorophenyl)boronic acid (1 equiv.)b
55 34
a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt (0.204 mmol), MeCN (6 mL). b 2-step procedure with NEt3 (0.204 mmol) as the Lewis base and DCM as the solvent. c Determined by 1H NMR of the crude reaction mixture after elution through a silica gel plug.
These results suggest that the organoboron species is participating in the reaction and is
promoting intramolecular rearrangement of erythromycin A to its enol ether form. Based
on this observation, it was difficult to say whether benzoylation or enol ether formation
occurred first. Regardless, the organoboron reagents did not alter the regiochemical
outcome of the reaction, nor increase the yield of C2’-monobenzoylated erythromycin A
relative to the control reaction. Another interesting result was that
(pentafluorophenyl)boronic acid reaction yielded less of 3.34 and nearly 10% more C2’-
monobenzoylated erythromycin A enol ether (3.35) compared to the borinic acid reaction
when used in equivalent stoichiometric amounts. Perhaps the thermally promoted
condensation of 3.33 with erythromycin A in the two-step stoichiometric method
promoted enol ether formation even before benzoyl chloride was introduced to the
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3O
(3.1) (3.34)
BzBoron source (x equiv.)BzCl, i-Pr2NEt
MeCN, 23 oC24 hr
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3O
OCH3
OHCH3
OCH3
CH3
(3.35)
Bz
40
reaction. This could explain the decreased ratio of 3.34:3.35 in the boronic acid-mediated
reaction relative to the borinic acid reaction.
Although interesting results were obtained from the organoboron-mediated reactions, the
desired O-11 selectivity was not achieved. The extent of erythromycin A
functionalization increased significantly for benzoylation compared to glycosylation but
the regiochemical outcome remained the same. Perhaps, like the work of Miller, we
would require difunctionalization to observe differences in regioselectivity between the
control and organoboron-mediated reactions. In attempt to accomplish this, the reaction
temperature was increased to 80 °C, with the remaining parameters unchanged (Table
3.4).
At 80 °C, the extent of C2’-monobenzoylated enol ether formation increased significantly
for the control and organoboron-mediated reactions. Moreover, when stoichiometric
boronic/borinic acid was used, C2’-monobenzoylated erythromycin A (3.34) was not
observed. Furthermore, a new product was observed when boronic or borinic acids were
employed. In the cases where stoichiometric organoboron reagent was used, nearly 50%
of the reaction mixture contained unfunctionalized erythromycin A enol ether (3.21).
This was an interesting observation because the presence of organoboron reagent resulted
in a significant decrease in benzoylation compared to the control reaction. This result
provided insight to the question of whether benzoylation or enol ether formation occurs
first. Perhaps the organoboron reagent promoted formation of erythromycin A enol ether
(3.21), which discouraged functionalization of O-2’ relative to native erythromycin A.
41
Table 3.4 – Organoboron-mediated benzoylation at 80 °Ca
Entry Boron source Yield (%)c [3.34]
Yield (%)c [3.35]
Yield (%)c [3.21]
1 none 57 35 0
2 2-aminoethyl diphenylborinate (0.25
equiv)
28 54 9
3 2-aminoethyl diphenylborinate (1 equiv.)
0 52 44
4 (pentafluorophenyl)boronic acid (1 equiv.)b
0 49 47
a Reaction conditions: erythromycin A (0.068 mmol), BzCl (0.204 mmol), i-Pr2NEt (0.204 mmol), MeCN (6 mL). b 2-step procedure with NEt3 (0.204 mmol) as the Lewis base and MeCN as the solvent. c Determined by 1H NMR of the crude reaction mixture after elution through a silica gel plug.
To test this hypothesis, erythromycin A enol ether (3.21) was prepared according to a
literature procedure.86 Then, it was subjected to benzoyl chloride (3 equiv.) and DIPEA
(3 equiv.) in MeCN for 24 hours at 23 °C (Scheme 3.10). After purification by silica gel
chromatography, C2’-monobenzoylated enol ether (3.35) was isolated in 32% yield, with
65% recovered starting material. Under equivalent conditions, erythromycin A formed
the C2’-monobenzoylated product (3.4) in 87% yield, with 5% recovered starting
material (Table 3.3). Comparing these results illustrates that the formation of
erythromycin A enol ether discourages benzoylation. Furthermore, the presence of
86 Alam, P.; Buxton, C. P.; Parkinson, J. A.; Barber, J. J. Chem. Soc. Perkin Trans. 2. 1995, 1163–1167.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3O
(3.34)
BzBoron source (x equiv.)BzCl, i-Pr2NEt
MeCN, 80 oC24 hr
3.1O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3O
OCH3
OHCH3
OCH3
CH3
(3.35)
Bz
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
(3.21)
42
organoboron reagent, whether catalytic or stoichiometric, appears to accelerate
erythromycin A enol ether formation at both 23 °C and 80 °C.
Scheme 3.10 – Monobenzoylation of erythromycin A enol ether under boron-free conditions
Evidently, when subjected to benzoyl chloride under thermal conditions, erythromycin A
was significantly less stable than at room temperature. Addition of organoboron-reagent
further complicated the reaction by promoting the formation of undesired by products,
which discouraged benzoylation. From here, the goal was to reduce enol ether formation,
while attempting to increase the extent of benzoylation.
To accomplish this goal, erythromycin A was subjected to the same organoboron reagent
screen as described in Table 3.3 at 23 °C for 72 hours. Though only preliminary results
were obtained, it was noted that C2’-monobenzoylated erythromycin A (3.34) was the
major product in the control and boronic acid-mediated reactions. Formation of
dibenzoylated products was not observed. Interestingly, the reaction with stoichiometric
2-aminoethyl diphenylborinate (3.30) gave a complex mixture of products that were
inseparable by silica gel chromatography. In pursuit of purifying this reaction mixture,
semi-preparative reversed-phase HPLC and LC-MS were employed. Preliminary
screening of the crude reaction mixture with LC-MS showed m/z peaks corresponding to
3.35 and 3.21 but not C2’-monobenzoylated erythromycin A (3.34). Though this wasn’t
an entirely unexpected result, it seemed unusual that 3.34 was not observed – especially
considering that the boronic acid-mediated reaction gave 3.34 as the major product under
the same conditions. In attempt to troubleshoot this problem, pure erythromycin A was
subjected to semi-preparative reversed-phase HPLC and LC-MS. Information from mass
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3O
OCH3
OHCH3
OCH3
CH3
(3.35)
Bz
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
(3.21)
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
(3.36)
BzCl, i-Pr2NEt
MeCN, 23 oC24 hr
32% 65%
43
spectrometry alone was not sufficient to identify the isolated compound. This is because
the acid-catalyzed rearrangement products of erythromycin A have identical molecular
masses in certain instances (Scheme 3.11). After characterization by 1H and 13C NMR, it
was revealed that erythromycin A (3.1) was converted quantitatively to
anhydroerythromycin A (3.22), which gave a signal of (M-18)+. This result prompted
investigation of the conditions used for separation. A gradient of 80 → 5% H2O in MeCN
was employed with 0.1% formic acid as the buffer. The presence of buffer in the mobile
phase is imperative to obtain effective separation of ionizable compounds and its identity
should be based on the compounds being separated.87 Given the acid-sensitivity of
erythromycin A, we hypothesized that formic acid was likely the cause of
anhydroerythromycin A formation and, thus, was an incompatible buffer choice for the
separation.
Scheme 3.11 – Erythromycin A acid-catalyzed rearrangement products and their molecular masses
87 Unger, K. K.; Ditz, R.; Machtejevas, E.; Skudas, R. Angew. Chem. Int. Ed. 2010, 49, 2300–2312.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
O
O
Et
O
O O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
O
OHHO OH
O
O
Et
O
OCH3
OHCH3
OCH3
O O
HO
O O
N(CH3)2
CH3HO
erythromycin A (3.1) 9,12-hemiketal of erythromycin A (3.24)
erythromycin A enol ether (3.21) anhydroerythromycin A (3.22)
O
O
Et
O
O
OOH
HO
O
N(CH3)2
CH3HO
OCH3
OHCH3
OCH3
6,9-hemiketal of erythromycin (3.23)
HO
m/z = 733m/z = 733 m/z = 733
m/z = 715 m/z = 715
44
Notably, Miller and co-workers relied extensively on semi-preparative reversed-phase
HPLC for purification of acetylated erythromycin A regioisomers. Their choice of buffer
for separation of the regioisomers was potassium phosphate dibasic (K2HPO4), which has
an optimal buffering range of pH = 6.2–8.2. Future efforts in our group are directed
towards optimizing this separation by using similar HPLC conditions to those employed
by Miller and co-workers.
3.7.3 NMR experiments with erythromycin A
Although regioselective functionalization was not accomplished using our methodology,
there was interest in gaining a better understanding of the interaction between boronic
and borinic acids with erythromycin A. Foremost, we wanted to establish whether the
organoboron reagents were capable of binding to the cis-vicinal diol in erythromycin A.
In light of the results obtained for benzoylation, we also wished to study the effect of
boron reagents 3.30 and 3.33 on enol ether formation in absence of electrophile.
NMR spectroscopy has been instrumental in our group for observing boron-diol
complexation in organic solvents. In terms of boronic ester formation, monitoring
chemical shift changes in 1H NMR is a useful method for determining which site(s) of the
substrate the boronic acid complexes. Furthermore, analyzing chemical shift changes in 19F NMR can be beneficial when using boronic acids containing fluorine groups. To
begin, erythromycin A was complexed with (pentafluorophenyl)boronic acid (3.33) in
toluene at 110 °C. One of the first challenges was finding a suitable deuterated solvent
that would dissolve the reaction mixture after complexation. The use of protic solvents
would interfere with the boron-diol equilibria, thus polar protic solvents such as methanol
and water were not suitable. Additionally, acetonitrile, acetone, chloroform,
dichloromethane, and toluene were unable to solubilize the reaction mixture at room
temperature. Deuterated DMSO was effective in this regard but led to inconclusive
results by 1H and 19F NMR. When comparing 19F NMR spectra of 3.33 in CDCl3 to that
of DMSO, we noted that the expected 3 signals were now more than 10 separate signals,
suggesting interaction of DMSO with the boronic acid or that trace water in the solvent
45
could have caused protodeborylation. Despite being the only solvent to dissolve the
reaction mixture, DMSO was unsuitable for this experiment.
Next, we turned our attention to studying the interaction between borinic acids and
erythromycin A. Previous studies in our group revealed that simple diol substrates such
as cis-1,2-cyclohexanediol were incapable of displacing the ethanolamine ligand from
precatalyst 3.30. In contrast, the free base of diphenylborinic acid (3.37) was effective in
complexing with cis-1,2-cyclohexanediol in the presence of DIPEA as shown by 1H and 11B NMR. Although 11B NMR does not give relevant information in terms of
integrations, it is useful for distinguishing between tricoordinate and tetracoordinate
boron species. Peaks corresponding to tetracoordinate boron exhibit an upfield shift
relative to those of tricoordinate boron and appear sharper.88
88 Solovyev, A.; Chu, Q.; Geib, S. J.; Fensterbank, L.; Malacria, M.; Lacôte, E.; Curran, D. P. J. Am. Chem. Soc. 2010, 132, 15072–15080.
46
Figure 3.8 – (a) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of Ph2BOH (3.37) (b) 11B NMR (128 MHz, decouple 1H 400 MHz, CD3CN, 295 K) of erythromycin A (3.1) upon addition of Ph2BOH (3.37)
Figure 3.8 shows the 11B NMR spectra of free base 3.37 (Figure 3.8a) and erythromycin
A with free base 3.37 (3 equiv.) and DIPEA (5 equiv.) (Figure 3.8b). The 11B NMR
spectrum of free diphenylborinic acid (3.37) showed a sharp peak at 45.16 ppm (top).
Upon addition of one equivalent of erythromycin A, we observed no signal at 45.16 ppm
and appearance of two sharp peaks at 7.24 ppm and 3.34 ppm (bottom). Brown and co-
workers have reported the 11B NMR signal corresponding to the “ate” complex of
diphenylborinic acid and 2-propanol at 6.41 ppm.89 Therefore, these results suggest that
erythromycin A was indeed complexing with diphenylborinic acid to form a
tetracoordinate borinate complex. 1H NMR of this interaction revealed spectra that were
not interpretable.
89 Brown, H.C.; Srebnik, M.; Cole, T. E. Organometallics. 1986, 5, 2300–2303.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
HO
(3.1)
i-Pr2NEt
CD3CNPh2BOH
(3.37)
O
O
OOH
O
Et
O
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
HO
BPh Ph
(3.1–3.37)
H2O
i-Pr2NEtH+
(a) 3.37
(b) 3.1–3.37
45.16 ppm
7.24 ppm 3.34 ppm
47
Evidently, studying the presence of reversible covalent interactions between
erythromycin A and boronic/borinic acids was challenging. Though, this was not entirely
surprising given the structural complexity of erythromycin A. Next, our goal was to
observe the effect of organoboron reagents on erythromycin A in the absence of an
electrophile. To do so, we subjected erythromycin A to (pentafluorophenyl) boronic acid
(3.33) (1 equiv.) and DIPEA (3 equiv.) in acetonitrile for 24 hours at 80 °C. After eluting
through a silica plug to remove the boronic acid, the crude sample was analyzed by TLC, 1H NMR and 13C NMR. Several products were observed by TLC and 1H NMR, which
made identification of the products a difficult task without further purification. However, 13C NMR was useful for identifying individual products in the crude reaction mixture.
In 13C NMR, the C-1 carbonyl signal of erythromycin A appears at 178 ppm. This region
of the spectrum is useful for determining how many erythromycin-related products are
present in the reaction mixture. In the case of the reaction with
(pentafluorophenyl)boronic acid (3.33), three peaks were observed in this region. Further
analysis of the 13C NMR spectrum showed the presence of the C-9 ketone peak for
erythromycin A (3.1) at 220 ppm, the C-9 signal for 6,9-hemiketal 3.23 at 111 ppm and
the C-9 peak for 9,12-hemiketal 3.24 at 108 ppm. This was also completed for
erythromycin A and free diphenylborinic acid (3.37) under the same conditions. In this
case, three peaks were observed between 172–178 ppm. Further analysis revealed the
absence of the C-9 ketone peak for erythromycin A at 220 ppm, which was replaced by
three signals corresponding to C-9 of erythromycin A enol ether (3.21) at 150 ppm, the
6,9-hemiketal (3.23) at 111 ppm and the 9,12-hemiketal (3.24) at 108 ppm. Although
relative ratios of these products were not obtained due to overlapping signals in the 1H
NMR, these results indicate that erythromycin A enol ether is more likely to form in the
presence of diphenylborinic acid than (pentafluorophenyl)boronic acid when reacted
under the same conditions.
Although the two-step stoichiometric boronic acid procedure produced comparable
results to the stoichiometric borinic acid conditions for benzoylation of erythromycin A,
these NMR studies suggest that (pentafluorophenyl)boronic acid could be a more suitable
48
reagent to discourage enol ether formation. With that said, both of the organoboron
reagents employed in this work promoted formation of enol ether 3.35 in the presence of
benzoyl chloride at 23 °C and 80 °C. This suggests that we may be putting ourselves at a
disadvantage by using Lewis acidic organoboron reagents to selectively functionalize
erythromycin A. Attempts made to study the boron-diol equilibria of boronic and borinic
acids with erythromycin A proved difficult. As a result, it is hard to conclude whether the
acid-promoted rearrangements are influenced by complexation of boron with the cis-
vicinal diol at C11–C12 or are a result of the organoboron reagents’ Lewis acidity.
Preference for enol ether formation could suggest that the C11–C12 diol is participating
in complexation, which would eliminate the stabilizing hydrogen bond between the 11-
OH group and the C-9 ketone. This could result in bias towards formation of the 6,9-
hemiketal and, subsequently, the enol ether.
3.8 Conclusions and outlook
As described herein, efficient and selective functionalization of complex natural products
can be a very difficult task. The regioselective functionalization of erythromycin A
presented the challenge of competing reaction pathways with unequal activation barriers.
This was further complicated because the desired C-11 hydroxyl group was the least
reactive secondary hydroxyl group in erythromycin A. Furthermore, the presence of
borinic and boronic acids promoted rearrangement to the enol ether form of erythromycin
A in the presence and absence of an electrophile. The source of this issue arises from the
C-9 ketone of erythromycin A. Previous efforts to avoid acid-catalyzed intramolecular
rearrangements have focused on selectively capping the hydroxyl groups that participate
in the rearrangements [see clarithromycin (3.26)] or removing the C-9 ketone
functionality all together [see azithromycin (3.28)]. Perhaps our methodology may be
better suited for acid-stable erythromycin A derivatives such as these.
49
3.9 Experimental details
General Procedures: All reactions were carried out in oven-dried glassware fitted with
rubber septa. Stainless steel syringes were used to transfer air- and moisture-sensitive
liquids. Analytical TLC was performed using EMD aluminum-backed silica gel 60 F254
plates and visualized using UV light and/or KMnO4 stain with heat. Flash
chromatography was performed using silica gel 60 (230–400 mesh) from Silicycle.
Materials: HPLC grade acetonitrile, dichloromethane and toluene were dried and
purified using a solvent purification system (Innovative Technology, Inc.). Distilled
water was obtained from an in-house supply. Nuclear magnetic resonance (NMR)
solvents were purchased from Cambridge Isotope Laboratories. The remaining reagents
were purchased from Sigma-Aldrich or ACROS Organics and were used without further
modification.
Instrumentation: 1H and 13C NMR spectra were recorded in CDCl3, CD3OD and
(CD3)2SO using Agilent DD2-500 (500 MHz) and DD2-700 (700 MHz) spectrometers
equipped with a XSens cryogenic probe or using a Varian Mercury 400 MHz
spectrometer. Chemical shifts are reported in parts per million (ppm) relative to
tetramethylsilane and are referenced to residual protium in the solvent. For 1H NMR:
CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for 13C NMR: CDCl3 -
77.16 ppm, CD3OD - 49.00 ppm, (CD3)2SO - 39.52 ppm. Spectral information is
tabulated in the following order: chemical shift (δ, ppm); multiplicity (s-singlet, d-
doublet, t-triplet, q-quartet, m-complex multiplet); coupling constant (J, Hz); number of
protons; assignment. Assignments for proton and carbon resonances were based on two-
dimensional 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC correlation experiments.
High-resolution mass spectra (HRMS) were obtained on a VS 70-250S (double focusing)
mass spectrometer at 70 eV. Fourier transform infrared (FTIR) spectra were obtained on
a Perkin-Elmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe
ATR accessory in a solid or liquid state as indicated. Data are tabulated as follows:
wavenumber (cm-1); intensity (s-strong, m-medium, w-weak, br-broad).
50
3.10 Characterization data 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31)
Compound 3.31 was synthesized according to a modified literature procedure.90
1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose (2.50 g, 6.41 mmol) was dissolved in
dichloromethane (1.5 M) and added to a round-bottom flask under an argon atmosphere
containing a stir bar. The solution was cooled to 0 °C in an ice bath followed by drop
wise addition of HBr (33 wt.%) in acetic acid (5.10 mL, 28.82 mmol, 4.5 equiv.). The
reaction was slowly warmed to 23 °C and then stirred at this temperature for 4 hours. The
reaction mixture was diluted with dichloromethane and poured into ice-cold water. The
aqueous layer was extracted with dichloromethane three times. The combined organic
layers were washed with water, saturated NaHCO3 (aq) and brine. The organic layers were
dried over MgSO4, filtered and concentrated under vacuum. The resulting crude product
was recrystallized from ethanol to give a white solid (2.21 g, 5.38 mmol, 84% yield). Rƒ
= 0.36 (EtOAc/pentane; 20/80). Spectral data are in agreement with previous reports.91
1H NMR (400 MHz, Chloroform-d): δ 6.60 (d, J = 4.0 Hz, 1H, H-1), 5.59–5.51 (m, 1H,
H-3), 5.15 (dd, J = 10.3, 9.4 Hz, 1H, H-4), 4.83 (dd, J = 10.0, 4.0 Hz, 1H, H-2), 4.37–
4.25 (m, 2H, H-6, H-5), 4.16–4.09 (m, 1H, H-6’), 2.10 (s, 3H, -OCOCH3), 2.09 (s, 3H, -
OCOCH3), 2.05 (s, 3H, -OCOCH3), 2.03 (s, 3H, -OCOCH3). 13C NMR (101 MHz Chloroform-d): δ 170.6, 170.0, 169.9, 169.6, 86.7, 72.3, 70.7,
70.3, 67.3, 61.1, 20.8, 20.8, 20.8, 20.7.
90,91 Brown, H.C.; Srebnik, M.; Cole, T. E. Organometallics. 1986, 5, 2300–2303.
O
Br
OAc
AcOAcO
OAc
51
2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32)
To a 20 mL scintillation vial equipped with a stir bar were added erythromycin A (100
mg, 0.136 mmol), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl bromide (62 mg, 0.149
mmol, 1.1 equiv.), silver(I) oxide (63 mg, 0.272 mmol, 2 equiv.), 4Å molecular sieves
(500 mg), and dichloromethane (6 mL). The resulting suspension was stirred at 23 °C for
30 hours. The reaction was then filtered through Celite® and eluted with
dichloromethane. The filtrate was concentrated in vacuo and the resulting crude solid was
purified by silica gel chromatography (0 → 20% methanol in dichloromethane) to give a
white solid. Rƒ = 0.60 (DCM/MeOH; 75/25).
1H NMR (700 MHz, Chloroform-d): δ 6.48 (d, J = 8.9 Hz, 1H), 5.51–5.43 (m, 1H),
5.34–5.26 (m, 1H), 5.20–5.16 (m, 1H), 5.02 (dd, J = 11.1, 2.3 Hz, 1H), 4.88–4.82 (m,
1H), 4.63 (d, J = 6.8 Hz, 1H), 4.53–4.48 (m, 1H), 4.40–4.29 (m, 1H), 4.27–4.18 (m, 2H),
4.06–3.89 (m, 3H), 3.84 (s, 1H), 3.75–3.61 (m, 1H), 3.55 (d, J = 7.4 Hz, 1H), 3.29 (s,
3H), 3.22 (s, 3H), 3.15–3.08 (m, 1H), 3.07–2.97 (m, 4H), 2.89–2.79 (m, 1H), 2.73–2.61
(m, 1H), 2.36–2.28 (m, 1H), 2.18 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.98–
1.81 (m, 4H), 1.78–1.66 (m, 1H), 1.66–1.47 (m, 3H), 1.43 (s, 3H), 1.40–1.35 (m, 1H),
1.35–1.11 (m, 22H), 1.08 (d, J = 7.3 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H).
HRMS (ESI, m/z): Calculated for [C51H85NO22] (M+H)+ 1064.5636; found 1064.5648.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2CH3
O
O
OOAc
OAc
AcOAc
52
2’-(O-benzoyl)erythromycin A (3.34)
Erythromycin A (25 mg, 0.034 mmol) was added to a 30 mL screw cap test tube
containing a magnetic stir bar. The tube was sealed with a rubber septum and purged with
a balloon of argon. Anhydrous acetonitrile (2 mL) was added to the tube, followed by
DIPEA (6 µL, 0.034 mmol, 1 equiv.) and benzoyl chloride (4 µL, 0.034 mmol, 1 equiv.).
The resulting mixture was capped and stirred at 23 °C for 24 hours. The solvent was then
removed in vacuo and the crude mixture was purified by silica gel chromatography (0 →
15% methanol in chloroform with 1% NH4OH) to give a white solid. Rƒ = 0.50
(CHCl3/MeOH; 90/10). [𝛼]!!"= -66.7 (c0.53, CHCl3).
1H NMR (700 MHz, Chloroform-d): δ 8.06–7.99 (m, 2H, ortho), 7.59–7.52 (m, 1H,
para), 7.47–7.39 (m, 2H, meta), 5.04 (dd, J = 10.6, 7.5 Hz, 1H, H-2’), 4.98 (dd, J = 10.9,
2.3 Hz, 1H, H-13), 4.86 (d, J = 4.9 Hz, 1H, H-1”), 4.68 (d, J = 7.5 Hz, 1H, H-1’), 3.99
(dq, J = 9.3, 6.2 Hz, 1H, H-5”), 3.93 (dd, J = 9.3, 1.4 Hz, 1H, H-3), 3.74 (d, J = 1.5 Hz,
1H, H-11), 3.60–3.53 (m, 1H, H-5’), 3.51 (d, J = 7.6 Hz, 1H, H-5), 3.40 (s, 3H, -OCH3),
3.07–2.99 (m, 2H, H-4”, H-10), 2.85–2.76 (m, 1H, H-3’), 2.73–2.68 (m, 1H, H-2), 2.38–
2.32 (m, 1H, H-2”eq), 2.28 (s, 6H, -N(CH3)2), 1.90–1.68 (m, 4H, H-14eq, H-4’eq, H-4,
H-7eq), 1.67–1.62 (m, 1H, H-7ax), 1.61–1.56 (m, 1H, H-2”ax), 1.47 (s, 3H, H-18), 1.44–
1.36 (m, 2H, H-4’ax, H-14ax), 1.31–1.23 (m, 9H, H-19, H-21, H-6’), 1.17 (d, J = 7.0 Hz,
3H, H-6”), 1.14–1.09 (m, 6H, H-20, H-16), 1.02 (s, 3H, H-7”), 0.79 (t, J = 7.4 Hz, 3H, H-
15), 0.71 (d, J = 7.5 Hz, 3H, H-17).
13C NMR (126 MHz Chloroform-d): δ 222.3 (C-9), 175.6 (C-1), 165.3 (C=O Benzoyl),
132.6 (para), 130.7 (ipso), 129.7 (ortho), 128.2 (meta), 101.0 (C-1’), 96.0 (C-1”), 83.2
(C-5), 79.6 (C-3), 77.9 (C-4”), 76.8 (C-13), 75.0 (C-6), 74.5 (C-12), 72.8 (C-3”), 72.2 (C-
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
53
2’), 68.9 (C-11), 68.5 (C-5’), 65.7 (C-5”), 63.7 (C-3’), 49.5 (-OCH3), 44.7 (C-2), 40.8 (-
N(CH3)2), 39.3 (C-4), 38.0 (C-7), 37.7 (C-10), 35.0 (C-2”), 31.1 (C-4’), 27.1 (C-18), 21.5
(C-21), 21.2 (C-6”), 21.0 (C-14), 18.6 (C-19), 18.1 (C-16), 16.2 (C-7”), 15.8 (C-20), 12.0
(C-6’), 10.6 (C-15), 9.3 (C-17).
FTIR (powder, cm-1): 3414 (w, br), 2971 (w), 2940 (w), 1733 (m), 1718 (w), 1695 (w),
1456 (w), 1271 (s), 1052 (s), 995 (s), 734 (s), 711 (s).
HRMS (ESI, m/z): Calculated for [C44H71NO14] (M+H)+ 838.4948; found 838.4942.
2’-(O-benzoyl)erythromycin A 6,9-enol ether (3.35)
Compound 3.35 was synthesized according to a modified literature procedure.92
Erythromycin A (25 mg, 0.034 mmol) was added to a 30 mL screw cap test tube
containing a magnetic stir bar. The tube was sealed with a rubber septum and purged with
a balloon of argon. Anhydrous acetonitrile (2 mL) was added to the tube, followed by
boronic or borinic acid (0.034 mmol, 1 equiv.), DIPEA (6 µL, 0.034 mmol, 1 equiv.) and
benzoyl chloride (4 µL, 0.034 mmol, 1 equiv.). The resulting mixture was capped and
stirred at 80 °C for 24 hours. The solvent was then removed in vacuo and the crude
mixture was purified by silica gel chromatography (0 → 15% methanol in chloroform
with 1% NH4OH) to give a white solid. Rƒ = 0.55 (CHCl3/MeOH; 90/10). [𝛼]!!"= -43.3
(c0.55, CHCl3).
92 Lee, D.; Williamson, C.L.; Chan, L.; Taylor, M. J. Am. Chem. Soc. 2012, 134, 8260–8267.
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
54
1H NMR (500 MHz, Chloroform-d): δ 8.06–7.95 (m, 2H, ortho), 7.57–7.49 (m, 1H,
para), 7.45–7.35 (m, 2H, meta), 5.12–5.03 (m, 2H, H-2’, H-1”), 4.82 (dd, J = 10.5, 2.4
Hz, 1H, H-13), 4.66 (d, J = 7.5 Hz, 1H, H-1’), 4.14–4.05 (m, 1H, H-5”), 4.05–3.98 (m,
1H, H-3), 3.84 (d, J = 7.5 Hz, 1H, H-5), 3.67–3.52 (m, 1H, H-5’), 3.46 (s, 3H, -OCH3),
3.37 (d, J = 7.9 Hz, 1H, H-11), 3.12–3.00 (m, 1H, H-4”), 2.92–2.80 (m, 1H, H-3’), 2.80–
2.69 (m, 1H, H-10), 2.60–2.50 (m, 1H, H-2), 2.49–2.37 (m, 2H, H-7eq, H-2”eq), 2.30 (s,
6H, -N(CH3)2), 1.97–1.92 (m, 1H, H-7ax), 1.89–1.77 (m, 2H, H-14eq, H-4’eq), 1.75–1.65
(m, 1H, H-4), 1.66–1.55 (m, 1H, H-2”ax), 1.52 (s, 3H, H-19), 1.49–1.37 (m, 2H, H-4’ax,
H-14ax), 1.37–1.21 (m, 12H, H-18, H-6”, H-21, H-6’), 1.08 (d, J = 7.4 Hz, 3H, H-16),
1.02 (d, J = 7.1 Hz, 3H, H-20), 0.96 (s, 3H, H-7”), 0.83 (t, J = 7.4 Hz, 3H, H-15), 0.71 (d,
J = 7.4 Hz, 3H, H-17).
13C NMR (126 MHz Chloroform-d): δ 178.4 (C-1), 165.4 (C=O Benzoyl), 151.7 (C-9),
132.8 (para), 130.8 (ipso), 129.9 (ortho), 128.3 (meta), 101.8 (C-8), 101.1 (C-1’), 94.7
(C-1”), 85.6 (C-6), 79.8 (C-5), 78.4 (C-13), 78.2 (C-4”), 76.3 (C-3), 75.4 (C-12), 73.3 (C-
3”), 72.4 (C-2’), 70.0 (C-11), 68.7 (C-5’), 65.9 (C-5”), 63.9 (C-3’), 49.7 (-OCH3), 44.7
(C-2), 43.3 (C-4), 42.4 (C-7), 41.0 (-N(CH3)2), 34.8 (C-2”), 31.8 (C-4’), 30.6 (C-10), 26.4
(C-18), 21.8 (C-21), 21.4 (C-6’), 21.1 (C-14), 18.4 (C-6”), 16.1 (C-7”), 15.2 (C-20), 13.6
(C-16), 12.1 (C-19), 11.0 (C-15), 9.0 (C-17).
FTIR (powder, cm-1): 3425 (w, br), 2970 (w), 1728 (m), 1703 (w), 1451 (w), 1267 (m),
1062 (s), 999 (s), 737 (s), 710 (s).
HRMS (ESI, m/z): Calculated for [C44H69NO13] (M+H)+ 820.4842; found 820.4858.
55
Erythromycin A 6,9-enol ether (3.21)
Compound 3.21 was synthesized according to a literature procedure.93 Erythromycin A
(200 mg, 0.273 mmol) was dissolved in glacial acetic acid (5 mL) and allowed to stir in a
round-bottom flask at 23 °C for 4 hours. The reaction was then quenched with saturated
NaHCO3 (aq), followed by addition of dichloromethane. The two layers were separated
and the aqueous layer was further extracted twice with dichloromethane. The combined
organic layers were washed with saturated NaHCO3 (aq) to remove any trace acetic acid.
The organic layers were combined, dried over Na2SO4, filtered, and concentrated under
vacuum. The resulting crude product was recrystallized from hexane-ethanol to give a
white powder (95 mg, 0.133 mmol, 49% yield). Rƒ = 0.42 (CHCl3/MeOH; 85/15).
Spectral data are in agreement with previous reports.94
1H NMR (500 MHz, Chloroform-d): δ 5.02–4.95 (m, 2H), 4.48 (d, J = 7.3 Hz, 1H),
4.27–4.16 (m, 1H), 4.06–4.02 (m, 1H), 3.92 (d, J = 7.4 Hz, 1H), 3.79–3.69 (m, 1H), 3.41
(d, J = 8.9 Hz, 1H), 3.38 (s, 3H), 3.25 (dd, J = 10.3, 7.3 Hz, 1H), 3.05 (d, J = 9.5 Hz, 1H),
2.78–2.62 (m, 4H), 2.51–2.44 (m, 1H), 2.34 (s, 6H), 1.97–1.81 (m, 3H), 1.78–1.71 (m,
1H), 1.59 (s, 3H), 1.52–1.41 (m, 1H), 1.34 (s, 3H), 1.28–1.23 (m, 7H), 1.18 (d, J = 6.0
Hz, 3H), 1.15 (d, J = 7.5 Hz, 3H), 1.10 (d, J = 7.6 Hz, 3H), 1.08–1.04 (m, 6H), 0.93–0.87
(m, 3H).
13C NMR (126 MHz Chloroform-d): δ 177.8, 152.2, 102.9, 100.9, 95.1, 85.4, 79.9,
78.0, 77.6, 76.8, 75.2, 73.0, 71.2, 69.7, 67.7, 65.3, 64.2, 48.7, 44.6, 43.5, 39.4, 34.4, 31.3,
31.0, 30.6, 25.5, 22.3, 20.5, 20.2, 17.5, 15.8, 14.7, 13.0, 12.6, 10.8, 9.8.
93,94 Alam, P.; Buxton, C.; Parkinson, J. A.; Barber, J. J. Chem. Soc., Perkin Trans. 2. 1995, 6, 1163–1167.
O
O O
O
OOH
HO
ON(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
56
Diphenylborinic acid (3.37)
Compound 3.37 was synthesized according to a modified literature procedure.95 In a 2-
dram vial equipped with a stir bar were added 2-aminoethyl diphenylborinate (200 mg,
0.889 mmol), acetone (0.5 mL) and methanol (0.5 mL). 1M HCl (aq) (1 mL) was added to
the solution and the reaction was stirred at 23 °C for 1 hour. The mixture was then diluted
in diethyl ether, washed with water and extracted three times with diethyl ether. The
combined organic layers were dried over MgSO4, filtered and concentrated under vacuum
to give a white solid (125 mg, 0.687 mmol, 77% yield). Spectral data are in agreement
with previous reports.96
1H NMR (400 MHz, DMSO-d6): δ 9.97 (s, 1H, OH), 7.74–7.66 (m, 4H, ArH), 7.54–7.45
(m, 2H, ArH), 7.45–7.37 (m, 4H, ArH).
13C NMR (101 MHz DMSO-d6): δ 134.5, 130.2, 127.5.
95 Hosoya, T.; Uekusa, H.; Ohashi, Y.; Ohhara, T.; Kuroki, R. Bull. Chem. Soc. Jpn. 2006, 79, 692–701. 96 Chen, X.; Ke, H.; Chen, Y.; Guan, C.; Zou, G. J Org. Chem. 2012, 77, 7572–7578.
B OHPh
Ph
57
4
Semisynthesis of Erythronolide A
4.0 Introduction
Several total syntheses of the erythromycin aglycons have been reported over the past 35
years (see Figure 3.4).97 Although extensive efforts have been made to decrease the step
count of these syntheses, accessing large quantities of the aglycons through total
synthesis remains an inefficient process. 6-deoxyerythronolide B (3.4) and erythronolide
B (3.5) can be obtained by fermentation because they are intermediates in the
erythromycin biosynthetic pathway (see Scheme 3.2) but the aglycon of erythromycin A,
known as erythronolide A, has only been attainable by total- or semisynthesis.98,99
4.1 Semisynthesis of erythronolide A
In 1974, LeMahieu and co-workers from Hoffmann-La Roche reported a 4-step
procedure to synthesize erythronolide A from erythromycin A 9-oxime (Scheme 4.1).100
The purpose of their work was to illustrate that selective cleavage of the cladinose sugar
was possible, in addition to removal of both sugars to furnish erythronolide A.
Furthermore, the biological activity of erythronolide A was compared to that of
erythromycin A. Despite obtaining disappointing results in terms of biological activity,
this semisynthesis was the first, and remains the only, known practical method to obtain
erythronolide A in sufficient quantities.
97 Gao, X.; Woo, S. K.; Krische, M. J. J. Am. Chem. Soc. 2013, 135, 4223–4226. 98 Muri, D.; Carreira, E. J. Org. Chem. 2009, 74, 8695–8712. 99 Staunton, J.; Wilkinson, B. Chem. Rev. 1997, 97, 2611–2629. 100 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.
58
Scheme 4.1 – Semisynthesis of erythronolide A (LeMahieu, 1974)
Prior to this synthesis, the acid sensitivity of erythromycin A had been well documented,
including characterization of erythromycin A enol ether and anhydroerythromycin A.
Thus, the first step in LeMahieu and co-workers’ semisynthesis of erythronolide A
involved the protection of the C-9 ketone, which was accomplished with an oxime. The
use of oximes for carbonyl protection has become quite rare in recent times because they
contain an acidic hydrogen and a somewhat reactive C=N functionality.101 With that said,
carbonyl protection is often limited to acetals and ketals, which would likely be cleaved
under the conditions necessary to hydrolyze L-cladinose and D-desosamine. Surprisingly,
LeMahieu did not report the conditions employed for oxime formation in their report. As
a result, the conditions described in Scheme 4.1 for oxime protection were adopted from
a more recent literature procedure by Ma and co-workers.102
Next, LeMahieu and co-workers attempted to cleave both sugars with 1% hydrochloric
acid in methanol. They noticed that L-cladinose had been hydrolyzed but the desosamine
moiety was left intact. Treatment of 4.1 under more vigorous acidic conditions failed to
cleave desosamine from the aglycon. The group of Celmer had previously completed
work with the macrolide antibiotic oleandomycin, which also has a desosamine sugar,
101 Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007; pp 515. 102 Zhang, L.; Jiao, B.; Yang, X.; Liu, L.; Ma, S. J. Antibiot. 2011, 64, 243–247.
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
N
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
OHN
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N
CH3HO
OH
CH3H3C O
N
O
OHOH
O
Et
HO
OH
OH
OHO
O
OHOH
O
Et
HO
OH
OH
NH2OH⋅HClNaOAc, AcOH
MeOH, 55 oC, 24 hr
98%
3% H2O2
MeOH, 23 oC, 19 hr
81%
155 oC (0.1 mm Hg)
3 hr
56%
3% HCl
MeOH, 23 oC, 21 hr
69%
NaNO2, 1M HCl
MeOH, 0 oC, 6 hr
40%
(3.1) (4.1) (4.2)
(4.5)(4.4)(4.3)
N
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
HO
OHO CH3
59
and experienced similar problems when attempting to cleave the sugar moieties from the
aglycon. Celmer later employed a Cope elimination procedure,103 whereby the tertiary
amine of desosamine was converted to an N-oxide, followed by thermally induced syn
elimination to form an alkene-containing neutral sugar and N,N-dimethyl hydroxylamine
(Scheme 4.2).104 This newly formed neutral sugar was cleaved under much milder acidic
conditions than those needed to cleave a basic sugar such as desosamine.
Scheme 4.2 – Cope elimination procedure employed by Celmer for removal of the tertiary amine from D-desosamine in oleandomycin
LeMahieu and co-workers adopted this procedure for the 9-oxime protected erythromycin
A substrate (4.1), which furnished 4.3 in moderate yield. Cleavage of 4.3 with 3%
hydrochloric acid in methanol smoothly removed both sugars and yielded erythronolide
A 9-oxime (4.4) in good yield.
Typically, carbonyl compounds are regenerated from oximes by oxidation, reduction, or
hydrolysis. The hydrolytic methods often involve a strong Lewis- or Brønsted acid.
Alternatively, the oxidative and reductive procedures are generally unsuitable for highly
functionalized molecules. Aware of the possibility of acid-promoted intramolecular
rearrangements, LeMahieu and co-workers opted for a milder hydrolytic approach
wherein nitrous acid was generated in situ with sodium nitrite and 1M hydrochloric acid.
The nitrous acid that is formed then decomposes and results in nitrosonium ion
formation, which promotes nucleophilic attack upon the carbon-nitrogen double bond
such that hydrolytic cleavage can occur.105 Adopting this protocol resulted in cleavage of
the oxime from 4.4 to afford erythronolide A (4.5), albeit in a 40% yield.
103 Cope, A. C.; Ciganek, E.; Howell, C. F.; Schweizer, E. E. J. Am. Chem. Soc. 1960, 82, 4663–4669. 104 W. D. Celmer.; Biogenesis of Antibiotic Substances.; Z.Vanek and Z. Hostalek, Ed., Academic Press.: New York, NY, 1965; pp 103-105. 105 Balaban, T. S.; et al. Science of Synthesis: Houben-Weyl Methods of Molecular Transformations, Vol. 26: Ketones., Georg Thieme Verlag.: Göttingen, Germany, 2009. pp 317.
RO O
N
CH3HO
RO O
N
CH3HO
H3CCH3
HH3CH3C
O
(CH3)2NOHH2O2 150 °C
RO OHO CH3
60
4.2 Research goals
Our goal was to reproduce LeMahieu and co-worker’s literature procedure for
preparation of erythronolide A. In doing so, we hoped to access a new substrate to
showcase our group’s organoboron-mediated methodology and to synthesize novel
antibiotic analogues for biological evaluation. Erythronolide A presented the opportunity
for a unique intramolecular competition experiment because it contains a 1,2-cis diol, a
1,2-trans diol and a 1,3-cis diol. Like our work with erythromycin A, our goal was to
selectively functionalize the 11-OH group on the aglycon, which is present within the
only cis-vicinal diol of erythronolide A.
4.3 Results and discussion
The semisynthesis reported by LeMahieu and co-workers was completed on a relatively
large scale when compared to modern syntheses. For example, synthesis of erythromycin
A 9-oxime N-oxide (4.2) was accomplished using 50 grams of starting material. The only
step in their synthesis that was not completed on multi-gram scale was that of the oxime
deprotection, which was optimized using 300 milligrams of erythronolide A 9-oxime
(4.4). The purification strategies employed in their work were also notable: all of the
reported steps involved several two-solvent recrystallizations. Ultimately, our goal was to
obtain a significant amount of erythronolide A but on a smaller scale than LeMahieu and
co-workers. This presented the opportunity to simplify difficult purifications using silica
gel chromatography as opposed to using recrystallization.
To begin, erythromycin A 9-oxime (4.1) was synthesized according to the protocol
described in Scheme 4.1, using 5 grams of erythromycin A. The only purification
described in the procedure by Ma and co-workers was an aqueous workup with 2M
sodium hydroxide. Although a 98% yield was reported for 4.1 in the literature procedure,
we found our product to be impure by 1H NMR. Subsequent reaction of the crude
material with a 3% solution of H2O2 in methanol furnished the desired N-oxide 4.2 in
67% yield over two steps (Scheme 4.3).
61
Scheme 4.3 – Synthesis of erythromycin A 9-oxime N-oxide (4.2)
The next step was the pyrolysis of 4.2 to obtain 3’-de(dimethylamino)-3’,4’-
dehydroerythromycin A 9-oxime (4.3) (Scheme 4.4). Using a Kugelrohr glass oven, N-
oxide 4.2 was heated under high vacuum at 150 °C in solvent-free conditions for 3 hours.
Upon investigation by 1H NMR, we noticed that the crude material still contained a
significant quantity of starting material. This could have been a consequence of the
difference in pressure within the reaction vessel between our method and the literature
procedure. To solve this problem, the temperature was increased to 170 °C, which
resulted in full conversion of 4.2.
Scheme 4.4 – Synthesis of 3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) via Cope elimination
The resulting brown solid was purified by recrystallization from acetone-hexanes but
several impurities remained in the recovered product. Thus, silica gel chromatography
was attempted and proved to be very effective for isolation of the desired product.
Optimal yields were obtained when the reaction was performed using 800 mg of starting
O
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N(CH3)2
CH3HO
N
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N
CH3HO
OH
CH3H3C O
(3.1) (4.2)67%
1) NH2OH•HCl, NaOAc, AcOH MeOH, 55 oC, 24 hr
2) 3% H2O2 (aq), MeOH 23 oC, 19 hr
N
O
OHOH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
O
N
CH3HO
OH
CH3H3C O
(4.2)
59%
170 °C, high vacuum
2 hr
(4.3)
N
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
HO
OHO CH3
62
material. Notably, our 59% yield was comparable to the 57% yield obtained by
LeMahieu and co-workers.
The step involving hydrolysis of cladinose and desosamine from the aglycon was the first
challenge we experienced in our synthesis of erythronolide A. Using a 37% (w/w) source
of hydrochloric acid, we prepared a 3% solution of methanolic hydrochloric acid. Upon
reaction with 4.3, a complex mixture of products was observed. Attempts at purifying
individual products by recrystallization and silica gel chromatography were unsuccessful
and none of the desired erythronolide A 9-oxime was observed. Notably, 1H NMR
spectra of the partially purified reaction mixture showed signals corresponding to alkene
protons (5.8–5.6 ppm) that were of a different chemical shift than those of the starting
material. Since we did not observe any of the desired product by NMR or mass
spectrometry, it was possible that the cladinose sugar was cleaved while the desosamine
sugar remained linked to the aglycon. We then decided to prepare anhydrous
hydrochloric acid in situ through reaction of acetyl chloride in methanol. After allowing
this mixture to stir at room temperature for 15 minutes, the solution was transferred via
cannula to a new flask containing 4.3. This procedure proved effective and gave
erythronolide A 9-oxime (4.4) in 63% yield after purification by silica gel
chromatography when performed with 1.3 g of starting material (Scheme 4.5).
Scheme 4.5 – Synthesis of erythronolide A 9-oxime (4.4) under acidic conditions
The last step in the synthesis involved regeneration of the C-9 ketone through hydrolytic
cleavage of the oxime using sodium nitrite and 1M hydrochloric acid. Our first attempt at
this procedure yielded none of the desired erythronolide A (4.5). Instead, we observed a
complex mixture of compounds with erythronolide A enol ether (4.6) as the major
63%
0.78 M AcCl
MeOH, 23 o C, 4 hr
N
O
OHOH
O
Et
HO
OH
OH
OH
(4.4)(4.3)
N
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
HO
OHO CH3
63
product in 32% yield (Scheme 4.6). Unlike erythromycin A, the preferred cyclization
product of erythronolide A is the 5,9-enol ether as opposed to the 6,9-enol ether. This has
been attributed to the increased stability of the resulting 6-membered ring in the 5,9-enol
ether.106 Additionally, the C5 secondary hydroxyl group was found to be more prone to
acetylation than the C6 tertiary hydroxyl group, suggesting increased reactivity of the 5-
OH group.107
Scheme 4.6 – Nitrous acid-mediated oxime cleavage to give erythronolide A 5,9-enol ether (4.6)
Slow addition of the 1M hydrochloric acid solution by syringe pump was attempted but
resulted in a similar product distribution with none of the desired product observed.
During this stage of troubleshooting, we questioned whether erythronolide A was
decomposing during purification by column chromatography. To ensure this was not the
case, crude reaction mixtures were screened by 1H and 13C NMR. Additionally, HMBC
experiments were instrumental for this task and provided increased sensitivity relative to 13C NMR. Since the 1H NMR of oxime protected erythronolide A (4.4) and deprotected
erythronolide A (4.5) were known to be relatively similar, HMBC NMR experiments
were used to determine if the characteristic C-9 ketone signal (220 ppm) of erythronolide
A was present. Still, erythronolide A was not observed when nitrous acid was used to
cleave the oxime.
At this point, it became evident that a new strategy would have to be taken to cleave the
oxime. Many of the literature protocols for oxime removal have only been tested on
simple substrates and their functional group tolerance is relatively unexplored. Thus, we
106 Schomburg, D.; Hopkins, P. B.; Lipscomb, W. N.; Corey, E. J. J. Org. Chem. 1980, 45, 1544–1546. 107 Woodward, R. B.; et al. J. Am. Chem. Soc. 1981, 103, 3213–3215.
N
O
OHOH
O
Et
HO
OH
OH
OH
(4.4)
32%
O
H3C
OH
OHOEtOH
HO
O
5
9
NaNO2, 1M HCl(aq)
MeOH, 0 oC, 6 hr
(4.6)
64
made hypotheses as to which methods would be tolerant of a polyol such as erythronolide
A.
The first set of conditions employed involved reaction of 4.4 with sodium bisulfite
(NaHSO3) in a 1:1 mixture of ethanol and water at reflux. This procedure was used for
cleavage of the oxime in the semisynthesis of clarithromycin (see Scheme 3.5), and
therefore, appeared to be a suitable starting point. Similar to the nitrous acid protocol,
only enol ether 4.6 was observed. Lowering the temperature to both 50 °C and room
temperature resulted in sluggish reactivity, with none of the desired product obtained
after silica gel chromatography. We then tried an oxidative procedure with NBS in a 10:1
mixture of acetone and water, which was performed at room temperature. Analysis of the
crude and partially purified reaction mixtures by 1H NMR and HMBC experiments
revealed a complex mixture of products with none of the desired product formed.
Another interesting set of conditions were those used in Carreira and co-workers’ total
synthesis of erythronolide A for the cleavage of an isoxazoline. In this step, the
isoxazoline was reductively opened using Raney Nickel, hydrogen gas and acetic acid in
methanol (Scheme 4.7). In addition to isoxazoline cleavage, Scheme 4.6 shows the last
step of Carreira and co-workers’ total synthesis of erythronolide A. This is provided to
illustrate the point that the final deprotection of erythronolide A is challenging, regardless
of the protecting group strategy used.
Scheme 4.7 – Final steps of the erythronolide A total synthesis (Carreira, 2009)
Typically, Raney Nickel and H2(g) will result in reduction of an oxime to the
corresponding amine but the presence of acetic acid promotes hydrolysis of the imine
N
O
OH
O
Et
HO
O
O
Ph
ORa-Ni, AcOH, H2
MeOH, 23 oC, 20 min
O
O
OH
O
Et
HO
O
O
Ph
OH
93%
Pd(OAc)2, H2O, H2
MeOH, 23 oC, 6 hr
40%
O
O
OH
O
Et
HO
OH
OH
OH
65
formed in situ to regenerate the carbonyl compound.108 Thus, we adopted these reductive
conditions for deprotection of oxime 4.4, which gave erythronolide A in 38% yield
(Scheme 4.8).
Scheme 4.8 – Oxime cleavage with Raney Nickel in the semisynthesis of erythronolide A (4.5)
Although a relatively poor yield was obtained for oxime deprotection, we were not overly
surprised given that LeMahieu and co-workers achieved a 40% yield in this step. Of note,
this was the only step of our semisynthesis that was sensitive to reaction scale. Optimal
yields were observed when using 80 mg of starting material. Increasing the amount of 4.4
to 200 mg resulted in a complex reaction mixture with none of the desired product
observed.
4.4 Conclusions and outlook
In summary, a semisynthesis of erythronolide A is reported herein, which was modified
from the synthesis developed previously by LeMahieu and co-workers. Aside from the
oxime removal, we found their synthesis to be entirely reproducible. Like erythromycin
A, acid-promoted intramolecular rearrangements pose a problem for erythronolide A.
Thus, care must be taken when handling these substrates. Based on the results from
chapter 3 and those obtained from our attempts at cleaving the oxime, using Lewis acidic
organoboron reagents may cause problems when attempting to selectively functionalize
erythronolide A. Perhaps an erythronolide A 9-oxime derivative with the hydroxyl group
108 Trost, B. M.; Fleming, I. Comprehensive Organic Synthesis – Selectivity, Strategy & Efficiency in Modern Organic Chemistry, Vol. 8: Reduction., Elsevier Ltd.: Kidlington, Oxford, 1991. pp 143.
N
O
OHOH
O
Et
HO
OH
OH
OHO
O
OHOH
O
Et
HO
OH
OH
Ra-Ni, AcOH, H2
MeOH, 23 oC, 10 hr
38%
(4.4) (4.5)
66
of the oxime protected would be more suitable for the organoboron-mediated
methodology developed in our group. Alternatively, the aglycon of azithromycin (3.28)
would also be an interesting substrate to apply our methodology.
67
4.5 Experimental details
General Procedures: All reactions were carried out in oven-dried glassware fitted with
rubber septa. Stainless steel syringes were used to transfer air- and moisture-sensitive
liquids. Analytical TLC was performed using EMD aluminum-backed silica gel 60 F254
plates and visualized using UV light and/or KMnO4 stain with heat. Flash
chromatography was performed using silica gel 60 (230–400 mesh) from Silicycle.
Materials: HPLC grade acetonitrile, dichloromethane and toluene were dried and
purified using a solvent purification system (Innovative Technology, Inc.). Distilled
water was obtained from an in-house supply. Nuclear magnetic resonance (NMR)
solvents were purchased from Cambridge Isotope Laboratories. The remaining reagents
were purchased from Sigma-Aldrich or ACROS Organics and were used without further
modification.
Instrumentation: 1H and 13C NMR spectra were recorded in CDCl3, CD3OD and
(CD3)2SO using Agilent DD2-500 (500 MHz) and DD2-700 (700 MHz) spectrometers
equipped with a XSens cryogenic probe or using a Varian Mercury 400 MHz
spectrometer. Chemical shifts are reported in parts per million (ppm) relative to
tetramethylsilane and are referenced to residual protium in the solvent. For 1H NMR:
CDCl3 - 7.26 ppm, CD3OD - 3.31 ppm, (CD3)2SO - 2.50 ppm; for 13C NMR: CDCl3 -
77.16 ppm, CD3OD - 49.00 ppm, (CD3)2SO - 39.52 ppm. Spectral information is
tabulated in the following order: chemical shift (δ, ppm); multiplicity (s-singlet, d-
doublet, t-triplet, q-quartet, m-complex multiplet); coupling constant (J, Hz); number of
protons; assignment. Assignments for proton and carbon resonances were based on two-
dimensional 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC correlation experiments.
High-resolution mass spectra (HRMS) were obtained on a VS 70-250S (double focusing)
mass spectrometer at 70 eV. Fourier transform infrared (FTIR) spectra were obtained on
a Perkin-Elmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe
ATR accessory in a solid or liquid state as indicated. Data are tabulated as follows:
wavenumber (cm-1); intensity (s-strong, m-medium, w-weak, br-broad).
68
4.6 Characterization data Erythromycin A 9-oxime N-oxide (4.2)
Compound 4.2 was synthesized according to modified literature procedures.108 ,109 To a
solution of erythromycin A (5.00 g, 6.81 mmol) in methanol (80 mL) were added
hydroxylamine hydrochloride (2.20 g, 34.06 mmol, 5 equiv.), sodium acetate (3.91 g,
47.69 mmol, 7 equiv.) and acetic acid (351 µL, 6.13 mmol, 0.9 equiv.). The mixture was
heated to 55 °C and stirred for 24 hours. After solvent was removed under vacuum, the
residue was taken up in ethyl acetate and water and was adjusted to pH 11–12 with 2M
sodium hydroxide. The resulting solution was extracted three times with ethyl acetate.
The organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo to give a white solid. The crude product was added to methanol (150 mL) and
H2O2 (3% [v/v]) in water (150 mL) and stirred for 20 hours. Most of the methanol was
removed in vacuo and the precipitate that separated was filtered, rinsed with 10 mL of
cold deionized water and air dried to give a pure white solid (3.49 g, 4.56 mmol, 67%
yield). Rƒ = 0.44 (DCM/MeOH/NH4OH; 75/25/1 v/v/v).
1H NMR (500 MHz, Methanol-d4): δ 5.20 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 4.92 (d, J =
5.0 Hz, 1H, H-1”), 4.63 (d, J = 7.0 Hz, 1H, H-1’), 4.13 (dq, J = 9.4, 6.2 Hz, 1H, H-5”),
3.98–3.92 (m, 1H, H-3), 3.83 (dqd, J = 12.2, 6.1, 1.4 Hz, 1H, H-8), 3.72–3.66 (m, 2H, H-
2’, H-5’), 3.59 (d, J = 6.9 Hz, 1H, H-5), 3.52 (ddd, J = 12.4, 10.2, 4.1 Hz, 1H, H-3’), 3.38
108 Zhang, L.; Jiao, B.; Yang, X.; Liu, L.; Ma, S. J. Antibiot. 2011, 64, 243-247. 109 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953– 956.
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O CH3HO
OH
N+ CH3H3C O-
69
(s, 3H, -OCH3), 3.22 (d, J = 8.3 Hz, 6H, -N(CH3)2), 3.04 (d, J = 9.4 Hz, 1H, H-4”), 2.99–
2.90 (m, 1H, H-2), 2.74 (q, J = 7.4 Hz, 1H, H-10), 2.45 (d, J = 15.1 Hz, 1H, H-2”eq),
2.14 (ddd, J = 12.4, 4.1, 2.1 Hz, 1H, H-4’eq), 2.08–1.97 (m, 1H, H-4), 1.90 (m, 1H, H-
14eq), 1.67–1.56 (m, 2H, H-7eq, H-2”ax), 1.55–1.47 (m, 1H, H-14ax), 1.45 (s, 3H, H-
18), 1.43–1.39 (m, 1H, H-4’ax), 1.30–1.25 (m, 9H), 1.23 (d, J = 6.0 Hz, 3H), 1.20 (d, J =
7.1 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (s, 3H), 1.12 (d, J = 7.6 Hz, 3H), 0.85 (t, J =
7.4 Hz, 3H, H-15).
13C NMR (126 MHz, Methanol-d4): δ 177.4 (C-1), 171.6 (C-9), 103.0 (C-1’), 97.8 (C-
1”), 84.6 (C-5), 81.0 (C-3), 79.2 (C-4”), 78.3 (C-13), 77.9 (C-6), 77.5 (C-3’), 76.6 (C-12),
76.1 (C-11), 74.1 (C-3”), 73.6 (C-2’), 72.2 (C-5’), 67.8 (C-8), 66.6 (C-5”), 58.2 (-
N(CH3)2), 54.5 (-N(CH3)2), 50.1 (-OCH3), 49.3, 46.2 (C-2), 39.0 (C-4), 36.2 (C-2”), 35.3
(C-4’), 27.4 (C-18), 26.6, 22.3, 21.8, 21.6, 19.2, 19.1, 17.2, 16.7, 14.8, 11.1 (C-15), 10.1.
HRMS (ESI, m/z): Calculated for [C37H68N2O14] (M+H)+ 765.4744; found 765.4742.
3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3)
Compound 4.3 was synthesized according to a modified literature procedure.110 N-oxide
4.2 (778 mg, 1.02 mmol) was added to a round bottom flask and placed in a Büchi® Glass
Oven B-585 Kugelrohr under high vacuum. The substrate was pyrolyzed at 170 °C for 2
hours at 35 rpm. The resulting dark brown solid was purified by silica gel
110 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
OH
OHO CH3
70
chromatography (0 → 10% methanol in diethyl ether) to give a light brown foam (422
mg, 0.599 mmol, 59% yield). Rƒ = 0.66 (Et2O/MeOH; 95/5).
1H NMR (500 MHz, Methanol-d4): δ 5.69 (ddd, J = 10.1, 2.2, 1.4 Hz, 1H, H-3’), 5.56–
5.51 (m, 1H, H-4’), 5.20 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 4.87 (d, J = 5.0 Hz, 1H, H-1”),
4.57 (d, J = 6.6 Hz, 1H, H-1’), 4.49–4.43 (m, 1H, H-5’), 4.22 (dq, J = 9.5, 6.2 Hz, 1H, H-
5”), 4.12–4.07 (m, 1H, H-3), 3.96–3.92 (m, 1H, H-2’), 3.71–3.65 (m, 2H, H-5, H-11),
3.30 (s, 3H, -OCH3), 3.03 (d, J = 9.5 Hz, 1H, H-4”), 2.96–2.88 (m, 1H, H-2), 2.77–2.70
(m, 1H, H-10), 2.41 (d, J = 15.1 Hz 1H, H-2”eq), 2.07–1.98 (m, 1H, H-4), 1.95–1.85 (m,
1H, H-14eq), 1.68–1.54 (m, 2H, H-7eq, H-2”ax), 1.53–1.44 (m, 4H, , H-14ax, H-18),
1.28 (d, J = 6.2 Hz, 3H), 1.25 (s, 1H), 1.23 (s, 3H), 1.21–1.16 (m, 12H), 1.15 (s, 3H),
1.13 (d, J = 7.5 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H, H-15).
13C NMR (126 MHz, Methanol-d4): δ 177.6 (C-1), 171.7 (C-9), 133.9 (C-3’), 127.8 (C-
4’), 102.9 (C-1’), 98.4 (C-1”), 82.7 (C-5), 81.5 (C-3), 79.3 (C-4”), 78.3 (C-13), 76.4 (C-
6), 74.1 (C-12), 72.2 (C-11), 70.8 (C-5’), 69.6 (C-2’), 66.6 (C-5”), 50.1 (-OCH3), 46.2
(C-2), 40.7 (C-4), 39.3 (C-7), 36.3 (C-2”), 32.1, 29.5, 27.5 (C-18), 26.6, 22.2, 21.6, 21.5,
19.2, 18.9, 17.2, 16.6, 14.8, 11.2 (C-15), 9.6.
HRMS (ESI, m/z): Calculated for [C35H61NO13] (M+H)+ 704.4216; found 704.4214.
71
Erythronolide A 9-oxime (4.4)
Compound 4.4 was synthesized according to a modified literature procedure.111 A
solution of 0.78 M acetyl chloride (4.20 mL, 59.10 mmol, 32 equiv.) in methanol (75
mL) was stirred in a round-bottom flask for 15 minutes. The solution was then transferred
via cannula to a round bottom flask containing 4.3 (1.30 g, 1.85 mmol) dissolved in
methanol (5 mL) and was stirred at 23 °C for 4 hours. After solvent was removed in
vacuo, the residue was taken up in ethyl acetate and washed with 1M NaHCO3 (aq). The
organic layers were combined, dried over Na2SO4, filtered, and concentrated under
vacuum. The resulting crude product was purified by silica gel chromatography (0 →
10% methanol in diethyl ether) to give a light brown solid (0.504 g, 1.16 mmol, 63%
yield). Rƒ = 0.60 (Et2O/MeOH; 95/5).
1H NMR (500 MHz, Methanol-d4): δ 5.29 (dd, J = 11.3, 2.4 Hz, 1H, H-13), 3.80–3.71
(m, 1H, H-8), 3.67 (d, J = 1.3 Hz, 1H, H-11), 3.47 (dd, J = 10.5, 1.6 Hz, 1H, H-3), 3.42
(d, J = 3.6 Hz, 1H, H-5), 2.75 (qd, J = 7.0, 1.3 Hz, 1H, H-10), 2.67 (dq, J = 10.5, 6.6 Hz,
1H, H-2), 2.06–1.97 (m, 1H, H-4), 1.96–1.86 (m, 1H, H-14eq), 1.66–1.57 (m, 1H, H-
7eq), 1.55–1.44 (m, 1H, H-14ax), 1.35 (s, 3H, H-18), 1.34–1.30 (m, 1H, H-7ax), 1.22–
1.15 (m, 9H, H-16, H-20, H-21), 1.06 (d, J = 7.0 Hz, 3H, H-19), 0.96 (d, J = 7.4 Hz, 3H,
H-17), 0.84 (t, J = 7.4 Hz, 3H, H-15). 13C NMR (126 MHz, Methanol-d4): δ 177.0 (C-1), 171.9 (C-9), 81.3 (C-5), 79.3 (C-3),
78.3 (C-13), 76.4 (C-6), 75.7 (C-12), 72.7 (C-11), 45.2 (C-2), 37.9 (C-4), 37.8 (C-7), 34.1
111 LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern, L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956.
N
O
OHOH
O
HO
OH
OH
OH
72
(C-10), 30.9 (C-14), 26.8 (C-18), 26.7 (C-8), 19.0 (C-19), 17.3 (C-21), 15.9 (C-20), 14.9
(C-16), 11.0 (C-15), 8.4 (C-17).
HRMS (ESI, m/z): Calculated for [C21H39NO8] (M+H)+ 434.2749; found 434.2748.
Erythronolide A (4.5)
To a solution of Oxime 4.5 (80 mg, 0.184 mmol) in methanol (6 mL) were added acetic
acid (21 µL, 0.369 mmol, 2 equiv.) and Raney®-Nickel (100 mg, 2800 mesh). The round-
bottom flask was purged twice with H2 and the resulting black suspension was stirred
rapidly at 23 °C under an atmosphere of H2 for 10 hours. The reaction mixture was
filtered through Celite® and eluted with methanol. The filtrate was concentrated in vacuo
and the resulting brown residue was purified by silica gel chromatography (20 → 0%
pentanes in diethyl ether, 0 → 10% methanol in diethyl ether) to give a white solid (29
mg, 0.070 mmol, 38% yield). Rƒ = 0.51 (Et2O/MeOH; 95/5). Spectral data are in
agreement with previous reports.112
1H NMR (500 MHz, Methanol-d4): δ 5.19 (dd, J = 11.1, 2.3 Hz, 1H, H-13), 3.87 (d, J =
1.7 Hz, 1H, H-11), 3.56 (dd, J = 10.5, 1.3 Hz, 1H, H-3), 3.51 (d, J = 3.3 Hz, 1H, H-5),
3.15 (qd, J = 6.8, 1.7 Hz, 1H, H-10), 2.76–2.65 (m, 2H, H-8, H-2), 2.06–1.99 (m, 1H, H-
4), 1.95–1.86 (m, 2H, H-14eq, H-7eq), 1.55–1.47 (m, 1H, H-14ax), 1.44–1.41 (m, 1H, H-
7ax), 1.29 (s, 3H, H-18), 1.22–1.12 (m, 12H, H-16, H-21, H-19, H-20), 0.99 (d, J = 7.3
Hz, 3H, H-17), 0.85 (t, J = 7.4 Hz, 3H, H-15).
112 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712.
O
OHOH
O
HO
OH
OH
O
73
13C NMR (126 MHz, Methanol-d4): δ 221.3 (C-9), 177.3 (C-1), 82.3 (C-5), 79.9 (C-3),
78.2 (C-13), 76.3 (C-6), 75.5 (C-12), 70.7 (C-11), 45.2 (C-2), 45.1 (C-8), 41.0 (C-10),
39.3 (C-7), 37.6 (C-4), 26.3 (C-18), 22.5 (C-14), 18.3 (C-19), 17.4 (C-21), 15.7 (C-16),
12.1 (C-20), 11.1 (C-15), 8.1 (C-17).
HRMS (ESI, m/z): Calculated for [C21H38O8] (M+Na)+ 441.2459; found 441.2453.
Erythronolide A 5,9-enol ether (4.6)
Compound 4.6 was synthesized according to a modified literature procedure.113 A
solution of sodium nitrite (382 mg, 5.55 mmol, 50 equiv.) and water (2 mL) was stirred in
a round-bottom flask for 10 min and transferred via cannula to a round bottom-flask
containing Oxime 4.4 (48 mg, 0.111 mmol) dissolved in methanol (3 mL). After cooling
the mixture in an ice bath, 1M hydrochloric acid (5.6 mL, 5.55 mmol, 50 equiv.) was
added over 3 hours using a syringe pump while keeping the reaction at 0 °C. The reaction
was then quenched with saturated NaHCO3 (aq) and the methanol was removed in vacuo.
The product was extracted three times with ethyl acetate. The organic layers were
combined, dried over Na2SO4, filtered, and concentrated under vacuum. The resulting
crude product was purified by silica gel chromatography (20 → 0% pentanes in diethyl
ether, 0 → 10% methanol in diethyl ether) to give a yellow glass (16 mg, 0.038 mmol,
32% yield). Rƒ = 0.65 (Et2O/MeOH; 95/5). Spectral data are in agreement with previous
reports.114
113 Corey, E. J.; Hopkins, P. B.; Kim, S.; Yoo, S.; Nambiar, K. P.; Falck, J. R. J. Am. Chem. Soc. 1979. 101, 7131–7134. 114 Muri, D.; Carreira, E. M. J. Org. Chem. 2009, 74, 8695–8712.
O
H3C
OH
OHOOH
HO
O
74
1H NMR (500 MHz, Methanol-d4): δ 5.18 (dd, J = 11.2, 2.4 Hz, 1H), 3.59–3.53 (m,
2H), 3.50 (dd, J = 10.4, 1.3 Hz, 1H), 2.84–2.73 (m, 2H), 2.67 (dq, J = 10.4, 6.7 Hz, 1H),
2.09 (dd, J = 15.7, 1.3 Hz, 1H), 2.00–1.77 (m, 2H), 1.58–1.46 (m, 4H), 1.35 (s, 3H), 1.17
(d, J = 6.7 Hz, 3H), 1.09 (s, 3H), 1.04 (d, J = 7.2 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H), 0.84
(t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, Methanol-d4): δ 176.9, 152.7, 102.5, 84.7, 82.8, 82.3, 79.2, 76.5,
71.2, 44.7, 43.0, 36.0, 31.7, 28.8, 22.0, 17.2, 16.0, 14.7, 12.5, 10.8, 7.0.
HRMS (ESI, m/z): Calculated for [C21H36O7] (M+K)+ 439.2093; found 439.2089.
75
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (3.31) 1H NMR (400 MHz, Chloroform-d)
13C NMR (101 MHz, Chloroform-d)
O
Br
OAc
AcOAcO
OAc
O
Br
OAc
AcOAcO
OAc
76
2’-(O-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl])erythromycin A (3.32) 1H NMR (700 MHz, Chloroform-d)
1H–1H COSY (700 MHz, Chloroform-d)
O
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3O
O
(OAc)4
O
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3O
O
(OAc)4
77
1H–13C HSQC (700 MHz, Chloroform-d)
1H–13C HMBC (700 MHz, Chloroform-d)
O
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3O
O
(OAc)4
O
O
OH OH
O
Et
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3O
O
(OAc)4
78
2’-(O-benzoyl)erythromycin A (3.34) 1H NMR (700 MHz, Chloroform-d)
13C NMR (126 MHz, Chloroform-d)
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
79
1H–1H COSY (700 MHz, Chloroform-d)
1H–13C HSQC (700 MHz, Chloroform-d)
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
80
1H–13C HMBC (700 MHz, Chloroform-d)
2’-(O-benzoyl)erythromycin A 6,9-enol ether (3.35) 1H NMR (500 MHz, Chloroform-d)
O
O
OH OH
O
HO
O
O
OCH3
OHCH3
OCH3
ON(CH3)2
CH3OBz
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
81
13C NMR (126 MHz, Chloroform-d)
1H–1H COSY (700 MHz, Chloroform-d)
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
82
1H–13C HSQC (700 MHz, Chloroform-d)
1H–13C HMBC (700 MHz, Chloroform-d)
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
O
O O
O
OOH
HO
ON(CH3)2
CH3O
OCH3
OHCH3
OCH3
Bz
CH3
83
Erythromycin A 6,9-enol ether (3.21) 1H NMR (500 MHz, Chloroform-d)
13C NMR (126 MHz, Chloroform-d)
O
O O
O
OOH
HO
ON(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
O
O O
O
OOH
HO
ON(CH3)2
CH3HO
OCH3
OHCH3
OCH3
CH3
84
Diphenylborinic acid (3.37) 1H NMR (400 MHz, DMSO-d6)
13C NMR (101 MHz, DMSO-d6)
B OHPh
Ph
B OHPh
Ph
85
Erythromycin A 9-oxime N-oxide (4.2)
1H NMR (500 MHz, Methanol-d4)
13C NMR (126 MHz, Methanol-d4)
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O CH3HO
OH
N+ CH3H3C O-
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O CH3HO
OH
N+ CH3H3C O-
86
1H–1H COSY (500 MHz, Methanol-d4)
1H–13C HSQC (500 MHz, Methanol-d4)
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O CH3HO
OH
N+ CH3H3C O-
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
O CH3HO
OH
N+ CH3H3C O-
87
3’-de(dimethylamino)-3’,4’-dehydroerythromycin A 9-oxime (4.3) 1H NMR (500 MHz, Methanol-d4)
13C NMR (126 MHz, Methanol-d4)
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
OH
OHO CH3
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
OH
OHO CH3
88
1H–1H COSY (500 MHz, Methanol-d4)
1H–13C HSQC (500 MHz, Methanol-d4)
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
OH
OHO CH3
N
O
OHOH
O
HO
O
O
OCH3
OHCH3
OCH3
OH
OHO CH3
89
Erythronolide A 9-oxime (4.4) 1H NMR (500 MHz, Methanol-d4)
13C NMR (126 MHz, Methanol-d4)
N
O
OHOH
O
HO
OH
OH
OH
N
O
OHOH
O
HO
OH
OH
OH
90
1H–1H COSY (500 MHz, Methanol-d4)
1H–13C HSQC (500 MHz, Methanol-d4)
N
O
OHOH
O
HO
OH
OH
OH
N
O
OHOH
O
HO
OH
OH
OH
91
Erythronolide A (4.5) 1H NMR (500 MHz, Methanol-d4)
13C NMR (126 MHz, Methanol-d4)
O
OHOH
O
HO
OH
OH
O
O
OHOH
O
HO
OH
OH
O
92
1H–1H COSY (500 MHz, Methanol-d4)
1H–13C HSQC (500 MHz, Methanol-d4)
O
OHOH
O
HO
OH
OH
O
O
OHOH
O
HO
OH
OH
O
93
Erythronolide A 5,9-enol ether (4.6) 1H NMR (500 MHz, Methanol-d4)
13C NMR (126 MHz, Methanol-d4)
O
H3C
OH
OHOOH
HO
O
O
H3C
OH
OHOOH
HO
O