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CHOLESTEROL
This page intentionally left blank
 CHOLESTEROL
From Chemistry and Biophysics to
the Clinic

Edited by

Anna N. Bukiya
Professor, Department of Pharmacology, Addiction Science and Toxicology, College of Medicine,
The University of Tennessee Health Science Center, Memphis, TN, United States

Alex M. Dopico
Van Vleet Chair of Excellence, Department of Pharmacology, Addiction Science and Toxicology, College of Medicine,
The University of Tennessee Health Science Center, Memphis, TN, United States
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 Contents

Contributors xiii Cholesterol and membrane evolution 36

Preface xvii References 38

Afterword xix 3. Role of cholesterol in maintaining the

physical properties of the plasma membrane
Witold K. Subczynski, Marta Pasenkiewicz-Gierula,
SECTION 1 Justyna Widomska, and Natalia Stein

Cholesterol chemistry and cell Introduction 42

function
1. Cholesterol chemistry and laboratory
synthesis
Hélio M.T. Albuquerque, Clementina M.M. Santos,
and Artur M.S. Silva
Introduction 4
Cholesterol structural characterization 6
Cholesterol laboratory synthesis 9
Concluding remarks 21
References 22
2. Molecular evolution of cholesterol and
other higher sterols in relation to membrane
structure
Ole G. Mouritsen
Introduction: The overlooked lipids 25
Key features of the cholesterol molecule 27
Evolution and streamlining of a molecule 27
Phase equilibria in lipid membranes 29
Cholesterol and lipid membrane phase equilibria:
The liquid-ordered phase 30
Cholesterol, transverse membrane order, permeability,
and mechanics 32
Cholesterol and lateral membrane organization 33
Other higher sterols: Universality in sterol
function 35
A case study: Cholesterol, lateral membrane structure,
and the functioning of Na+/K+-ATPase 36
Phase diagram for cholesterol/phospholipid mixtures 43
Basic membrane physical properties and how
cholesterol regulates them 45
Lateral organization of membranes: Effect of
cholesterol 57
New information from the saturation recovery
electron paramagnetic resonance stretched
exponential function approach 59
Concluding remarks 61
Summary 64
References 64
4. The fundamental interaction
of cholesterol with lipid membranes:
The umbrella model
Juyang Huang
Introduction 73
Maximum solubility of cholesterol
in phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) bilayers 74
The umbrella model 77
Monte Carlo simulation of chemical potential of
cholesterol using multibody interactions 81
A family of small headgroup molecules: Sterols,
ceramides, and diacylglycerols 85
Molecular dynamics (MD) simulation of the umbrella
effect 87
Measurement of the chemical potential of cholesterol
in PC bilayers 90
Concluding remarks 95
References 96

v
vi Contents

5. Model peptides and cholesterol Molecular dynamics (MD) simulations on AT1R

István P. Sugár and Parkson Lee-Gau Chong embedded in a DPPC:Cholesterol (60:40mol%)
bilayer reveal a putative binding site for AT1R
On the energy and entropy parameters of the free blockers on the receptor 161
energy of the membrane 107 Conclusions 162
References 109 References 163

6. Cholesterol and ceramide: 9. Principles of cholesterol regulation

An unlikely pair of ion channels
Qiu-Xing Jiang and Irena Levitan
Aritz B. García-Arribas, Alicia Alonso, and Félix M. Goñi

Introduction: Membrane heterogeneity 111 Introduction 169

Sphingolipids and cholesterol: Biological Lipids as structural components and functional
impact 112 regulators of membrane proteins 173
Sphingolipids and cholesterol: Membrane A thermodynamic consideration of lipid-binding sites
biophysics 114 on ion channels and their generic effects on the
Cholesterol and ceramide in fluid membranes 117 gating of the channels 175
Conclusions and future perspectives 123 Cholesterol-binding sites on ion channels 177
References 123 Functional impact of cholesterol on the activities
of ion channels 186
Technical limitations and potential solutions for
7. Cholesterol-recognizing amino acid further developments 195
consensus motifs in transmembrane Conclusions 197
proteins: Comparative analysis of in silico References 198
studies and structural data
Fodil Azzaz, Henri Chahinian, Nouara Yahi, Coralie Di Scala, 10. Fluorescent probes for microscopy
Carlos J. Baier, Francisco J. Barrantes, and Jacques Fantini visualization of cholesterol topography
and dynamics in membranes
Introduction 127
Francisco J. Barrantes
Cholesterol: Bifacial structure, multifaceted
functions 128
Introduction 206
Cholesterol footprint on a membrane protein:
The elusive ideal probe 208
Starting the investigation 130
Cholesterol probes 208
CARC and CRAC algorithms: Basic principles
Indirect cholesterol probes 209
at work 132
Direct imaging of intrinsic fluorescent cholesterol
Cholesterol-binding motifs in 3D: What structural
analogs 211
studies reveal 134
Concluding remarks 219
Moving to the 3rd dimension 139
References 220
Conclusion 140
References 141
11. Cholesterol transport in blood,
lipoproteins, and cholesterol metabolism
8. Effects of cholesterol on the GPCR
Mark T. Mc Auley and Amy E. Morgan
AT1 receptor and its interplay with AT1
antagonists Introduction 228
Sofia Kiriakidi, Zoe Cournia, and Thomas Mavromoustakos Lipoproteins an overview 230
Low-density lipoprotein cholesterol 235
Introduction 148 High-density lipoprotein cholesterol 236
Experimental results and their discussion 156 How does aging impact cholesterol metabolism? 238
Cholesterol influence on the physicochemical Obesity and aging: Two sides of the same coin? 240
properties of the cell membrane in the presence Using mathematical modeling to explore cholesterol
of AT1R and sartans 160 metabolism 242
 vii
Contents  

Discussion of future perspectives 245 Control diets 358

References 247 References 359

12. Common laboratory research methods
for detection and quantification of
cholesterol
Anna N. Bukiya, Hanxuan Li, Steven Mysiewicz, and Wei Li
Introduction to cholesterol detection and
quantification 259
The early era of cholesterol detection and
quantification: Colorimetric approaches 262
MS-based approaches 264
Imaging-based approaches 270
Enzymatic methods 276
Summary and concluding remarks 279
References 280
13. Approaches for modifying cellular
cholesterol levels and their application to
mechanistic studies: Examples from the ion
channel field
Avia Rosenhouse-Dantsker, Alexandria Slayden, and Anna N. Bukiya
Introduction 290
In vitro methods for modifying cholesterol levels in
cell membranes 291
In vivo modification of cholesterol levels 303
Applications to mechanistic studies on the effect of
cholesterol on ion channel targets 312
Outlook 319
References 320
SECTION 2
Cholesterol homeostasis
and its disruption
15. Nutrition and cholesterol metabolism
Ghada A. Soliman
Introduction 372
Cholesterol structure 373
Nutritional recommendations for maintaining
healthy blood cholesterol levels 374
Dietary cholesterol intake 375
Saturated fat intake 377
Cholesterol functions 381
Digestion and absorption of cholesterol 381
Transport of cholesterol 382
Endogenous cholesterol synthesis 384
Blood cholesterol and atherosclerosis 385
Guinea pigs as a preclinical model 386
Advances in cholesterol research 387
Explanatory and predictive models approach 393
Conclusions 394
References 396
16. Cholesterol and early development
Isabella Ellinger and Waranya Chatuphonprasert
Introduction 404
The fetus and cholesterol 406
Steroid hormones in reproduction and early life 414
Extraembryonic structures, secondary yolk sac
and placenta, and materno-fetal cholesterol
transport 422
Maternal cholesterol levels in healthy pregnancies,
maternal hyper- and hypocholesterolemia, and
consequences for the fetus 426
Effect of selected medicines related to cholesterol
metabolism on the fetal development 430
Summary 436
References 437

14. Diet-induced hypercholesterolemia in 17. Clinical and biochemical diagnostic

small laboratory animal models methods: What do blood lipid levels tell us?
Tina Herfel Marshall B. Elam

Introduction 343 Introduction 452

Mouse models 344 Use of conventional laboratory methods to measure
Rat models 348 lipoproteins by determining their cholesterol
Hamsters 349 content 454
Guinea pigs 352 Beyond standard lipid profiles: Determination of
Rabbits 354 atherogenic lipoproteins by advanced lipoprotein
Additional diet considerations 356 testing 454
viii Contents

Fasting versus nonfasting lipid measurements 458 Management of homozygous familial

Lipid and lipoprotein testing in laboratory hypercholesterolemia 514
animals 460 Familial hypercholesterolemia-related
What do lipid/lipoprotein levels tell us? 460 diseases 514
References 462 Familial hypercholesterolemia current status and
future perspectives 516
18. Familial hypobetalipoproteinemia References 517
and abetalipoproteinemia
21. Niemann-Pick type C disease (NPC)
Francine K. Welty
Agnieszka Ługowska
Introduction 465
ApoB gene mutations causing familial Introduction 525
hypobetalipoproteinemia 466 Clinical picture of NPC disease 527
Familial hypobetalipoproteinemia (FHBL) and Genetic background of NPC 528
coronary heart disease (CHD) 468 Biochemical aspects of NPC1 and NPC2
FHBL and hepatic steatosis 468 proteins 532
FHBL and insulin sensitivity 469 Pathomechanisms underlying NPC disease 535
FHBL, hepatic cirrhosis, and hepatocarcinoma 470 Diagnostics 538
FHBL and psychiatric disease 470 Treatment 543
Heterozygous FHBL and neurological References 544
manifestations 471
Proprotein convertase subtilisin kexin 9 gene
22. Rare monogenic disorders of cholesterol
(PCSK9) mutations 471 metabolism
Familial combined hypolipidemia (FHBL2) 471 Małgorzata Bednarska-Makaruk and Agnieszka Ługowska
ANGPTL3 S17X 472
Abetalipoproteinemia (ABL) and homozygous Introduction 555
hypobetalipoproteinemia (HHBL) 473 Monogenic hypercholesterolemia 555
Diagnosis and management 473 Sterol storage diseases 569
Heterozygous FHBL 474 Bile acid biosynthesis disorders 576
Pregnancy management 477 Hypocholesterolemia 583
Conclusion 477 References 598
References 477
23. Secondary (acquired)
19. Critical illness and cholesterol levels hypercholesterolemia
Zdenek Zadak Arrigo F.G. Cicero and Ivan R. Cincione

Introduction 481 Introduction 609

Clinical implications of hypocholesterolemia 488 Causes of secondary hyperlipidemias and their
References 496 treatment 610
Conclusions 616
20. Familial hypercholesterolemia References 616
Shifa Jebari-Benslaiman, Unai Galicia-Garcia, Asier Larrea-Sebal,
Kepa B. Uribe, Cesar Martin, and Asier Benito-Vicente
24. Blood lipids and molecular pathways
of atherogenesis
Introduction 502 Ricardo Stein, Filipe Ferrari, and Vítor M. Martins
Genetic and molecular background of FH 502
Familial hypercholesterolemia diagnosis 507 Introduction 624
Familial hypercholesterolemia treatments 509 Cholesterol metabolism 625
Nutrition and familial hypercholesterolemia 513 Pathophysiology of atherosclerosis 626
 ix
Contents  

Low-density lipoprotein cholesterol and
cardiovascular disease 626
Oxidized low-density lipoprotein cholesterol and
cardiovascular disease 627
High-density lipoprotein cholesterol: Marker or risk
factor? 630
Triglycerides and cardiovascular disease 631
Genetics and dyslipidemia 632
Conclusions 633
References 633
25. Lysosomal acid lipase: Roles in rare
deficiency diseases, myeloid cell biology, innate
immunity, and common neutral lipid diseases
Gregory A. Grabowski and Hong Du
The amyloid cascade as a central cause for
Alzheimer’s disease 718
Cellular domains important for the formation
of Aβ and cholesterol 720
Interactions of Aβ with the neuronal membrane
and the initiation of synaptic failure 723
Participation of membrane lipids in the initiation
of Aβ-mediated neurotoxicity 725
How Aβ and cholesterol might lead to
neurodegeneration 729
Cholesterol affects a number of membrane proteins
that fine tune neuronal excitability 730
The ε4 isoform (ApoE4) factor in disease onset and
progression 733
References 736

Historical background: Lysosomal acid lipase

28. Cholesterol and alcohol
(LAL) 640 Andrew S. Bell, Emma M. O’Connell, and Falk W. Lohoff

Structure, properties, and biology of LAL 641

Introduction 747
LAL roles in disease states 652
Alcohol pharmacology 748
Molecular biology and genetics 657
Alcohol use disorder 749
LAL in diseases beyond the LALDs 662
Alcohol and cholesterol interactions 750
References 664
Alcohol and lipoproteins 751
Alcohol and PCSK9 753
26. Cholesterol and pathogens Fetal alcohol spectrum disorders (FASDs) and
Tatiana M. Clemente and Stacey D. Gilk cholesterol 755
Targeting lipids for treatment of alcohol-related
Introduction 676 diseases 757
The role of cholesterol-rich membrane microdomains Conclusion 760
in infectious diseases 676 References 760
Cholesterol recruitment to pathogen-containing
vacuoles 681
Targeting cellular cholesterol metabolism 684
Systemic cholesterol levels and pathogens 687 SECTION 3
Cholesterol-lowering agents as potential therapeutics
in infectious diseases 690 Pharmacological considerations
Cholesterol and immune response 692 and perspectives
Summary 696
References 697 29. Cholesterol stiffening of lipid
membranes and drug interactions: Insights
27. Involvement of cholesterol and from neutron spin echo and deuterium
β-amyloid in the initiation and progression NMR spectroscopy
of Alzheimer’s disease Sudipta Gupta, Fathima T. Doole, Teshani Kumarage, Milka
Luis G. Aguayo, Jorge P. Roa, Carlos F. Burgos, and Doktorova, George Khelashvili, Rana Ashkar, and Michael F. Brown
Juliana Gonzalez-Sanmiguel
Introduction 772
Neurodegenerative diseases are a significant health Neutron spin echo spectroscopy of lipid
problem: Alzheimer’s disease (AD) 716 membranes 775
x Contents

Solid-state 2H NMR spectroscopy of lipid Pharmacologic treatment of lipids 864

membranes 782 Other lipid-lowering therapies 883
Membrane stiffening effect of cholesterol from Nonprescription and dietary supplements 886
molecular dynamics simulations 788 Patient education 890
Cholesterol effects on drug uptake and drug delivery Clinical pearls 890
applications 789 Screening for lipid disorders 891
Conclusions 790 Summary 892
References 791 References 893

30. Cholesterol in drug delivery 33. Medicinal chemistry and pharmacology

systems of statins
DanRong Hu and ZhiYong Qian Bob M. Moore, II and George A. Cook

Introduction 798 Introduction 903

The common synthesis chemistry of modifying Medicinal chemistry of statins 905
cholesterol into the polymers 799 Pharmacology 919
Cholesterol in the form of drug delivery Future drugs and concluding remarks 922
vehicles 801 References 922
Conclusion 817
References 817
34. Cyclodextrins as promising
31. Modification of vascular receptor therapeutics against cholesterol
pharmacology by cholesterol: From overload
Florina Zakany, Tamas Kovacs, Lajos Szente, and Zoltan Varga
molecular determinants to impact on
arterial function Introduction 929
Alex M. Dopico, Anna N. Bukiya, and Kelsey C. North Cyclodextrins in general 930
Cyclodextrins in the treatment of Niemann-Pick type
Introduction 826 C disease 934
Cholesterol modulation of vasoactive drug action Cyclodextrins with great potential in the treatment of
in which the underlying pharmacodynamic neurodegenerative diseases 941
process(es) is not fully determined 827 Cyclodextrins as promising therapeutics in
Cholesterol modulation of vasoactive drug atherosclerosis 947
action occurring at the cell membrane where Role of cyclodextrins in the treatment of kidney
the vasoactive drug receptor of interest is diseases 951
embedded 834 Role of cyclodextrins in the treatment of eye
Cholesterol modulation of vasoactive drug action disorders 952
occurring at the vasoactive drug receptor protein Potential effects of cyclodextrins against
itself 839 coronavirus 953
Conclusions and prospective 844 Concluding remarks 954
References 845 References 955

32. Clinical strategies for reducing 35. Hyperlipidemia and rheumatoid arthritis
cholesterol levels Aliki I. Venetsanopoulou, Paraskevi V. Voulgari, and
Claude K. Lardinois and Samantha Karr Alexandros A. Drosos

Introduction 854 Introduction 970

Epidemiology 854 Pathophysiologic mechanisms in rheumatoid
Major guidelines for lipid-lowering therapy in the arthritis (RA) 971
United States 856 Articular and extra-articular manifestations of RA 972
 xi
Contents  

Rheumatoid arthritis treatment 973 Other agents 984

Comorbidities in RA 974 Mediterranean diet and RA 986
CVD: A major comorbidity in RA 974 The role of exercise in RA 986
CVD risk assessment in RA 975 Conclusions 987
CVD and atherosclerosis in RA 975 References 987
Lipid profile in RA patients 978
Mechanisms related to dyslipidemia in RA 979 36. Management of hypercholesterolemia
Lipid concentration and inflammatory markers 979 in individuals living with HIV/AIDS
Atherosclerosis and inflammation 979 Musaab Ahmed, Marium Ahmed, Dushyant Mital, and
Lipid metabolism and inflammation 980 Mohamed H. Ahmed
The impact of cytokines on LDL 980
Lipid peroxidation 980 Introduction 1000
Altered HDL function and structure 981 Nucleoside reverse transcriptase inhibitors
Effects of antirheumatic therapy on serum lipid (NRTIs) 1002
levels 981 Nonnucleoside reverse transcriptase inhibitors
Glucocorticoids (GCs) 981 (NNRTIs) 1002
DMARDs 982 Diabetes mellitus, metabolic syndrome and HIV 1004
Antitumor necrosis factor-alpha (anti-TNF-a) Conclusion 1012
agents 983 References 1013
Anti-interleukin-6 (IL-6) agents 984
Janus kinase inhibitors (JAK inhibitors) 984 Index 1021
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 Contributors
Luis G. Aguayo Department of Physiology,
Universidad de Concepción, Barrio Universi-
tario, Concepción, Chile
Marium Ahmed Department of Rehabilitation,
Saint Agnes Hospital, Baltimore, MD, United
States
Mohamed H. Ahmed Department of Medicine
and HIV Metabolic Clinic, Milton Keynes Uni-
versity Hospital NHS Foundation Trust, Milton
Keynes, United Kingdom
Musaab Ahmed College of Medicine; Center
of Medical and Bio-allied Health Sciences
Research, Ajman University, Ajman, United
Arab Emirates
Hélio M.T. Albuquerque LAQV-REQUIMTE,
Department of Chemistry, University of Aveiro,
Aveiro, Portugal
Alicia Alonso Instituto Biofisika (UPV/EHU,
CSIC); Departamento de Bioquímica y Biología
Molecular (UPV/EHU), Leioa, Spain
Rana Ashkar Department of Physics and Center
for Soft Matter and Biological Physics, Virginia
Tech, Blacksburg, VA, United States
Fodil Azzaz INSERM UMR_S 1072; Aix-­
Marseille Université, Marseille, France
Carlos J. Baier Laboratory of Toxicology, Insti-
tuto de Ciencias Biológicas y Biomédicas del
Sur (INBIOSUR), Universidad Nacional del
Sur-CONICET, Bahía Blanca, Argentina
Francisco J. Barrantes Laboratory of Molecular
Neurobiology, Biomedical Research Institute
(BIOMED), Catholic University of Argen-
tina (UCA), National Scientific and Technical
Research Council (CONICET), Buenos Aires,
Argentina
Małgorzata Bednarska-Makaruk Department
of Genetics, Institute of Psychiatry and Neu-
rology, Warsaw, Poland
xiii
Andrew S. Bell Section on Clinical Genom-
ics and Experimental Therapeutics, National
Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, Bethesda, MD,
United States
Asier Benito-Vicente Department of Molecu-
lar Biophysics, Biofisika Institute (University
of Basque Country and Consejo Superior de
Investigaciones Científicas (UPV/EHU, CSIC));
Department of Biochemistry and Molecular
Biology, University of the Basque Country
(UPV/EHU), Leioa, Spain
Michael F. Brown Department of Chemistry
and Biochemistry; Department of Physics, Uni-
versity of Arizona, Tucson, AZ, United States
Anna N. Bukiya Department of Pharmacology,
Addiction Science, and Toxicology, College of
Medicine, The University of Tennessee Health
Science Center, Memphis, TN, United States
Carlos F. Burgos Department of Physiology,
Universidad de Concepción, Barrio Universi-
tario, Concepción, Chile
Henri Chahinian INSERM UMR_S 1072;
Aix-Marseille Université, Marseille, France
Waranya Chatuphonprasert Faculty of Med-
icine, Mahasarakham University, Maha Sara-
kham, Thailand
Parkson Lee-Gau Chong Department of
Medical Genetics and Molecular Biochemistry,
The Lewis Katz School of Medicine at Temple
University, Philadelphia, PA, United States
Arrigo F.G. Cicero Medicine and Surgery Sci-
ences Department, Alma Mater Studiorum
University of Bologna; S. O
­ rsola-Malpighi Uni-
versity Hospital, Bologna, Italy
Ivan R. Cincione Department of Clinical and
Experimental Medicine, University of Foggia;
A.O.U. Ospedali Riuniti, Foggia, Italy
xiv
Tatiana M. Clemente Department of Pathol-
ogy and Microbiology, University of Nebraska
Medical Center, Omaha, NE, United States
George A. Cook College of Medicine, Uni-
versity of Tennessee Health Science Center,
Memphis, TN, United States
Zoe Cournia Biomedical Research Foundation,
Academy of Athens, Athens, Greece
Coralie Di Scala Neuroscience Center-HiLIFE,
University of Helsinki, Helsinki, Finland
Milka Doktorova Department of Molecular
Physiology and Biological Physics, University
of Virginia School of Medicine, Charlottesville,
VA, United States
Fathima T. Doole Department of Chemistry and
Biochemistry, University of Arizona, Tucson,
AZ, United States
Alex M. Dopico Department of Pharmacology,
Addiction Science, and Toxicology, College of
Medicine, The University of Tennessee Health
Science Center, Memphis, TN, United States
Alexandros A. Drosos Rheumatology Clinic,
Department of Internal Medicine, Medical
School, University of Ioannina, Ioannina,
Greece
Hong Du Department of Pathology and Labora-
tory Medicine; Simon Comprehensive Cancer
Center, Indiana University School of Medicine,
Indianapolis, IN, United States
Marshall B. Elam Department of Pharmacol-
ogy and Medicine, Cardiovascular Diseases,
University of Tennessee HSC, Memphis, TN,
United States
Isabella Ellinger Institute for Pathophys-
iology and Allergy Research, Center for
Pathophysiology, Infectiology and Immunol-
ogy, Medical University of Vienna, Vienna,
Austria
Jacques Fantini INSERM UMR_S 1072;
Aix-Marseille Université, Marseille, France
Filipe Ferrari Postgraduate Program in Cardi-
ology and Cardiovascular Sciences, School of
Medicine, Federal University of Rio Grande do
Sul; Hospital de Clínicas de Porto Alegre, Porto
Alegre, Brazil
Contributors
Unai Galicia-Garcia Department of Molecu-
lar Biophysics, Biofisika Institute (University
of Basque Country and Consejo Superior de
Investigaciones Científicas (UPV/EHU, CSIC));
Department of Molecular Biophysics, Fun-
dación Biofísica Bizkaia, Leioa, Spain
Aritz B. García-Arribas Instituto Biofisika
(UPV/EHU, CSIC); Departamento de Bio-
química y Biología Molecular (UPV/EHU),
Leioa, Spain
Stacey D. Gilk Department of Pathology and
Microbiology, University of Nebraska Medical
Center, Omaha, NE, United States
Félix M. Goñi Instituto Biofisika (UPV/EHU,
CSIC); Departamento de Bioquímica y Biología
Molecular (UPV/EHU), Leioa, Spain
Juliana Gonzalez-Sanmiguel Department of
Physiology, Universidad de Concepción, Barrio
Universitario, Concepción, Chile
Gregory A. Grabowski Department of Pediat-
rics; Department of Molecular Genetics, Bio-
chemistry and Microbiology, University of
Cincinnati College of Medicine; The Division
of Human Genetics, Cincinnati Children’s Hos-
pital Research Foundation, Cincinnati, OH,
United States
Sudipta Gupta Department of Physics and
Center for Soft Matter and Biological Physics,
Virginia Tech, Blacksburg, VA, United States
Tina Herfel Technical Services, Envigo, Teklad
Diets, Madison, WI, United States
DanRong Hu State Key Laboratory of Biother-
apy and Cancer Center, West China Hospital,
Sichuan University, and Collaborative Innova-
tion Center, Chengdu, Sichuan, PR China
Juyang Huang Department of Physics and
Astronomy, Texas Tech University, Lubbock,
TX, United States
Shifa Jebari-Benslaiman Department of Molec-
ular Biophysics, Biofisika Institute (University
of Basque Country and Consejo Superior de
Investigaciones Científicas (UPV/EHU, CSIC));
Department of Biochemistry and Molecular
Biology, University of the Basque Country
(UPV/EHU), Leioa, Spain
 Contributors
Qiu-Xing Jiang Laboratory of Molecular Physi-
ology and Biophysics, and the Cryo-EM Center,
Hauptman-Woodward Medical Research Insti-
tute; Departments of Materials Design and
Innovation, and Physiology and Biophysics,
University of Buffalo, Buffalo, NY; Department of
Medicinal Chemistry, College of Pharmacy, Uni-
versity of Florida, Gainesville, FL, United States
Samantha Karr Midwestern University, College
of Pharmacy—Glendale, Glendale, AZ;
Medical Science Liaison, Sanofi, Bridgewater,
NJ, United States
George Khelashvili Department of Physiology
and Biophysics; Institute of Computational Bio-
medicine, Weill Cornell Medical College, New
York, NY, United States
Sofia Kiriakidi Department of Chemistry,
National and Kapodistrian University of
Athens; Biomedical Research Foundation,
Academy of Athens, Athens, Greece
Tamas Kovacs Department of Biophysics and
Cell Biology, Faculty of Medicine, University
of Debrecen, Debrecen, Hungary
Teshani Kumarage Department of Physics and
Center for Soft Matter and Biological Physics,
Virginia Tech, Blacksburg, VA, United States
Claude K. Lardinois University of Nevada
School of Medicine, Reno, NV, United States
Asier Larrea-Sebal Department of Molecu-
lar Biophysics, Biofisika Institute (University
of Basque Country and Consejo Superior de
Investigaciones Científicas (UPV/EHU, CSIC));
Department of Molecular Biophysics, Fun-
dación Biofísica Bizkaia, Leioa, Spain
Irena Levitan Department of Medicine, Phar-
macology and Bioengineering, University
of Illinois College of Medicine, Chicago, IL,
United States
Hanxuan Li Department of Pharmaceutical
Science, College of Pharmacy, The University
of Tennessee Health Science Center, Memphis,
TN, United States
Wei Li Department of Pharmaceutical Science,
College of Pharmacy, The University of Ten-
nessee Health Science Center, Memphis, TN,
United States
xv
Falk W. Lohoff Section on Clinical Genom-
ics and Experimental Therapeutics, National
Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, Bethesda, MD,
United States
Agnieszka Ługowska Department of Genetics,
Institute of Psychiatry and Neurology, Warsaw,
Poland
Cesar Martin Department of Molecular Biophys-
ics, Biofisika Institute (University of Basque
Country and Consejo Superior de Investigaciones
Científicas (UPV/EHU, CSIC)); Department of
Biochemistry and Molecular Biology, University
of the Basque Country (UPV/EHU), Leioa, Spain
Vítor M. Martins Hospital de Clínicas de Porto
Alegre, Porto Alegre, Brazil
Thomas Mavromoustakos Department of
Chemistry, National and Kapodistrian Univer-
sity of Athens, Athens, Greece
Mark T. Mc Auley University of Chester,
Chester, United Kingdom
Dushyant Mital Department of HIV and Blood
Borne Viruses, Milton Keynes University Hos-
pital NHS Foundation Trust, Milton Keynes,
United Kingdom
Bob M. Moore, II College of Pharmacy, Uni-
versity of Tennessee Health Science Center,
Memphis, TN, United States
Amy E. Morgan University of Chester, Chester,
United Kingdom
Ole G. Mouritsen Department of Food Science,
University of Copenhagen, Frederiksberg,
Denmark
Steven Mysiewicz Department of Pharmacol-
ogy, Addiction Science, and Toxicology, College
of Medicine, The University of Tennessee Health
Science Center, Memphis, TN, United States
Kelsey C. North Department of Pharmacology,
Addiction Science, and Toxicology, College of
Medicine, The University of Tennessee Health
Science Center, Memphis, TN, United States
Emma M. O’Connell Section on Clinical
Genomics and Experimental Therapeutics,
National Institute on Alcohol Abuse and Alco-
holism, National Institutes of Health, Bethesda,
MD, United States
xvi Contributors
Marta Pasenkiewicz-Gierula Department of
Computational Biophysics and Bioinformatics,
Jagiellonian University, Krakow, Poland
ZhiYong Qian State Key Laboratory of Biother-
apy and Cancer Center, West China Hospital,
Sichuan University, and Collaborative Innova-
tion Center, Chengdu, Sichuan, PR China
Jorge P. Roa Department of Physiology, Uni-
versidad de Concepción, Barrio Universitario,
Concepción, Chile
Avia Rosenhouse-Dantsker Department of
Chemistry, University of Illinois at Chicago,
Chicago, IL, United States
Clementina M.M. Santos LAQV-REQUIMTE,
Department of Chemistry, University of Aveiro,
Aveiro; Centro de Investigação de Montanha
(CIMO), Instituto Politécnico de Bragança, Bra-
gança, Portugal
Artur M.S. Silva LAQV-REQUIMTE, Depart-
ment of Chemistry, University of Aveiro,
Aveiro, Portugal
Alexandria Slayden Department of Pharma-
cology, Addiction Science, and Toxicology,
College of Medicine, The University of Ten-
nessee Health Science Center, Memphis, TN,
United States
Ghada A. Soliman Department of Environmen-
tal, Occupational, and Geospatial Health Sci-
ences, City University of New York (CUNY)
Graduate School of Public Health and Health
Policy, New York, NY, United States
Natalia Stein Department of Biophysics,
Medical College on Wisconsin, Milwaukee, WI,
United States
Ricardo Stein Postgraduate Program in Cardi-
ology and Cardiovascular Sciences, School of
Medicine, Federal University of Rio Grande do
Sul, Porto Alegre, Brazil
Witold K. Subczynski Department of Biophys-
ics, Medical College on Wisconsin, Milwaukee,
WI, United States
István P. Sugár Department of Neurology, Icahn
School of Medicine at Mount Sinai, New York,
NY, United States
Lajos Szente CycloLab Cyclodextrin R&D Lab-
oratory Ltd., Budapest, Hungary
Kepa B. Uribe Center for Cooperative Research
in Biomaterials (CIC biomaGUNE), Basque
Research and Technology Alliance (BRTA),
Donostia San Sebastián, Spain
Zoltan Varga Department of Biophysics and
Cell Biology, Faculty of Medicine, University
of Debrecen, Debrecen, Hungary
Aliki I. Venetsanopoulou Rheumatology
Clinic, Department of Internal Medicine,
Medical School, University of Ioannina, Ioan-
nina, Greece
Paraskevi V. Voulgari Rheumatology Clinic,
Department of Internal Medicine, Medical
School, University of Ioannina, Ioannina,
Greece
Francine K. Welty Division of Cardiology, Beth
Israel Deaconess Medical Center, Boston, MA,
United States
Justyna Widomska Department of Biophysics,
Medical University of Lublin, Lublin, Poland
Nouara Yahi INSERM UMR_S 1072; Aix-­
Marseille Université, Marseille, France
Zdenek Zadak University Hospital Hradec
Kralove and Charles University in Prague,
Faculty of Medicine in Hradec Kralove, Hradec
Kralove, Czech Republic
Florina Zakany Department of Biophysics and
Cell Biology, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary
 Preface
This project started on an otherwise unevent-
ful day when an Elsevier invitation to edit a
book on “cholesterol” landed in the pile of our
daily emails. We have edited several books
throughout our scientific careers, and working
on yet another book in the midst of grant re-
newal, paper resubmission, fellow mentoring,
and other regular loads in the daily life of a sci-
entist sounded quite challenging. Our initial
hesitancy to pitch in drastically changed when
the first details of the book came to light. Upon
discussions with Elsevier, it became apparent
that there was not an updated volume that
could be called “The Book of Cholesterol” ex-
panding from very basic scientific questions to
clinical problems associated with cholesterol
dyshomeostasis in humans. Thus, while cho-
lesterol is generously covered in many sources
from popular literature to the most highly
scholastic research articles, there is no single
source of knowledge that is available to the
wide audience. This gap constituted the “call
of the wild” for us to start and organize this
book; we expect that this single volume will
connect chemical and biophysical knowledge
on cholesterol structure and the function of
this vital lipid as a regulator of protein-driven
signaling with its relevance to tissue and sys-
tem physiology, pathology, and medicine. We
also expect that the writing style, while driven
by experts in the field, would be appealing
to a wide readership that starts with curious
undergraduate students and culminates with
seasoned professionals who work on choles-
terol whether at the lab bench or by the hos-
pital bed.
The initial draft of the book chapters’
layout underwent a rigorous review by
xvii
Elsevier-recruited reviewers. While they
expressed high enthusiasm for the book,
­
they also pointed at several weak spots in
our initial vision of the list of topics to in-
clude. Following the reviewers’ feedback,
the book layout has been significantly ex-
panded. From a collection of chapters, it
grew to its current structure comprising
three sections. The first introduces readers to
“Cholesterol Chemistry and Cell Function.”
The section covers fundamental knowledge
about cholesterol endogenous synthesis and
laboratory bench approaches that led to
the current understanding of cholesterol’s
role in maintaining biophysical properties
of cellular membranes. The second section
deals with “Cholesterol Homeostasis and Its
Disruption” and provides a detailed account
of currently known diseases and conditions
associated with disruption of cholesterol
biosynthesis, availability, trafficking and
metabolism. Finally, the third section pro-
vides “Pharmacological Considerations
and Perspectives” on currently avail-
able therapeutic options for treating
cholesterol-related disorders. Our integra-
­
tive effort is summarized in the book’s title
Cholesterol: From Chemistry and Biophysics to
the Clinic. As the book covers a huge land-
scape across many disciplines, some omis-
sions are unavoidable. Conversely, as each
chapter is supposed to “stand on its own,”
so are the overlaps. It should be noted, how-
ever, that repeated topics (e.g., structure
of cholesterol, pharmacological actions of
statins) are presented from a different stand-
point and with a drastically different per-
spective between chapters.
xviii Preface
We are very grateful to all authors who
made their contribution to this book for their
enthusiasm, spark, and dedicated work on re-
spective chapters. Some of them even helped
us by recommending or directly inviting their
colleagues to pitch in. We would also like to
thank the book proposal reviewers who re-
mained anonymous yet must be entitled to
share this book’s success if any. Last but not
least, we would like to express our endless
gratitude to the Elsevier support team, in par-
ticular Barbara Makinster, for their round-the-
clock work and engagement on this project.
Before the first page is flipped, we apologize
to the many brilliant scientists and clinicians
that have worked in the cholesterol field
who did not author a chapter. We hope that
most are cited throughout the book. It is their
passion for the cholesterol field that built a
­decades-long foundation for this book. As for
the reader, we hope that you enjoy the jour-
ney through this book on cholesterol as much
as we did during our writing and editing.
Anna N. Bukiya, Editor
Alex M. Dopico Editor
Memphis, TN, United States
 Afterword
The main objective of this book has been
to present a comprehensive state-of-the-art
evaluation of a variety of topics dealing with
cholesterol, from its synthetic pathway to
therapeutic approaches aimed at fighting dis-
ease and conditions associated with choles-
terol dyshomeostasis in humans. In doing so,
we tried to set the tone to a level that, while
delivered by experts on highly specific top-
ics, could be accessible for cross-reading by
both specialists and the general readership.
Now 36 chapters in the making, we can only
hope that we have achieved this goal. One
thing to be sure of, however, is that as many
hypotheses have been tested in the content
of these pages, many more will be raised.
Even questions that appeared to be settled
keep delivering fertile ground for discov-
ery. Thus, while the biosynthetic paths going
from a simple linear structure to the complex
heterocyclic molecule that makes cholesterol
have been largely resolved, their study is
still of great interest for the purpose of creat-
ing new drug delivery systems, or selective
and sensitive cholesterol-sensing probes.
Likewise, cholesterol’s well-­ understood
hydrophobic character is a stepping stone
when including sterols as novel carriers to
improve drug bioavailability. Collectively,
examination of cholesterol biosynthetic
pathways, structure, and/or its receptor tar-
gets and downstream signaling keeps deliv-
ering an increasing set of novel agents aimed
at controlling cholesterol levels in the body,
whether by inhibiting specific biosynthetic
steps, sterol-­ sequestering, or other meth-
ods. Detailed knowledge of cholesterol bio-
synthetic pathways and h ­ omeostasis in the
xix
body has helped researchers and clinicians
alike to understand the differences between
animal models and humans and the lim-
itation of findings in the former to directly
translate onto human diagnosis and thera-
peutics. Finally, the multiple enzymatic steps
involved in cholesterol biosynthesis led to
the discovery of a wide variety of genetic
disorders with poor prognosis. While genetic
therapy has a major role in dealing with these
conditions, understanding the underlying
common pathophysiological process will
eventually help in delivering therapeutic ap-
proaches when gene therapy is not amenable
or recommended.
Cholesterol’s unique role in animal evo-
lution and cell function, as well as its cen-
tral place in common human pathologies,
stems from the particular chemical features
of this sterol, which allows it to organize
cell membranes, determine lipid phase
equilibria, form lipid domains and myelin,
act as a precursor of steroid hormones and
vitamin D, regulate protein receptor func-
tion and trafficking, and affect both patho-
physiology and the biological responses to
drugs used to treat human disease, in some
cases by directly controlling drug passage
through membranes and living cells. So far,
the study of cholesterol interactions with
signaling molecules has been limited by
resolution limitations (e.g., cholesterol con-
centrations that exert regulatory control of
proteins are high enough to challenge con-
ventional binding methods, absence of high-­
resolution protein structures), so a multitude
of approaches including magnetic resonance
methods, spectroscopy, fluorescence probes,
xx Afterword
c­omputational modeling, and systematic
mutations in receptor targets have been used
to tackle many key interactions between cho-
lesterol and other lipids or proteins; in many
cases, each of these methods has been found
insufficient yet necessary to answer a given
question. As methodologies of higher ana-
lytical resolution become available and al-
low us to explore lipid and protein function
in more detail, newer parameters and vari-
ables will become more accurate readers of
the presence of small amounts of cholesterol
in any given biological entity. Unavoidably,
newer models of cholesterol interaction with
other biological players will be advanced.
Should other sterols, such as lanosterol and
sitosterol, interact with biological entities in
ways different from cholesterol, newer stud-
ies might also inform us on whether and
how incorporation of such sterols into an-
imal organisms through diet affect normal
physiology.
A thorough understanding of ­cholesterol-
related conditions constitutes an urgent
­public-health matter that affects all mankind:
We are witness to an increasing gap between
the haves and haves-not, and while many
transmitted diseases have been eradicated or
drastically reduced, noncommunicable dis-
eases linked to cholesterol dyshomeostasis are
all but growing; they include nutritional defi-
cits leading to demyelinization and poor brain
development, obesity and type II diabetes,
­
cardiovascular disease and stroke, colon can-
cer, and some forms of dementia. Understand-
ing the role of cholesterol in human disease
and eventual therapeutic approaches become
particularly challenging when cholesterol
dyshomeostasis coexists with another major
biological process of its own, such as human
body colonization by pathogens (e.g., HIV or
SARS-CoV-2 viruses), systemic inflammation
(e.g., patients with rheumatoid arthritis at risk
for cardiovascular disease), some forms of
dementia (Alzheimer’s disease), or substance
use disorders. Not only therapeutics become
multifactorial but human conditions may also
lead to potentiation of cholesterol action and
other noxa (e.g., cholesterol levels and alcohol
presence during intra utero development).
Since the diseases and conditions stem-
ming from cholesterol dyshomeostasis may
present, at least during their early stages,
without symptoms, genetic mapping and
biochemical profiling (e.g., blood lipid panels
that enable accurate detection and quantifi-
cation of blood cholesterol level and various
lipoprotein fractions and subfractions) become
an increasing necessity for early intervention
and to implement dietary and lifestyle rec-
ommendations that adjust to each particular
individual. This exemplifies the increasing
practice of personalized medicine.
In closing, we also hope that the pages
herein conceived will help to further trigger
intellectual spark and human health con-
cern around this always intriguing molecule
termed cholesterol.
Anna N. Bukiya, Editor
Alex M. Dopico, Editor
 S E C T I O N 1

Cholesterol chemistry and cell

function
This page intentionally left blank
 C H A P T E R

1
Cholesterol chemistry and laboratory
synthesis
Hélio M.T. Albuquerquea, Clementina M.M. Santosa,b,
and Artur M.S. Silvaa
a
LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal bCentro
de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Bragança, Portugal

Abbreviations
Ac acetyl
ABSA acetamidobenzenesulfonyl azide
BBN 9-borabicyclo[3.3.1]nonane
Bn benzyl
Bu butyl
COSY correlation spectroscopy
DEPT distortionless enhancement by polarization transfer
DMSO dimethylsulfoxide
DMF N,N-dimethylformamide
DMAP 4-dimethylaminopyridine
Et ethyl
HMBC Heteronuclear Multiple Bond Correlation
HSQC Heteronuclear Single Quantum Coherence
HMPA hexamethylphosphoramide
IUPAC International Union of Pure and Applied Chemistry
LDA lithium diisopropylamide
Me methyl
MMC magnesium methyl carbonate
Ms methanesulfonyl (often shortened to mesyl)
NMR nuclear magnetic resonance
NOESY Nuclear Overhauser Effect Spectroscopy
p para
PCC pyridinium chlorochromate
Pd/C palladium on carbon
Ph phenyl
Py pyridine
t tert
TBSCl tert-butyldimethylsilyl chloride

Cholesterol 3 Copyright © 2022 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-323-85857-1.00007-9
4 1. Cholesterol chemistry and laboratory synthesis

THF tetrahydrofuran
TMS tetramethylsilane
Ts toluenesulfonyl (often shortened to tosyl)

Introduction

The name cholesterol derives from the Ancient Greek chole- (bile) and stereos (solid), fol-
lowed by the chemical suffix of the functional group alcohol (-ol). Known also by the name
cholesterin, cholesteryl alcohol, cholest-5-en-3β-ol, among others, this interesting natural
molecule is a type of modified sterol belonging to the heterogeneous group of organic com-
pounds known as lipids. With a bulky, rigid, and asymmetric structure, the cholesterol skel-
eton possesses four fused rings aligned from A to D, corresponding to three six-membered
and one five-membered. As a whole, the four rings comprise the 1,2-cyclopentane perhy-
drophenanthrene system (Fig. 1A) (Nes, 2011). The rings are trans-connected and create an
almost planar structure (Fig. 1C). The C-18 and C-19 methyl substituents are linked at C-10
and C-13, in relative cis configuration. Due to this structural prolife, the flat face of cholesterol
is called the smooth α-face, and all substituents located on this face (in trans-conformation rel-
ative to C-19) are called α, while the substituents located on the rough β-face (presence of the
two methyl substituents) are called β (in cis-conformation relative to C-19). The cholesterol
moiety bears an additional polar 3β-hydroxy group and a C5C6 double bond in B-ring (Róg,
Pasenkiewicz-Gierula, Vattulainen, & Karttunen, 2009).
From a chemical point of view, the cholesterol molecule comprises four essential domains
(Fig. 1B). The 3-hydroxy group of domain I constitutes not only an important active site
for hydrogen bond interactions with several biological molecules but also a versatile func-
tional group for derivatization. In domain II, the absence of methyl groups at C-4 and C-14
influences the planarity of the molecule, and the C5C6 double bond is an attractive car-
bon center to several addition reactions. The natural (R)-configuration at C-20 observed in
domain III determines the “right-handed” conformation of the side chain, while in domain
IV, the conformation and length of the side chain are of high importance to intermolecular
contacts (Cerqueira et al., 2016). The recommended name by the International Union of
Pure and Applied Chemistry (IUPAC) for natural ­cholesterol is (3S,8S,9S,10R,13R,14S,17R)-
10,13-dimethyl-17-[(R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-­dodecahydro-1H-
cyclopenta[a]phenanthren-3-ol. In its pure state, it is a white and crystalline powder

21
(A) (B) III (C)
20 22
18 24 20 IV
12 17 II
11 H 23 26
25 H
19 13
1 9 C D 16
2 27
14 15
3 A10 B 8 I 14
7 3
HO 5
4 6 HO
4
- chiral centers

FIG. 1 (A) Cholesterol tetracyclic nucleus with numbering of carbon atoms and rings labelling; (B) cholesterol
four structural domains; (C) cholesterol crystal structure obtained from https://www.ccdc.cam.ac.uk/structures/
search?id=doi:10.5517/cc66d1t&sid=DataCite.

1. Cholesterol chemistry and cell function

 Introduction 5
that is odorless and tasteless, with a melting point of 148–149°C (“[cholesterol],”, 2016;
Barton, 1976).
Historically, the first identification of cholesterol is attributed to the French chemist François
Poulletier de la Salle, who collected it as a crystalline component from human gallstones, in 1769.
In 1815, the chemist Michel Eugène Chevreul isolated a crystalline compound of bile stones
and named it cholesterine, which was renamed to cholesterol after knowing that the substance
was a secondary alcohol. The correct chemical formula of C27H45O was only proposed in 1888
by F. Reinitze, and the first steric representations of the molecule were published by Heinrich
Wieland and Adolf Windaus, their efforts leading the two scientists to win the Nobel Prize in
Chemistry in 1927 and 1928, respectively (Nes, 2011). The steroid nucleus proposed by Wieland
in his Nobel lecture presented some limitations. In 1932, however, his research group corrected
it to the skeleton known nowadays (Vaupel, 2007). The research in steroids by Konrad Bloch
and Feodor Lynen granted them the Nobel Prize in Physiology or Medicine in 1964, for their
discoveries concerning the mechanism and regulation of cholesterol and fatty acid metabolism.
Later in 1985, Michael S. Brown and Joseph L. Goldstein were also awarded with the Nobel
Prize in Physiology or Medicine for their findings relating to the regulation mechanisms of cho-
lesterol metabolism (“Feodor Lynen—Biographical, 2021,” “Joseph L. Goldstein—Biographical,
2021,” “Konrad Bloch—Biographical, 2021,” “Michael S. Brown—Biographical, 2021”).
Cholesterol is synthesized by all animal cells and is an essential structural component of
animal cell membranes, where it contributes to the order of phospholipid chains and over-
all membrane (dis)order, integrity and heterogeneity. It is also used as a precursor for the
biosynthesis of steroid hormones, bile acids and vitamin D (Cerqueira et al., 2016; Ercole,
Whittaker, Quinn, & Davis, 2015; Róg et al., 2009). Although cholesterol has eight stereo-
centers (Fig. 1B) that could rise to 256 stereoisomers, only the natural enantiomer with the
(3R,20R)-configurations, is used as a membrane constituent (Xu et al., 2005).
As an amphiphilic molecule, having a hydrophobic hydrocarbon body and a hydrophilic
hydroxy headgroup, cholesterol occupies a position at polar-nonpolar interfaces, as observed
in cell membranes. The crystal structure of one form of cholesterol monohydrate published
by Craven (1976) is based on a local pseudosymmetry arrangement of eight independent mol-
ecules in the triclinic cells, similar to the structure reported by Shieh, Hoard, and Nordman
(1977) for anhydrous cholesterol crystals at room temperature (25°C). This molecular packing
in the crystal structures is in some way in line to the tendency toward double layer struc-
tures with an end-for-end arrangement of nearly parallel molecules (Bernal, Crowfoot, &
Fankuchen, 1940). On the other hand, cholesterol crystals at 37°C have a remarkably large
unit cell containing 16 independent cholesterol molecules, and the transition preserves a
closely obeyed pseudosymmetry present in the structure (Hsu & Nordman, 1983). Garti et al.
studied phase transitions in cholesterol crystallized from various solvents, characterizing the
effect of several solvents (e.g., carbon tetrachloride, acetonitrile, methanol, ethanol) and con-
ditions of crystallization (Garti, Karpuj, & Sarig, 1980). Using differential thermal analysis,
infrared spectroscopy and polarization microscopy, Barton had found that the phase transi-
tions of cholesterol and other sterols subjected to heating and cooling in a range of − 20°C to
+ 150°C were dependent on the state of hydration and on the structure of the aliphatic side
chain (Barton, 1976).
Below, we will review major milestones in characterization of cholesterol structure, cho-
lesterol laboratory synthesis, and synthetic routes for production of enantiomeric cholesterol.

1. Cholesterol chemistry and cell function

6 1. Cholesterol chemistry and laboratory synthesis

Cholesterol structural characterization

In 1973, Barry et al. used 1D 1H nuclear magnetic resonance (NMR) experiments to assign
unequivocally the chemical shifts of the A and B ring protons of cholesterol using deuterated
chloroform (CDCl3) as solvent (Table 1) (Barry et al., 1973). Years later, Sawan et al. performed
1
H NMR spectrum of cholesterol in pyridine‑d5 to accomplish the same goal (Sawan et al.,
1979). Since then, several 1D and 2D NMR techniques have been used to complete ring proton
assignment of various steroids by comparison with cholesterol data (Drew, Brisson, Morand,
& Szabo, 1987; Zipser et al., 1998).
The latest NMR characterization of cholesterol dates back to 1998, and therefore, with more
than 20 years passed by, we were encouraged to get our own 1D (1H, 13C, DEPT 90, and DEPT
135) and 2D NMR (HSQC, HMBC, COSY, and NOESY) data for the commercial cholesterol
molecule, presenting the 1H and 13C NMR spectra as standard reference (Figs. 2 and 3). Our
own interpretation of NMR data, based on the obtained 1D and 2D NMR, is listed in Tables
1 and 2, with unequivocal assignments of almost all carbons. Carbons C-7, C-11, C-13, C-15,
C-16, and C-23 were assigned by analogy with previous reported data (*) (Table 2).
Although the assignment of 13C NMR chemical shifts in a molecule as large as choles-
terol is a challenging task, some research groups dedicated their efforts to achieve this goal
(ApSimon, Beierbeck, & Saunders, 1973; Mantsch & Smith, 1973; Reich, Jautelat, Messe,

1
TABLE 1 H chemical shifts of cholesterol in several deuterated solvents.a
H CDCl3b,c Pyridine-d5b,c CDCl3d CDCl3e
1α, 1β – 1.83 – –

2α 1.90 2.07 1.50 –

2β 1.58 1.80 –

3α 3.47 3.82 3.39 3.47–3.57 (m)

4α, 4β 2.3 2.60 2.13 2.17–2.33 (m)

6 5.30 5.41 5.34 5.34–5.36 (m)

7α, 7β 2.05 2.03 1.95 –

8 – – 1.5 –
c
18-CH3 0.68 – 0.72 0.67 (s)
c
19-CH3 1.02 – 1.02 1.00 (s)

21-CH3 – – 0.94 0.91 (d, J = 6.5 Hz)

26-CH3 – – 0.87 0.862 (d, J = 6.6 Hz) or 0.858 (d, J = 6.6 Hz)

27-CH3 – –
a
Chemical shifts in ppm relative to the internal standard, tetramethylsilane (TMS).
b
Barry, Dobson, Sweigart, Ford, and Williams (1973).
c
Sawan, James, Gruenke, and Craig (1979).
d
Zipser, Bradford, and Hollingsworth (1998).
e
Our own data 1H NMR (300 MHz).

1. Cholesterol chemistry and cell function

 Cholesterol structural characterization 7

H3C
H3 C
H CH3
H 3C H H3 C
H H
HO

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5
ppm

1
FIG. 2 H NMR spectrum of cholesterol (CDCl3, 300 MHz).

H3 C
H 3C
H CH3
H3C H H 3C
H H
HO

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
ppm

13
FIG. 3 C NMR spectrum of cholesterol (CDCl3, 75 MHz).

1. Cholesterol chemistry and cell function

8 1. Cholesterol chemistry and laboratory synthesis

13
TABLE 2 C chemical shifts of cholesterol in several deuterated solvents.a
CDCl3e (DEPT Pyridine- 1,4-Dioxane-
C CDCl3b,c CDCl3c CDCl3d 135, DEPT 90) CCl4b,c Benzene-d6b,c d5b,c d4b,c
1 38.6 37.3 37.2 37.2 (CH2) 38.6 38.0 38.4 38.2

2 32.8 31.6 31.6 31.7 (CH2) 32.6 32.4 32.7 32.7

3 72.8 71.6 71.8 71.8 (CH) 72.1 71.9 71.6 71.8

4 43.5 42.2 42.3 42.3 (CH2) 43.5 43.2 43.8 43.5

5 142.1 140.6 140.6 140.7 142.1 141.6 142.4 142.4

6 122.7 121.4 121.4 121.7 (CH) 122.3 121.8 121.5 121.7

f
7 33.1 31.9 31.9 31.9 (CH2) 33.1 32.6 32.7 32.7

8 33.1 31.9 31.9 31.9 (CH) 33.1 32.6 33.0 32.7

9 51.4 50.2 50.2 50.1 (CH) 51.4 50.9 51.0 51.3

10 37.6 36.5 36.5 36.5 37.6 37.0 37.4 37.4

f
11 22.3 21.1 21.1 21.1 (CH2) 22.3 21.7 21.9 21.9

12 41.0 39.8 39.8 39.8 (CH2) 41.0 40.4 40.8 40.7

f
13 43.4 42.3 42.3 42.3 43.3 43.0 43.0 43.2

14 58.0 56.8 56.8 56.8 (CH) 58.0 57.2 57.4 57.8

f
15 25.5 24.3 24.3 24.3 (CH2) 25.4 24.8 25.0 25.0
f
16 29.4 28.3 28.3 28.2 (CH2) 29.4 24.8 29.0 29.0

17 57.4 56.2 56.1 56.1 (CH) 57.4 56.9 57.0 57.2

18 13.1 11.9 11.9 11.9 (CH3) 13.1 12.3 12.5 12.4

19 20.6 19.4 19.4 19.4 (CH3) 20.6 19.8 20.1 19.9

20 37.0 36.8 35.8 35.8 (CH) 37.0 36.5 36.8 36.8

21 20.0 18.8 18.7 18.7 (CH3) 20.0 19.3 19.5 19.4

22 37.4 36.2 36.2 36.2 (CH2) 37.4 37.0 37.0 37.2

f
23 25.1 23.9 23.8 23.8 (CH2) 25.1 24.6 24.7 24.7

24 40.7 39.5 39.5 39.5 (CH2) 40.7 40.2 40.2 40.3

25 29.2 28.0 28.0 28.0 (CH) 29.1 28.6 28.7 29.0

26 23.9 22.6 22.6 22.6 or 22.8 23.9 23.1 23.1 23.1

(CH3)

27 24.1 22.8 22.8 22.8 or 22.6 24.1 23.3 23.5 23.3

(CH3)
a
Chemical shifts in ppm relative to the internal standard, tetramethylsilane (TMS).
b
Mantsch and Smith (1973).
c
Blunt and Stothers (1977).
d
Smith (1978).
e
Our own data 13C NMR (75 MHz).
f
Assigned by analogy to previous publications.

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 9
Weigert, & Roberts, 1969; Smith, 1978; Smith, Deavenport, Swanzy, & Pate, 1973). Blunt and
Stothers covered the 13C NMR assignments of cholesterol in several deuterated solvents
(Blunt & Stothers, 1977) and the chemical shifts are presented in Table 2. The 27 carbon atoms
of cholesterol are characterized to possess mainly nonpolar atoms (24 out of the 27), a polar
atom corresponding to C-3 and the unsaturated carbons corresponding to C5C6 double
bond, in a total range of 130 ppm. From analyzing the data shown in Table 2 we can state that
the chemical shifts vary slightly with the solvent used in the acquisition and even using the
same solvent, the data can be quite different according to the research group.
Based on a more detailed analyzes on the data provided by Mantsc et al., the most
­solvent-sensitive positions are the 3-OH and C-6, both of which are shifted by about 1 ppm in
CDCI3 and about 0.5 ppm in CCl4, for higher frequency values, when compared to ­benzene‑d6,
pyridine‑d5, and 1,4‑dioxane‑d4 (Table 2).

Cholesterol laboratory synthesis

Cholesterol total synthesis—Historical perspective

The total synthesis of cholesterol was one of the most remarkable achievements of 20th
century Chemistry. An upmost historical curiosity is that the laboratory synthesis of choles-
terol was some sort of a mental competition between Robinson in Oxford and Woodward
at Harvard. The interesting outcome was that both research groups, simultaneously and
independently, achieved the cholesterol total synthesis in 1951 (Cardwell, Cornforth, Duff,
Holtermann, & Robinson, 1951; Woodward, Sondheimer, & Taub, 1951a). According to chem-
istry historian Mulheirn (2000), the preliminary notice of Robinson’s total synthesis was pub-
lished in Chemistry and Industry in 1951 (Cardwell et al., 1951), only a couple of weeks after
Woodward’s announcement of his own synthesis at the Chemical Society Centenary Lecture
(subsequent preliminary notice of the synthesis was published in the Journal of the American
Chemical Society) (Woodward, Sondheimer, Taub, Heusler, & McLamore, 1952). Despite
Robinson substantial contributions to synthetic organic chemistry (Robinson annulation is
perhaps the most well-known), Woodward was able to complete his project in a remarkably
short period (around 2 years), which was testimony both to his brilliance and to the pharma-
ceutical industry financial support.
The Woodward synthesis itself can be described as a C → CD → BCD → ABCD route (Fig. 4),
rather than the BC → ABC → ABCD route (Fig. 5) used by Robinson. Woodward was able to
gather support of industry to not only fund human resources but also supply key interme-
diates; Robinson’s synthesis in turn had to resort to using relays. Many of chemical inter-
mediates of Robinson’s synthesis were already known and available from natural sources,
and therefore, Robinson’s challenge was to proof that these intermediates could be linked
to each other via chemical synthesis, in order to develop a formal cholesterol total synthesis.
From a practical point of view, and despite that all steroid intermediates of Robinson’s re-
lay approach were already known, his linear cholesterol synthesis requires 68 reaction steps,
(Cardwell et al., 1951; Cardwell, Cornforth, Duff, Holtermann, & Robinson, 1953; Cornforth
& Robinson, 1946, 1949) in opposition to Woodward’s with “only” 35 steps (Woodward
et al., 1952; Woodward, Sondheimer, and Taub, 1951a, 1951b; Woodward, Sondheimer, Taub,
Heusler, & McLamore, 1951).

1. Cholesterol chemistry and cell function

10 1. Cholesterol chemistry and laboratory synthesis

Woodward’s cholesterol total synthesis

Steroid ring system

C D

A B

C D C D
C D C
A B B

FIG. 4 Retrosynthetic analysis of Woodward’s cholesterol total synthesis.

Steroid ring system

C D

A B

C D C C

A B A B B

FIG. 5 Retrosynthetic analysis of Robinson’s cholesterol total synthesis.

Woodward’s cholesterol total synthesis

The retrosynthetic analysis of Woodward’s approach could sometimes be misunderstood,
since the D ring remains D-homo until the last step of ring construction (Scheme 2) and the
required 5-membered ring was obtained only after a ring contraction; it could also be termed
as C → BC → ABC → ABCD. Whatever the case, Woodward’s starting point was 5-methoxy-2-
methyl-1,4-quinone 1, used to form ring C in the final structure. The Diels-Alder reaction of hy-
droquinone 1 with butadiene 2 gave the cis-bicycle 3, which was converted to the trans-isomer
4 through sodium enolate followed by acidification (Scheme 1). Reduction with lithium alu-
minum hydride (LiAlH4) followed by dehydration gave ketol 6, which upon deoxygenation
of its acetate with zinc gave enone 8 (Scheme 1). Claisen condensation of enone 9 followed
by Michael addition of ethyl vinyl ketone originates dione 10, which undergoes cyclization
with KOH to produce tricycle 11 (Scheme 1). The following steps of Woodward’s synthesis
involve the diol 12 formation with osmium tetroxide (OsO4), subsequent diol protection with
acetone and copper(II) sulfate (CuSO4), hydrogenation and Claisen condensation to give 15

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 11
O O O
OH

NaOH LiAlH4
2
+
MeO Dioxane, H3O THF, reflux
PhH, 100 ºC MeO MeO 65 min, 64%
MeO
96 h, 86% H 40-50 ºC, 90% H H
O O O
OH
1 3 4 5

Dioxane, H2O
H2SO4
rt, 24 h, 99%

HCO2Et, NaOMe Zn Ac2O

PhH, rt Ac2O, reflux Pyridine, rt
O O O
H 30 min, 94% O 8 min, 63% H 12 h, 96% H
H AcO
CHO
OH
9 8 7 6
O t t
BuOK, NaOH, H2O, BuOH
Et 0 ºC to rt, 10 h, 53%

OH O
H KOH OsO4 CuSO4
H H H
O Dioxane, H2O Et2O, 0 ºC, OH Acetone, rt, O
CHO 0 ºC, 3 h, 88% H 5-8 days, 57% H 36 h, 83% H
Et O O O
10 11 12 13
O

Pd/SrCO3, H2, 1 atm PhH, rt, 3.5 h,

85%
NC O O
H NC H O
O +
Me3N BnHO - O H HCO2Et, O
H PhNHMe
H O H
NaOMe
O H2O, PhH, tBuOH, O MeOH, rt, H O
24 h, 88% H
Me 50 ºC, 45 h Me O PhH, rt
N N O
17 CHO 14
Ph Ph 16
15
KOH H2O, reflux, 46%

HO2C O O MeOC O
NaOAc MeMgBr KOH
H H H
Ac2O, reflux, Et2O, PhH, H2O, MeOH,
O O O
H 2 h, 28% H -18 ºC, 3 h H reflux, 2 h,
O O O O 58%
18 19 20

"Christmasterone"

O
H
O
H
O
21

SCHEME 1 Woodward’s cholesterol total synthesis: preparation of Christmasterone 21.

(Scheme 1). The enamine protection followed by Michael addition of cyanoethylene and sub-
sequent nitrile hydrolysis gave the carboxylic acid 18 (Scheme 1). Lactonization of carbox-
ylic acid 19, followed by Grignard reaction with methylmagnesium bromide (MeMgBr), and
subsequent aldol condensation gave the tetracyclic ketone 21 (Scheme 1), which completes
the four-ring structure required for cholesterol synthesis. Tetracyclic ketone 21 (nicknamed
“Christmasterone”) was obtained on Christmas Day in 1950 by Sondheimer, and it was a top-
most example of Woodward’s high-pressure style of leadership combined with the sense of
success being just around the corner. At this point, the final hurdle was the contraction of ring
D from a six-membered to a five-membered ring.

1. Cholesterol chemistry and cell function

12 1. Cholesterol chemistry and laboratory synthesis

"Christmasterone"
CHO
O Piperidine, AcOH,
HIO4 CHO Na2Cr2O7
H H Dean-Stark
CHO H
O 1,4-Dioxane, H2O, AcOH, H2O, rt
H 0 ºC, 14 h H PhH, 60 ºC, 10 h
60 min, 66% H
O O O
21 22 23

CO2H CO2Me CO2Me

CH2N2
Et2O, PhH, rt, H2, PtO2, 1 atm H
H H
20 min, 30% AcOH, rt
H H H H
(2 steps) HO
O O H CrO3
24 25 26
AcOH, rt, ON, CO2Me
2.4 % (2 steps)
CO2H H
CO2H
CO2Me
H H
H Ac2O NaBH4
H KOH O
H H
H H Pyr, 60 ºC, 3 h, H2O, MeOH,
27
92% H H EtOH, rt,
AcO reflux, 12 h, H H 4 h, 64%
H HO
H 100% HO
30 H
29 28

SOCl2 0 ºC to rt,
15.5 h, 90%
O O
Cl Me OH

H MeCd BrMg H
H
H
PhH, reflux, Et2O, PhH, rt,
H H 90 min, 92% H H
60 min H H
AcO AcO Ac2O
H H HO
H AcOH, reflux,
31 32 33 2.5 h H

H H
AcO 34
H

PtO2, H2
H H
KOH AcOH, rt, 30 min,
H H 14% (3 steps)
H2O, MeOH,
H H reflux, 2 h, H H
HO 85% AcO
H H
36 35

SCHEME 2 Woodward’s cholesterol total synthesis: preparation of cholestanol 36.

Treatment of Christmasterone 21 with periodic acid (HIO4) in 1,4-dioxane followed by heat-

ing the product 22 in the presence of a catalytic amount of piperidine acetate gave DL-Δ9(11),16-
bisdehydro-20-norprogesterone 23 (Scheme 2), from which a route to cholesterol was known.
The sodium dichromate (Na2Cr2O7) oxidation of the aldehyde function of 23 gave carboxylic
acid 24, which upon diazomethane esterification, hydrogenation and oxidation gave ketone
27 (Scheme 2). The sodium borohydride (NaBH4) ketone reduction, ester hydrolysis, and sec-
ondary alcohol acetylation with acetic anhydride gave carboxylic acid 30 (Scheme 2). The final
stages of Woodward’s synthesis were focused on the preparation of C-17 aliphatic side chain.
The thionyl chloride (SOCl2) treatment of carboxylic acid 30 gave the corresponding acyl chlo-
ride 31, which upon methyl cadmium (MeCd) and Grignard reaction with isohexylmagne-
sium bromide afforded diol 33 (Scheme 2). Three reaction steps later, involving dehydration,
hydrogenation and ester hydrolysis, cholestanol 36 was obtained (Scheme 2). The conversion
of cholestanol 36 into cholesterol 41 was already demonstrated, involving five additional

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 13

Jones C5H5N×HBr×Br2 H
H H
reagent H
H H AcOH

H H H H H H
O O
HO H
H H Br
36 37 38

1. NaOAc, NH2CONHNH2·HCl,
AcOH, 70 ºC, 2 h
2. Pyruvic acid, 70 ºC, 2 h

NaBH4 MeCOCl
H H H
KOH, iPrOH Ac2O
H H H

H H H H H H
HO AcO O
41 40 39

SCHEME 3 Cholestanol 36 conversion into cholesterol 41 following Dauben and Eastham method.

­reaction steps (Scheme 3). The oxidation of cholestanol 36 to the corresponding ketone 37 and
further selective C-4 bromination and elimination gave cholestenone 39 (Scheme 3).
The conversion of cholestenone 39 into cholesterol 41 was accomplished by the method of
Dauben and Eastham reported in 1950 (Dauben & Eastham, 1950). The treatment of choles-
tenone 39 with acetyl chloride in acetic anhydride gave the enol acetate 40 which, without
purification, was reduced by sodium borohydride and potassium hydroxide to yield natu-
ral cholesterol 41, upon fractionation with digitonin for the isolation of the correct isomer
(Scheme 3) (Birch, 1950; Dauben & Eastham, 1950; Djerassi & Scholz, 1948; Kritchevsky,
Garmaise, & Gallagher, 1952; Ruzicka, Plattner, & Aeschbacher, 1938).

Robinson’s cholesterol total synthesis

As Robinson used a BC → ABC → ABCD synthetic approach (Fig. 5), his starting material
was 1,6-dihydroxynaphthalene 42 (corresponding to B and C rings in the final cholesterol
structure), which was converted in the tricyclic structure 43 in a five reaction steps protocol
(addition of A ring) (Scheme 4).
The synthesis of the first relay molecule 44 (also known as Reich diketone) (Reich, 1945) was
completed 12 steps later (Scheme 4). The second relay molecule 45 was prepared resorting to
eight additional reaction steps. Interestingly, this differed from the first one in “only” a dou-
ble bond in ring B and the 3-hydroxy group replacing the original carbonyl group (Scheme 4).
Resorting to another 12 reaction steps, Robinson prepared his third relay molecule 46, en route
to the fourth relay 47, which has already ring D of the final cholesterol structure (Scheme 4).

1. Cholesterol chemistry and cell function

14 1. Cholesterol chemistry and laboratory synthesis

BC ABC

12 steps H
5 steps
OH OMe O
H
HO O O
H 8 steps
42 43 44
First relay molecule
ABCD
O
CO2Me
H 8 steps H 12 steps H
CO2Me
O
H H H H
AcO BzO HO
H 47 H 46 45

Third relay molecule Second relay molecule

Fourth relay molecule

5 steps

O O
Cholestanol
6 steps 12 steps
H H H
H
H H H H
O HO H H
48 49 HO
H 36
Fifth relay molecule Sixth relay molecule

SCHEME 4 Robinson’s cholestanol 36 total synthesis, based on a relay approach.

Two more relays were synthesized, molecules 48 and 49, being the final 12 reaction steps used
to add the cholesterol tail. Thus, he resorted to a similar strategy to that used by Woodward
(Scheme 4).a The conversion of cholestanol 36 into cholesterol 41, followed the same already
known methodology depicted in Scheme 3.

Cholesterol hemisynthesis
The introduction of cholesterol side chain at C-20 is quite a challenge, as can be understood
either from Woodward’s or Robinson’s total syntheses. An interesting approach, however,
was developed by Schmuff and Trost (1983), based on organocuprate-mediated methods.
This strategy started from the natural dehydroepiandrosterone 50 which was further con-
verted in alcohol 51 in three reaction steps (Scheme 5). Then, the Moffatt-type oxidation gave
the (E)-enone 52, which upon reaction with lithium diisohexylcuprate gave cholestanone 53

a
A relay molecule can be defined as a compound which needs to be synthesized for the first time in total
synthesis methodologies, but once synthesized, it is necessary to have it available in larger quantities
from natural sources. This strategy saves many valuable man-hours synthesizing the relay substrates in
the laboratory, because for every experiment that is successful, there are many that are not, and so a large
amount of substrate is needed at each stage in the synthesis.

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 15

H 3 steps H OH (COCl)2 H O
DMSO
H H H H H H
HO
OMe OMe 52
50 51
i
( C6H12)2CuLi Et2O

H H
TsOH N2H4, K2CO3
H H O
cholesterol aqueous triethylene
dioxane H H glycol H H
54 53
OMe OMe

SCHEME 5 Alkylcuprate-mediated synthetic route to cholesterol from dehydroepiandrosterone 50.

as the only detectable C-20 isomer (Scheme 5). Subsequent Wolff-Kishner reduction gave the
isocholesterol methyl ether 54, which was further converted into cholesterol (Scheme 5).

Synthesis of ent-cholesterol: The unnatural enantiomer

All known natural sterols have the same absolute configuration at the C-10 and C-13 qua-
ternary centers, and so there is no simple way to convert readily available natural sterols into
their enantiomeric series. Therefore, the preparation of ent-cholesterol (the unnatural enantio-
mer of cholesterol) (Fig. 6) is only possible through enantioselective total synthesis.
The ent-cholesterol 55 total synthesis was reported for the first time in 1992 by Rychnovsky
and Mickus (1992). They took as inspiration an elegant stereoselective synthesis of 19-nor steroids
by a group at Hoffmann-La Roche, and prepared ent-testosterone 64 as chemical intermediate for
the synthesis of ent-cholesterol (Scheme 6). The achiral triketone 56 was used as starting mate-
rial for the enantioselective intramolecular aldol reaction followed by acid-catalyzed elimination
to give the chiral enedione 57 (Scheme 6). The stereogenic center in dione 57 was employed
to control the remaining stereocenters in the final ent-cholesterol. The NaBH4 reduction of the
saturated ketone followed by protection with isobutylene gave enone 58, which upon treatment
with Stile’s reagent delivers the carboxylic acid 59 (Scheme 6). Hydrogenation and reaction with

H H
H H

H H H H
HO HO
cholesterol ent-cholesterol (55)

FIG. 6 Structures of cholesterol and ent-cholesterol.

1. Cholesterol chemistry and cell function

16 1. Cholesterol chemistry and laboratory synthesis

O OtBu
O 1. D-proline, DMF, O 1. NaBH4, EtOH, -5 ºC
rt, 7 days
2. Isobutylene, H3PO4/BF3OEt2, -78 ºC
2. H2SO4, DMF, 95 ºC O
O O
R
56 57
58 R = H
Mg(OMe)O2COMe, DMF, 125 ºC 59 R = CO2H

1. H2, Pd/BaSO4, MeOH

O O 2. CH2O, piperidine,
O OtBu CO2Et OtBu DMSO/H2O
O
O 61

1. 61, NaOMe, MeOH O

62 2. H3O+, 80 ºC
O 60

Li, NH3, THF, -33 ºC;

MeI, -78 to -33 ºC

O OtBu OH
O
HCl, MeOH, reflux

63 O 64
O
ent-testosterone

SCHEME 6 Synthesis of ent-testosterone 64, intermediate in the synthesis of ent-cholesterol 55.

aqueous formaldehyde gave the enone 60, which upon Robinson annulation with β-keto ester
61 gave the tricyclic intermediate 62 (Scheme 6). The 19-methyl group was introduced through
enone reduction followed by treatment with iodomethane to give ketone 63 (Scheme 6). The acid
catalyzed cyclization of ketone 63 gave the required ent-testosterone 64 (Scheme 6).
Once ent-testosterone 64 was obtained, Rychnovsky and Mickus were able to reach ent-­
cholesterol in a few reaction steps (Scheme 7). They obtained the β,γ-unsaturated ketone 65
in acidic media, which upon reduction with LiAl(OtBu)3H followed by OH-protection with
tert-butyldimethylsilyl chloride (TBSCl) gave the monosilyl diol 67 (Scheme 7). The stereochem-
istry at C-17 and C-20 was set by hydroboration with 9-borabicyclo[3.3.1]nonane (9-BBN) which
enters from the top face of the alkene. Coupling the resulting hindered trialkylborane with chlo-
roacetonitrile in the presence of a hindered base gave nitrile 69 as a single isomer (Scheme 7).
The side chain was completed by nitrile alkylation with 1-bromo-3-methylbutane and reductive
decyanation followed by desilylation to afford ent-cholesterol (Scheme 7).
An alternative methodology to convert ent-testosterone into ent-cholesterol was reported
later in 1999 by Kumar and Covey (1999). To do so, Kumar and Covey used the previously re-
ported methodology to prepare steroid 67 from ent-testosterone 64 (Scheme 7). However, they
faced successive experimental failures building up the side chain of ent-cholesterol, and there-
fore they were forced to consider an alternative strategy to complete the synthesis (Scheme 8).
The Kumar and Covey strategy relied on the ene reaction of (Z)-olefin 70 with
4-­methylpent-1-enal which gave the epimeric alcohol 71 (Scheme 8). The selective reduction

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 17
OH OH OH

tBuOK, tBuOH, rt LiAl(OtBu)3H

AcOH THF

O O RO
64 65 66 R = H
TBSCl, DMAP, Et3N, CH2Cl2
67 R = TBS

1. PCC, 4A sieves,
1. LDA, Br(CH2)2CH(CH3)2,
CH2Cl2
THF, -78 ºC 2. EtPPh3Br, tBuOK, THF
2. K, dicyclohexyl-18-crown-6, H CN
toluene
ent-cholesterol
3. Bu4NF, THF 9-BBN, ClCH2CN, KOAr
THF
TBSO
69 TBSO
68

SCHEME 7 Synthesis of ent-cholesterol from ent-testosterone 64 precursor.

OH
OH
5 steps 4-methylpent-1-enal
H
Me2AlCl, CH2Cl2 H
H H
H H
O TBSO
TBSO
ent-testosterone 70
71

Pt/C (5%), H2 atm,

EtOH/Et20 (1:1)
R
OH
H
H H
Bu4NF H
H 1. MeSO2Cl, Py
H H THF H
HO H H 2. LiB(Et3)H, THF
55 H H
TBSO
ent-cholesterol TBSO
73 R = OSO2Me 72
74 R = H

SCHEME 8 Alternative method for the preparation of ent-cholesterol from ent-testosterone.

of Δ16-double bond of 71 gave the C-22 epimers of steroid 72, which upon a tosylation/deto-
sylation method gave steroid 74 (Scheme 8). The final removal of TBS protecting group with
Bu4NF gave ent-cholesterol 55 (Scheme 8).
As demonstrated earlier in this chapter, the common synthetic strategies for the synthesis
of either cholesterol or ent-cholesterol proceed via the initial construction of the steroid ring
system followed by the subsequent introduction of the C-17 side chain. This type of synthetic
strategies is not suitable for preparing 13C-labeled ent-cholesterols because the isotopic labels
have to be incorporated before the multiple steps involved in construction of the side chain are
initiated. In this sense, Jiang and Covey proposed in 2002 the total synthesis of ent-cholesterol
by a route which starts with construction of the sterol D-ring containing the cholesterol side
chain and then proceeds via elaboration of the sterol C, B, and A rings, respectively (Jiang &
Covey, 2002). Accordingly, they started with methyl acetoacetate 75 which was converted in
three steps into racemic compound 76 (Scheme 9). The addition of 4-methylpentylmagnesium

1. Cholesterol chemistry and cell function

18 1. Cholesterol chemistry and laboratory synthesis

1. NaH, nBuLi, crotyl bromide,

Mg, CuI,
O O THF, 25 ºC
MeO2C H (CH3)2CH(CH2)3Br MeO2C H R
2. p-TsN3, Et3N, MeCN, 25 ºC
OMe THF, -15 ºC
3. bis(N-tBu-salicylaldiminato)Cu(II), O 68% O
toluene, reflux 76 77
75
60%
(R)-pentanolactone, DMAP,
toluene, reflux 82%

R
R 1O R 1O 2 C H O O
2C O
H R p-TsOH
O H R
toluene, reflux
O
NaOMe, MeOH, 0 ºC
O O O
80 79 59% O
78
90% ethylene glycol R = isopentyl; R1 = (R)-pentanolactone
p-TsOH, toluene, reflux

R 1O HO
2C 1. MsCl, Et3N, CH2Cl2, 25 ºC
H R LiAlH4 H R H R
2. LiI, NaHCO3, 1,4-dioxane, reflux
O THF, reflux O O
3. Superhydride 1 M in THF, reflux
95%
O O O 83
81 82
2 N aqueous HCl,
91%
THF, 25 ºC

H R H R
5% Pd/BaSO4, H2 1. 2 M MMC--DMF, 125 ºC
H R
MeOH, 50 psi, 25 ºC 2. CH2N2, ether, 0 ºC
O O
H 95% 69% O
C CO2Me
MeO 2 86 84
85 O O
O O
97% ethylene glycol OMe
p-TsOH, PhH, reflux 91

1. 91, NaOMe,
H R H R H R
2 N HCl MeOH, 25 ºC O
H
O
acetone, 25 ºC O 2. 5 N aq. NaOH, O
O H H 25 ºC H
R 2
R 3O O 92
LiAlH4, ether, 87 R2 = CO2Me
MsCl, Et3N, 89 R3 = H
reflux 88 R2 = CH2OH 1. LiI, NH3, THF, -78 ºC
CH2Cl2, 25 ºC 90 R3 = Ms 83%
2. MeI, -40 to 25 ºC

H R
H
1. Me3SiCl, NaI, H R H R
Ac2O, 25 ºC O
H 6 N HCl H
H H 2. NaBH4, EtOH, MeOH, reflux O
H H H H
HO 25 ºC 95%
O O
ent-cholesterol (55) 93
94

SCHEME 9 ent-Cholesterol total synthesis reported by Jiang and Covey.

1. Cholesterol chemistry and cell function

 Cholesterol laboratory synthesis 19
bromide to β-keto ester 76 gave racemic product 77, which upon transesterification with (R)-
pantolactone gave a diastereomeric mixture from which diastereomer 78 was easily separated
(Scheme 9). At this point, the side chain of ent-cholesterol was already incorporated, and subse-
quent reaction with methyl vinyl ketone gave the intermediate compound 79, thus setting the
proper chemical features of what will become the C-ring, and also formation of the 18-methyl
group (Scheme 9). p-Toluenesulfonic acid (p-TsOH) catalyzes the cyclization of intermediate
79 to give enone 80, which upon reaction with ethylene glycol was converted into ketal 81
(Scheme 9). The (R)-pantolactone group of compound 81 was then reduced using LiAlH4 and
upon three additional reaction steps, the 18-methyl group with proper stereochemistry was
established in compound 83 (Scheme 9). The removal of the ketal protecting group from 83
yielded the desired C,D ring-side chain fragment, indenone 84 (Scheme 9). The reaction be-
tween indenone 84 and magnesium methyl carbonate (MMC) in DMF, followed by COOH
methylation gave the keto ester 85, used to stabilize the keto acid obtained from reaction with
MMC (Scheme 9). Hydrogenation using 5% Pd/BaSO4 gave saturated keto ester 86, which
was subsequently converted in compound 90, in four steps so that the remaining rings of
ent-cholesterol could be built (Scheme 9). Next, displacement of the mesylate group of com-
pound 90 by the anion formed from methyl 6-(2-methyl-1,3-dioxolan-2-yl)-3-oxohexanoate
91, followed by cyclization gave compound 92 (Scheme 9). The introduction of the 19-methyl
group of ent-cholesterol into precursor enone 92 was made by reduction followed by lithium
enolate intermediate reaction with excess iodomethane to give compound 93, which upon
cyclization rendered ent-­cholestenone 94 (Scheme 9). The conversion of ent-cholestenone 94 to
ent-cholesterol 55 was achieved via the dienol acetate, which was then reduced with NaBH4
to give ent-cholesterol 55 (Scheme 9).
Sixteen years later from Rychnovsky first synthesis of ent-cholesterol 55, his group reported
a new concise and scalable synthesis of the unnatural enantiomer of cholesterol, starting from
(S)-citronellol (Fig. 7) (Belani & Rychnovsky, 2008). The Rychnovsky new synthesis of ent-­
cholesterol 55 is based on a ring D to C to B to A approach and incorporates the cholesterol side
chain early in the synthetic procedure, as in the strategy reported in 2002 by Jiang and Covey.
The first key intermediates C and B were synthesized following a CH insertion strat-
egy (Fig. 7 and Scheme 10). Commercially available (S)-citronellol was converted to the
corresponding benzenesulfonate and subsequently alkylated with the dianion of methyl

H CO2Me
H O
H

H H O H
HO A B
ent-cholesterol
O O

OMe OH
N2

C (S)-citronellol

FIG. 7 Retrosynthetic analysis of ent-cholesterol starting from (S)-citronellol.

1. Cholesterol chemistry and cell function

20 1. Cholesterol chemistry and laboratory synthesis

O O O O
1. PhSO2Cl, Et3N
OH OMe p-ABSA, Et3N OMe Rh2(PTV)4
2. OLi ONa
95 MeCN, 23 ºC N2 CH2Cl2, 23 ºC
OMe 96% 76%
65%
(S)-citronellol 96 97

O O O
CO2Me CO2Me CO2Me
H2, Pd/BaSO4 MeI, K2CO3 1. NaCN, HMPA, 80 ºC
H MeOH, 23 ºC H Acetone H 2. KOH, MeOH
99% 95% 93%
98 99 100

O
1. O O

MeO OMg(OMe)
H NaOMe, 23 ºC H H
2. TsOH, PhMe, 110 ºC DMF, 125 ºC
O 90% O
101 84%
102 103
COOH

O O
O O
OMe
91

1. H2, Pd/BaSO4, MeOH, -5 ºC 1. 91, NaOMe, 10 h

2. i) CH2O, DMSO, piperidine, 23 ªC H 2. 5 N NaOH, 2 h H
ii) PhSH, Et3N, 3. 80 ºC, 0.2 torr O O H
57% O 73%
H
SPh O
104 105

1. 6M HCl, MeOH, reflux

1. Li/NH3, THF, -78 ºC H 2. i) tBuOK, tBuOH H
O O H
2. MeI, THF, -33 ºC ii) AcOH, H2O, 23 ªC H
81% H H 3. Li(OtBu)3 AlH, THF, 23 ºC
H H
O 80%
106 HO
ent-cholesterol

SCHEME 10 Synthesis of ent-cholesterol from (S)-citronellol.

1. Cholesterol chemistry and cell function

 Concluding remarks 21
a­ cetoacetate to give β-keto ester 96 (Scheme 10). A diazo transfer reaction allowed the con-
version of β-keto ester 96 into α-diazo-β-keto ester 97, which upon diastereoselective CH
insertion reaction gave the keto ester 98 (Scheme 10). The CH insertion strategy drastically
shortens the synthesis of the sterol side chain and allows the C-20 stereogenic center to be
introduced from a chiral pool source. Hydrogenation of keto ester 98 using palladium on car-
bon (Pd/C) provided compound 99 with a saturated side chain, which subsequently under-
went methylation followed by decarbomethoxylation to give α-methyl ketone 101 as a single
diastereomer (Scheme 10). The Robinson annulation of ketone 101 with methyl vinyl ketone
gave the corresponding Michael adduct which, upon treatment with p-TsOH, provided the
enone 102 (CD rings completed) (Scheme 10). The strategy for the conversion of enone 102 to
ent-cholesterol was the same double annulation strategy developed by Hoffmann La Roche,
similar to that used by Rychnovsky in his first ent-cholesterol synthesis (Rychnovsky &
Mickus, 1992). Therefore, the treatment of enone 102 with Stile’s reagent gave the carboxylic
acid 103, which upon hydrogenation and subsequent reaction with formaldehyde followed
by the addition of thiophenol gave thioether 104 (Scheme 10). The annulation of thioether 104
with β-keto ester 91 provided the tricyclic enone 105, which upon reduction and alkylation
installed the C-19 methyl group stereoselectively (Scheme 10). Acid-catalyzed deprotection
of the ketal followed by aldol condensation provided the A ring of ent-cholestenone. The AB
ring functionality was modified by deprotonation using tBuOK, followed by kinetic proton-
ation to provide the deconjugated ketone and diastereoselective reduction of the ketone with
Li(OtBu)3AlH gave ent-cholesterol (Scheme 10) (Belani & Rychnovsky, 2008).

Concluding remarks

Cholesterol is an essential component of animal cell membranes and the precursor for the
synthesis of steroid hormones and bile acids. The interest of scientist and industry in steroids,
particularly cholesterol, dates back to the 1930s as these compounds were widely used in medi-
cine. Steroids were big business in the pharmaceutical industry and a company that discovered
viable ways to produce them stood to make huge profits. At that time, steroids were exclusively
obtained through chemical conversion of steroid precursors extracted from natural sources in
very expensive and unproductive processes. As a consequence, a general belief that completes
synthesis might provide a cheaper and quicker method of production of steroids started to grow,
even though complete synthesis might require over 30 stages. It is no coincidence, though, that
one of the most significant chemical problems of that time drew the attention of two of the great-
est chemists of the 20th century: Sir Robert Robinson at Oxford and R.B. Woodward at Harvard.
Cholesterol was the most complex organic molecule synthesized up to that time, and its total
synthesis paved the way for the synthesis of many related steroid hormones. Since 1951, there
was no significant developments in cholesterol synthesis, with only one example of hemisyn-
thesis from dehydroepiandrosterone through organocuprate-mediated methods. Interestingly
enough, in recent years, cholesterol unnatural enantiomer—ent-­cholesterol, has drawn much
more attention than cholesterol itself. In fact, the scientific applications of ent-cholesterol as
a tool to study the enantioselectivity of cholesterol interactions or the molecular recognition
of cholesterol stereoisomers by monoclonal antibodies, for example, drove the development
of three total synthetic routes to it within 18 years’ time lapse. Noteworthy is the synthesis of

1. Cholesterol chemistry and cell function

22 1. Cholesterol chemistry and laboratory synthesis

Jiang and Covey which allows the preparation of 13C- and 2H-labeled forms of ent-cholesterol,
introduced near the end of the reaction sequence. This route is of particular importance for
NMR studies of ent-cholesterol interactions. Apart from that, cholesterol has more interest in
Chemistry as synthon to create cholesterol-based new molecules for a wide range of applica-
tions ranging from drug delivery or bioimaging applications to cholesterol-based liquid crys-
tals and gelators (Albuquerque, Santos, & Silva, 2019).

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1. Cholesterol chemistry and cell function

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Title: Histoire du Bas-Empire. Tome 01

Author: Charles Le Beau

Editor: J. Saint-Martin

Release date: December 14, 2023 [eBook #72414]

Language: French

Original publication: Paris: F. Didot, 1836

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*** START OF THE PROJECT GUTENBERG EBOOK HISTOIRE

DU BAS-EMPIRE. TOME 01 ***
 HISTOIRE
DU
BAS-EMPIRE.
 TOME I.
A PARIS,

CHEZ

FIRMIN DIDOT père et fils, Libraires, rue Jacob, no 24;

BOSSANGE père, Libraire, rue de Richelieu, no 60;

VERDIÈRE, Libraire, quai des Augustins, no 25.

HISTOIRE DU BAS-EMPIRE,
PAR LEBEAU.
NOUVELLE ÉDITION, REVUE
ENTIÈREMENT, CORRIGÉE,
ET AUGMENTÉE D'APRÈS LES
HISTORIENS ORIENTAUX,
 Par M. DE SAINT-MARTIN,
MEMBRE DE L'INSTITUT (ACADÉMIE DES
INSCRIPTIONS ET BELLES-LETTRES).
 TOME I.
PARIS,

DE L'IMPRIMERIE DE FIRMIN DIDOT,

IMPRIMEUR DU ROI ET DE L'INSTITUT, RUE JACOB, No 24.

M. DCCC. XXIV.
 AVERTISSEMENT DE L'ÉDITEUR.
L'Histoire romaine, commencée par Rollin et achevée par Crevier;
l'Histoire des Empereurs, composée par ce dernier, et qui n'est
réellement qu'une continuation du travail entrepris par Rollin; et
l'Histoire du Bas-Empire, de Lebeau, terminée par Ameilhon, seront
toujours, malgré les jugements très-divers qu'on a pu en porter, trois
ouvrages recommandables et propres à honorer la littérature
française. Ils ne brillent pas toujours et partout par les mêmes
qualités, mais on ne peut leur refuser un mérite assez rare dans les
grandes compilations: c'est d'offrir le recueil le plus complet, et en
même temps le plus clair et le plus méthodique, de tous les
renseignements que les auteurs anciens nous ont transmis sur
l'histoire du peuple-roi, dont le nom et les souvenirs remplissaient
encore le monde à l'époque même où son empire avait depuis long-
temps cessé d'être redoutable.
L'Histoire romaine et celle des Empereurs sont encore parmi nous
les seuls livres que l'on puisse consulter pour ce qui concerne cette
partie de l'histoire ancienne jusqu'au temps de Constantin. Il n'est
guère probable que de nouveaux ouvrages les fassent oublier. On y
trouve tout ce que l'antiquité nous a laissé, et on y prend une idée
plus juste de la liaison des faits et de la succession des événements,
qu'on ne pourrait le faire en lisant les auteurs originaux eux-mêmes.
Rollin et Crevier ont mis à profit toutes les observations publiées
avant eux par les savants modernes; les découvertes plus récentes
et les travaux scientifiques publiés de nos jours ajouteraient peu de
choses à leurs recherches.
Il n'en est pas de même pour l'Histoire du Bas-Empire, de Lebeau;
on le concevra sans peine. L'histoire de la République et celle du
Haut-Empire est tout entière dans les écrits des Grecs et des
Romains, ou dans les monuments que le temps a épargnés. Les
puissantes nations qui luttèrent contre la fortune de Rome ont été
anéanties avec toutes leurs productions littéraires, et il n'est pas
présumable que de nouvelles découvertes nous révèlent encore des
faits d'une grande importance. Depuis Constantin, au contraire,
l'empire romain et celui de Constantinople furent toujours en relation
avec des peuples qui ont raconté eux-mêmes, dans une multitude
d'ouvrages encore inédits et dans des langues très-diverses,
l'histoire de leurs rapports et de leurs démêlés avec les Romains et
les Grecs du Bas-Empire. Les livres écrits en arménien, en syriaque,
en arabe, en persan et en turc, doivent donc contenir et contiennent
effectivement beaucoup de renseignements précieux, propres à
compléter, à modifier ou même à changer entièrement ce que nous
savons déja.
Lebeau est le premier et même le seul qui ait songé à classer, dans
un ordre facile à saisir, tous les faits contenus dans la vaste
collection des auteurs byzantins; il y a joint tout ce que les écrivains
grecs et latins, les ouvrages des jurisconsultes et les chroniques du
moyen âge ont pu lui fournir; et il est résulté du tout, un corps
d'annales aussi complet qu'il était possible de le faire de son temps.
Si d'autres, comme Gibbon, par exemple, sont parvenus à donner à
leur récit une forme quelquefois plus agréable, ils n'ont aucun
avantage sur Lebeau pour la connaissance des sources originales;
ils n'eurent pas d'autres moyens à leur disposition: on doit donc leur
reprocher les mêmes défauts. Si Lebeau avait pu joindre à ses
autres connaissances celle des langues orientales, ou si un plus
grand nombre d'auteurs orientaux avaient été publiés à l'époque où
il écrivait, il aurait fait sans doute à son ouvrage des additions
considérables, et il lui aurait donné dans plusieurs parties un plus
haut degré de perfection.
Il a bien cherché, il est vrai, à profiter de quelques ouvrages
orientaux traduits en latin; mais comme il était dépourvu de notions
personnelles sur les langues et la littérature orientales, il n'a su
comment combiner les renseignements qu'il trouvait dans ces
ouvrages avec ceux qui sont consignés dans les auteurs byzantins.
Ces derniers écrivains sont pour la plupart assez obscurs dans leurs
narrations, et extrêmement concis sur ce qui concerne les relations
de leurs empereurs avec les princes de l'Asie. Ils défigurent
étrangement les noms d'hommes ou de lieux. Ils furent aussi
toujours très-mal instruits des révolutions arrivées chez les peuples
de l'Asie. Les confondant tous sous les noms de Sarrasins,
d'Ismaélites ou d'Agaréniens, ils attribuent souvent aux califes,
successeurs de Mahomet, ou aux musulmans de l'Asie, des faits
militaires ou politiques qui appartiennent aux souverains particuliers
de la Syrie, de l'Égypte, de l'Afrique, ou même de l'Espagne. Il devait
résulter, et il est résulté effectivement de toutes ces imperfections,
une multitude de petites erreurs de détail qui affectent sensiblement
l'ensemble de la narration, et donnent de fausses idées des choses.
Il est facile d'y remédier. La forme de rédaction qui a été adoptée par
Lebeau, et qui est peut-être la meilleure qu'on puisse suivre pour un
vaste corps d'annales, le soin qu'il a pris de raconter les événements
sans anticiper jamais sur l'ordre des temps, fournissent les moyens
d'améliorer sans peine son ouvrage. Il suffit de faire ce qu'il aurait
certainement fait lui-même s'il l'avait pu, c'est-à-dire qu'il faut
intercaler dans sa narration, selon leur ordre chronologique, les faits
et les indications nouvelles que fournissent les auteurs orientaux.
Quant à ceux des récits de cet historien qui seraient inexacts ou
susceptibles d'être considérablement augmentés, changés ou
modifiés, ils doivent être retranchés, ou soumis à une rédaction plus
conforme au résultat que présentent les ouvrages originaux. Partout
il faut rétablir les noms altérés, et joindre au texte les notes et les
éclaircissements nécessaires à l'instruction du lecteur.
Pour les temps qui précédèrent l'avénement d'Héraclius au trône
impérial, ces additions et ces rectifications ne sont pas à beaucoup
près aussi nombreuses que pour la relation des événements
postérieurs. Les auteurs arabes et persans nous apprennent peu de
choses de ces époques anciennes: heureusement les écrivains
arméniens suppléent à leur silence. Placés entre les deux grands
empires de Perse et de Constantinople, et compromis dans tous les
démêlés de ces puissances, ils connurent mieux la plupart des faits;
et leurs récits éclaircissent souvent les narrations imparfaites et
confuses des écrivains de Byzance, généralement mal informés de
l'histoire des Orientaux.
Ainsi, par exemple, deux siècles avant Héraclius, l'empire romain
reçut un accroissement de territoire dont on chercherait vainement
l'indication dans les auteurs que nous possédons. Le royaume
d'Arménie, qui, depuis quatre cents ans, était le rempart de l'empire
du côté de l'Orient, cessa d'exister par l'imprudente politique de
Théodose le Jeune, qui souscrivit avec le roi de Perse un traité de
partage, dont tout l'avantage fut pour les Persans. Ce grand
événement fut précédé et suivi de guerres et de révolutions qui nous
sont restées inconnues, mais qui doivent se retrouver dans une
histoire complète du Bas-Empire. C'est par le secours seul des
auteurs arméniens qu'il est possible de suppléer à cette lacune. Il
serait facile d'indiquer un grand nombre d'autres faits aussi
importants et également ignorés, mais qui se retrouveront dans cette
nouvelle édition.
Depuis l'époque d'Héraclius jusqu'à la destruction de l'empire, les
modifications qu'il faut apporter à l'ouvrage de Lebeau sont
continuelles. Dès lors, les empereurs furent toujours en relation avec
les puissances de l'Orient; et c'est justement au point le plus
intéressant de cette période, du VIIe au XIIe siècle, que les annales
byzantines présentent la plus grande disette d'écrivains. Il faut
nécessairement substituer les Arabes et les Arméniens, aux maigres
et ineptes annalistes que Lebeau a été obligé de consulter. Leurs
récits doivent donc trouver place dans cette édition. Les exploits des
conquérants arabes, qui chassèrent de l'Orient les successeurs
d'Héraclius; la formation d'une nouvelle monarchie arménienne; les
expéditions glorieuses entreprises par Théophile, Nicéphore Phocas
et Jean Zimiscès; les guerres opiniâtres que l'empire soutint contre
les Arabes, maîtres de la Sicile et de l'île de Crète; les règnes si
brillants et cependant si désastreux de Basile II et de Constantin
Monomaque: tous ces événements, dont il est facile d'apprécier
l'importance, sont à peine indiqués dans l'histoire de Lebeau. Les
renseignements que les auteurs arabes et arméniens fournissent
pour cette époque, augmenteront du double cette partie de l'histoire
du Bas-Empire. Après les croisades, on trouve les écrivains turcs qui
ont raconté les victoires de leurs souverains sur les derniers
successeurs de Constantin: les ouvrages qu'ils ont composés, et les
lettres originales des sultans othomans, dont il existe plusieurs
copies manuscrites dans nos bibliothèques, doivent être aussi
consultés, et ils fourniront des indications souvent plus exactes et
plus authentiques que les narrations passionnées des derniers
auteurs byzantins.
Il est hors de doute que, depuis le temps où Lebeau a écrit,
beaucoup de savantes recherches, et la publication de plusieurs
ouvrages estimables, nous ont mieux fait connaître l'histoire de
plusieurs états et de divers peuples de l'Europe qui eurent des
rapports avec l'empire de Constantinople. Le grand nombre de faits
qu'ils contiennent devront donc être ajoutés à l'histoire du Bas-
Empire, surtout pour ce qui concerne les relations des Grecs avec
les Russes, la république de Venise, et les princes croisés.
Ce court exposé suffira pour faire voir que ce n'est pas seulement
une nouvelle édition de l'Histoire du Bas-Empire par Lebeau que
nous annonçons, mais qu'il s'agit d'un ouvrage nouveau dont
l'importance ne saurait être contestée par aucune des personnes qui
s'intéressent au progrès des études historiques.
La géographie fut toujours la compagne inséparable de l'histoire.
Dans les ouvrages où les récits sont un peu détaillés, les lecteurs
aiment à pouvoir les suivre sur la carte: sans un tel secours, un livre
ne serait trop souvent qu'un amas de faits incohérents et
inintelligibles. C'est surtout pour l'histoire du Bas-Empire qu'on sent
à chaque instant le besoin d'un pareil secours. Pour l'histoire
ancienne de Rome on pourrait, à la rigueur, s'en passer; les recueils
de cartes, les traités de géographie, qui font connaître l'état du
monde ancien, sont assez nombreux et suffisamment exacts pour
qu'ils puissent suffire. Tout avait changé et changea plusieurs fois
pendant la longue période du Bas-Empire: les divisions
géographiques et politiques de l'antiquité furent détruites; les
dénominations classiques disparurent, et furent remplacées par des
noms barbares de toute espèce: aucun livre, aucune carte ne les
indique; cependant sans ces connaissances diverses l'histoire serait
un chaos inextricable, et on ne peut les acquérir que par un travail
considérable et très-pénible.
Il faut donc, pour compléter l'Histoire du Bas-Empire par Lebeau, y
joindre un certain nombre de cartes et de dissertations destinées à
faire connaître tous les changements survenus dans la géographie
et les divisions civiles, politiques, militaires, ecclésiastiques et
administratives de l'empire de Constantinople pendant toute sa
durée.
INDICATION DES CARTES.
1. Carte destinée à faire connaître l'empire d'Occident sous le règne
de Constantin. 2. Une autre pour l'empire d'Orient à la même
époque. 3. Une pour l'expédition de Julien contre les Perses. 4. Une
pour l'empire d'Occident après l'invasion des Barbares.
DEPUIS THÉODOSE JUSQU'A HÉRACLIUS:
5. Carte particulière de la Grèce. 6. Carte particulière de l'Italie. 7.
Illyrie et provinces sur le Danube jusqu'à la mer Noire. 8. Asie-
Mineure. 9. Syrie et provinces orientales. 10. Égypte. 11. Carte pour
l'expédition d'Héraclius en Perse.
Pour faire connaître les divisions militaires en usage au Xe siècle
dans l'empire de Constantinople, et les états qui étaient alors dans la
dépendance de cet empire, ou en relation avec lui, il faudra six
cartes particulières:
12. L'Italie et la Sicile. 13. La Grèce proprement dite. 14. L'Illyrie et
les rives du Danube. 15. L'Asie-Mineure. 16. L'Arménie et les
régions orientales. 17. La Syrie.
Pour bien comprendre la dernière période de l'Histoire du Bas-
Empire après la conquête de Constantinople par les Français, il faut
encore ajouter trois cartes à ce recueil:
18. L'Asie-Mineure au XIIIe siècle, après les conquêtes des Turcs
Seldjoukides. 19. La Grèce et la mer Egée, après l'établissement de
l'empire des Latins. 20. La Thrace, l'Illyrie, et les régions limitrophes
du Danube, pour les derniers temps de l'empire.
On joindra à ces cartes un plan de Constantinople telle qu'elle était
sous les empereurs.
Tous les passages intercalés dans la narration de Lebeau, ou
rajustés en note, seront placés entre crochets [ ] précédés d'un tiret,
et suivis de cette signature [S.-M.]
J. S.-M.
 ÉLOGE DE LEBEAU,
Par DUPUY,

SECRÉTAIRE DE L'ACADÉMIE DES

INSCRIPTIONS ET BELLES-LETTRES,
Prononcé le 11 novembre 1778 dans la séance publique de cette
Académie.
Charles Lebeau naquit à Paris, le 15 octobre 1701, de parents
honnêtes, mais peu favorisés de la fortune. Avec les qualités solides
et brillantes qui promettent les plus grands succès en divers genres,
la nature avait jeté dans son cœur le germe d'une passion pour les
lettres qui s'enflamma de bonne heure, et s'empara impérieusement
de toutes les facultés de son ame; mais d'abord, telle qu'un feu
enseveli sous la cendre, et comme captive au milieu d'une famille
chargée de l'éducation de cinq enfants, elle n'eut pas la facilité de se
faire jour et de s'élancer à son gré. Le foyer paternel lui paraissait un
lieu d'esclavage: il lui fallait un air libre, tranquille et serein, où,
maîtresse d'elle-même, elle pût prendre un essor qu'aucun obstacle
ne fût capable d'arrêter. L'atmosphère qui lui convenait, elle la trouva
dans le collége de Sainte-Barbe, célèbre par des phénomènes qui
attiraient les regards de la capitale et des provinces.
Dans un séjour si favorable à ses vues, Lebeau, respirant en liberté
et selon son goût, vit des maîtres zélés, vigilants, éclairés; des
disciples actifs, diligents, laborieux, toujours en haleine, toujours se
disputant à l'envi la gloire des succès. En fallait-il davantage pour
exciter chez lui une émulation dont il n'avait jamais encore senti si
puissamment l'aiguillon? Il se livre donc tout entier aux exercices
prescrits à son âge; et bientôt une application forte et constante le
rendant supérieur à tout ce qu'on exigeait de lui, ces exercices ne
suffisent plus ni à son activité, ni à ses désirs. Attristé de voir que le
travail commun et ordonné le laisse comme dans un état de
langueur et d'inaction, il se ménage secrètement une étude
particulière, et s'enfonçant dans la lecture des meilleurs écrivains
grecs et latins, il se nourrit en silence de leur suc le plus pur et le
plus substantiel.
Si la vigueur qu'il y puisa ne put rester long-temps inconnue, elle ne
se montra pas sans causer la plus grande surprise. On ne concevait
point que le temps assigné par la règle aux études et aux
occupations ordinaires eût pu permettre une si abondante récolte de
fruits de toute espèce, qui leur étaient comme étrangers. Ce n'était
point non plus le seul temps consacré aux devoirs de la journée, qu'il
avait mis à profit: loin d'en souffrir, tous ces devoirs avaient été
remplis avec la plus scrupuleuse exactitude; d'autres d'une espèce
différente ne l'avaient pas été de même. Une loi sage, nécessaire
même, autant pour la santé de la jeunesse, que pour la sûreté du
lieu, fixant les heures destinées au repos, marquait celle où partout
devait cesser la lumière. Aussi semblait-elle disparaître dans la
chambre de Lebeau, comme dans les autres; mais elle n'y était pour
ainsi dire, qu'éclipsée: cachée furtivement sous un vase pour
tromper, au mépris de la règle, la vigilance des maîtres, elle
reparaissait impunément à une heure indue, lorsque tout était
assoupi, pour éclairer les larcins que faisait au sommeil le jeune
téméraire; tandis que les Muses indulgentes, souriant à cette ruse
dangereuse, et secondant ses veilles, lui payaient amplement les
sacrifices faits en leur faveur. Une constitution vigoureuse, un
tempérament fort et robuste l'enhardissaient à les réitérer
fréquemment, et le garantissaient des suites funestes de cette
espèce d'intempérance.
Les richesses acquises par ce commerce nocturne avec les anciens,
ne pouvaient manquer de lui assurer une supériorité décidée sur
tous ses rivaux; mais cette supériorité ne fut jamais pour eux, ni un
principe de jalousie, ni un motif de haine. Il ne la leur faisait point
sentir: à peine s'en apercevait-il lui-même: ce fut au contraire un
nouveau lien pour s'attacher à lui, pour briguer son estime, et pour
lui vouer une amitié mêlée d'une sorte de respect, parce que leurs