preparation of compounds labeled with tritium and carbon...
TRANSCRIPT
Preparation of Compounds Labeledwith Tritium and Carbon-14
ROLF VOGES
J. RICHARD HEYS
THOMAS MOENIUS
Preparation of Compounds Labeledwith Tritium and Carbon-14
Preparation of Compounds Labeledwith Tritium and Carbon-14
ROLF VOGES
J. RICHARD HEYS
THOMAS MOENIUS
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Library of Congress Cataloging-in-Publication Data
Voges, R. (Rolf)Preparation of compounds labeled with tritium and carbon-14 / Rolf Voges,
J. Richard Heys, Thomas Moenius.p. cm.
Includes bibliographical references and index.ISBN 978-0-470-51607-21. Organic compounds–Synthesis. 2. Radiolabeling. 3. Tritium. 4. Carbon–Isotopes.I. Heys, J. R. (J. Richard) II. Moenius, Thomas. III. Title.QD262.V54 2009572’.36–dc22 2008046982
A catalogue record for this book is available from the British Library.
ISBN 9780470516072 (Hbk)
Typeset in 10/12pt Times by Thomson Digital, Noida, India.Printed and bound in Great Britain by CPI Antony Rowe, Chippenham, Wiltshire
Contents
Preface xi
Glossary xiii
Author Biographies xvii
1 Introduction 1
1.1 Physical Properties of Tritium and Carbon-14 3
1.2 Purification 5
1.3 Analysis 6
1.3.1 Chemical Identity 6
1.3.2 Chemical (and Enantiomeric) Purity 7
1.3.3 Radiochemical (and Radionuclidic) Purity 8
1.3.4 Specific Activity 9
1.3.5 Position of Label 10
1.4 Stability and Storage of Compounds Labeled with Tritium
or Carbon-14 11
1.5 Specialist Techniques and Equipment 15
References 21
2 Strategies for Target Preparation 25
2.1 Formulating Target Specifications 26
2.2 Planning Radiotracer Preparations 31
2.2.1 The Construction Strategy 31
2.2.2 Reconstitution Strategies 32
2.2.3 The Derivatization Strategy 34
2.2.4 Selection of an Appropriate Strategy 34
2.2.5 Case Studies of Strategy Development 36
References 44
3 Preparation of Tritium-Labeled Compounds by Isotope Exchange
Reactions 47
3.1 Homogeneous Acid- or Base-Catalyzed Exchange 49
3.1.1 Exchange without Added Acid or Base 49
3.1.2 Exchange under Acidic Conditions 51
3.1.3 Exchange under Basic Conditions 56
3.2 Heterogeneous Catalysis with Tritium in Solvent 60
3.2.1 Metals 61
3.2.2 Other Catalysts 65
3.3 Heterogeneous Catalysis in Solution with Tritium Gas 66
3.3.1 Metal Catalysts with Nonreducible Substrates
in Aqueous Solution 67
3.3.2 Metal Catalysts with Nonreducible Substrates in Organic Solvents 68
3.3.3 Other Catalysts 69
3.3.4 Metal Catalysts with Reducible Substrates 70
3.4 Homogeneous Catalysis in Solution with Tritiated Water 71
3.4.1 Exchange Catalyzed by Metal Salts 71
3.4.2 Exchange Catalyzed by Organoruthenium Complexes 73
3.4.3 Exchange Catalyzed by Iridium Dionates 74
3.4.4 Exchange Catalyzed by Iridium Cyclopentadienides 76
3.5 Homogeneous Catalysis with Tritium Gas 77
3.5.1 Iridium Phosphines 77
3.5.2 Iridium Dionate Complexes 90
3.5.3 Iridium Cyclopentadienide Complexes 91
3.6 Solvent-Free Catalytic Exchange 93
3.6.1 High-Temperature Solid-State Catalytic Isotope Exchange 93
3.6.2 Thermal Tritium Atom Bombardment 96
3.6.3 Other Radiation-Induced Labeling Methods 97
References 98
4 Preparation of Tritium-Labeled Compounds by Chemical Synthesis 109
4.1 Catalytic Tritiations 110
4.1.1 Tritiation of Carbon–Carbon Multiple Bonds 111
4.1.2 Tritiation of Carbon–Heteroatom Multiple Bonds 125
4.1.3 Homogeneously Catalyzed Reactions 126
4.2 Catalytic Tritiolyses 132
4.2.1 Tritiodehalogenations 133
4.2.2 Tritiolyses of Benzylic N- and O-Functions 144
4.2.3 Tritiodesulfurizations 145
4.3 Tritide Reductions 146
4.3.1 Sodium Borotritide (NaB3H4) 148
4.3.2 Sodium Cyanoborotritide (NaB3H3CN) 157
4.3.3 Sodium/Tetramethylammonium Triacetoxyborotritide
[Na/NMe4B3H(OAc)3] 159
4.3.4 Lithium Tritide (Li3H) 160
4.3.5 Lithium Borotritide (LiB3H4) 161
4.3.6 Lithium Triethylborotritide (LiEt3B3H, Li-Super-Tritide) 163
4.3.7 Lithium Tri-sec-Butylborotritide [Li(sec-Bu3)B3H,
Li T-Selectride] 165
4.3.8 Lithium [3H2]Boratabicyclo[3.3.1]nonane 166
vi Contents
4.3.9 Tritiated Borane (THF-Complex) (B23H6; B
3H3.THF) 167
4.3.10 Tritiated Alkylboranes 169
4.3.11 Lithium Aluminum Tritide (LiAl3H4) 170
4.3.12 Tri-n-Butyltin Tritide (n-Bu3Sn3H) 172
4.3.13 Tritiated Schwartz’s Reagent (ZrCp2Cl3H) 176
4.3.14 Tritiated Triethylsilane and Trihexylsilane 177
4.4 Small Tritiated Building Blocks 178
4.4.1 Tritiated Water (3H2O;3HHO) 179
4.4.2 Tritiated Diimide (3HN ¼ N3H) 182
4.4.3 Tritiated Methyl Iodide (C3H3I; C3HH2I) 183
4.4.4 Tritiated Diiodomethane (C3HHI2) 190
4.4.5 Tritiated Formaldehyde (3HCHO, 3HC3HO) 191
4.4.6 Dimethyl[3H]formamide (3HCONMe2), Acetic
[3H]Formic Anhydride (3HCOOCOMe) 192
4.4.7 Tritiated Diazomethane (C3HHN2) 193
4.4.8 N-Tritioacetoxyphthalimide 194
4.4.9 N-Succinimidyl [2,3-3H]Propionate ([3H]NSP) 195
References 195
5 Barium [14C]Carbonate and the Preparation of Carbon-14-Labeled
Compounds via One-Carbon Building Blocks of the [14C]CarbonDioxide Tree 211
5.1 [14C]Carbon Dioxide (14CO2) 212
5.1.1 [14C]Carboxylations of Organometallic Compounds 212
5.1.2 Manipulations of [14C]Carboxylation Products 218
5.1.3 N-[14C]Acyl Building Blocks 219
5.1.4 Preparation of Other Building Blocks from [14C]Carbon Dioxide 221
5.2 [14C]Carbon Monoxide (14CO) 222
5.2.1 [14C]Phosgene 229
5.3 [14C]Formic Acid (H14COOH) 233
5.4 [14C]Formaldehyde (H14CHO) 240
5.4.1 Carbanion-Mediated Hydroxy[14C]methylation
and [14C]Methylenenation 242
5.4.2 Acid-Catalyzed Hydroxy[14C]methylations 246
5.4.3 Amino[14C]methylations 248
5.4.4 Reductive Methylations 254
5.4.5 Polycondensations 255
5.4.6 Thio[14C]methylations 256
5.5 [14C]Methyl Iodide (14CH3I) 256
5.5.1 [14C]Methyl Iodide as an Electrophilic One-[14C]Carbon
Building Block 257
5.5.2 [14C]Methyl Iodide as a Source of Nucleophilic [14C]Methyl
and [14C]Methylene Building Blocks 262
5.5.3 Further Building Blocks Derived from [14C]Methyl Iodide 268
5.6 [14C]Nitromethane (14CH3NO2) 270
References 277
Contents vii
6 Preparation of Carbon-14-Labeled Compounds via Multi-Carbon
Building Blocks of the [14C]Carbon Dioxide Tree 287
6.1 [14C]Acetic Acid and Its Derivatives 287
6.1.1 [14C]Acetic Acid 287
6.1.2 [14C]Acetyl Chloride 289
6.1.3 [14C]Acetic Anhydride 298
6.1.4 [14C]Acetic Acid Alkyl/Aryl Esters 301
6.2 Halo[14C]acetates 307
6.2.1 Reaction at the Carboxyl Group 309
6.2.2 Reactions at the Methylene Group 312
6.2.3 Reactions at the Halogen Atom 314
6.3 [14C]Acetone 337
6.3.1 Reaction at the Carbonyl Group 338
6.3.2 Reaction at the Methyl Group 343
6.4 Alkyl [14C]Acetoacetate 346
6.4.1 Alkylation Reactions 348
6.4.2 Acylation Reactions 351
6.4.3 Aldol Reactions 352
6.4.4 Knoevenagel–Michael Reactions 353
6.4.5 Reactions at the Functional Groups 356
6.5 [14C]Malonates 357
6.5.1 Reactions at the Methylene Group 359
6.5.2 Reactions at the Carboxyl Functions 374
References 381
7 Preparation of Carbon-14-Labeled Compounds
via the [14C]Cyanide Tree 393
7.1 Metal [14C]Cyanides 393
7.1.1 Preparation 393
7.1.2 Introduction of [14C]Cyanide into Organic Substrates 394
7.1.3 Synthetic Transformations of Organic [14C]Nitriles 399
7.2 Preparation of Other Building Blocks from [14C]Cyanide 411
7.2.1 Trimethylsilyl[14C]cyanide (TMS14CN) 412
7.2.2 [14C]Cyanogen Bromide (Br14CN) 413
7.2.3 Alkali Metal [14C]Cyanates (M14CNO; M ¼ Na, K) 415
7.2.4 Alkali Metal Thio[14C]cyanate (M14CNS; M ¼ Na, K) 417
7.2.5 Triethyl [14C]Orthoformate [H14C(OEt)3] 419
7.2.6 [14C]Cyanoacetic Acid [14CNCH2COOH] 420
7.2.7 [14C]Diazomethane (14CH2N2) 431
References 433
8 Preparation of Carbon-14-Labeled Compounds
via the [14C2]Acetylene Tree 441
8.1 [14C2]Acetylene (H14C:14CH) 441
8.2 [14C2]Acetaldehyde (14CH314CHO) 445
8.3 [1,2-14C2]Acetic Acid (14CH314COOH) 446
viii Contents
8.4 2-[2,3-14C2]Propyne-1-ol ([2,3-14C2]Propargyl Alcohol)
and 2-[2,3-14C2]Butyne-1,4-diol 447
8.5 Methyl [2,3-14C2]Propiolate (H14C�14CCOOMe) and Dimethyl
[2,3-14C2]Acetylenedicarboxylate (HOOC14C�14CCOOH) 447
8.6 1,2-[14C2]Dibromoethane (Br14CH214CH2Br) 448
8.7 [14C2]Ethylene Oxide 448
8.8 [14Cn]Benzene and the Synthesis of Ring-Labeled Aromatic
Compounds 448
8.8.1 Nitrobenzene Branch 451
8.8.2 Phenol Branch 454
8.8.3 Bromobenzene Branch 456
8.8.4 Iodobenzene Branch 457
8.8.5 Benzoic Acid Branch 458
8.8.6 Alkyl Phenyl Ketone Branch 459
8.8.7 Sulfonylbenzene Branch 459
References 460
9 Preparation of Carbon-14-Labeled Compounds
via the [14C]Cyanamide Tree 465
9.1 [14C]Cyanamide (H2N14C�N) 465
9.2 [14C]Guanidine (H2N14C(¼NH)NH2) 467
9.3 [14C]Urea, H2N14CONH2 468
9.4 [14C]Thiourea, H2N14CSNH2 472
References 477
10 Reconstitution Strategies 479
10.1 Replacement Strategies 479
10.1.1 1H/3H Replacement Strategies 479
10.1.2 12C/14C Replacement Strategies 485
10.2 Disconnection–Reconnection Strategies 488
10.2.1 Dealkylation–Re[3H/14C]alkylation Procedures 488
10.2.2 CO2/14CO2 Replacement Strategies 492
10.2.3 CO/14CO Replacement Strategy 501
10.2.4 Oxidative Cleavage of C¼C Bonds in the Reconstitution
Approach 502
References 517
11 Preparation of Enantiomerically Pure Compounds Labeled
with Isotopes of Hydrogen and Carbon 523
11.1 Resolution of Racemates 524
11.2 Enantioselective Synthetic Methods 529
11.2.1 Hydrogenation/Tritiation of Labeled/Unlabeled �2,3-Amino
Acid Derivatives 530
11.2.2 Reduction of Labeled Prochiral Carbonyl Compounds
and Oximes 535
11.2.3 Enantioselective Oxidation of Olefins and Allylic Alcohols 541
Contents ix
11.3 Diastereoselective Synthetic Procedures 546
11.3.1 �-Alkylation of Chiral Imide Enolates 551
11.3.2 Aldol Reactions of Chiral Imides and Ester Enolates 558
11.3.3 1,4-Additions of Chiral Imide Enolates to Michael Acceptors 564
11.3.4 �-Amination of Chiral Imide Enolates 566
11.3.5 �-Hydroxylation of Chiral Imide Enolates 571
11.3.6 �-Alkylation of Chiral Glycinates 571
11.3.7 Aldol Reactions of Chiral Glycinates 583
11.3.8 Aldol Reactions of Chiral Glycolates 586
11.3.9 Aldol Reactions of Chiral Haloacetates 586
11.3.10 Reactions on Chiral �,�-Unsaturated Imides and Esters 591
References 596
12 Biotransformations in the Preparation of Compounds Labeled
with Carbon and Hydrogen Isotopes 607
12.1 Applications of Isolated Enzymes 608
12.1.1 Optical Resolutions via Derivatives 608
12.1.2 Synthesis of Isotopically Labeled, Enantiomerically
Pure Compounds 612
12.1.3 Conjugation Reactions 618
12.2 Application of Cell-Containing Systems 618
12.2.1 Transformations of Functional Groups 619
12.2.2 Fermentative Synthesis of Structurally Complex Molecules
by Incorporation of Labeled Precursors 621
12.2.3 Specific Requirements for Fermentations Using Isotopically
Labeled Compounds 623
12.3 Biocatalyzed Synthesis of Key Intermediates for Reconstitution
Approaches 630
12.3.1 Oxidation–Reduction Approach 631
12.3.2 Dealkylation–Realkylation Approach 632
References 634
Index 639
x Contents
Preface
The field of organic radiochemical synthesis, like any scientific discipline, grows over timewith the development of new knowledge, while remaining rooted in the old fromwhich it iscontinually nourished. Despite the recent appearance of substantive accounts of newer,related fields such as synthesis with short-lived isotopes, no comprehensive account of thesynthesis of tritium- and carbon-14-labeled compounds has appeared for a long time. Yettritium and carbon-14 isotopes remain the cornerstone isotopes for research in the lifesciences, broadly defined. Previous texts on synthesis with carbon-14 or tritium, or both,achieved their different goals well, but the newest of these is 25 years old. Many specialistreviews have been written since then, but by their nature they are narrowly focused or lackdepth and are therefore of limited utility.
Despite the lack of an up-to-date comprehensive text in this area, one may question thevalue a newbookmay have today, given the increasing extent towhich organic chemists relyon electronic databases for synthetic information. In the present case, there are at least twoaffirmative answers to such a question. First, it is intended that this book will be availablefrom the publisher electronically in several forms, some searchable. Second and moreimportant, in contrast to one-question-one-answer information sources, this book isintended to provide thorough discussions of strategies and methods, to show connectionsbetween different elements, exemplifying, comparing and contrasting them. Thereby, wehope to provide the reader with not only a source of information, but also opportunities toacquire deeper understanding, perspective and a stimulus to more creative thinking in thefield. This ambition is reflected in the inclusion of chapters devoted to some of theways thatthe generation of synthetic strategies for tritium and carbon-14-labeled compounds aredifferent from those of standard (nonisotopic) organic synthesis.
A second important organizing principle of this book is its focus on labeled reagents andbuilding blocks, in contrast to the more reaction-based orientation that characterizes mostsources of standard organic synthetic chemistry information (although reaction-basedinformation is thoroughly addressed here as well). Accordingly, this book is intended tocomplement, not to replace, these other resources. In standard organic synthesis, theconception and ‘desktop’ evaluation of candidate synthetic routes are carried out throughconsideration of possible retrosynthetic disconnections or forward synthetic transforma-tions relevant to the target molecule. Generally, the reagents and small building blocksrequired for these transformations tend to be regarded as incidental, because they areusually available from commercial sources at reasonable costs. However, in organic
radiochemical synthesis it is precisely the choice of the most appropriate isotopicallylabeled reagent or small building block that is critical to optimal route planning. Therefore,it is through consideration of both the available nonisotopic reactions and routes, and theavailable isotopic reagents and building blocks and their reactions, that good radiochemicalsynthetic planning can be accomplished.
Many times throughout the book, reaction conditions described in the original publica-tions arementioned either in the text or beneath the reaction schemes.We have included thisinformation to assist the reader’s understanding of the practical dimensions of thechemistry, and as an aid to comparison of different reactions. This information shouldnot be used as directions for actual work in the laboratory. Instead, the reader shouldrefer to the original cited literature for the relevant details.
Writing this book has only been possible because of the efforts of the many, manycolleagueswho devised, carried out and reported on the chemistry of which this text is built.We thank them. The authors are further indebted to many colleagues for their kindness inanswering questions and offering suggestions and advice.Wewish to give special thanks tothosewho generously devoted their time and expertise to review parts of themanuscript andoffer helpful comments and corrections. They include Hendrik Andres, A. Jon Bloom, KarlCable, Grazyna Ciszewska, Chad Elmore, Crist Filer, Albrecht Gl€anzel, Thomas Hartung,John Herbert, Barry Kent and his colleagues at GE Healthcare, Scott Landvatter, BillLockley, Franz Maier, Ulrich Pleiss, Tapan Ray, Ines Rodriguez, Sebastien Roy, RhysSalter, Piet Swart, Alain Schweitzer, Bill Wheeler, Chris Willis and Markus Zollinger.Finally, our deepest gratitude goes individually to Anna, Elisabeth, Ulrike, Hanna andMoritz who have been generous, loving, accommodating, and as patient as reasonablepeople can be, during our work on this book.
xii Preface
Glossary
ADME absorption, distribution, metabolism, excretion
AFA acetic formic anhydride
AIBN �,�0-azo-bis(isobutyronitrile); �,�0-azobis (2-methyl-propionitrile)
Alpine-Borane lithium B-isopinocampheyl-9-borabicyclo[3.3.1]nonane
BABS N-bromoacetylbornane-2,10-sultam
9-BBN 9-borabicyclo[3.3.1]nonane
BINAP 2,20-bis-(diphenylphosphino)-1,10-binaphthylBn benzyl
Boc tert-butyloxycarbonyl
Boc-BMI 1-(tert-butoxycarbonyl)-2-tert-butyl-3-methyl-4-imidazolidinone
BOP-Cl bis(2-oxo-3-oxzolidinyl)phosphinic chloride
BSA bis(trimethylsilyl)acetamide
Burgess reagent (methoxycarbonylsulfamoyl)triethylammonium hydroxide
Bz benzoyl
CBS catalysts substituted 1,3,2-oxazaborolidine (Corey-Bakshi-Shibata) catalysts
CCD charge-coupled device
CDI 1,10-cabonyldiimidazole
CDT 1,20-carbonyldi-1,2,4-triazoleCi Curie; 1 Ci¼ 3.7x1010 Bq
CNC 1-chloro-1-nitrosocyclohexane
COD, cod 1,5-cyclooctadiene
Cp* pentamethylcyclopentadiene
Cy, cy cyclohexyl
CyTMG N-cyclohexyl-N0, N0, N00, N00-tetramethyl guanidine
d.e. diastereomeric excess
d.p. diastereomeric purity
DABCO 1,4-diazabicyclo[2.2.2]octane
DAST (diethylamino)sulfur trifluoride
Davies reagent 10-camphorsulfonyloxaziridine
dba dibenzylideneacetone
DBAC di-tert-butyl azodicarboxylate
DBH 1,3-dibromo-5,5-dimethylhydantoin
DBN 1,5-diazabicyclo[4,3,0]non-5-ene
DBU 1,8-diazabicyclo[5,4,0]undec-7-ene
DCC, DCCI 1,3-dicyclohexylcarbodiimide
DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DEAD diethyl azodicarboxylate
(þ)-or (�)-DET diethyl (L)- or (D)-tartrate
DIAD diisopropyl azodicarboxylate
DIBAL diisobutylaluminum hydride
DIEA diisopropylethylamine
DIH 1,3-diiodo-5,5-dimethylhydantoin
DIP-Cl B-chlorodiisopinocampheylborane
DMA, DMAC N,N-dimethylacetamide
DMAP 4-(dimethylamino)pyridine
DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
DMS dimethylsulfide
DPMGBS N-(N0-diphenylmethylideneglycyl)bornane-10,2-sultam
DPPA diphenylphosphoryl azide
dppe 1,2-bis(diphenylphosphino)ethane
dppf 1,10-bis(diphenylphosphino)ferroceneEC Enzyme Commission (enzyme nomenclature)
e.e. enantiomeric excess
e.p. enantiomeric purity
EDC, EDCI 1-[3-(dimethylamino]propyl]-3-ethylcarbodiimide
Et-DUPHOS 1,2-bis(2,5-diethylphosphino)ethane
Evans reagent 4-substituted-1,3-oxazolidin-2-one
galvinoxyl [2,6-di-tert-butyl-�-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)p-tolyloxy
GPE N-glycylpseudoephedrine
hfpd 1,1,1,5,5,5-hexafluoropentane-2,4-dionate
HMDS hexamethyldisilazane
HMPA hexamethylphosphoamide
HOBt 1-hydroxybenztriazole
homochiral enantiomerically pure, enantiopure
HOSu N-hydroxysuccinimide
HSCIE high-temperature solid-state catalytic isotope exchange
HYTRA 2-hydroxy-1,2,2-triphenylacetate
Ipc2BCl B-chlorodiisopinocampheylborane (DIP-Cl)
isotopomer the isotopically labeled version of a compound; one of a set of
compounds differing from one another only in the number
and/or distribution of isotopic atoms
KIE kinetic isotope effect
Lawesson’s reagent 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-
disulfide
Lindlar catalyst 5% palladium on calcium carbonate poisoned with lead
MCPBA 3-chloroperbenzoic acid
MOM methoxymethyl
MPEG mono-methyl polyethylene glycol
xiv Glossary
MsCl methanesulfonyl chloride
NBS N-bromosuccinimide
NCS N-chlorosuccinimide
NMM 4-methylmorpholine
NMO 4-methylmorpholine N-oxide
NMP 1-methyl-2-pyrrolidinone
Oppolzer reagent (2R)- or (2S)-bornane-10,2-sultam
Pa, kPa pascal, kilopascal; 1 atm¼ 101,325 Pa
PABS N-phosphonoacetylbornane-2,10-sultam
PCy3 tricyclohexylphosphine
Periodinane 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one
(Dess-Martin reagent)
PET positron emission tomography
PPTS pyridinium p-toluenesulfonate
proton sponge N,N,N0,N0-tetramethyl-1,8-naphthalenediamine
py pyridine
QWBA quantitative whole body autoradiography
RAMP (R)-1-amino-2-(methoxymethyl)pyrrolidine
salen N,N0-bis(3,5-di-tert-butylsalicylidine)-1,2-cyclohexanediamine
SAMP (S)-1-amino-2-(methoxymethyl)pyrrolidine
Seebach reagent 1-(tert-butoxycarbonyl)-3-methyl-4-imidazolidinone
TBAF tetrabutylammonium fluoride
TBDMS tert-butyldimethylsilyl
TBDPS tert-butyldiphenylsilyl
TBTU O-benzotriazol-1-yl-N,N,N0,N0-bis(tetramethylene) uronium salt
TFA trifluoroacetic acid
TFAA trifluoroacetic anhydride
TIPS triisopropylsilyl
TMEDA N,N,N0N0-tetramethylethylenediamine
TMS trimethylsilyl
TPAP tetrapropylammonium perruthenate
Trioxane c-(HCHO)3trisyl 2,4,6-triisopropylbenzenesulforyl
triton B benzyltrimethylammonium hydroxide
UDP uridine 50-diphosphateXc chiral auxiliary
VOC vinyloxycarbonyl
Z benzyloxycarbonyl
Isotopic designators in chemical names: Naming conventions generally follow IUPAC rules
(Pure and Applied Chemistry, 1979, 51, 353–380; www.iupac.org/publications/pac/)
(‘‘brackets preceding name of labeled unit’’)
Isotopic designators in chemical formulas: These generally follow common usage,
which is to place a superscript number before the atom to indicate the mass of the isotope
that replaces the natural isotope at that position, regardless whether it replaces the natural
isotope in all molecules (isotopic substitution) or only some molecules (isotope labeling).
Glossary xv
Isotopic designators in chemical structures:
* preferred symbol to indicate that site is labeled or substituted with
tritium or carbon-14 (3H may be used instead to indicate
stereochemistry)o indicated site is labeled or substituted with deuterium, carbon-11
or carbon-13 (2H may be used instead to indicate
stereochemistry)� #^ indicated site is an alternative/optional/conditional position for a
label (identity of isotope will be noted in Figure)( ) when used with an isotopic designator, indicates minor isotopic
content
Examples of the use of isotopic names, designators and symbols:
[14C]Methyl cyanoacetate N:CCH2COO14CH3
OCH3
ON *
Methyl [14C]cyanoacetate N:14CCH2COOCH3
OCH3
ON *
Methyl cyano[1-13C]acetate N:CCH213COOCH3
OCH3
ON
o
Methyl cyano[2-13C]acetate N:C13CH2COOH3OCH3
ON
o
Sodium [3H]borohydride NaB3H4 (regardless of specific activity)
CH3
CH3
N N
CH3 CH3CH3 CH3
CH3
CH3
O
OH
O
OH
H3
H3
3H2, catalyst
3H2, catalyst
3H2, catalyst
2H2, catalyst
* *
* *
*
o
o
cis-[3,4-3H]hex-3-ene
2-[2,6-2H]phenylpyridine
[1 ,2 -3H]testosterone
* = positions labeled in reduction without isotope scrambling(2-methyl-1-phenyl[1,2-3H]propane)
= positions labeled in reduction with isotope scrambling(2-[3H]methyl-1-phenyl[1,2,3- 3H]propane)
xvi Glossary
Author Biographies
Rolf Voges studied chemistry at the Universities of Marburg and Freiburg, where he received
his Ph.D. in organic chemistry on investigations into steric isotope effects, for which he received
the Godeke Award. After postdoctoral research on stereoselective syntheses he joined the
isotope group of Sandoz Pharma AG (now Novartis AG) being involved for thirty years in
organic radiochemical synthesis as Head of the Isotope Laboratories and then Head of Isotope
Section. He is author or coauthor of about 40 publications, one patent, and coeditor of two
previous conference proceeding volumes in the field, co-organizer of two international symposia
on the synthesis and application of isotopically labeled compounds, founder and co-organizer of
eleven Bad Soden meetings of the Central European Division of the International Isotope
Society (CED-IIS), and co-editor of the proceedings. For three years he held a leadership
position in the IIS, serving as its 2001 president. In recognition of his scientific achievements and
his service to the isotope society he received the IIS-CED Award in 1995 and in 2003 the IIS-
Award. He is now retired and lives in southwestern Germany, near the Swiss and French borders.
Contact: Oberer Birkenweg 18, 79540 Lorrach, Germany; [email protected]
Richard Heys received his Ph.D. in organic chemistry from Stanford University in 1976
and conducted postdoctoral research in the chemistry department at Yale; both involved the
synthesis of radiolabeled compounds and their use in elucidation of biosynthetic pathways.
His subsequent 29-year career in organic radiochemical synthesis both in the laboratory and
as a manager took him to the Radiochemistry Department of Midwest Research Institute
(now part of Aptuit, Inc.), Smith Kline & French Laboratories/SmithKline Beecham
Pharmaceuticals (now GlaxoSmithKline) and AstraZeneca Pharmaceuticals. Author or
coauthor of over 85 publications, 8 patents and a previous conference proceedings volume
in the field, organizer of an international symposium on the synthesis of isotopically labeled
compound and holder of leadership positions (including president and CFO) in the
International Isotope Society for 9 years, he is retired and lives in northwestern Connecticut.
Contact: P.O. Box 576, Litchfield, CT 06759; [email protected]
Thomas Moenius received his Ph.D. in organic chemistry from the University of
Erlangen-Nurnberg in 1986. He is a member of the isotope group of Novartis Pharma
AG, working in the field of carbon-14 and tritium labeling. Since 2007 he is also
European Editor for Journal for Labelled Compounds and Radiopharmaceuticals.
Contact: Novartis Pharma AG, WSJ.507.951, Novartis Campus CH-4056, Basel,
Switzerland; [email protected]
1
Introduction
Compounds labeled with carbon-14 or tritium have for decades been used in a vast numberand wide range of applications, especially in the life sciences1,2, including research anddevelopment of human and animal pharmaceuticals and crop protection agents. Notwith-standing new technological developments, and in some cases because of them, the value ofthese isotopes in various research areas continues to be great.
In studies of the interactions of small molecules (both synthetic and natural) withreceptors, enzymes and other complex biological molecules and systems, compoundslabeled with tritium are indispensable because they can be detected and quantified at thenanomolar level of concentration. The tritium label facilitates measurements of the affinityof labeled ligands to their cognate receptors, the densities of receptors in tissue preparations,and the development of high-throughput assays for assessment of the interactions of testcompounds with receptors. Analogously, the activity of enzymes can be studied, andpotential nonnative substrates screened, by use of a test substrate labeled in such away as tosignal (e.g., by release of radioactivity or change in chromatographic mobility of thesubstrate) the chemical transformation catalyzed by the enzyme.
Similarly, carbon-14-labeled compounds have no equal for assessment of their metabo-lism in vitro (such as with hepatocytes, cytochrome P450 subtypes or other enzyme orsubcellular tissue preparations), or for in vivo characterization of their absorption, distri-bution,metabolism and excretion (ADME) in animals and humans3, as they can be detectedby several different methods and accurately quantified in complex biological matrices. Oneof the newer of these methods is accelerator mass spectrometry (AMS)4, whose exquisitesensitivity allows the use of far smaller quantities of carbon-14 than standard ADMEstudies, therefore providing increased safety margins with regard to radiation exposure tohuman volunteers.
Compounds labeled with isotopes such as carbon-14 or tritium have also contributed tonumerous advances in studies of biochemistry5, biosynthetic pathways6, enzyme mechan-isms7, elucidation of organic reaction mechanisms8 and environmental sciences.
Preparation of Compounds Labeled with Tritium and Carbon-14 Rolf Voges, J. Richard Heys and Thomas Moenius� 2009 John Wiley & Sons, Ltd
Clearly, the value of carbon-14 and tritium isotopes in research is dependent upon theirincorporation into compounds of interest. This ismade possible by the availability of awidevariety of preparative methods capable of furnishing study compounds possessing thedesired isotope(s) in specific locationswithin the chemical structure and in suitable levels ofenrichment.
The successful practitioner in standard (nonisotopic) synthetic organic chemistry needsto possess a broad knowledge of reactions and reagents, the ability to plan a practicablesequence of reactions starting from readily available starting materials and ending with thesynthetic target, a facility in executing chemical laboratory operations efficiently andsafely, and aworking knowledge of analyticalmethods sufficient to ensure that the progressof a synthesis can be adequately assessed and to obtain information helpful in improvingreaction parameters.
The synthesis of compounds labeled with isotopes requires the synthetic chemist to haveadditional expertise because the synthetic target must be assembled so as to contain one ormore isotopic atoms. The preparation of compounds labeled with carbon-14 and tritiumrequires the ability to deal with a far smaller selection of starting materials comparedwith standard synthetic chemistry, the ability to plan reaction sequences that generate thecorrect chemical structures required isotopes in the appropriate positions, knowledge of thecircumstances under which these isotopes’ b� emissions may lend additional instability tocompounds and of ways to avoid or mitigate these effects. Moreover, work with tritiumrequires mindfulness of how this isotope’s vulnerability to loss by exchange processes canbe affected by its position in the chemical structure and by the conditions to which thecompound is subjected. These are properties relevant not only to predictions about thestability and utility of the tritiated products, but also to the practicability of preparing themby tritium-for-hydrogen exchange when it would be advantageous to do so. Lastly, the factthat carbon-14 and tritium are unstable nuclei means that the practitioner must be welltrained and familiar with the proper handling of radioactive materials.
Given all this, it is fair to say that organic synthesis with isotopes is a demandingspecialty field within organic synthesis. This book is intended to be both a learning tool forscientists new to the field, and a continuing resource for radiochemical synthesis chemiststhroughout their careers. It is assumed that the reader has, at a minimum, a practicalknowledge of synthetic organic chemistry and a good working knowledge of the chemistrylaboratory.
The authors emphasize the importance of safe working practices and expect that readersmake themselves familiar with, and take care to work at all times in accordance with, theirnational, local and institutional radiation safety protocols regarding carbon-14 and tritium,to maintain good practices of contamination monitoring, and are competent in the controland remediation of radioactive contamination. Some general guidelines have beenpublished9.
The organization of this book is as follows. The remainder of this chapter provides shortaccounts of purification, analysis and storage and stability of compounds labeled withcarbon-14 and tritium, and descriptions of some common techniques and technologiesunique to work with these isotopes. Chapter 2 discusses some strategies particularlyappropriate for planning syntheses of compounds labeled with carbon-14 and tritium, anappropriate topic for inclusion because there are distinct differences vis-�a-vis the waysnonisotopic synthetic problems are approached, and an appreciation of these differences is
2 Preparation of Compounds Labeled with Tritium and Carbon-14
key to effective work in the field. The discussion of one strategy unique to organicradiochemical synthesis, reconstitution, is considered worthy of its own chapter, and iselaborated in Chapter 10.
The main parts of the book are devoted to presentations and critical discussion of the useof building blocks, reactions and reagents. These sections are arranged in ways appropriatefor each isotope: preparation of tritium-labeled compounds is in large part organized bymethodological approach, while preparation of carbon-14-labeled compounds is organizedby the various isotopically labeled building blocks. Though most labeling reactions withtritium involve incorporation of the isotope from tritium gas or tritiated water sourcesinto the intact carbon frameworks of final products or late stage synthetic intermediates,sometimes the use of tritiated building blocks is more appropriate. The aim of planning istherefore to identify appropriate substrates andmethods for introducing the label. Chapter 3discusses methods of exchange labeling with tritium gas or tritiated water, and Chapter 4presents methods of synthesis utilizing tritiated reagents and the relatively small number ofreadily available tritiated building blocks.
On the other hand, the preparation of compounds labeled with carbon-14 usuallyinvolves some amount of carbon framework construction, and a number of carbon-14-labeled building blocks are available for this purpose. Therefore the planning process forsyntheses of carbon-14-labeled compounds involves evaluation of synthetic pathways andselection of building blocks, including one or more containing the carbon-14 label.Chapters 5–9 present the most frequently used carbon-14-labeled building blocks anddiscuss their use.
Finally, two chapters cover, inmethodologically oriented fashion, the chemical synthesisof enantiomerically pure 3H- and 14C-labeled compounds (Chapter 11) and biologicalmethods of preparation (Chapter 12).
This book is intended to be useful for the researcher in any of several ways. It can be usedas a text, which by study in the entirety can bring a newcomer in this field up to a reasonablelevel of competence. It can be used by scientists faced with specific labeling tasks as asource of reference for comprehensive and critical information on the utility of particularmethods, reagents and building blocks. And finally it is hoped that scientists working in thefield will find that browsing the book will stimulate new ideas for labeling, providereminders of methods that can be productively employed in future projects, or sparkcreative thinking for problem solving in the field.
At times throughout the book the authors have included examples using deuterium orcarbon-13 (or even carbon-11) when, in their opinion, the methods are likely to beapplicable to tritium and carbon-14, respectively. In particular we note the growingimportance of synthesis of carbon-11-labeled compounds as the utility of positron emissiontomography grows rapidly10; however, synthesis with short-lived isotopes such as carbon-11 and fluorine-18 is a subfield in itself and is covered elsewhere11.
1.1 Physical Properties of Tritium and Carbon-14
The properties of tritium and carbon-14 are well suited for use as tracers in many lifesciences and chemistry applications. Table 1.1 lists the important physical properties of theisotopes.
Introduction 3
Tritium is prepared in a 6Li(n,a)3H-reaction by irradiation of appropriate lithium-6-enriched compounds (e.g. LiF) or alloys (Li–Al, Li–Mg) with a high flux of neutrons in anuclear reactor. Some of the tritium evolves as 3H2 gas from the target through recoil duringthe generation process, and the rest is retained in the solid from which it is liberated bychemical methods.
Carbon-14 isproduced ina14N(n,p)14C reaction, also inanuclear reactor,by irradiationofsolid beryllium or aluminum nitride or a saturated solution of ammonium nitrate for periodsranging from1to3years.Afterwards the target isdissolved inhalf-concentratedsulfuric acidand the effluent gases are oxidized over an appropriate catalyst. [14C]Carbon dioxideresulting from this procedure is absorbed by an aqueous solution of sodium hydroxide andBa14CO3 isprecipitatedbyadditionofaqueousbariumhydroxide.Barium[14C]carbonate isthe standard chemical formfor storageandcommerce, and it is theuniversal startingmaterialfromwhich all other carbon-14-labeled compounds are prepared. Because of the omnipres-ence of environmental carbon, the isotopic purity of Ba14CO3 is normally in the range of80–90%, corresponding to specific activities of 50–56mCi/mmol. Material of higherspecific activities up to 62mCi/mmol is commercially available, but it is considerablymoreexpensive and only needed in exceptional cases.
These isotopes emit low-energy b� particle (electron) radiation that does not requireshielding for worker safety, as the radiation cannot penetrate the skin. Only with largeamounts of carbon-14 can detectable secondary X-radiation occur. This radiation, Brems-strahlung12, is produced when electrons are decelerated in the Coulomb fields of atomicnuclei. As the energy of Bremsstrahlung is proportional to both the energy of the electronand the atomic number of the matter through which it passes, it is very low for carbon-14used or stored in normal laboratory vessels. Routine precautions must be taken, however, toavoid internal exposure to these isotopes through ingestion, inhalation, contact with openwounds, or topical contact with compounds that may be absorbed transdermally. This iseasily accomplished by working in fume hoods or glove boxes when there is any possibilityof airborne radioactivity, by wearing suitable gloves at all times and by refraining fromeating, drinking or smoking in the laboratory. Monitoring of laboratory spaces, equipmentand personnel for contamination is easily accomplished in the case of carbon-14 using thin-window Geiger counters; analogous monitoring for tritium can only be accomplished byusing windowless gas proportional counting devices. Usually the most expedient methodfor monitoring of surfaces for removable tritium or carbon-14 contamination is by wiping
Table 1.1 Physical properties of tritium and carbon-14
Tritium Carbon-14
Half-life 12.3 years 5730 yearsSpecific activity 29.2Ci/milliatom 62.4mCi/milliatomMaximum energy of radiation (b�) 18.6 keV 156 keVMean energy of radiation 5.7 keV 56 keVDecay product 3Heþ (stable) 14Nþ (stable)
Maximum penetration of radiationAir ca 6mm ca 20 cmWater ca 6mm ca 250mmGlass/concrete ca 2mm ca 170mm
4 Preparation of Compounds Labeled with Tritium and Carbon-14
the surface with a moist cotton swab or filter paper disk and measuring the radioactivity onthe wiper material by liquid scintillation counting. Internal exposure of personnel is mosteasily monitored by regular urine radioanalysis.
As carbon and hydrogen are fundamental components of every organic compound, theycan be replaced with carbon-14 and tritium without changing compounds’ chemicalmakeup. Therefore, the chemical and physical properties of compounds labeled withtritium or carbon-14 are very similar to those of their unlabeled counterparts.Metabolically,they behave the same with one exception: if a metabolic transformation involves oxidationat a carbon atomwhose hydrogen has been replaced by tritium, thatmetabolic pathwaymaybe slowed because of the greater energy that is required to break a carbon–tritium bondcompared with a carbon–hydrogen bond (primary isotope effect), and in rare cases this cancause significant alterations in the ratio of two or more different metabolites (‘metabolicswitching�13, see also Chapter 2). Also, in rare cases the small differences in polarity and/orpKa caused by tritium isotopic substitution can become apparent during the use ofespecially sensitive separation methods, such as high performance liquid chromatography(HPLC), in which the retention times of labeled and unlabeled congeners may be different(see Section 1.3.1 below) (secondary isotope effect). Because of the small difference inmass between carbon-14 and carbon-12 these effects are very small, and in life sciencesexperiments they can usually be neglected.
The long half-lives of these isotopes rarelymake it necessary to correct for natural decay;an exception is the long-term ( > 1 year) storage of tritium-labeled compounds.
The range of specific activities available in compounds labeled with these isotopes issuitable for tracer applications extending from mass balance studies in drug metabolismresearch to detailed investigations of the interactions of small and medium-sizedmolecules with biological macromolecules such as receptors and enzymes, and fromtracing of biosynthetic pathways to the elucidation of chemical reaction mechanisms.Both isotopes can be detected with high sensitivity by a variety of instrumentsreadily available in life science laboratories (liquid scintillation counters, 3H nuclearmagnetic resonance (NMR) instruments, mass spectrometers, phosphorimagers14),allowing discrimination and measurement of labeled compounds in complex biologicalsamples.
1.2 Purification
The methods suitable for purifying compounds labeled with tritium or carbon-14 arefundamentally the same as those for similar nonlabeled compounds on the samemass scale,which is typically in the tens to hundreds of milligrams for carbon-14-labeled compounds,and micrograms to a few milligrams for tritiated compounds. Books such as MicroscaleManipulations in Chemistry title as appropriate15 are useful guides to techniques anddevices for manipulating small quantities of compounds. Besides small mass scales, themost important source of constraint on laboratory methods is the need to control thematerial so as to minimize exposure of the worker and contamination of the laboratory.Approaches to purifying any material should take into consideration the possibility ofradiation damage to compounds, which can produce impurities different from thoseencountered in a corresponding unlabeled compound.
Introduction 5
Chromatographic methods are by far the most useful ones for the purification ofcompounds labeled with carbon-14 and tritium. There is a variety of methods havingmedium to high resolving power, the most common of which are flash chromatography16
and its automated cousins, closed columnmethods such asHPLCormediumpressure liquidchromatography (MPLC), the more recently emerging supercritical fluid chromatography(SFC), and the more classical planar techniques of preparative radial flow chromatographyand thin layer chromatography (TLC). The choice of method depends on the equipmentavailable, the mass scale, and the ease of separation of the impurities17.
Another common purification method is recrystallization. It is operationally simpleand can be done on quantities down to the milligram scale using conventional micro-scale techniques and apparatus. It is relatively easy to conduct the requiredmanipulations soas to avoid the inadvertent dispersal of particulates. Compounds sensitive to radiation-generated oxygen radicals in solution can be protected by working under an inert gasatmosphere18.
1.3 Analysis
Analytical characterization of tritium and carbon-14-labeled compounds used in lifesciences usually includes the following aims:
(a) To provide evidence of chemical identity;(b) To measure chemical, and if appropriate enantiomeric, purity;(c) To measure radiochemical purity (and, rarely, radionuclidic purity);(d) To determine the specific activity;(e) To determine or confirm the site(s) of labeling within the molecule.
The analysesmost pertinent to each compound are determined by its intended use and themethod of its synthesis. The specifications or acceptable numerical limits will depend uponthe intended use and the requirements of applicable local procedures, institutional standardsor government regulations. The level of detail with which analytical procedures areprescribed, the skill and care with which they are conducted and the quality of datainterpretation all vary significantly, according to the expertise of the analyst and localstandards of practice. Such differences can be expected to result in correspondingly higheror lower risk to the success of the studies in which the compounds are used. The goal for allanalytical measurements should be to minimize subjectivity.
The standards for thoroughness of analysis, degree of procedural rigor, etc. are generallyflexible for compounds used in research and early drug discovery studies (where the settingof specifications may be relatively informal), somewhat more formal for studies such asADME in animals (where some institutional specifications or standards of practice usuallyexist). The standards are highest for compounds intended for human radiolabel studies(where extensive and detailed prescriptions must be adhered to and formal oversight ofprocedures and independent review of written records and data are common).
1.3.1 Chemical Identity
Analyses pertaining to chemical identity are intended to provide evidence that the structureand, if appropriate, stereochemistry, of a compound are in accordance with that claimed.
6 Preparation of Compounds Labeled with Tritium and Carbon-14
NMR is a useful analytical method because it can provide quite detailed information aboutchemical structure; current widely available instruments are sensitive enough so that1H NMR analysis is feasible for all carbon-14-labeled compounds and all tritium-labeledcompounds except the cases where very limited quantities of high specific activity samplesare available. 13C NMR should also be routinely run when possible, especially for carbon-14-labeled compounds (see below). 2D-NMR methods, which are within reach of mostlaboratories, are very powerful for assessment of structural details, and should be con-sidered whenever 1D methods leave ambiguity. Mass spectrometric analysis is universallyrecommended: it can provide not only confirmation of molecular weight, but also datasuitable for calculation of specific activity (see Section 1.3.4 below). Classical methodsshould not be discounted; for example, infrared analysis provides a detailed fingerprintwhen a reference standard is available for comparison. The potential for isotope-inducedchanges in NMR and IR spectra should also be recognized, but these changes arequantitatively predictable and need not detract from the quality of the analysis.
Matching of chromatographic retention times is an unreliable indicator of chemicalidentity, for two reasons. Firstly, it is not uncommon for closely related compounds to haveindistinguishable retention characteristics, even in chromatographic systems of highresolving power. Neither is it uncommon that a byproduct or analog of the intendedsynthesis product is closely related to it, and therefore to the reference standard. Secondly,the higher the resolving power of the method, the more likely it is that the presence ofisotopes can alter the retention characteristics of a compound, causing the labeledcompound and its unlabeled reference standard to appear chromatographically nonidenti-cal. This phenomenon, called isotopic fractionation19 has been recognized for decades andhas more recently been the subject of a review20. Furthermore, the pKas of amines may bealtered by substitution of tritium (or deuterium) for hydrogen on adjacent carbon atoms,resulting in a significant change in retention time if the HPLCmobile phase has a pH valuenear the pKa of the amine21.
1.3.2 Chemical (and Enantiomeric) Purity
Chemical purity is defined as the weight of the compound of interest contained in a sampleof given weight, usually expressed in percent. For radiolabeled compounds, it is usuallycalculated arithmetically from measurements of the respective HPLC/UV peak areasobtained after multiple injections of known weights of a sample and of a referencestandard of known chemical purity. In the absence of a suitable reference standard, it isonly possible to establish a chemical purity relative to an available sample of the authenticcompound. The ratio of the HPLC/UV peak area of the analyte to the total area of all peaksin an HPLC chromatogram is most definitely not a measure of the chemical purity, becausesuch a measurement fails to take into account either the absorbance characteristics of theobserved impurities or any impurities that do not absorb light at thewavelength of detection,nor do they account for any impurities not eluting from the column, or any solvents orinorganic salts that may be present in the sample.
Chemical purity measurements are often not performed on tritiated compounds of highspecific activity. Such materials are usually prepared in submilligram quantities, and, evenif it were feasible to weigh samples accurately enough to prepare solutions of known massconcentration, the required manipulations would increase the risk of decomposition of thecompound (see Section 1.5). Fortunately, high specific activity tritiated compounds are
Introduction 7
used in such small (mass) quantities that unlabeled impurities are unlikely to causeproblems. The absence of major impurities can be assessed to a modest degree of certaintyby HPLC analysis with UV detection.
Measurements of enantiomeric purity are most conveniently accomplished using chiralHPLC analysis against authentic samples of enantiomerically pure and racemic materials.Alternative methods include NMR with a chiral shift reagent and optical rotationmeasurements.
1.3.3 Radiochemical (and Radionuclidic) Purity
Radiochemical purity, in analogy to chemical purity, is the ratio of radioactivity containedin the compound of interest to the total radioactivity of the sample. Radiochemical purity isusually measured chromatographically in order to exploit its separation power, and HPLCwith online radioactivity detection is the most preferred method because of its superiorresolution and detail-rich radioactivity profiling. Prior to the advent of modern HPLCradiodetectors, the eluate stream was collected in fractions that were counted by liquidscintillation counting (LSC) in order to construct a histogram fromwhich the radiochemicalpurity could be extracted. This method is sometimes used with extremely low specificactivity samples; however, the newer technique of automated fraction collection inmultiwell microplates followed by high-throughput solid scintillation counting allows adegree of fractionation high enough to approach the resolution of on-line detection22.
Thin layer chromatography continues to be used in spite of its relatively lower resolvingpower compared with HPLC, and potential problems in quantitation, such as self-absorption of radiation within the layer, especially with tritium (ca 2mm path length ofb� in solid materials, see Section 1.1 above). One advantage compared with HPLC radio-detection is that impurities not eluting through HPLC columnsmay be detected on the TLCplate. In this technique, radiometry is conducted after development and drying of the TLCplate; available methods are of two types, linear and two-dimensional. Linear methods arebased on single- or multi-wire gas proportional counters sensitive to a narrow band parallelto the direction of plate development. Two-dimensional methods include film-basedautoradiography, which is of relatively low quantitative power, and the more moderntechniques based on phosphor imaging screens or high-resolution crossed-wire propor-tional counters and high sensitivity CCD cameras, both of which are supported bycomputerized measurement systems.
Since it is relatively common that compounds coelute or nearly coelute in even high-resolution chromatographic systems, it is always recommended that radiochemical purityanalyses be carried out in two different chromatographic systems, as unlike one another aspossible. For example, the combination of one reverse-phaseHPLCorTLCmethod and onenormal-phaseHPLCor TLCmethod is usually recommended, but two reverse-phaseHPLCanalyses using different column types and mobile phases may also be acceptable.
It should be noted that these chromatographic–radiometric methods have inherentshortcomings that must be understood and taken into account both in the chromatographycomponent and the radiodetection component of the assay.
The chromatographic part: in HPLC, there is the possibility that one or more radioactivecomponents are not detected because they fail to exit the column by the end of themonitoring period. An effective check for this possibility is to measure the quantity of
8 Preparation of Compounds Labeled with Tritium and Carbon-14
radioactivity exiting the column and to compare itwith the amount injected. In TLC, there isthe possibility that one or more components may be lost or diminished through volatiliza-tion before scanning can be completed. Before running a TLC analysis, the absence ofvolatile components may be confirmed by measuring the radioactivity in a sample beforeand after drying in vacuo.
The radiometric part: there are two phenomena involved. First, unlike UV detectors forHPLC, which have virtually no baseline noise, in HPLC radiodetection and TLC radio-scanning there is always a significant baseline noise, resulting from a combination ofenvironmental radiation, detector noise and the decay statistics of low-level radioactivity.The presence of this noise makes it difficult to distinguish between baseline and minorradioactiveimpurities, andevenmoredifficult tomeasure themaccurately.Thesecondis thatradiodetector peaks of eluting components tend to be broader and to havemore pronouncedtails than the UV detector peaks for the same components. This reduction in resolution iscaused by the additional internal volume of the in-line radiodetector cell and associatedplumbing that the samplemust travel throughafter it exits theUVdetector.Resolutioncanbefurther degraded by suboptimal scintillant flow rates (for liquid scintillant radiodetectorcells) and peak tailing characteristic of many solid scintillant cells. There is an unavoidabletradeoff between maximization of radiodetector resolution and sensitivity (signal-to-noiseratio). These characteristics of radiodetector performance may make it more difficult torecognize and accurately quantify impurities running close to the compound of interest, andmoredifficult to judge thepoint atwhich the tailof thepeakofinterest returns tobaseline.Theuncertainties involved make accurate and consistent interpretation difficult, and make thedata reduction process vulnerable to subjective judgments and therefore person-to-personvariability. It is not unusual formeasurements by two scientists using the same instrumenta-tion for the analysis of the same sample to vary by 2%, and by 1% between successiveinjections by the same person. It is therefore not surprising that it is sometimes difficult to becertainwhether a98%radiochemical purity level (typically specified forADMEstudies)hasbeenmet.Most radiometric instruments allow the operator to establish settings for control ofa variety of parameters for data processing, such as baseline correction, peak detection andpeak deconvolution, but correctly establishing these parameters requires skill, and the sameparameter settings may not be optimal for all analyses.
Radionuclidic purity is only of concern in the context of dual-isotope labeling, or if cross-contamination from a laboratory mishap is suspected. Radionuclidic purity is bestmeasured by liquid scintillation counting; modern LSC instruments have detectors andanalysis software designed to discriminate quantitatively between the different isotopesused in the life sciences, except at very low counting levels. Radionuclidic purity is entirelydistinct from isotopic purity, or content, of compounds labeled with stable isotopes, such asdeuterium or carbon-13. Such information may be very important to the utility of stable-labeled compounds such as internal standards for mass spectrometric quantitation assays23.
1.3.4 Specific Activity
There are twoways in which specific activity is expressed, radioactivity per unit mass (e.g.,mCi/mg) and radioactivity per molar unit (e.g., Ci/mmole or mCi/mmole).
The former, used more frequently for carbon-14-labeled compounds and low specificactivity tritiated compounds, is most simply determined by preparing a solution of known
Introduction 9
mass concentration and measuring the radioactivity of defined volumes by LSC. Solutionsalready made up for chemical purity determinations can be used for this assay.
The latter expression of specific activity is used almost always for high specific activitytritiated compounds, and often for carbon-14-labeled compounds. Measures of radioactiv-ity per molar unit can be calculated from mass spectrometry data24. In this analysis, thedistribution of isotopic species (e.g., 3H0,
3H1,3H2, . . .) is determined by measuring the
relevant peak intensities in the molecular ion envelope and correcting them for naturallyoccurring isotopes (e.g., 13C, 34S) present in the molecule; this can be done manually or byuse of readily available computer algorithms. The contribution of each isotopic species tothe total can be used to calculate the average number of isotopic atoms per molecule andthence, from themolar specific activity of the pure isotope, themolar specific activity of thecompound.
Interconversion between expressions of radioactivity per unit mass and radioactivity permolar unit must take into account the fact that the former has to be corrected for thechemical purity of the sample, whereas the latter does not.
1.3.5 Position of Label
The importance of knowing the location of carbon-14 or tritium atoms within compoundsdepends primarily upon their intended use, but may be of interest also in studies of reactionmechanisms, molecular rearrangements and mechanisms of isotope exchange.
In most syntheses the location of carbon-14 atoms follows logically from the route ofsynthesis.However, there are cases inwhich the position of a labelwas altered by previouslyunrecognized or incompletely understood reaction pathways, as illustrated by the reactionsof 1 to 225 and 3 to 426 in Figure 1.1. In these cases, the carbon-13 labels and 13C NMRwere
N
CH3
F
+
HN
NH
F
ClHN
F
N
CH3
OSO2Ph
F
N
CH3
O O
O
HN
N
F
Cl
NO2HN
N
F
Cl
NH2
**
*
1. sesamol aq. NaOH
4-Me-2-pentanol toluene 100°C, 8.5 h2. chromatography
*
mostly cis mostly trans
via10%
90%
* *
H2, Ra-Ni
EtOH/THF
* *via
90%
10%
1 2
43
Figure 1.1 Isotope scrambling in carbon-14 syntheses
10 Preparation of Compounds Labeled with Tritium and Carbon-14