National Stable Isotope Resource at Los Alamos

Transcription

1 ational table Isotope Resource at Los Alamos I ational Institute for Biological Imaging and Bioengineering lifford J. Unkefer 2,, 15, 17,18 1

2 utline ynthetic apabilities Example ynthesis of chiral methyl labeled valine Properties of table Isotopes eparation Methods orrelated Labels for Metabolic Analysis ynthesis of Labeled ompounds 2

3 tereochemistry R R L-amino acid -amino acid 3

4 Racemic Mixtures L-arnitine -arnitine Acyl-oA X R

5 Methyl Labeling iastereotopic Methyl Groups in Amino Acids L-Leucine L-Valine 3 3 L-Isoleucine 3-1 and he mical hifts of Methyl Groups AA 1 (ppm) (ppm) ILE LEU VAL

6 hiral L-Valine 2 (3R,2)-[4-, 2, 4 -, 2 2, 2-15, 2 ]Valine 6

7 ynthesis of 2 or 3 Methyl Iodide I l T 2 or 2 T () I T () eat aljoughian, M., Monouchehr, M.. and Rapoport,., J. rg. hem. (1989) 54(19)

8 ynthesis of Labeled Methyl Iodide I 96% I Pha Ph 1. sec BuLi 2. 2 Ph (99%) (83%) I I I I Pha Ph 1. sec BuLi 2. 2 (99%) Ph I (80%) I 94% 8

9 Alkylation of Propionylsultam 3 1. n-buli 0.5 h -78 o 2. 3 I (99%) 3 3 9

10 ynthesis of hiral Methyl-labeled Valine 1. n-buli 36 h -78 o 2. 2 I -78 o to -30 o (63%) 1. n-buli 36 h -78 o 2. MPA 3. 2 I (70%) (98% de) 1. LiAl 4 2. p-tsl (75% over 2 steps) Ts Pa (92%) Ph 10

11 hiral Methyl-Labeled Valine cont. Ph 1. Li o (l) 2 2. ultam (49% over 3 steps) 1. BuLi 2. itrosyl hloride 3. Reduction / ydrolysis 15 l

12 2- pectrum of hiral Valine 12

13 hiral Methyl Labeling of aloalkane ehalogenase aloalkane ehalogenase 310 Amino acids 35,000 a 14 Valines Valine Auxotroph of BL21(E3) 20 mg of labeled valine Perdeuterated background

14 hiral Methyl Labeling of aloalkane ehalogenase I 2 I I 2 I I 2 + I Methyl Groups I Methyl Groups LeMaster, ernández, Velarde and Martinez 14

15 table Isotope Labeling for MIM Isotope atural Abundance (%) Available Enrichment (%) $/Mole ~ ~ ~ ~ ~

16 euterium Advantage - heap Isotopic Precursors 2 2, 2 2, LAl 2, ab 2 4 isadvantage - Label can wash out Kinetic Isotope Effects 16

17 Kinetic Isotope Effects A + A L-lactate pyruvate Rate = X A + 2 A L-[2-2 ]lactate 3 - pyruvate Rate = X/4 to X/8 17

18 Wash ut transamination L-[3-2 ]alanine 3 pyruvate L-[3-2 ]alanine transamination

19 hemistry ab

20 Low Abundance Isotopes Isotope atural Abundance (%) Available Enrichment (%) $/Mole ~ ~ ~ ~ ~

21 15 eparated by cryogenic distillation of 15 3, R K Br 15 R ucleophilic isplacement l Br Electrophilic amination L-[ 15 ]labeled amino acids are available 21

22 tereoselective synthesis of 15 - labeled amino acids ppolzer ynthesis R a(ime 3 ) 2 15 l 15 R Zn / aq l Ac, o 15 2 Li in 15 2 R 2 /TF R 22

23 ommercial ynthesis of L-[1- ]- and L-[α- 15 ]amino acids -acetyl--[1- ]- or [α- 15 ]amino acid R (excess) 15 o() R 6 3 -acetyl-,l-[1- ]- or [α- 15 ]amino acid Acylase 15 R 3 15 R L-[1- ]- or [α- 15 ]amino acid 23

24 Unified Route to -, and 15 -labeled Purines Et - a + 2 Pl 3 l l l 2 Et 1. Br 2 2. Reduction 1. i-amylnitrite, MF, TF 2. Enzymatic Ribosylation 3. 4, 120 o 2 l [ ]triethylorthoformate l '-deoxy adenosine 1. Enzymatic Ribosylation 2. 4, 120 o 2 adenosine deaminase 'deoxy guanosine 24

25 Transamination of amino acids L-α-amino acid α-keto acid Transaminase 3-3 α-keto acid 15 2 L-α-amino acid 25

26 Low Abundance Isotopes Isotope atural Abundance (%) Available Enrichment (%) $/Mole ~ ~ ~ ~ ~

27 18 eparated by cryogenic distillation of 2 18 ; 18 2, l l l Ph 3 P 18 2 R EA R R

28 Wash out of 18 -labels 18 2 R 18 2 R 18 R 2 18 R 28

29 Low Abundance Isotopes Isotope atural Abundance (%) Available Enrichment (%) $/Mole ~ ~ ~ ~ ~

30 eparated by cryogenic distillation of 1.1% natural abundance - high background Multiple carbon labeling hallenging hemistry to make carbon-carbon bonds xidize to 2 grow algae isolate compounds Labels stay where you put them 30

31 -labeled precursors 2 u 3 2, 225 o 2 4 I a 3, Pt, 1000 o 3 I a K 31

32 ew Labeled Precursors Precursors from [ ]methyl phenyl sulfide i( 3 ) 3 "ource for Labeled Acetals" "Alkenyl ulfides and yclopropanes" "ource of Li x y i( 3 ) 3 " l l x y x y l x y x y x y Ph "Aldehyde arbon omologation ource" x y Ph x y "Alkenes and Alkynes" "Masked Formaldehyde ource" "1.hloroform, 2. Triethylortho formate, 3. arbon Tetrachloride ource" l l l Li x y "Anion source of for various addtions of a methyl group (i.e. - conjugate additions)" n(bu 3 ) 3 x y "Radical oupling ynthon" 32

33 Precursors ew Labeled from [ ]methyl Precursors phenyl sulfide 1) I - a ) 3 Li 2 X 2 RX R R - M + 2 R R R R 2 Li 33

34 ynthesis of -labeled xamate 3 [ ]methyl phenyl sulfide 1) secbuli 2) % yield Ph 1) (l) 2 2) ( 3 ) 2 ( 3 ) 2 ( 3 ) l 2 l 99+% yield 99+% yield Ph Ph Et/ l 2 5% Et ( 3 ) 2 88% Yield Ethyl,-dimethyl [1,2-2 ]oxamate 34

35 Asymmetric Labeling of Glycerol 2 Li () methyl phenyl sulfoxide IBAL TF 1) Ac 2 /aac Ethyl -dimethyl [1,2- ]oxamate 2) LiB 4 2 () 2 (2) [1,2-2 ]glycerol 35

36 haracterization of hiral [1,2-2 ]glycerol 2 2 ATP AP Glycerol Kinase 2 2 P 3-2 ()-[1,2-2 ]Glycerol L-[1,2-2 ]glycerolphosphate 2 2 ATP AP Glycerol Kinase 2 2 P 3-2 (R)-[1,2-2 ]Glycerol L-[2,3-2 ]glycerolphosphate 36

37 MR analysis of hiral Glycerol 1 J - 1 J P J - (2R)-glycerol-3-phosphate 3 J P- 3 J P

38 Amino Acid Labeling 2 ()-[1,2-2 ]Glycerol ATP AP Glycerol Kinase 2 L-[1,2-2, ]amino acids 2 2 P L-[1,2-2 ]glycerolphosphate Biosynthesis R L-[1,2-2, ]amino acids Glycine Alanine erine ysteine Aspartic acid Asparagine Threonine Methionine Lysine Tryptophan Tryosine Phenylalanine Glutamate Glutamine Arginine Proline Valine Leucine Isoleucine istidine 38

39 orrelated Labeling ato Gould and ane 81% 10% 9% 90% 9% 1% 39

40 Biosynthetic Precursors of PQQ - Glutamate ' ' PQQ 9 9a 3 3a 4 5a 5 2' + 3 Tyrosine - 40

41 Incorporation of L-[2',6'- 2,α- 15 ]Tyrosine into PQQ MR pectrum of -1a 41

42 Incorporation of L-[2',6'- 2,α- 15 ]Tyrosine into PQQ 42

43 hemistry of secondary - production

44 hemistry of secondary - production '-deoxyadenosine 15 44

45 Protein Turnover 2 18 / = ATP 2 amino acyl tra synthase P - 2 tra PE tra PE - AMP - P % 50% R R 1 18 R R 1 protein synthesis protein degradation R 1 R 2 R 4 18 protein synthesis R 3 18 / = 1 18 / =

46 Ribonucleotide Reductase Activity R 18 R= Ribonucleotide Reductase P P - - R

47 ynthesis of [2-18, 15 3]cytosine Molybdate Bz Bz Me 18 Bz 47

48 ynthesis of -Labeled Lipids Et 2 Et 1) a 2) RX 3) Raney i V 1) 2 Li II Ph Li % Ph X 3) a 4) l 2 R XI VII R = - 2 ( 2 ) 10 3 (Palmitic Acid) R = - 2 ( 2 ) 3 =( 2 ) 7 3 (leic Acid) Li Ph XII 2 2 Et 2 2 Ph 1) Raney i 2) Et/ + 3) wern xidation Et XIV icolaou Procedure Arachidonic and ocosahexaenoic Acid XIII 48

49 icolaou Procedure Br 4, PP 3, 2 l 2 Br pp 3, 3 P + Ph 3 Br - Me a(i( 3 ) 3 ) 2, Me 89% 49

50 Triglyceride Biosynthesis R R 18 R 18 R R 2 R R

51 Biosynthesis of L-glycerol = P 3 Glucose-6-phosphate = 2 P 3 Glucose-6-phosphate 2 = 2 P 3 Fructose-6-phosphate ATP = 1 2 P 3 1 = 2 P = 2 P 3 aldolase 1 = 2 P = 2 P = 2 P 3 L-glycerol-3-P = 2 P = AP 2 P 2 triose-phosphate 3 isomerase -Glyce raldehyde-3-p = 2 P 3 51

52 ynthesis of -[6-18 ]Glucose Bn Bn Bn Bn Me PPh 3, EA, Bn Bn Bn Me 18 Me 52

53 ational table Isotope Resource at Los Alamos taff Members Rodolfo A. Martinez Louis (Pete) A. ilks III Jürgen chmidt Ryszard Michalczyk iegfried. Lodwig Robert Williams Philip totter Rulian Wu Post octoral Researchers Rachel M. Williamson avid B. Kimball Technicians Marc A. Alvarez Morgane V. llivault-hiflett Erick G. rtiz Penelope A. aranjo Administrative Assistant MaryAnn. Martinez 53