LIPIDS and RELATED COMPOUNDS

Transcription

1 LIIDS and RELATED CMUDS 1. Waxes esters of long chain carboxylic acids (fatty acids) and long chain alcohols 2. Fats (animal) and oils (vegetable) triacylglycerols: triesters of glycerol and fatty acids 3. hospholipids a) phosphoglycerides phosphoryldiacylglycerols: esters of glycerol with 3 4 and fatty acids b) sphingolipids esters of sphingosine with 3 4 and fatty acids 4. rostaglandins mono or bicyclic products of oxygenation of arachidonic acid 5. Terpenes diverse structures derived from isoprene unit 6. Steroids tetracyclic structures (derived from triterpenes) 73

2 FATTY ACIDS Saturated 3 C-[C 2 ] n -C lauric C o C myristic C o C palmitic C o C stearic C o C arachidic C o C Unsaturated palmitoleic C 16 (9-cis) 32 o C oleic C 18 (9-cis) 16 o C ricinoleic C 18 (9-cis, 12-) 5 o C linoleic C 18 (9,12-cis) -5 o C linolenic C 18 (9,12,15-cis) -11 o C arachidonic C 20 (5,8,11,14-cis) -50 o C

3 WAXES FATS and ILS 75

4 SLIIDS hosphoglycerides phosphatidylcholine (lecithin) phosphatidylethanolamine (cephalin) 3 76

5 SLIIDS Sphingolipids sphingomyelin sphingosine 2 77

6 ETERCYCLIC BASES pyridine pyrrole indole pyrimidine purine ucleic-acid s: 1. urines 2 2 adenine (A) guanine (G) 2. yrimidines 2 cytosine (C) thymine (T) uracil (U) 78

7 UCLEIC ACIDS 1. DA (deoxyribonucleic acid) sugar amine sugar amine 3 4 phosphate sugar amine nucleic acid UCLESIDE UCLETIDE 2 2 2'-deoxyadenosine 5'-phosphate 2'-deoxyguanosine 5'-phosphate 2 2'-deoxycytidine 5'-phosphate 79 2'-deoxythymidine 5'-phosphate

8 UCLEIC ACIDS 2. RA (ribonucleic acid) 2 2 adenosine 5'-phosphate guanosine 5'-phosphate 2 cytidine 5'-phosphate uridine 5'-phosphate 80

9 UCLEIC ACIDS 2 5' A 2 C 2 G T 3' one strand of DA (A-C-G-T) 81

10 BASE AIRIG A T G C 82

11 UCLEIC ACIDS replication DA transcription RA translation RTEIS 1. Replication: copying of DA during cell division synthesis of complementary strands (enzyme DA polymerase, from 5' 3') in humans: 3 billion pairs are copied in minutes with ppb error rate! 2. Transcription: transferring of genetic message from DA (in the nucleus) into RA the template (antisense) strand of DA is transcribed into messenger RA copying starts at promoter sites and stops at "stop" signals; a "bubble" forms (5' 3' ) final mra is formed after introns are cut (only exons) 83

12 UCLEIC ACIDS 3. Translation: decoding of the genetic message from RA to protein sequences three letter codons are used to translate the code into amino acid structure; 64 codons; 61 codes for amino acids (redundant!) and 3 code for "stop" the translation takes place in ribosomes (60% ribosomal RA, rra; 40% protein) individual amino acids are introduced by transfer RA (tra); there are 61 of them 5' 3' A U A G A C G G A U A C G C C mra (codons) U A U C U G C C U A U G C G G tra (anticodons) Ile Asp Gly Tyr Ala amino acid bound to tra Ile-Asp-Gly-Tyr-Ala protein product 84

13 DA SEQUECIG 1. Maxim-Gilbert (chemical cleavage) i) DA is cleaved at specific sites by restriction enzymes ii) the restriction fragments are labeled with radioactive 32 at 5' end, heated to get single strands, and the strands are separated iii) each strand is treated with four parallel set of chemicals that cleave the strand at specific sites (next to A, G, C and T and C) under conditions that only a few (one) splits occurs iv) each of the four mixtures is separated in parallel by electrophoresis (smaller pieces migrate faster) and the gel is exposed to a photographic plate v) the bands allow the sequence to be read directly from the gel (up to 600 s per gel can be analyzed) 2. Sanger (dideoxy method; enzymatic) i) restriction fragments are produced as above ii) small DA fragments, called primers, labeled at 5' with radioactive 32 or 35 S are added iv) in four separate batches four different deoxyribonucleoside triphosphates are added containing small amount of the corresponding dideoxynucleoside triphosphate; the enzyme (DA polymerase) synthesizes the complementary sequences that are terminated by the dideoxynucleoside v) the four reactions are analyzed on the gel, as described above (most recent version use fluorescent dyes) 85

14 DA SYTESIS 1. Attachment of the deoxynucleoside to silica (Si 2 ) by an ester linkage DMT Si silica rotection schemes: h h Me R DMT R Me 86

15 DA SYTESIS 2. Deprotection of 5' hydroxyl group DMT Si silica C 2 ClC/C 2 Cl 2 Si silica 87

16 DA SYTESIS 3. Coupling with deoxynucleoside containing phosphoramidite group Si silica DMT C (ir)2 DMT C Si silica 88

17 DA SYTESIS 4. xidation of the phosphite to the phosphate with I 2 DMT C Si silica I 2 2 /TF 2,6-dimethylpyridine DMT C Si silica 89

18 DA SYTESIS 5. Repeat DMT deprotection, coupling, and oxidation (steps 2, 3, 4) per each added nucleotide 6. Final deprotection of s, removal from the solid support and purification by electrophoresis DMT C polynucleotide chain Si silica 3 / 2 polynucleotide chain 90

19 LYMERASE CAI REACTI (CR) A method to produce many copies of a DA sequence 16 copies 95 o C 8 copies 4 copies primers 50 o C repeat sequence polymerase, Mg 2+, 72 o C dts 91