Global Journal of Science Frontier Research Chemistry Volume 12 Issue 2 Version 1.0 February 2012 Type : Double Blind Peer Reviewed International Research Journal Publisher: Global Journals Inc. (USA) Online ISSN: 2249-4626 & Print ISSN: 0975-5896 Synthesis of Biologically Important Pyrrole Derivatives in Any 13C and 15N Isotope Enriched Form By Prativa B. S. Dawadi & Johan Lugtenburg Leiden University, Leiden Abstract - Recently the synthesis of [3-13C]-, [4-13C]-, and [11-13C]- porphobilinogen, [15N,13C4]-1H - pyrrole-2,3,5 - tricar - boxylic acid, [1-15N]-3-cyano-4-methyl-1H-pyrrole and [2-13C]- and [3-13C]-cyano-4- methyl-3-pyrrolin-2-one have been published. Incorporation of 13C and 15N in these systems at any position and combination of positions has become accessible. Also mild alkylations of active methylene compounds with α-halo carbonyl compounds open up many 3-pyrrolin-2- ones and pyrrole systems based on stable isotope building blocks that have been published. This gives the access to a whole new library of stable isotope enriched pyrroles in any stable isotope enriched form. This is also the case for biliverdin IXα which after enzymatic treatment has been converted into (2R)-phytochromobilin that reacts with its apoprotein to form intact active phytochrome. Keywords : [1-15N]-3-Cyano-4-methyl-1H-pyrrole, [3-13C], [4-13C]-, and [11-13C]-porphobilinogen, [ 15N, 13C4,] -1H-pyrrole-2,3,5-tricarboxylic acid and biliverdin IXα. GJSFR -B Classification: FOR Code: 030503, 040203, Synthesis Of Biologically Important Pyrrole Derivatives In Any 13C And 15N Isotope Enriched Form Strictly as per the compliance and regulations of: © 2012. Prativa B. S. Dawadi, Johan Lugtenburg.This is a research/review paper, distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License http://creativecommons.org/licenses/by-nc/3.0/), permitting all non commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Synthesis of Biologically Important Pyrrole Derivatives in Any 13C and 15N Isotope Enriched Form Prativa B. S. Dawadiα & Johan Lugtenburg σ 12 0 13 13 2 Abstract - Recently the synthesis of [3- C]-, [4- C]-, and of membrane fractions of Heliobacillus mobilis that was 13 15 13 13 [11- C]- porphobilinogen, [ N, C4]-1H -pyrrole -2,3,5 - tricar - grown on media containing [4- C]-aminolevulinic acid 15 13 13 boxylic acid, [1- N]-3-cyano-4-methyl-1H-pyrrole and [2- C]- have been obtained. and [3-13C]-cyano-4-methyl-3-pyrrolin-2-one have been ebruary Besides NMR spectroscopy, vibrational F 13 15 published. Incorporation of C and N in these systems at techniques such as resonance raman spectroscopy any position and combination of positions has become have been applied in heme protein research.14 In this 23 accessible. Also mild alkylations of active methylene compounds case some of the vibrations coupled to an electronic I with -halo carbonyl compounds open up many 3-pyrrolin-2- transition of the chromophore showed enhanced α 6 ones and pyrrole systems based on stable isotope building inelastic scattering up to 10 fold. on blocks that have been published. This gives the access to a Access to pyrroles enriched on each position whole new library of stable isotope enriched pyrroles in any and any combination of positions with stable isotopes V I stable isotope enriched form. This is also the case for such as 2H, 13C and 15N is essential to study the I biliverdin IXα which after enzymatic treatment has been metabolism of important pyrrole derivatives using non- ersi ue ss converted into (2R)-phytochromobilin that reacts with its invasive isotope sensitive techniques.The chromophores apoprotein to form intact active phytochrome. of heme proteins and photosynthetic reaction centres XII I 15 13 Keywords :[1- N]-3-Cyano-4-methyl-1H-pyrrole, [3- C], have been prepared with stable isotope enriched pyrrole 13 13 15 13 [4- C]-, and [11- C]-porphobilinogen, [ N, C4,] -1H- building blocks. pyrrole-2,3,5-tricarboxylic acid and biliverdin IXα. Recently, we have published a review paper about the stable isotope enriched systems in heme and ) I. INTRODUCTION B ) (bacterio)chlorophyll protein systems that were known at 15 yrroles and their derivatives are one of the most that time. In the meantime a number of important 1 stable isotope enriched pyrrole systems have been important classes of heterocyclic compounds. arch Volume They exhibit extensive biological and published together with a new method to prepare se P 2 pyrroles and stable isotope enriched building blocks pharmacological properties. Many pyrrole derivatives have shown interesting biological properties such as that allow access to a whole new range of stable isotope 3 4 5 enriched pyrroles. tier Re antibacterial , antiinflammatory , antioxidant , antitumor, on antifungal6 and immune suppressant activities.7 Highly In this paper we focus on those new functionalized pyrroles are subunits of heme, possibilities that allow access to biliverdin IXα which can chlorophyll, bile pigments, vitamin B12 and pyrrole be converted into (2R)-phytochromobilin, the alkaloids isolated from marine source.8 Atrovastatin chromophore of phytochrome via one enzymatic 16 13 15 (Lipitor) is a drug for lowering cholesterol.9 conversion. We mainly focus on C and N enriched 2 building blocks leading to the labels at all atoms in the Access to stable isotope enriched systems ( H, l of Science Fr 13C and 15N) allows the metabolic conversions of these molecular skeleton of the pyrroles and tetrapyrrole na systems. We have not focused on 2H systems because systems to be followed with mass spectroscopic Jour 2H occupies the peripheral positions on the molecular techniques when they have at least three stable l isotopes.10 13C-NMR Techniques have been used to system and is more prone to isotope loss and 13 scrambling during the synthetic process. However, the study the conversion of [5- C]-aminolevulinic acid into Globa 13 porphobilinogen in vivo in living Rhodobacter schemes for C incorporation can easily be adjusted to 2 sphaerhoides cells.11 H incorporation as well. Similarly, the conversion of [2-13C]- and [11- 13C]-porphobilinogen in the body into uroporphyrinogen II. SYNTHESI S AND DISCUSSION 12 III and coproporphyrinogen III has been investigated. 13 13 13 13 a) Synthesis Of [3- C]- , [4- C]- And [11- C]- Very recently the C photo-CIDNAP MAS NMR spectra Porphobilinogen 1. 13 Enzymatic incorporation of [11- C]- and [2,11- α σ 13 Author : Leiden Institute of Chemistry, Leiden University, P.O. Box C2]-porphobilinogen 1 (fig. 1) into uroporphyrinogen I 9502, 2300 RA, Leiden. E-mail : [email protected] and III has been reported.17,18 © 2012 Global Journals Inc. (US) Synthesis Of Biologically Important Pyrrole Derivatives In Any 13C And 15N Isotope Enriched Form 8 a scheme that allows access to any stable isotopomer CO2H 19 10 9 6 7 and isotopologue has been reported. HO C 2 4 3 Acetic acid 2 is treated with 1 eq of bromine in the presence of trifluoroacetic anhydride to afford a high 5 11 2 yield of the 2-bromoacetic acid which is esterified with N1 ethanol into ethyl bromoacetate 3. The bromine is easily H N H 2 substituted for the cyano group with KCN 4 to give ethyl 1 cyanoacetate 5. The ester function is reduced with NaBH to give an alcohol function in 3- 13 4 1a: [4- C] hydroxypropionitrile 6. Treatment of 6 with aqueous HBr 12 0 13 and subsequent esterification afforded methyl 3- 2 1b: [3- C] 13 bromopropionate 7 in high yield. The SN2 reaction of 1c: [11- C] reagent 7 with nitromethane 8 in the presence of 2 eq BuLi to obtain methyl 4-nitrobutanoate 9 is somewhat ebruary Figure 1 : Structure and numbering of porphobilinogen F difficult. 1 and its highly enriched isotopomers 1a, 1b and 1c. An alternative method to obtain the product 9 is 24 Porphobilinogen 1 is a biosynthetic precursor of to treat reagents 3 and 5 in the presence of NaOEt to tetrapyrrole chromophores in heme proteins, afford diethyl 2-cyanopentanedioate 10.20 Selective I photosynthetic antennae proteins, photosynthetic removal of one of the ester functions in NaCl, DMSO, on reaction centres and phytochromes. The synthetic H2O gave ethyl 3-cyanopropionate 11. Subsequent 13 15 access to C and N enriched porphobilinogen will reduction of the ester function with NaBH4 afforded 4- V allow access to enrich stable isotopes in the above I hydroxybutyronitrile 12 which is further converted into 4- I mentioned systems at any possible position. With 13C iodobutyronitrile 13. SN2 substitution of the iodo function ue ersi ue and 15N isotope incorporation in the chromophores of ss with NaNO2 and subsequent conversion of the nitrile these biologically important proteins can be investigated function into ethyl carboxylate is expected to give ethyl XII I with noninvasive isotope sensitive techniques. 4-nitrobutanoate 9 without problem. In porphobilinogen In figure 1 the structure and numbering of 1 (fig. 1) the carbon atoms 3, 6, 7 and 8 are derived 13 porphobilinogen 1 is depicted. The synthesis of [3- C]-, from the compound 9 and carbon atoms 4, 9 and 10 are [4-13C]- and [11-13C]-porphobilinogen 1a, 1b and 1c via derived from 3-hydroxypropionitrile 6. ) B ) a KCN O O H3C OH 4 a a arch Volume NaBH TFAA, Br2 Br 4a: [13CN] NC 4 NC se OEt + OEt OH O EtOH , H 6 2 3 5 tier Re 13 13 on 5a: [3- C] 6a: [1- C] b CH3NO 2 O O 8 13 NO HBr, MeOH 8b: [ C] 2 6 MeO Br MeO 2 eq BuLi b 7 9 l of Science Fr 13 na 9b: [4- C] O Jour O l NC 5 OEt NaCl NaBH4 NC OH 3 NC OEt DMF, H2O Globa CO2Et 10 11 12 TsCl, NaI 12 NC I 13 Scheme 1.