SYNLETT0936-52141437-2096 Georg Thieme Verlag Stuttgart · New York 2019, 30, 263–274 account 263 en Syn lett B. Kräutler Account Chlorophyll Breakdown – How Chemistry Has Helped to Decipher a Striking Biological Enigma Bernhard Kräutler* Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria [email protected] Dedicated to Professor Franz-Peter Monforts on the occasion of his 70th birthday. Received: 07.08.2018 etation in spring are probably the most visual signs of life, Accepted after revision: 20.09.2018 observable on Earth from outer space.1 Indeed, the annual Published online: 31.10.2018 DOI: 10.1055/s-0037-1611063; Art ID: st-2018-a0501-a apparent ‘recycling’ of the green pigment Chl is a massive License terms: biological process involving about 1000 million tons, world- wide, and occurring by seasonally alternating de novo Chl 2 Abstract How the fall colors arise and how chlorophyll (Chl) break- biosynthesis and Chl degradation. Strikingly, until about down occurs in higher plants has remained enigmatic until three de- 30 years ago Chl seemed to disappear without leaving a cades ago. Fundamental insights into this fascinating puzzle have been trace, as non-green products of Chl breakdown remained gained, meanwhile, by basic contributions from plant biology and elusive.2,3 In fact, all searches for genuine Chl catabolites chemistry. This short review is a personal account of key advances from synthetic, mechanistic, and structural chemistry that led to the discov- were futile, since they concentrated on the detection of col- ery of the bilin-type Chl catabolites and helped elucidate the metabolic ored remains of the Chls. However, as is now well known, processes that generated them from Chl. Chl breakdown products in higher plants accumulate as col- 1 Introduction orless linear tetrapyrroles, primarily.1,4–7 All the same, the 2 Discovery and Structure Elucidation of a First Non-Green Chl bilin-type Chl catabolites, named phyllobilins (PBs),8 turn Catabolite 9 3 Structure Elucidation of Fleetingly Existent Blue-Fluorescent Chl out to be remarkably related, structurally, to bilins, the col- Catabolites ored products from heme breakdown.10 4 The Red Chl Catabolite – Key Ring-Opened Tetrapyrrole Accessed by Partial Synthesis 5 Synthesis of ‘Primary’ Fluorescent Chl Catabolites by Reduction 2 Discovery and Structure Elucidation of a of Red Chl Catabolite 6 Nonfluorescent Chl Catabolites from Isomerization of Fluores- First Non-Green Chl Catabolite cent Chl Catabolites 7 Persistent Fluorescent Chl Catabolites and Blue-Luminescent Chl breakdown has for a long time been considered to Bananas play a particular role in the recuperation of nitrogen.3 First 8 Discovery, Structure Elucidation, and Biological Formation of Dioxobilin-Type Chl Catabolites traces of non-green Chl breakdown products were traced by 9 Occurrence, Partial Synthesis, and Structure of Phyllochromobi- Matile, Thomas and coworkers by comparing the pigment lins, the Colored Bilin-Type Chl Catabolites pattern of senescent leaves of wild type Festuca pratensis 10 Conclusion and Outlook with those in ‘stay green’ mutants of this grass.11 Remark- ably, colorless compounds could be spotted in the wild type Keywords antioxidants, bilin, catabolite, chlorophyll, fluorescence, glycoside, heterocycles, pigments, porphyrinoids, phyllobilin, senes- that were absent in the green mutant, and which were pre- cence, tetrapyrroles sumed to represent Chl catabolites. These polar compounds were called ‘rusty pigments’, as they rapidly converted into rust-colored products.12 We learned to isolate one such 1 Introduction ‘rusty pigment’ without degradation and color formation, and elucidated its chemical constitution in 1991 with the The appearance of the fall colors is an enchanting and help of heteronuclear NMR spectroscopy and soft ionization puzzling phenomenon. The widespread disappearance of mass spectrometry.4,13 It turned out to be fruitful, to solve chlorophyll (Chl) in autumn and the re-greening of the veg- Georg Thieme Verlag Stuttgart · New York — Synlett 2019, 30, 263–274 264 Syn lett B. Kräutler Account the desired structure directly by purely spectroscopic O H O 5 H means,4 rather than submitting this polar Chl catabolite to 4 O O 19 1 1 NH N 19 H NH HN NH HN R chemical modifications, e.g., in order to decrease its polarity. 20 10 n H n 15 5 15 5 N HN N HN NH HN HO H R OH 5 4 H O H A B 18 O O O 1 3 O N N 19 A CO2Me CO2Me D HO C HO C CO2H HN OH 2 2 HO2C Mg H n NH N N 5 D C NH HN Pheo a (6) pFCC (5) Bn-NCCs 2,3,4 C B E CH3 E Scheme 2 Structural formulae of Pheo a (6), pFCC (5), and of NCCs 2 O H C (R = malonate), 3 (R = 1’--D-glucopyranose), and 4 (R = OH) from se- C CO2Me 3 O CO2Me O O CH3 CH3 HO2C nescent leaves of oil seed rape (Brassica napus) CH3 chlorophyll a (Chl a, R = CH3) Hv-NCC-1(1) 3 Structure Elucidation of Fleetingly Exis- chlorophyll b (Chl b, R = HC=O) tent Blue-Fluorescent Chl Catabolites Scheme 1 Structural formulae of chlorophylls a and b and of Hv-NCC-1 (1) The repeated observation of fleetingly existent blue-flu- The colorless ‘rusty pigment’ from de-greened leaves of orescent compounds alongside of the NCCs in extracts of barley (Hordeum vulgare) was unambiguously revealed to senescent leaves suggested their relevance in Chl break- be a bilane-type linear tetrapyrrole that carried structural down.17 In vitro experiments by Hörtensteiner and Matile hallmarks of the Chls, such as their characteristic ring C/E- showed the requirement for molecular oxygen and identi- portion.4 It was classified as a nonfluorescent Chl catabolite fied the Mg-free and dephytylated Chl derivative pheophor- (NCC) and given the phenomenological name Hv-NCC-1 bide a (Pheo a, 6) as a precursor for the presumed Chl (1).14 More recently, it was classified semisystematically as catabolites.18 From a preparation with Pheo a as the sub- a 1-formyl-19-oxo-16,19-dihydrophyllobilane,5,8 see strate by using an enzyme active extract of senescent leaves Scheme 1. The structure of 1 indicated oxidative ring open- of oil seed rape (Brassica napus), a rather instable blue-fluo- ing of the Chl macrocycle at the ‘northern’ meso position, rescent compound became available in small quantities, be- between C4 and C5, thus, representing a 4,5-secophytopor- lieved to be a Chl catabolite. By spectroscopic means its phyrinoid.4,13 This cleavage site contrasted with all expecta- structure was, again, revealed as a linear tetrapyrrole, 19 tions from Chl chemistry,15 but turned out to be strikingly closely related to 1, and to three polar Brassica napus NCCs reminiscent of the site of ring opening in heme break- that had meanwhile been found in senescent cotyledons of down.16 However, in contrast to the typical colored bilins oil seed rape20 (typical UV/Vis absorption spectra are col- from heme breakdown,9,16 the meso-carbon C5 of Chl a was lected in the reviews5,8). The fluorescent compound was, retained in the formyl group of the NCC 1, and its three re- therefore, phenomenologically named Bn-FCC-2 (5) and maining meso positions were saturated. Additional periph- classified as a fluorescent Chl catabolite (FCC) (see Scheme eral polar functionality of 1 pointed to puzzling catabolic 2).19 The chemical constitution of 5 supported its hypothet- reactions in its formation, raising further questions to its ical role as a biological precursor of the Bn-NCCs 2–4. The biochemical formation and to the general relevance of its molecular formula of the FCC 5 also indicated a close rela- structure for the general problem of Chl breakdown in tionship to 6, involving the mere formal incorporation of higher plants. two oxygen atoms and four hydrogen atoms in the course of the presumed formation of 5 from 6. Hence, the moderately polar FCC 5 was deduced to represent a straight forward Biographical Sketch Bernhard Kräutler studied lumbia University, New York University of Innsbruck, where chemistry at the ETH in Zürich, City) he returned to the ETH to he has been Professor Emeritus where he received his PhD start his own research group. In since October 2015. His current working with Prof. Albert the fall of 1985 he was a visiting research interests include the Eschenmoser. After postdoctor- Professor at the Roger Adams chemistry and chemical biology al studies with Prof. Allen J. Bard Labs of the University of Illinois. of chlorophyll and vitamin B12. (University of Texas, Austin) and In 1991 he became Full Profes- with Prof. Nicholas J. Turro (Co- sor of Organic Chemistry at the Georg Thieme Verlag Stuttgart · New York — Synlett 2019, 30, 263–274 265 Syn lett B. Kräutler Account (‘primary’) product of the breakdown of 6, and was as- 4 The Red Chl Catabolite – Key Ring-Opened signed the specific role as ‘primary’ FCC (pFCC).19 As the rel- Tetrapyrrole Accessed by Partial Synthesis ative configuration of the three stereocenters of pFCC (5) at C82, C12, and C13 was the same as that in Pheo a (6), their Since a red Chl catabolite was unknown in higher plants, absolute configuration was also inferred to be retained. we set out to prepare the likely candidate by partial synthe- Hence, pFCC (5) is a 82R,12S,13S,10Z,16n-1-formyl-19-oxo- sis from the methyl ester of Pheo a (6), in order to help test 19,12,13,16-tetrahydrophyllobilene-b.5,8 the suggested intermediacy of such a red bilin-type Chl However, in extracts of senescent leaves of sweet pep- catabolite in higher plants.