amino acids

163 Pages·2013·9.08 MB·English

by  Francesco Zamberlan

Università degli Studi di Ferrara

DOTTORATO DI RICERCA IN
Scienza Chimiche

CICLO XXV

COORDINATORE Prof. Carlo Alberto Bignozzi

Synthesis and biological applications of non
natural α-amino acids

Settore Scientifico Disciplinare CHIM/06

Dottorando Tutore
Dott. Zamberlan Francesco Prof. Benetti Simonetta

_______________________________ _____________________________
(firma) (firma)

Anni 2009/2013
ii
iii
Allofold. Nothingelseever. Evertried. Everfailed.
Nomatter. Tryagain. Failagain. Failbetter.
–SamuelBeckett
Thereisexcitement,adventure,andchallenge,
andtherecanbegreatartinorganicsynthesis.
—R.B.Woodward
Allamiafamiglia.
Eachihasemprecredutoinme.
iv
SOMMARIO
InquestolavorodiTesièstataaffrontatalasintesiel’applicazioneincampobiome-
dicodi α-amminoacidinonnaturaliintredifferentiprogetti.
Èstatamessaapuntolasintesi“one-pot”diN-acetamidoacrilatodimentileot-
ticamente attivo e puro a partire da reattivi commerciali a basso costo, ottenendolo
in buone rese ed identificando il maggiore sottoprodotto di reazione. Tale acrilato
è stato utilizzato come accettore in reazioni di Michael per la formazione di nuovi
legami C-C e C-S, valutando l’induzione asimmetrica apportata dal (−)-mentolo.
Diversinucleofilisonostatiutilizzatiemolteplicibasitestatepercercarediottenere
una conversione completa. Si è dunque indagata la possibilità di reazione della
molecola con opportuni nucleofili a dare cicli a cinque termini tetraidrotiofenici ed
altri intermedi a struttura di biciclo[3.1.0]esano e biciclo[4.1.0]eptano. Tutto ciò è
statofattonell’otticadiotteneremetodialternativiperlasintesidianaloghiastrut-
tura costretta del glutammato, in precedenza identificati da Ely Lilly come potenti,
selettivieattiviagonistideirecettorimGluR.
Il secondo progetto, svolto presso il laboratorio del Professor Matteo Zanda
all’UniversitàdiAberdeen,riguardalasintesidipiccolipeptidicontenentiarginina
ed analoghi dello stesso amminoacido. I piccoli peptidi sono costituiti dal residuo
argininico accoppiato con differenti basi a struttura picolinica, mentre gli analoghi
contengono un sostituente alfa-metilico ed un triplo legame, od un gruppo triflu-
orometilico ed il triplo legame – purtroppo questa parte del progetto non è stata
conclusa per motivi di tempo. L’interesse per questo tipo di analoghi e peptidi
risale al fatto che l’arginina è il substrato per le Nitric Oxide Synthase, classe di en-
zimi coinvolti nella regolazione del rilassamento muscolare e della pressione san-
guigna attraverso la sintesi di ossido nitrico a partire dal summenzionato ammi-
noacido. I substrati progettati e sintetizzati, grazie alla loro struttura, risultano po-
larizzabili per mezzo di spettroscopia NMR e tecniche particolari con molecole di
paraidrogeno. Così, sarebbe possibile preparare specifici “tracers” per Risonanza
Magnetica, permettendo una facile e sicura rivelazione di malattie attraverso una
sempliceevelocescansione. Infatti,alcunideipeptidisintetizzatiepolarizzatisono
stati testati in vitro e risultano compatibili con l’enzima. Infine, è stato messo a
punto un sistema per la determinazione del contenuto di controione trifluoroac-
etatoall’internodellemolecole.
Il terzo progetto ha riguardato l’individuazione e la messa a punto di una via
alternativaallasintesiapococostodell’amminoacido2,6-dimetil-L-tirosina,unpo-
tenteanalogodellatirosinausatoinpeptidiattivisuirecettorideglioppiacei. Come
reagente di partenza è stato utilizzato un derivato analogo dell’estere di Hage-
mann. Tale reagente è stato utilizzato come punto di partenza per due vie sin-
tetiche: una stereoselettiva grazie all’impiego di un sintone chirale a struttura 2,5-
dichetopiperazinica,edunasecondachehavistocomereazione-chiaveunareazione
diMichaelperl’introduzionedelresiduoamminoacidico.
vi
ABSTRACT
In this PhD Thesis work, the synthesis and the biomedical application of non natu-
ral α-aminoacidswasaccomplishedinthreedifferentprojects.
A “one-pot” procedure for the synthesis of optically active and pure methyl
N-acetamido acrylate starting from cheap and commercial sources was developed
and fine-tuned, obtaining the target compound in good yields and identifying the
major reaction by product. This acrylate was used as acceptor in Michael reaction
conditionsfortheformationofnewC-CandC-Sbonds,evaluatingtheasymmetry
induced by the (−)-menthol. Different nucleophiles were used and different bases
weretestedinordertoobtainafullconversion. Afterthat,reactionoftheaforemen-
tionedmoleculewithopportunenucleophilestosynthesisefive-memberedtetrahy-
drothiophene rings and other intermediates with a bicyclo[3.1.0]hexane and bicy-
clo[4.1.0]heptane structure was investigated. The project was undertaken in order
to find new synthetic ways obtain constrained glutamate analogues, which have
beenidentifiedbyEliLillyaspotent,selectiveandactiveagonistsofmGluRrecep-
tors.
The second project, developed in the laboratory of Professor Matteo Zanda at
theUnivesityofAberdeen,isfocusedonthesynthesisofsmallarginine-containing
peptides and arginine analogues. The small peptides are obtained by simple cou-
pling of the amino acid residue with different picolyl amines, while analogues of
the parent amino acid will contain a triple bond and a methyl or trifluoromethyl
substituent on the α-carbon of the amino acid skeleton. Interest in these two type
of compounds was risen because arginine is a substrate for Nitric Oxide Synthase,
aclassofenzymesinvolvedintheregulationofmuscularrelaxandbloodpressure
through nitric oxide synthesis starting from the aforementioned amino acid. The
designedandsynthesisedsubstratesresultbeingabletopolarisetheirsignalunder
specificNMRconditionsinthepresenceofparahydrogen. Inthisway,apossibility
towards the synthesis of of specific and target-focused tracers for Magnetic Reso-
nance Imaging would be achieved, allowing for a simple and secure detection of
illness via a simple and quick MRI scan. Indeed, some of the synthesised com-
pounds polarise and are substrates for the Enzyme. On the last note, a method for
thedeterminationoftheamountoftrifluoroaceticcounter-ionwasdeveloped.
Thethirdprojectisfocusedonthedevelopmentofanalternativesyntheticroute
forthecheapsynthesisof2,6-dimethyl-L-tyrosine,apotentaminoacidanalogueto
tyrosine and employed in peptides which target opioid receptors. An analogue of
Hagemann’s ester ’s was used as starting point for the entire process and it was
used in two synthetic pathways. The first one is stereoselective in the fact that
involves alkylation of the chiral synthon 2,5-diketopiperazine, while in the sec-
ondonethekey-stepinvolvesaMichaeladditionfortheintroductionoftheamino
acidicresidue.
Contents
Contents vii
ListofFigures xi
ListofTables xvii
Acronyms xix
1 Introduction 1
1.1 Aminoacids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2 Thesynthetictarget–Project1 . . . . . . . . . . . . . . . . . . . . . . . 10
1.2.1 CyclopropanationReaction . . . . . . . . . . . . . . . . . . . . 13
1.2.2 Tandemreactions . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.3 Thesynthetictarget–Project2 . . . . . . . . . . . . . . . . . . . . . . . 19
1.3.1 MRIcontrastingagents . . . . . . . . . . . . . . . . . . . . . . . 19
1.3.2 para-HydrogenInducedPolarisation . . . . . . . . . . . . . . 20
1.3.3 ArginineandNOS . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.4 Thesynthetictarget–Project3 . . . . . . . . . . . . . . . . . . . . . . . 23
1.4.1 Precedingenantioselectivesynthesis . . . . . . . . . . . . . . . 25
1.4.2 Pharmacologicalactivity . . . . . . . . . . . . . . . . . . . . . . 27
1.4.3 Hagemann’sEsterasbuildingblock . . . . . . . . . . . . . . . 29
I FirstProject: SynthesisofBicyclo[3.1.0]hexanesAminoAcids
33
2 Aim 35
2.1 StudiesTowardstheSynthesisofCompoundLY404039 . . . . . . . . 35
2.1.1 StudiestowardtheSynthesisofAppropriateAminoAcrylate 36
2.1.2 Reaction of the N-Acetamido acrylate of (−)-Menthol with
NucleophilesasMichaelAcceptor . . . . . . . . . . . . . . . . 37
CONTENTS viii
2.1.3 SynthesisoftheTetrahydrothiophene-compound . . . . . . . 39
2.2 CyclicketonesandMIRCReactions . . . . . . . . . . . . . . . . . . . . 41
2.2.1 Cyclopentenone . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.2.2 Cyclohexenone . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3 ConclusionsandPerspectives 45
3.1 OnthesynthesisofLY404039 . . . . . . . . . . . . . . . . . . . . . . . 45
3.2 CycloenonesandMichaelReactions . . . . . . . . . . . . . . . . . . . 47
4 Experiments 49
4.1 Onepotpreparationof(1S,2R,5S)-2-isopropyl-5-methylcyclohexyl2-
acetamido-2-propenoate . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2 Synthesisof(−)-Menthyl2-acetamido-4-nitrobutanoate . . . . . . . . 50
4.3 Synthesisof(−)-Menthyl2-acetamido-4-nitropentanoate . . . . . . . 51
4.4 Synthesisof1-ethyl6-(−)-Menthyl5-acetamido-3-acetyl-2-oxohexane-
dioate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.5 Synthesisof(−)-Menthyl3-acetamido-4-hydroxythiophene-3-carboxy-
late . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.6 Synthesisof(−)-Menthyl3-acetamido-4-acetoxythiophene-3-carboxy-
late . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.7 Synthesisof(−)-Menthyl-3-acetamido-4-acetoxysulpholan-3-carboxy-
late . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.8 Synthesisof(−)-Menthyl -3-acetamido-2,3-disulpholan-3-carboxylate 54
4.9 SynthesisofN-tert-butoxycarbonylserinemethylester . . . . . . . . 55
4.10 Synthesisofmethyl2-(bis(tert-butoxycarbonyl)amino)acrylate . . . . 55
4.11 Synthesis of methyl 3-((di-Boc)amino)-4-(2-ethoxy-2-oxoethyl)tetra-
hydrothiophene-3-carboxylate . . . . . . . . . . . . . . . . . . . . . . . 56
4.12 Synthesisofethyl2-nitro-2-(3-oxocyclopentyl)acetate . . . . . . . . . 56
4.13 Synthesisofdiethyl2-(3-oxocyclopentyl)malonate . . . . . . . . . . . 57
4.14 Synthesisofethyl3-oxo-2-(3-oxocyclopentyl)butanoate . . . . . . . . 57
4.15 Synthesisof3-(nitromethyl)cyclopentanone . . . . . . . . . . . . . . . 58
4.16 Synthesisof2-bromocyclopent-2-enone . . . . . . . . . . . . . . . . . 59
4.17 Synthesisofdiethyl2-oxobicyclo[3.1.0]hexane-6,6-dicarboxylate . . . 59
4.18 Synthesisofethyl2-nitro-2-(3-oxocyclohexyl)acetate . . . . . . . . . . 60
4.19 Synthesisofdiethyl2-(3-oxocyclohexyl)malonate . . . . . . . . . . . . 60
4.20 Synthesisofethyl3-oxo-2-(3-oxocyclohexyl)butanoate . . . . . . . . . 61
II Second Project: Synthesis of L-arginine analogues as po-
tential MRI contrast agents 63
5 Aim 65
5.1 Argininecoupledwithpicolylmoieties . . . . . . . . . . . . . . . . . 65
5.1.1 FirstGeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.1.2 PolarisationTests . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.1.3 BiologicalTests . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
CONTENTS ix
5.1.4 SecondGeneration . . . . . . . . . . . . . . . . . . . . . . . . . 69
5.1.5 DeterminationoftheMolecularWeightofthesynthesisedana-
logues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
5.2 α-Methyl-and α-trifluoromethylarginine . . . . . . . . . . . . . . . . 73
5.2.1 α-Methyldoublebondedarginine . . . . . . . . . . . . . . . . . 75
5.2.2 α-trifluoromethylarginine . . . . . . . . . . . . . . . . . . . . . 75
6 ConclusionsandPerspectives 79
6.1 Onthepicolyl-coupledarginines . . . . . . . . . . . . . . . . . . . . . 79
6.2 Onthedouble-andtriplebond-containingα-methyl-andα-trifluoromethyl
arginines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6.3 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
7 Experiments 81
7.1 Firstgenerationpicolyl-coupledarginineanalogues . . . . . . . . . . 82
7.1.1 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
7.1.2 Deprotectionreaction . . . . . . . . . . . . . . . . . . . . . . . . 83
7.2 Secondgenerationpicolyl-coupledarginineanalogues . . . . . . . . . 84
7.2.1 Synthesisoftheprotectedderivatives . . . . . . . . . . . . . . 84
7.2.2 DeprotectionReaction . . . . . . . . . . . . . . . . . . . . . . . 85
7.3 Synthesisof(R)-methyl2-amino-3-hydroxy-2-methylpropanoate . . 86
7.4 Synthesis of (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-
2-methylpropanoate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.5 Synthesis of (R)-methyl 2-((tert-butoxycarbonyl)amino)-2-methyl-3-
oxopropanoate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.6 Synthesisofdiethyl(2-(1,3-dioxoisoindolin-2-yl)ethyl)phosphonate . 88
7.7 Synthesisof(Benzyloxycarbonyl)aminotriphenylphosphorane . . . . 88
7.8 SynthesisofSulfinylamine . . . . . . . . . . . . . . . . . . . . . . . . . 89
7.9 Synthesisof α-trifluoromethylserine . . . . . . . . . . . . . . . . . . . 89
III ThirdProject: StudiestowardtheSynthesisof2’,6’-dimethyl-
L-tyrosine 91
8 Aim 93
8.1 RouteA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8.2 RouteB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8.3 RouteC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
9 ConclusionsandPerspectives 99
10 Experiments 101
10.1 Synthesisofethyl2,6-dimethyl-4-oxocyclohexen-2-encarboxylate . . 102
10.2 Synthesisof3,5-dimethylcyclohexen-2-one . . . . . . . . . . . . . . . 102
10.2.1 ProcedureA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
10.2.2 ProcedureB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
10.3 Synthesisof2,6-dimethyl-4-(pivaloxy)cyclohexen-2-encarboxylate . . 103
CONTENTS x
10.3.1 ProcedureA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
10.3.2 ProcedureB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
10.4 Synthesisof4-methoxy-2,6-dimethylbenzene . . . . . . . . . . . . . . 104
10.5 Synthesisof(4-methoxy-2,6-dimethyl)methanol . . . . . . . . . . . . 104
10.6 Synthesisof2-(chloromethyl)-5-methoxy-1,3-dimethylbenzene . . . . 105
10.7 Synthesisof2-(bromomethyl)-5-methoxy-1,3-dimethylbenzene . . . . 106
10.8 Synthesisof2-(iodomethyl)-5-methoxy-1,3-dimethylbenzene . . . . . 106
10.8.1 ProcedureA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
10.8.2 ProcedureB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
10.9 Synthesisof(3S,6S)-3,6-bis(4-methoxy-2,6-dimethylbenzyl)-1,4-bis((S)-
1-phenylethyl)piperazyn-2,5-dione . . . . . . . . . . . . . . . . . . . . 107
10.10Synthesisof2,6-dimethyl-L-tyrosine . . . . . . . . . . . . . . . . . . . 108
10.11Synthesisofethyl7,9-dimethyl-1,4-dioxaspiro[4.5]dec-7-ene-8-carboxy-
late . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
10.12Synthesisof3,5-dimethyl-4-methylenecyclohex-2-enone . . . . . . . . 109
10.13Synthesisofethyl3-(2,6-dimethyl-4-oxocyclohex-2-en-1-yl)-2-nitropro-
panoate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
10.14Synthesisofethyl3-(4-ethoxy-2,6-dimethylphenyl)-2-nitropropanoate 110
10.15Synthesis of ethyl 2-amino-3-(4-ethoxy-2,6-dimethylphenyl)propan-
oate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
A Polarizationexperiments 113
A.1 CompoundBoc-Arg(Cbz)-N-(pyridin-4-ylmethyl)amide . . . . . . . . 113
A.2 CompoundBoc-Arg(Cbz)-N-methyl-N-(pyridin-4-ylmethyl)amide . 115
A.3 CompoundBoc-Arg(Cbz)-N-(1-(pyridin-4-yl)ethyl)amide . . . . . . . 116
A.4 Compound(R)-methyl2-Boc-Arg(Cbz)-2-(pyridin-4-yl)acetate . . . . 118
B BiologicalAssay 121
B.1 ProtocolfortestingL-arginineanalogues . . . . . . . . . . . . . . . . . 121
B.1.1 IngredientsfortheNOSreaction . . . . . . . . . . . . . . . . . 121
B.1.2 Stocksolutionspreparation . . . . . . . . . . . . . . . . . . . . 122
B.1.3 PreparationoftheAnaloguessolutions . . . . . . . . . . . . . 123
B.1.4 PreparationoftheStopsolution . . . . . . . . . . . . . . . . . . 124
Publication 125
Bibliography 131