Chemo-Enzymatic Synthesis of Enantiopure Aliphatic Amino Acids

130 Pages·2017·5.43 MB·Italian

POLITECNICO DI MILANO
Scuola di Ingegneria Industriale e dell’Informazione
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta”
Corso di Laurea Magistrale in Ingegneria Chimica

Chemo-Enzymatic Synthesis of
Enantiopure Aliphatic Amino Acids

Relatore: Prof. Davide Tessaro

Tesi di Laurea Magistrale di:
Nicolò Rossi
Matricola: 863867
A.A 2017-2018
ABSTRACT

The synthesis of aliphatic amino acids with high optical purity is of major importance in
synthetic chemistry, since such compounds find use in a variety of tailor-mad oligopeptides
with biological or pharmacological activity. Biocatalysis, being based on the exploitation of
enzymes with intrinsical high selectivity, may represent in this view a preferential technology.
In this thesis work, we carry out an enzyme-mediated selective hydrolysis of amino acid
thioesters based on a thienylic core in presence of an organic base (trioctylamine), which
promotes their racemization. In such conditions, a dynamic kinetic resolution takes place, and
we were able to synthesize a series of variously substituted thienylglycines presenting high
enantiomeric excess in high yield. The successive reduction of the thienylic core promoted by
Ni Raney in presence of hydrazine permits to obtain the corresponding long-chain alkylic
amino acid with retention of enantiomeric excess. In conclusion, we were able to devise and
put in practice a new synthetic strategy for the obtainment of long-chain alpha amino acids,
based on the consecutive action of a biocatalyst and an inorganic catalyst which furnishes
products in high yield and excellent enantiomeric excess.
ABSTRACT IN LINGUA ITALIANA

La sintesi di ammonoacidi alifatici ad elevata purezza ottica presenta una grossa importanza,
in quanto tali composti trovano uso, oltre che per se stessi, nella sintesi di oligopeptidi a
struttura nota aventi diverse applicazioni. La biocatalisi, fondata sull'uso di enzimi ad alta
selettività, può rapprentare una via preferenziale in quest'ottica. In questo lavoro di tesi si
sfrutta un'idrolisi enzimatica selettiva di tioesteri di amminoacidi basati su di un nucleo
tienilico in presenza di una base organica che ne favorisce la racemizzazione, in modo da
essere in condizioni di risoluzione cinetica dinamica. Tramite tale metodo, una serie di
tienilglicine variamente sostituite è stata ottenuta con elevato eccesso enantiomerico ed alta
conversione. Una successiva riduzione del nucleo tienilico mediata di Ni Raney ha permesso
di ottenere i corrispondenti amminoacidi alchilichi con ritenzione della purezza ottica. In
conclusione, è stata progettata e realizzata una nuova via di sintesi per amminoacidi a lunga
catena basata sull'azione successiva di un catalizzatore biologico e di un catalizzatore
inorganico.
SUMMARY
ESTRATTO IN LINGUA ITALIANA _________________________________________ 10 
1.1 Amino Acids ___________________________________________________________ 13 
1.2 Synthesis of α-Amino Acids ______________________________________________ 15 
1.2.1 Strecker Synthesis __________________________________________________ 16 
1.2.2 Amination of α-Halogen Acids ________________________________________ 17 
1.2.3 Amination via Molecular Rearrangement _______________________________ 19 
1.3 Synthesis of Entiomerically Pure Amino Acids _______________________________ 21 
1.4 Asymmetric Synthesis ___________________________________________________ 24 
1.4.1 Non-enzymatic Catalytic Asymmetric Synthesis _________________________ 25 
1.4.2 Enzymatic Asymmetric Synthesis _____________________________________ 26 
1.5 Kinetic Resolution ______________________________________________________ 27 
1.5 Deracemization Process _________________________________________________ 28 
1.5.1 Dynamic Kinetic Resolution __________________________________________ 29 
1.5.2 Stereoinversion of Crixivan __________________________________________ 30 
1.6 Fermentation __________________________________________________________ 31 
1.7 Other Resolution Methods ________________________________________________ 33 
1.7.1 Crystallization Procedures ___________________________________________ 33 
1.7.2 Diastereoisomeric Salts ______________________________________________ 35 
1.8 Paper Chromatographic Approaches _______________________________________ 37 
2.1 Introduction ___________________________________________________________ 38 
2.2 Dynamic Enzymatic Resolution of Thioesters ________________________________ 38 
2.2.1 Kinetic α-Proton Acidity of Thioesters _________________________________ 39 
2.2.2 Thioester as Substrates for Hydrolytic Enzyme __________________________ 42 
2.2.3 Demonstration of Dynamic Enzymatic Resolution of Thioesters ____________ 43 
2.2.4 Conclusion ________________________________________________________ 44 
2.3 Previous work _________________________________________________________ 44 
2.3.1 DKR of α-aryl Amino Acid Thioesters _________________________________ 47 
2.3.1 DKR of aryl and aliphatic Amino Acid Thioesters ________________________ 48 
3.1 introduction ___________________________________________________________ 50 
3.2 Preparation of Substrates ________________________________________________ 51 
3.2.1 Production of D,L-2-ThienylGlicyne ___________________________________ 52 
3.2.2 Production of D,L NBoc Thienyl-Glycine _______________________________ 53
3.2.3 Production of D,L NBoc Thinyl-Glycine thioester ________________________ 54 
3.3 Dynamic Kinetic Resolution of D,L NBoc Thienyl-Glycine-Thioester Mediated by
Solution of Subtilisin Carlsberg ______________________________________________ 55 
3.4 Deprotection of Enantiomerically Pure Amino Acids __________________________ 57 
3.5 Production of aliphatic amino acids by reduction with RaNi ____________________ 58 
3.6 Conclusion & Prospects _________________________________________________ 59 
4.1 Procedures for the production of enantiopure nor-Leucine _____________________ 60 
4.1.1 Production of D,L-2-Thienylglycine ____________________________________ 60 
4.1.2 Production of D,L-NBoc-2-Thienylglycine ______________________________ 62 
4.1.3 Production of D,L-NBoc-2-Thienylglycine Thioester ______________________ 63 
4.1.4 Enzymatic hydrolysis of D,L-NBoc-2-Thienylglycine Thioester _____________ 64 
4.1.5 Deprotection of enantiopure NBoc-2-Thienylglycine ______________________ 65 
4.1.6 Reduction of enantiopure 2-Thienylglycine______________________________ 65 
4.2 Procedures for the production of Isoleucina/Alloleucina _______________________ 66 
4.2.1 Production of D,L-3-Thienylglycine ____________________________________ 66 
4.2.2 Production of D,L-NBoc-3-Thienylglycine ______________________________ 68 
4.2.3 Production of D,L-NBoc-3-Thienylglycine Thioester ______________________ 69 
4.2.4 Enzymatic hydrolysis of D,L-NBoc-3-Thienylglycine Thioester _____________ 70 
4.2.5 Deprotection of enantiopure NBoc-3-Thienylglycine ______________________ 71 
4.2.6 Reduction of enantiopure 3-Thienylglycine______________________________ 71 
4.3 Procedures for the production of Heptanoic Amino Acid _______________________ 73 
4.3.1 Production of D,L-Me-2-Thienylglycine ________________________________ 73 
4.3.2 Production of D,L-NBoc-Me-2-Thienylglycine ___________________________ 75 
4.3.3 Production of D,L-NBoc-Me-2-Thienylglycine Thioester __________________ 76 
4.3.4 Enzymatic hydrolysis of D,L-NBoc-2-Thienylglycine Thioester _____________ 77 
4.3.5 Deprotection of enantiopure NBoc-Me-2-Thienylglycine __________________ 78 
4.3.6 Reduction of enantiopure Me-2-Thienylglycine __________________________ 78 
4.4 Procedures for the production of Octanoic Amino Acid ________________________ 79 
4.4.1 Production of D,L-Et-2-Thienylglycine _________________________________ 79 
4.4.2 Production of D,L-NBoc-Et-2-Thienylglycine ____________________________ 82 
4.4.3 Production of D,L-NBoc-Et-2-Thienylglycine Thioester ___________________ 83 
3.4.4 Enzymatic hydrolysis of D,L-NBoc-Et-2-Thienylglycine Thioester __________ 84 
4.4.5 Deprotection of enantiopure NBoc-Et-2-Thienylglycine ___________________ 84 
4.4.6 Reduction of enantiopure Et-2-Thienylglycine ___________________________ 85
4.5 Eluents Used for TLC ___________________________________________________ 86 
4.6 Preparation OPATBC ___________________________________________________ 86 
4.7 HPLC Analysis of the Biotrasformation Product _____________________________ 87 
4.8 HPLC Analysis of the Deprotection ________________________________________ 91 
4.9 HPLC Analysis of the Reduction __________________________________________ 98 
4.10 MS Spectra __________________________________________________________ 102 
4.11 NMR Spectra ________________________________________________________ 111
INDEX OF FIGURES
Figura 1 - Substrati utilizzati _________________________________________________ 12 
Figura 2 - Sintesi norLecina a partire da NBoc-2-ThGlySEt ________________________ 12 
Figure 3 - Amino Acids Classification __________________________________________ 13 
Figure 4 - Heminhendral Crystals _____________________________________________ 34 
Figure 5 - Phase Diagrams __________________________________________________ 36 
Figure 6 - Time course for α-proton exchange for thioesters of α-phenylpropionic acid as
monitored by 1H NMR. H = α- proton signal intensity at each time point. H0 = initial α-
t
proton signal intensity. ______________________________________________________ 41 
Figure 7 - Proton Exchange Rate of Thioester of α-Aryl-Amino Acids _________________ 45 
Figure 8 - Proton Exchange Rates of Thioesters of non-α-aryl-substituted-amino amino acids
________________________________________________________________________ 47 
Figure 9 - CLEA ___________________________________________________________ 49 
Figure 10 - Selected Substrates _______________________________________________ 52 

INDEX OF TABLES
Table 1 - α-Proton Exchange Rate of Different Thioesters (Drueckhammer) ____________ 40 
Table 2- Rates of R-Proton Exchange for R-Phenylpropionate Thioesters (1, X = Ph) ____ 41 
Table 3 - Rates of Enzymatic Hydrolysis of Ethyl Butyrate and Ethyl Thiobutyrate
(µmol/(min*g of enzyme))____________________________________________________ 43 
Table 4 - Resolution of 1l,m with Subtilisin Carlsberg under Nonracemizing and Racemizing
Conditions ________________________________________________________________ 43 
Table 5 summarized the dynamic kinetic resolution of compounds 1, 4-8 _______________ 48 
Table 6 - Results of DKR with CLEA ___________________________________________ 50 
Table 7 - Results of DKR of ThienylGlycine Thioesters _____________________________ 56 
Table 8 - Measured Optical Power of ThienylGlycines _____________________________ 56
INDEX OF SCHEMES
Scheme 1 - Strecker Synthesis ________________________________________________ 16
Scheme 2 - Modified Strecker Syntheses ________________________________________ 17
Scheme 3 - Cahours synthesis, X=Cl or Br ______________________________________ 17
Scheme 4 - Hell-Volhard-Zelinsky _____________________________________________ 18
Scheme 5 - Gabriel Synthesis _________________________________________________ 18
Scheme 6 - Curtius and Sieber Synthesis ________________________________________ 19
Scheme 7 - Darapsky Synthesis _______________________________________________ 20
Scheme 8 - Huang, Lin and Li Synthesis ________________________________________ 21
Scheme 9 - Asymmetric Synthesis ______________________________________________ 22
Scheme 10 - Kinetic Resolution _______________________________________________ 23
Scheme 11 - Deracemization Processes _________________________________________ 23
Scheme 12 - Asymmetric Synthesis of L-DOPA ___________________________________ 26
Scheme 13 - Enzymatic Asymmetric Synthesis of L-t-Leucine ________________________ 27
Scheme 14 - Asymmetric Oxidation (b) _________________________________________ 27
Scheme 15 - Kinetic Hydrolysis (d) ____________________________________________ 28
Scheme 16 - Acid Hydrolysis _________________________________________________ 28
Scheme 17 - DKR by Hydantoinase/Racemase ___________________________________ 29
Scheme 18 - Stereoinversion of Hafner and Wellner ______________________________ 30
Scheme 19 - Stereoinversion of Crixivan ________________________________________ 31
Scheme 20 - Diastereoisomeric Salts ___________________________________________ 36
Scheme 21 - Resonance of Thioester ___________________________________________ 39
Scheme 22 - Racemization of Thioester _________________________________________ 39
Scheme 23 - General System Used to Measure α-Proton Exchange Rate (Drueckhammer) _ 40
Scheme 24 - Hydrolysis of Ethyl butyrate and Ethyl Thiobutyrate with various enzyme ____ 42
Scheme 25 - General System to Measure α-Proton Exchange rate by 1H NMR (Previous
Work) ___________________________________________________________________ 45
Scheme 26 - System Used to Measure α-Proton Exchange Rate of Aryl and Aliphatic Amino
Acid Thioesters ____________________________________________________________ 46
Scheme 27 - DKR of α-Aryl Amino Acid Thioesters ________________________________ 47
Scheme 28 - DKR of Aryl and Aliphatic Amino Acid Thioester with CLEA and DBU _____ 50
Scheme 29 - General Procedure to produce Aliphatic α-Amino Acids _________________ 51
Scheme 30 - Production of D,L-2-ThGly ________________________________________ 52
Scheme 31 - Production of Ethyl-Thienyl Aldehyde ________________________________ 53
Scheme 32 - Protection Reaction ______________________________________________ 54
Scheme 33 - Production of D,L-2-NBoc-ThGly Thioester ___________________________ 54
Scheme 34 - DKR of NBoc-2-ThGlySEt _________________________________________ 55
Scheme 35 - Racemization mechanism __________________________________________ 55
Scheme 36 - Deprotection Reaction ____________________________________________ 57
Scheme 37 - Reduction of Thiophen Ring ________________________________________ 58
Scheme 38 - Synthesis of D,L-2-ThGly (1) _______________________________________ 60
Scheme 39 - Synthesis of D,L-2-ThGly (2) _______________________________________ 61
Scheme 40 - Synthesis of D,L-2-ThGly (3) _______________________________________ 61
Scheme 41 - Synthesis of D,L-NBoc-2-ThGly _____________________________________ 62
Scheme 42 - Synthesi of D,L NBoc-2-ThGly _____________________________________ 63
Scheme 43 - Enzymatic Hydrolysis of D,L NBoc-2-ThGly Thioester ___________________ 64
Scheme 44 - Deprotection Reaction of NBoc-2-ThGly ______________________________ 65
Schema 45 - Reduction of 2-ThGly _____________________________________________ 65
Scheme 46 - Synthesis of D,L-3-ThGly (1) _______________________________________ 66
Scheme 47 - Synthesis of D,L-3-ThGly (2) _______________________________________ 67
Scheme 48 - Synthesis of D,L-3-ThGly (3) _______________________________________ 68
Scheme 49 - Synthesis of D,L-NBoc-3-ThGly _____________________________________ 68
Scheme 50 - Synthesi of D,L NBoc-3-ThGly _____________________________________ 69
Scheme 51 - Enzymatic Hydrolysis of D,L NBoc-3-ThGly Thioester ___________________ 70
Scheme 52 - Deprotection Reaction of NBoc-3-ThGly ______________________________ 71
Scheme 53 - Reduction of 3-ThGly _____________________________________________ 72
Scheme 54 - Synthesis of D,L-Me-2-ThGly (1) ____________________________________ 73
Scheme 55 - Synthesis of D,L-Me-2-ThGly (3) ____________________________________ 74
Scheme 56 - Synthesis of D,L-Me-2-ThGly (3) ____________________________________ 74
Scheme 57 - Synthesis of D,L-NBoc-Me-2-ThGly _________________________________ 75
Scheme 58 - Synthesi of D,L NBoc-Me-2-ThGly __________________________________ 76
Scheme 59 - Enzymatic Hydrolysis of D,L NBoc-Me-2-ThGly Thioester _______________ 77
Scheme 60 - Deprotection Reaction of NBoc-Me-2-ThGly __________________________ 78
Scheme 61 - Reduction of Me-ThGly ___________________________________________ 78
Scheme 62 - Synthesis of D,L Et-2-ThGly (1) _____________________________________ 79
Scheme 63 - Synthesis of D,L Et-2-ThGly (2) _____________________________________ 80
Scheme 64 - Synthesis of D,L Et-2-ThGly (3) _____________________________________ 81
Scheme 65 - Synthesis of D,L-NBoc-Et-2-ThGly __________________________________ 82
Scheme 66 - Synthesi of D,L NBoc-Et-2-ThGly ___________________________________ 83
Scheme 67 - Enzymatic Hydrolysis of D,L NBoc-Et-2-ThGly Thioester ________________ 84
Scheme 68 - Deprotection Reaction of NBoc-Et-2-ThGly ___________________________ 84
Scheme 69 - Reduction of Et-2-ThGly __________________________________________ 85
ESTRATTO IN LINGUA ITALIANA

Gli amminoacidi sono composti organici caratterizzati dalla presenza simultanea di un gruppo
carbossilico (-COOH) e di un gruppo amminico (-NH2). Ne esistono diverse classi, ma la più
importante è quella degli α-amminoacidi, i quali possiedono i due gruppi funzionali connessi
allo stesso atomo di carbonio. La principale caratteristica che rende tali amminoacidi così
interessanti è la presenza, in posizione α, di un centro chirale. Essi trovano apllicazione in
diversi campi, ma vengono usati soprattutto come “building blocks” nella sintesi di svariati
farmaci. Nei primi anni del XX secolo, visto il continuo aumento della richiesta di
amminoacidi varie vie di sintesi furono sviluppate e migliorate. Si passò dall’estrazione alla
sintesi chimica o ai primi processi fermentativi.
Negli anni cinquanta il caso del talidomide, un farmaco usato dalle donne in gravidanza come
sedativo e anti-nausea, determinò un punto di svolta per la sintesi dei farmaci. Il talidomide
veniva fino a quel momento venduto in forma racema, solo che ci si accorse, dopo la nascita
di 10'000 bambini malformati, che effettivamente un enantiomero agiva da farmaco mentre
l’altro incideva negativamente sulla crescita del feto. Quindi furono imposti maggiori
controlli sui farmaci ed iniziò di conseguenza la ricerca a nuovi metodi per sintetizzare
composti enantiomericamemte puri.
Tra le tante tecniche scoperte una è particolarmente interessante: la risoluzione cinetica
dinamica. Essa si basa su un sistema composto da un substrato chirale capace di racemizzare
spontaneamente nelle condizioni di reazione e da un enzima capace di convertire
selettivamente uno solo dei due enantiomeri. In sostanza, da una miscela racema è possibile
ottenere un solo enantiomero convertendo tutto il composto iniziale.
Questo lavoro di tesi si inserisce nell’ampliamento della risoluzione cinetica dinamica
sviluppata dal gruppo di ricerca del prof. S. Servi. Tale tecnica è basata sull’uso di tioesteri di
amminoacidi N-protetti, i quali hanno l’idrogeno in posizione α sufficientemente acido da
permettere la racemizzazione tra i due enantiomeri del composto in presenza di una base.
Inizialmente tale tecnica venne provata su tioesteri di α-amminoacidi aromatici con ottimi
risultati: grande resa ed elevato eccesso enantiomerico. Essa fu condotta tramite l’utilizzo di
un sistema bifasico (acqua/MTBE), di una base organica idrofobica (TOA, triottilammina)
capace di estrarre il protone in posizione α e dell’Alcalasi© come enzima. Purtroppo, per
quanto riguarda i tioesteri di amminoacidi alifatici, tale sistema non risulta funzionare. Infatti,