Chemo-Enzymatic Synthesis of Enantiopure Aliphatic Amino Acids - 2018 - 4 - Rossi

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
4.2.5 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
4.3.5 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. Ht = α- proton signal intensity at each time point. H0 = initial α-
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 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 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 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 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,
a causa della minore acidità dell’idrogeno in posizione α (l’anione corrispondente è meno
delocalizzato), la TOA non si rivela abbastanza forte per estrarre il protone. Si decise allora di
usare la DBU (1,5-diazabiciclo(5.4.0)undec-7-ene) la quale, essendo una base decisamente
più forte, era in grado di promuovere la racemizzazione del composto, come venne dimostrato
da dati NMR. Tale base, però, a contatto con l’acqua si protona per la maggior parte,
rendendo cosi impossibile un’efficiente estrazione del protone dal composto e quindi la sua
racemizzazione. Si decise allora di eliminare la maggior parte dell’acqua dal sistema di
reazione passando ad un sistema di solvente monofasico (tert-butanolo) che potesse dissolvere
allo stesso tempo il substrato, la base, l’acqua necessaria all’idrolisied il prodotto. A tali
condizioni venne ritenuto più adatta una forma immobilizzata dello stesso enzima, detta
CLEA© (Cross-Linked Enzyme Aggregates). La risoluzione cinetica dinamica di tioesteri di
ammino acidi alifatici attraverso un sitema composto da DBU, CLEA e tert-butanolo come
solvente, portò effettivamente alla sintesi dei corrispondenti L-amminoacidi N-protetti con
alta resa ed elevato eccesso enantiomerico. La stessa tecnica è stata poi estesa alla sintesi delle
corrispondenti ammidi enantiomericamente pure partendo sempre dai tioesteri racemi,
impiegando diverse ammine come nucleofilo.
In questo lavoro di tesi è stata ideata e messa in pratica una via alternativa alla produzione di
amminoacidi alifatici, sempre basata però sulla risoluzione cinetica dinamica di tioesteri.
Nello studio precedente si è usato un approccio del tipo “reaction engineering” andando a
modificare le condizioni di reazione per ottenere gli amminoacidi alifatici, mentre nel nostro
caso è stato utilizzato un approccio di “substrate engineering”. L’idea è di “mascherare” la
catena alifatica attraverso un anello tienilico, anche sostituito, il quale, essendo aromatico,
possiede un’elevata acidità dell’idrogeno in posizione α cosi da permettere l’utilizzo del
sistema bifasico; in un secondo momento l’anello tienilico viene ridotto e desolforato,
trasformandosi nella corrispondente catena alchilica. Sono stati usati i seguenti substrati:
Figura 1 - Substrati utilizzati
Quindi, la risoluzione cinetica dinamica è stata eseguita a pH 9 a 45°C in un sistema bifasico
(acqua/MTBE) in presenza di TOA e Alcalasi. Dopo deprotezione dell’azoto, la riduzione
dell’anello è stata condotta in acqua a riflusso, catalizzata da Nichel Raney e impiegando
idrazina come fonte di idrogeno.
Figura 2 - Sintesi norLecina a partire da NBoc-2-ThGlySEt

La risoluzione cinetica dinamica così sviluppata ha ottimi risultati, resa superiore al 99% e
eccesso enantiomerico maggiore del 97%. Anche la riduzione finale conduce a ottimi risultati,
con la conservazione dell’eccesso enantiomerico e elevata resa.
1.Introduction
1.1 Amino Acids
Amino acids are organic compounds characterized by the simultaneous presence of

amino (-NH2) and carboxyl (-COOH) functional groups together with an organic side chain,
specific to each amino acid. In Nature, these chemicals are the monomers of proteins, very
complex molecules that play crucial functions in living organisms. They can be classified
according to the core structural functional groups locations in alpha- (α-), beta- (β-), gamma-
(γ-) amino acids.
Figure 3 - Amino Acids Classification

The α-amino acids, usually bearing four different substituents in position 2 (namely: the
carboxy-group, the amino group, a hydrogen and the side chain), are intrinsically chiral, with
the sole exception of glycine. It is notable that, even if there is the possibility of the existence
of two enantiomers, the D-isomers are uncommon in living organisms. In fact, the α-amino
acids include 22 proteinogenic amino acids, which are the building blocks of proteins and are
all in the L-form. The proteogenic amino acids are: L-Alanine, L-Asparagine, L-Arginine, L-
Aspartic acid, L-cysteine, L-Glutamine, L-Glutamic acid, Glycine , L-Proline , L-Serine ,
L-Tyrosine , L-Histidine , L-Isoleucine , L-Leucine , L-Lysine , L-Valine , L-Methionine , L-
Phenylalanine , L-Threonine , L-Tryptophan, L-Selenocysteine, L-Pyrrolysine (see Figure 1).
Twenty of the proteinogenic amino acids are encoded directly by triplet codons in the genetic
code and are known as "standard" amino acids.
Amino acids were discovered in 19th century, but only in the 1935 it was proven that nine of
them are “essential” to man because they can’t be synthesized by the human organism. This
shows that amino acids are necessary first of all as nutrients for the production of proteins.
Furthermore, many proteinogenic and non-proteinogenic amino acids have important
biological functions. For example, glutamate (standard glutamic acid) and γ-amino-butyric
acid ("GABA", non-standard γ-amino acid) are, respectively, the main excitatory and
inhibitory neurotransmitters1. Nowadays about 500 amino acids are known.
Historically, amino acids are mainly used as additives to animal feed due the fact that such
preparations have low levels or even lack some essential amino acids. For example, soybean
is poor of methionine. In the same field they are also used to chelate metal cations in order to
improve the absorption of minerals from supplements, which may be required to improve the
health or production of these animals2.
This last technology is used also in fertilizers for agriculture to facilitate the delivery of
minerals to plants in order to correct mineral deficiencies.
Amino acids find their use also in the food industry, not only to overcome the shortage of
minerals, as previously described, but also employing some of them, in particular glutamic
acid, aspartame (a dipeptide formed by L-Asp and L-Phe) and tryptophan, for particular
applications. The first one is used as flavour enhancer and the second as artificial sweetener.
The third, in association with histidine acts as an antioxidant to preserve milk powder.
Another simple amino acid, cysteine, is used for the preservation of fruit juices3,4.
In recent years amino acids are used more and more in pharmaceutical industry due to their
structural diversity and functional versatility. In particular, nonnatural amino acids find
application in the synthesis and improvement of drugs or to favour drug-delivery. Many
pharmaceuticals contain α-amino acids as critical components, such as L-Dopa (used for
Parkinson disease) and Taxol (an anticancer drug). For example, Taxol is loaded into protein
nanoparticles, appearing to be less toxic and more effective when used for metastatic breast
cancer treatments5,6. Amino acids can be also used as chemical building blocks for chiral
catalyst their stereogenic properties7.
The use of proteins and peptides as human therapeutics has recently expanded due to:
 discovery of novel peptides and proteins

 a better understanding of the mechanism of action in vivo
 improvements in expression or synthesis of proteins and peptides that closely
resemble fully human proteins and peptides
 improvements in formulation or molecule-altering technologies that have the ability
to deliver polypeptides in vivo with improved pharmacokinetic and
pharmacodynamic properties8.
Amino acids find application also in the field of biodegradable plastics as component of
polymer. They can be part of the main chain or bonded as side chain and these alter the
physical properties and reactivities of the polymers. Polyaspartate, for example, is a
poly(amino acids) with free carboxylic group and when is produced higly linear is fully
biodegradable. TPAs (thermal polyaspartate) are being targeted for three global markets:
performance chemicals, diapers, and agriculture. In agriculture, PAs have been found to
stimulate crop growth by enhancing root development9.
In conclusion, amino acids, in particular α-amino acids, are used for a variety of applications
in industry. In order to correspond at the market demand were developed vary different
methods were developed to efficiently produce theythem, from the chemical synthesis to the
extraction from natural sources
1.2 Synthesis of α-Amino Acids
Although isolations from acidic, alkaline, or enzymic digest of proteins, or from other natural
sources, have sufficed to supply a goodly portion of the amino acids available in the past, and
are in fact still exploited for the large-scale procurement of arginine, asparagine, cystine,
glutamic acid, histidine, hydroxyproline, proline, and tyrosine, such methods none the less do
not currently serve as major source of supply for most of the protein-derived amino acids.
However, a number of practicable chemical synthesis are presently known which permit ready
access to alanine, aspartic acid, glycine, isoleucine, leucine, lysine, methionine,
phenylalanine, serine, threonine, tryptophan, and valine, as well as to a host of other α-amino
acids, on both an industrial and laboratory scale. The more important procedures are as
follows:
 Strecker Synthesis
 Amination of α-Halogen Acids
 Amination via Molecular Rearrangement
1.2.1 Strecker Synthesis
In the 1850, Strecker, when he tried to prepare lactic acid via the acid hydrolysis of the
amino-nitrile intermediate CH3CH(NH2)CN, formed from the interaction of acetaldehyde first
with ammonia, then with hydrocyanic acid, produced alanine instead. So Strecker accidentally
reported the first route for α-amino acids synthesis that could be generally applied.
Scheme 1 - Strecker Synthesis

Already in 1900 many modifications of the Strecker procedure were introduced for the dual
purpose of generally increasing the relatively poor yields secured by the original procedure
and obviating the technical difficulties imposed by handling the higly toxic hydrogen cyanide.
In fact, the main modifications were the use of alkaline cyanides and ammonium salts instead
of hydrogen cyanide and ammonia.
An interesting modification was described in 1889 by Pinner and Spilker10, who obtained the
5-alkylhydantoin (VII) by digestion of the corresponding cyanohydrin (VI) with urea and
treating the reaction mixture with hydrochloric acid. Alkaline hydrolysis of (VII) then yielded
the desired amino acid (IV). This procedure was indeed ignored until 1934 when Bucherer
and his collaborator11,12 demonstrated that 5-substituted hydantoins (VII) could be readily
prepared in high yield upon heating an α-amino-nitrile (III) or a cyanohydrin (VI) with
ammonium carbonate. 5-substituted hydantoins (VII) with an alkaline hydrolysis were
converted into the corresponding amino acids.
During the 1940’s this procedure was extended to a wide variety of α-amino acids due to
commercial aviability of a number of hitherto inaccessible precursor aldehydes. Since the
Bucherer modification of the Strecker procedure has proved economical from the standpoints
of time, labor, and material, it constitutes one of the principal synthetic means currently
employed for large-scale production of α-amino acids13.
Scheme 2 - Modified Strecker Syntheses
The reaction mechanism of the Strecker synthesis start with the carbonyl oxygen of an
aldehyde which is protonated, followed by a nucleophilic attack of ammonia to the carbonyl
group. After subsequent proton exchange, water is cleaved from the iminium ion intermediate.
A cyanide ion then attacks the iminium carbon yielding an aminonitrile. In the second step
instead the nitrile nitrogen of the aminonitrile is protonated, and the nitrile carbon is attacked
by a water molecule. A 1,2-diamino-diol is then formed after proton exchange and a
nucleophilic attack of water to the former nitrile carbon. Ammonia is subsequently eliminated
after the protonation of the amino group, and finally the deprotonation of a hydroxyl group
produces an amino acid.
1.2.2 Amination of α-Halogen Acids
In 1858, Cahours14 treated α-chloroacetic acid with ethanolic ammonia thereby accomplishing
the first synthesis of glycine. Further investigation on this reaction and similar ones conducted
by others demonstrated that the formation of an α-amino acid, via the action of aqueous,
alcoholic, or liquid ammonia on the corresponding α-chloro or α-bromocarboxylic acid, is a
general pathway.
Scheme 3 - Cahours synthesis, X=Cl or Br

More comprehensive study revealed that α-bromo acids are more active than the
corresponding α-chloro analogs15. The ammoniolysis, due to its conveniency and respectable
over-all yields, remains a highly preferred method for the synthesis of α-amino acids.
Furthermore, if the requisite α-halo acids are unavailable, they may be synthesized through
the use of standard organic procedures, such as bromination of the appropriate malonic ester
or the Hell-Volhard-Zelinsky procedure.
Scheme 4 - Hell-Volhard-Zelinsky
Gabriel in the 1889 proposed a method in which ammonia is not used. In this regard, the
method involves the condensation of potassium phthalylamino acid ester with an α-halo ester,
by thermal fusion in the absence of solvent16. The so derived pththalylamino acid ester usually
yields the desired amino acid in good amount upon removal of the blocking substituents by
drastic acid hydrolysis, or by a two-step alkaline-acid hydrolysis. This method is applicable
not only to the introduction of α-amino moieties but to that of ω-amino substituents as well. It
has been extensively employed in the preparation of α,ω-diamino acids17.
Scheme 5 - Gabriel Synthesis

1.2.3 Amination via Molecular Rearrangement
The well-known rearrangement of acid azides to isocyanates, with the concomitant

elimination of nitrogen, was discovered by Curtius in 1890. Utilization of such reaction for
the preparation of α-amino acids was described by Curtius and Sieber18 in 1921 and extended
by the former19 nine years later.
Scheme 6 - Curtius and Sieber Synthesis
This sequence involves the rearrangement of an alkyl- or aryl-malonylazidic acid to the

corrensponding isocyanate upon heating in ether or chloroform solution. The latter, which
undergoes ready cyclization to the amino acid N-carbonic acid anhydride, is then acid-
hydrolyzed to the pertinent amino acid. Preparation of the crucial azidic acid intermediate is
affected by the action of nitrous acid on the half -hydrazide of the corresponding malonic acid
which, in turn, is derived from the treatment of the malonic half-ester with hydrazine. As the
preparation of the substrate from the suitably substituted malonic ester represents a
formidable chore in itself, it becomes apparent that such prolonged and tedious procedure, in
its present form, is of little value for the practicable preparation of α-amino acids20.
Ten years later, Darapsky proposed the use of substituted cyanoacetic esters instead of the
corresponding malonic esters. This permitted to him and coworkers21 to devise a comparable,
but more conveniently accomplished, synthesis of amino acids than that described by Curtius.
Thus, the requisite alkylcyanoacetic ester, which is easily accessible from the sodium
ethoxide-catalyzed condensation of cyanoacetic ester and an alkyl halide, readily affords the
monohydrazide analogous to on interaction with hydrazine in ethanol solution. Subsequent
treatment of with nitrous acid converts it to the azide, which upon heating in ethanol
undergoes a Curtius rearrangement to the corresponding urethane, presumably via a transitory
isocyanate intermediate. Acid hydrolysis of the latter then yields the desired amino acid.
Scheme 7 - Darapsky Synthesis

A procedure related to that just given, as described by Huang, Lin and Li22, is founded on the
use of a Hofmann degradation rather than a Curtius rearrangement as the key reaction. Thus,
saponification of thesuitably alkylated cyanoacetic ester results in the production of the
monoalkylcyanoacetic acid which, in turn, is transformed into the corresponding malonamic
acid by treatment with hot, concentrated sulfuric acid. The action of bromine on an alkaline
solution of then leads to a Hofmann rearrangement with the concominant formation of the
desired amino acid.
R
R
OEt RX OEt OH-
NaOEt COOH
N N
N
O O
H2SO4
R R
-
Br2,OH
H2N COOH H2NOC COOH
Scheme 8 - Huang, Lin and Li Synthesis
1.3 Synthesis of Entiomerically Pure Amino Acids
In the XX century, due to the rapid advances in medicine, a strong struggle to develop
convenient synthetic routes to yield the desidered new complex drugs took place. An aspect,
in particular, was studied as a crucial aspect for the synthesis of pharmaceutical compounds:
the difference of biological activity between enantiomers.
Two enantiomers have the same physical properties, except for their effect on the on the plane
polarized light, but not have the same biological activity. In 1962 Thalidomide, a racemic
drug used by pregnant women against nausea and to alleviate morning sickness, had to be
removed by the market because while an enantiomer had a sedative effect the other had a
strong teratogenic side effect. In fact, between 1957 and 1962, 10’000 infants were born with
malformation of the limbs. Events like this have stimulated legislation on drug regulation
including highly restrictive guidelines for the marketing of synthetic chiral drugs. The
marketing of racemates was indeed not completely interrupted, but rather more controlled
with the introduction, by the FDA (The United States Food and Drug Administration), of
mandatory investigations on the bioavailability and pharmacological effects of the drug with
the collection of all background information on each enantiomer and, possibly, on the racemic
mixture.
In order to avoid these costly investigations, the possibility to synthesize only a single
enantiomer was investigated. In particular, time has been spent on the synthesis of optically
pure amino acids due to the fact that they are important chemical building block of
pharmaceutical compounds, as described in chapter 1.1. Different procedures have been
proposed and developed; these, can be summarised as follows:
 Asymmetric synthesis or desymmetrization

 Kinetic resolution
 Deracemization
 Fermentation
 Other resolution methods
Desymmeritrization is a chemical reaction (or reaction sequence) in which one or more new
elements of chirality are formed in a substrate molecule and which produces the
stereoisomeric (enantiomeric or diastereoisomeric) products in unequal amounts. This type of
reaction starts from a pro-chiral or meso- compound and can obtain a theoretical yield of
100%.
kp P
M
Q
kQ
M = meso-substrate
kP,kQ = stereodivergent reaction rate
Scheme 9 - Asymmetric Synthesis
Kinetic resolution, instead, is based on the use of an enantioselective reaction so one entiomer
is not affected while the other is converted into the desired product. In this case, the
theoretical maximumyield is 50% due to the fact that only half of the starting material reacts.
Scheme 10 - Kinetic Resolution
Deracemization can be divided in three protocols and rely on the transformation of both
substrate enantiomers A and B into a single product of different structure (i.e., P or Q) via
independent enantioconvergent pathways through retention and inversion of configuration
(Scheme 11, enantioconvergent process). Alternatively, the non-reacting substrate enantiomer
may be racemized (in situ) to furnish a dynamic kinetic resolution (DKR) (Scheme 11,
dynamic kinetic resolution). In contrast, deracemization by enantioselective stereo inversion23
of enantiomer a yields substrate enantiomer B as the sole product. This process is sometimes
also denoted as enantiomerization. Stereoinversion is any reaction that inverts the chiral
centre of a compound.
Enantioconvergent process Dynamic kinetic resolution Stereoinversion

kR P
AR PR A
Retention
A
Racemization Inversion
kS
Inversion B
BS QS B Q
A,B = Substrate enantiomers

P,Q = Product enantiomers
Scheme 11 - Deracemization Processes

In a fermentation the desired product is produced from microorganisms. Nowadays it is an
important route to produce complex optically pure molecule because micorganisms can be
genetically modified with recombinant methods. The idea is to modify their genetic code to
obatain the well-defined coumpound in great quantity and high yield.
To the techniques just described other less important resolution methods can be added and be
summarised as follows:
 Spontaneous crystallization procedures

 Diastereoisomeric salts
 Paper chromatographic approaches
Before moving on, it is useful to state that many of these procedures can involve
biotrasformations. Such reactions can be defined as the specific modification of a definite
compound to a distinct product with structural similarity, by use of biological catalysts
(including microorganisms). The biological catalyst can be described as an enzyme, or a
whole, inactivated microorganism containing a single active enzyme or several enzymes
working together24. The essential difference between fermentation and biotransformation is
that in fermentation there are generally several catalytic steps between the substrate and the
product while in a biotrasformation there is only one or two steps. The distinction is also in
the fact that the chemical structures of the substrate and the product resemble one another in a
biotransformation, but not necessarily in fermentation. The use of biotrasformation have many
advantages as high performances beacause enzyme are very selective for reactions and are
able to work in mild conditions. This opportunity has been deeply investigated in the last fifty
years in particular due to development of recombinant method in which recombinant DNA,
molecules of DNA engineered in laboratory, is introduced in a microorganism in order to
improve and modify its activity. So, the application of recombinant DNA technology can
allow to rapidly screen and design biocatalysts for new application.
1.4 Asymmetric Synthesis

Asymmetric synthesis is the process by which a substrate containing no stereogenic elements
is transformed by means of an asymmetric step into the desired chiral product. The advantage
of this technique is the theoretical yield of 100%; obviously, it is necessary to consider also
the cost of the starting material and of the operating conditions.
There are many approaches to achieve the desired final chiral product, such as
enantioselective catalysis, the use of chiral auxiliaries, biotacatalysis, enantioselective
organocatalysis or chiral pool synthesis.
In the enantioselective catalysis, usually is based on a catalyst characterized by chiral ligands,

and this technique is often suitable for industrial scale synthesis. An alternative is constituted
by the use of chiral auxiliaries, which are organic compounds which couple to the starting
material to forman intermediate which can in turn then undergo enantioselective reactions via
intramolecular asymmetric induction. Chiral auxiliariesd must be used in stoichiometric
amounts to be effective and require additional synthetic steps to apply and remove the
auxiliary.
Biocatalysis makes use of biological compounds, ranging from isolated enzymes to living
cells, to perform chemical transformations. Enzymes are often very selective reagent and
work at mild condition with low environmental impact.
Chiral pool synthesis is one of the simplest and oldest approaches for enantioselective
synthesis. A readily available chiral starting material is manipulated through successive
reactions, often using achiral reagents, to obtain the desired target molecule. This route is
attractive in particular if the starting material is inexpensive and synthethic path is not
tortuous.
In general, the asymmetric synthesis can be split in two families: non-enzymatic methods and
enzymatic methods.
1.4.1 Non-enzymatic Catalytic Asymmetric Synthesis
Non-enzymatic catalytic asymmetric synthesis is based on the generation of chirality by the

interaction between a chiral ligand and the starting substrate. The first industrial non-
enzymatic catalytic asymmetric synthesis was developed by Dr William S. Knowles, at
Monsanto Company, who in the 2001 won the Noble Nobel Prize in Chemistry for his work
on asymmetric hydrogenation reactions. Knowles in the 1974 made up synthesis of L-DOPA,
an important amino acid used in the treatment of Parkinson’s desease, using catalytic
asymmetric hydrogenation. He discovered that a cationic rhodium complex containing
DiPAMP, a chelating diphosphine with two chiral phophorus atoms, catalyzes highly
enantioselective hydrogenations of enamides such as A, the enamide precursor of L-DOPA.
In the key step of the syntheses of L-DOPA enamide A is hydrogenated in the presence of a
catalytic amount of [Rh(R,R)-DiPAMP)COD]+BF4 affording the protected amino acid B in
quantitative yield and in 95% ee. A simple acid-catalyzed hydrolysis step completes the
syntheses of L-DOPA25,26.
Scheme 12 - Asymmetric Synthesis of L-DOPA
1.4.2 Enzymatic Asymmetric Synthesis
In recent years, the enzymatic asymmetric synthesis of chiral amino acids has attracted much
attention due to the high atom economy and brief reaction steps in fact using a chiral
biocatalyst, the prochiral starting material is directly transformed into a single enantiomer in
one step. Generally, four major routes for the enzymatic asymmetric synthesis of chiral amino
acids can be followed: (1) asymmetric reductive amination of keto acids; (2) asymmetric
transfer of an amino group to keto acids; (3) enantioselective addition of ammonia to a,b-
unsaturated acids; and (4) aldol condensation of an amino acid to aldehydes.
An important enzymatic asymmetric synthesis is the reductive amination that produce L-tert-
leucine27. This amino acid has been successfully produced on an industrial scale by Degussa
in an enzyme membrane reactor using LeuDH, isolated from Bacillus cereus, with formate
dehydrogenase (FDH), from Candida boidinii, for the regeneration of cofactor at a high
conversion rate (93%) and, enantiomeric excess (ee) (99%).
Scheme 13 - Enzymatic Asymmetric Synthesis of L-t-Leucine
1.5 Kinetic Resolution

In the kinetic resolution the starting material is a racemic mixtures, only one enantiomer is
transformed into the desired product, so a 50% conversion constitutes the maximum yield.
The selectivity of the reaction towards one enantiomer depends on the performances of the
used biocatalyst.
There are many biological procedures that can be applied: (a) the use of an animal to whom a
racemic mixture is fed, followed by the isolation from the urine of one or the other antipode;
(b) an asymmetric oxidation or decarboxylation through the action of microorganism,
whereby one of the enantiomorphs is unaffected and the other is metabolized; (c) the
asymmetric synthesis through the action of protease on N-acylated racemic amino acids,
whereby only the L-antipode in the most favourable cases partakes in a synthetic reaction
with a base to form an insoluble and hence separable derivative, leaving most of if not all the
D-antipode in solution; (d) asymmetric hydrolysis through the action of amidases, esterases
and acylases on the appropriately substituted racemic amino acids28.
Scheme 14 - Asymmetric Oxidation (b)

This last procedure for the resolution of amino acids is a very promising way, it is based on
the presence on the amino acid of a group which may be asymmetrically removed by
hydrolysis. The enzymes used for this purpose can be purified, immobilized, cell free, or in
whole-cell form. Several studies have been made and typically proteins such as amidases or
acylases, lipases and esterases were proven effective in the resolution of racemic mixtures for
the purpose of production of optically active α-amino acids. With a few exceptions, the
method of kinetic hydrolysis could be successfully extended to the resolution of some 60
racemic amino acids by the following procedure:
Scheme 15 - Kinetic Hydrolysis (d)

Where R is H, CH3, etc. Acid hydrolysis of the respective acylated D-amino acids or D-amino
acid amides yields the D-amino acids:
Scheme 16 - Acid Hydrolysis

Among the avantages of this procedure is the fact that it is applicable to the large-scale
production of both optical isomers27,29.
1.5 Deracemization Process

As it previously, deracemization can be split in enantioconvergent process, dynamic kinetic
resoluition and stereoinversion. In all the cases only one enantiomer is in the end obtained. In
order to achieve this purpose, the enantioconvergent procedure needs biocatalysts able to
convert the racemic starting mixture only in the desired enantiomer. On the other side,
apparently, a dynamic kinetic resolution needs a more complex system because, togeter with
an enzyme converting only one of the two enantiomers, an in situ racemization of the not
consumed enantiomer must occur.
Concerning steoreoinversion, this is a very attractive procedure since involves the complete
conversion of a stereisomer into the other. This is useful when in a racemic mixture only one
enantiomer is needed and there is a simple method to convert the other enantiomer into the
valuable one.
Even if, compared with the procedures just described, the traditional KR seems to belong to
an old chemistry era, the majority of chiral molecules of industrial interest are still prepared in
this way. This is mainly due to the cost associated to the replacement of an established
industrial process by a new one, even if intrinsically more convenient.
1.5.1 Dynamic Kinetic Resolution
A successful DKR must posses some fundamental characteristics. Other than a selective
enzyme and a substrate configuration that permits the in situ racemization, the interconversion
rate between the substrate enantiomers must be higher than the biotrasformation rate.
Moreover, the final product must not be prone to racemization.
One of the most interesting and famous DKR was employed by Dagussa after 70’s and todays
it’s commercially applied at a scale of >1000 tons per year for the production of D-
phenylglycine and p-OH-phenylglycine. The reaction starts from a racemic mixture of 5-
monosubstituted hydantois that can racemize spontaneously and, trough enantioselective
biostrasformations, can be converted to the desired amino acid with high yield and high ee. 5-
monosubstituted hydantois can be easily prepared from an aldehyde and isocyanate, or by the
Bucherer–Berg synthesis and similar methods. The system is of special interest because the
proton in the 5-position in the hydantoin ring (it will become the α-hydrogen in the α-amino
acid) is quite more acidic then conventional protons in amino acid esters and amides and
much more acidic then amino acid itself. Thus, the hydantoin can often be racemized in situ
at slightly basic pHs where the enzymes are still stable and active. If these conditions are met,
the amino acid can be obtained in one single enantiomeric form in yields higher than 97%.
R O R O
pH > 8
HN NH or racemase HN NH
R OH D-hydantoinase
L-hydantoinase
O O
R R
COOH HN NH COOH
HN NH2 HN NH2
O
O L-carbamoylase R O
R D-carbamoylase
COOH COOH
H2N H2N
L-AA D-AA
Scheme 17 - DKR by Hydantoinase/Racemase

Theoretically, both the enantiomers can be produced but in reality the commercial application
of the hydantoinase process is still limited to the production of D-amino acids. Processes for
the production of L-amino acids are limited by low space-time-yields and high biocatalyst
costs. It is useful to notice that, due to the attractiveness of the hydantoinase process, Dagussa
has started a R&D program to establish a commercially feasible process for the production of
natural and nonatural L-amino acids. Recently, a new generation of an L-hydantoinase
process was developed based on a tailor-made recombinant whole-cell biocatalyst. Further
reduction of biocatalyst cost by use of recombinant Escherichia coli cells over expressing a
hydantoinase, a carbamoylase and an hydantoin racemase from Arthrobacter sp. DSM 9771
was achieved. To improve the hydantoin-converting pathway, expression levels of the
different genes were balanced on the basis of differences in the specific activities of the
enzymes. This has been accomplished by using different gene dose coding for the respective
enzymes. This biocatalyst, has been successively modify to obtain very high performances for
the L-amino acids production. So, recombinant methods can be very useful to improve
existing biotrasformations30,31.
1.5.2 Stereoinversion of Crixivan
In 1971, Hafner and Wellner reported the generation of L-alanine 1a and L-leucine 1b from
the corresponding D-enantiomers through the use of porcine kidney D-amino acid oxidase
(DAAO) and NaBH4 (Scheme 18). Although the yield was low, the principle of
stereoinversion was established; the enzyme catalysed oxidation of the D-enantiomer to the
corresponding achiral imino acid 2 followed by in situ reduction by the borohydride
generating a mixture of D- and L-amino acids32.
Scheme 18 - Stereoinversion of Hafner and Wellner

A recent stereoinversion has been investigated by Beard and Turner33: the deracemisation of
DL-piperazine-2-carboxylic acid. It is a component of the HIV-protease inhibitor Crixivan,
and can be obtained in 86% yield and 99% ee with the use of NaCNBH3 rather than NaBH4
and DAAO.
Scheme 19 - Stereoinversion of Crixivan
1.6 Fermentation
Amino acids can be produce in large quantities with the use of microorganism. Historically,
man has used always microorganism for the fermentation in the production of wine and beer
but nowadays we can use them as an alternative to traditional chemical synthesis. In the 1950s
Dr Kinoschita discovered that Corynebacteriam glutamicum can produce easily large
quantities of amino acids. This microorganism was applied mainly for the production of L-
glutamic acid, used as food additive and flavour enhancer in the form of its sodium salt MSG.
Even if fermentation process may appear an easy task there are many aspects that must be
considered. Usally it occurs in batch reactors, and in the production of amino acids are used
bioreactor with sizes from 50 to 500 m3. Before reaching the production reactor a few other
steps have to be carried out. Preparation of inocula is the first crucial step in most
bioprocesses. The purpose of this lab procedure is to obtain a huge amount of stable inocula,
which can be used to run dozens of production batches under the same defined conditions
finally reaching the same result or yield. Inocula have to be validated after preparation in
terms of sterility and productivity before being transferred to production, and often influence
productivity and yield of the bioprocess significantly. Due to the importance of inocula
quality (inoculum size and stability) and quantity (amount of inocula and cell titer), they have
to be tested regularly during their use in order to avoid decrease in productivity. The inoculum
is afterwards propagated in a so called seed train. In the case of bioprocesses with Coryneform
bacteria this means usually 1:10 steps from seed step to seed step. Depending on the scale of
the production, this is performed in shaking flasks or more preferably in bioreactors of
different scale.
The fermentation is much affected by operating condition as pH, temperature, feed rate
carbon source and nirogen sources. For example, addition of CO2 to the process increased the
biomass yield and considerably decreased formation of organic acids in L-lysine production
with leucine and homoserine auxotrophic C. glutamicum. In particular selection of raw
material is essential not only for the final quality and quantity of the product, but also for the
economy of amino acids production. Especially the carbon source represents a major part of
variable production costs. So, an optimization of this parameter is always necessary.
Another aspect must be taken in account, namely the downstream technology. A cost-efficient
downstream process is crucial to reduce investment and production costs in amino acid
production. The separation of biomass is usually the first step of amino acid purification.
Removal of the cell mass is accomplished either by gravitation-based techniques
(centrifugation or decantation) or by filtration. A significant amount of product might be lost
in this biomass removal step which has furthermore the disadvantage of (A) high investment
costs and (B) biomass waste streams. Once the biomass is removed from the product stream
the purification of the product begins. Typical purification steps are chromatographic
technologies, combined concentration/crystallisation steps or combinations of both. What
method or what sequence of methods is used depends on a couple of effectors: the physico-
chemical ties of the amino acid (solubility, isoelectric point), composition of the process
liquid (quality and quantity of by-products), environmental regulations and on the application
of the product (feed or pharmaceutical use). Ion exchange resins are the base for
chromatographic methods. Disadvantages of this common technique are lower concentrations
and increased waste streams leading to higher costs in wasteliquor treatment. On the other
hand chromatographic methods provide usually higher product qualities. After treatment with
ion exchange resin, a further step usually consists in a single crystallisation. If only
crystallisation is applied in product purification, often two or more subsequent crystallisation
steps are necessary. Crystallisation techniques like cooling or vacuum crystallisation are
economically favourable but cannot be applied for all amino acids34.
Like in other processes that used biocatalysts, these procedures can be nowadays applied for
large vatiety of amino acids due to the possibility of genetically modified microorganism with
the recombinant method.
1.7 Other Resolution Methods
The term “resolution” may be defined as a procedure whereby both optical isomerides are
separated and prepared in pure form from a racemic mixture or compound. All the resolution
procedures stem from the studies of Louis Pasteur that are based on the following principles:
 The mechanical separation of crystals possessing requisite hemihedrism

 The differential solubility of diastereoisomeric salts of the racemate with optically
active compounds
 The action of living organisms or of enzymes derived from living organisms which
utilize or attack preferentially one of the two optical antipodes or its derivatives
To the latter principle belong for example Kinetic Resolution and stereo inversion procedure
that are previous described.
1.7.1 Crystallization Procedures
It is the oldest method to separate two enantiomers one from the other. It was discovered by
Louis Pasteur in the 1848 with the separation of the two forms of tartaric acid through
spontaneous crystallization. In his publication he investigated the crystallographic properties
of tartaric acid beacause it can exist in three different forms and, as many other organic
compounds, when it is dissolved in a solvent, has the ability to rotate the plane of linearly
polarized light. Pasteur observed that while tartaric acid crystals exhibited hemihendral faces,
crystals of racemic acid did not. He extended his studies to include many simple and double
salts of the same acids and verified that every case optical activity was accompanied by the
existence of hemihendral facets on the crystals. Pasteur then examined the crystals of
optically inactive sodium ammonium paratrate under the microscope and was very surprised
to note that these crystals did contain hemihendral facets. Moreover, he observed that the
overall assembly appeared to contain equivalent numbers of left-handed and right-handed
crystals. He manually separated the two crystal types and examined these using a polarimeter.
His observation was that the crystals heminhendral to the right rotated the place of linearly
polarized light in a clock-wise manner, while crystals hemihendral to the left rotated the
linearly polarized light in the opposite fashion. Pasteur concluded that while racemic acid
appeared to be a unique species, paratartaric acid actually consisted of a mixture of the two
optically active tartaric acid forms.
Figure 4 - Heminhendral Crystals
It usefull to note that today it’s known that there are four ways in which a racemate can be
crystallized, depending on the substance:
 Conglomerate: If the 'molecules' of the substance have a much greater affinity for the
same enantiomer than for the opposite one, a mechanical mixture of enantiomerically
pure crystals will result. Roughly 10% of racemic chiral compounds crystallize as
conglomerates
 Racemic compound: If molecules have a greater affinity for the opposite enantiomer
than for the same enantiomer, the substance forms a single crystalline phase in which
the two enantiomers are present in an ordered 1:1 ratio in the elementary cell.
 Pseudoracemate: When there is no big difference in affinity between the same and
opposite enantiomers, then in contrast to the racemic compound and the conglomerate,
the two enantiomers will coexist in an unordered manner in the crystal lattice.
 Quasiracemate: A quasiracemate is a mixture of two similar but distinct compounds,
one of which is left-handed and the other right-handed. Although chemically different,
they are sterically similar (isosteric) and are still able to form a racemic crystalline
phase.
The type of crystallization may be known from the phase diagram.
In particular compounds known to crystallize as conglomerates are much easier to resolve

than true racemates, since the resolution step takes place spontaneously upon crystallization.
There is no need to rely on the use of resolving agents, chromatographic solid supports, or the
use of biological or chemical activation process. When possible, the direct crystallization of
one or both enantiomers represent the most economically attractive method for generation of
enantiomerically pure substances.
Nowadays three procedures of separation can be used. The first method involves the
mechanical separation of enantiomorphic crystals, formed simultaneously while the mother
liquor remains racemic. This method is extremely time-consuming to perfom, and impossible
unless the crystals form with well-defined hemihendral faces but usaually is used to obtain the
seed crystals required for other direct crystallization process. In fact, a second method for
enantiomer separation entails the localized crystallization of each enantiomer from a racemic,
supersatured solution. Seed crystal are placed in geographically separated locations in the
crystallization vessel, and these serves as nuclei for the further crystallization of the like
enantiomer.
Another method used on the large industrial scale is known as resolution by entrainment. It is
based on the condition that solubility of a given enantiomer be less than that of the
corresponding racemate. To begin, a solution is prepared which contains a slight excess of
one enantiomer. Crystallization is induced, whereupon the desired enantiomer is obtained as
a solid and the mother liquor is enriched in the other isomer. In a second crystallization step,
the other enantiomer is obtained35,36.
1.7.2 Diastereoisomeric Salts
The previous technique can be applied only at a small quantity of compounds, for example
doesn’t work for chemical forming true racemates. So, another method was developed based
on transformation of the racemic compounds in a salt in which the enantiomers are more
easily separable. The idea is to perform a derivatization reaction that involves the formation of
dissociable diastomer species, which is often a simple salt. The produced salts have different
physical properties in particular, their solubility so that they can be resolved by differential
crystallisation. After the separation, the salt is decomposed to regenerate the enantiomerically
pure starting compound.
This method was applied for the first time again by Pasteur that used alkaloid bases to
separate the optical isomers of D,L-tartaric acid. Racemic bases have in turn been resolved
into their isomerides by the use of optically active acids.
The object is the formation of salts of optically active bases with racemic acids or of optically
active acids with racemic bases leads to diastereomeric mixtures which may be resolved by
the differential solubility of the two components of such mixtures.
(+B) + (D,L) A (+B)(L)A + (+B)(D)A
or
(+A) + (D,L) B (+A)(L)B + (+A)(D)B
Scheme 20 - Diastereoisomeric Salts

Although these may be separated on a small scale by preparative chromatography, the most
generally used method is the fractional crystallization because diastereoisomeric salts usually
present a binary melting point phase diagram with only one eutectic point not at equimolar
point (figure). If the eutectic point is close or at equimolar point can be applied entrainment
procedure due to the fact that diastereoisomeric salt froms a conglomerate.
Figure 5 - Phase Diagrams

After separation the salts may be decomposed by a metathetical reaction involving a stronger
base than (+)B or a stronger acid than (+)A, to yield thereby the free, optically active
isomerides of (D,L)A or (D,L)B.
In any case by means of these reciprocal procedures, a wider variety of optically active acids
and bases has become available as tools for resolution of racemates than were hitherto on
hand from natural sources.
In practical terms, this type of resolution is not so easy to perform. None of the conditions
necessary for a successful resolution can be predicted a priori, and the resolution of each
individual racemate constitutes a separate experimental problem. Moreover, complete
purification of each salt is thus accomplished by tedious sequence of crystallization and
polimetric observations, supplemented wherever possible by examination of crystal forms.
Despite these formidable difficulties, the procedure has in one form or another been
successfully applied to the resolution of a variety of α-amino acids. Emil Fischer converted,
for example, racemic alanine, glutamic acid and aspartic acid to their N-benzoyl derivatives
and mixed them with brucine or strychnine in molar proportions37. The diastereoisomeric salts
separted. The alkaloid was removed by alkaline treatment, and the enantiomorphic
benzoylamino acids were converted to the free amino acids by prolonged refluxing with
strong HCl. The hydrolysis of the benzoyl group occurs at drastic condition and can leads to
some racemization of the product so, Fisher, subsequently employed the more easily
hydrolazyble N-formyl group as substituent in the racemic amino acids.
The gerneral procedure, wether the free, racemic amino acid or some derivative therefor is
employed, is as follows. The racemate is dissolved or simply suspended in the desired
solvent, usually water or an alcohol-water mixture, and the solution warmed. If the racemate
is monobasic or monoacid, 1 molar equivalent of the optically active base or acid, is added.
The mixture is warmed until solution occurs, filtered clear and allowed to cool to some
desired temperature over a period of time. When the precipitation appears to have come to an
end, the crystals are filtered off and washed briefly with cold solvent, dried, and weighed.
The method just is described it’s a simple way to achieve good resolution of racemic mixture
and until today a large number of reactant and conditions have been proposed and
investigated36,38.
1.8 Paper Chromatographic Approaches

A very particular way to achieve a resolution is based on the possible different affinity
between enantiomer with the solid phase or with the solvent in a chromatography. Several
studies have been made on this procedure, one of the first was that of Mason and Berg. They
noted that, with Whatman paper No.1 and solvent mixture of four volumes of methanol plus
two volumes each of n-butanol, benzene, and water, L-kynurenine migrated somewhat faster
than did D-kynurenine, and that DL-kynurenine yielded two fluorescing spots of the same
color39.
2. DKR of Substituted and Unsubstituted Amino Acid
Thioesters
2.1 Introduction
One of the most interesting ways to match the market demand of enantiomerically pure amino
acids is the dynamic kinetic resolution. Enzymatic dynamic kinetic resolution is a very
attractive possibility due to environmental and economic reasons and especially because
nowadays it is easy to improve and modify the properties of enzyme by the use of
recombinant method.
In this chapter, a new path to produce enantiopure amino acids is reported, based on the use of
thioesters as substrates. They possess particular ability like D,L-5-substituted hydantoin used
by Degussa to produce unnatural amino acids at 100% chemical and optical yield (chapter
1.5.1).
2.2 Dynamic Enzymatic Resolution of Thioesters40
A successful DKR is based on the following boundaries: the substrate must be able to
racemize in the reaction conditions; the selective agent (e.g. the enzyme) must react with only
one of the two starting enantiomers; the racemization rate must be higher than the enzymatic
reaction rate.
Drueckhammer first proposed the thioesters of carboxylic acids having chiral centers at the α-
carbon as possible substrates for DKR. In contrast to an oxoester, the α-proton of the thioester
was sufficiently acidic to permit continuous racemization of the substrate by base-catalysed
deprotonation. This concept was demonstrated by the work of Tina L. Amyes and John P.
Richard, in which the exchange rate for deuterium of the α-protons of ethyl thioacetate and of
acetone in 3-quinuclidinone buffers in D2O at 25°C was followed by 1H-NMR spectroscopy.
From these experiments it was found that the resonance overlap of the lone-pair electrons on
the sulphur atom with the carbonyl group of the type Z, which would tend to decrease the ease
of formation of an adjacent carbanion, is an important factor41.
O O-
RS R' RS+ R'

Z
Scheme 21 - Resonance of Thioester
The difference between thioester and oxoester was also initially demonstrated by
Drueckhammer with an α-phenylthio propionate thioester 1a (R = Et, X = PhS), the
phenylthiol group also contributing to the acidity of the α-proton (Scheme 22). So, it was
expected that this procedure could be applicable to a variety of carboxylic acids having a
chiral center and a proton at the α-carbon.
Scheme 22 - Racemization of Thioester

However, several factors regarding the general applicability of this procedure were unknow,
then Drueckhammer started to study the real acidity of the α-proton of thioesters of other
carboxylic acids and the general utility of hydrolytic enzymes as catalysts for enantioselective
thioester hydrolysis.
2.2.1 Kinetic α-Proton Acidity of Thioesters
The major goal of the work of Drueckhammer has been to investigate the enforceability of
this procedure to thioesters of acids having inherently lower α-proton acidity. Studies of α -
proton exchange with deuterated solvent were undertaken with the ethyl thioesters of several
carboxylic acids to evaluate their potential for racemization during the course of enzymatic
resolution. These studies were conducted using triethylamine in toluene-d8 with CD3OD
added as a source of exchangeable deuterium (Scheme 23). Previous studies have shown the
rate of α-proton exchange under these conditions to be similar to that under biphasic
conditions which mimic the enzymatic resolution, in which racemization occurs in the organic
phase42. The ethyl thioesters of the α -thio, α -halo, and α -azido propionates all appear
sufficiently acidic for resolution coupled with racemization over 2-3 days. In contrast, the
dichlorophenoxy, 3-benzoylphenyl, and phenyl compounds all appear insufficiently acidic for
practical dynamic resolution.
Scheme 23 - General System Used to Measure α-Proton Exchange Rate (Drueckhammer)

Table 1 - α-Proton Exchange Rate of Different Thioesters (Drueckhammer)
Further investigations were made on phenylpropionate thioester, even if the lower α-proton
acidity it serves as a model for the α-arylpropionate class of nonstereoidal antiinfiammatory
drugs. In particular, studies were undertaken to determine if modification of the S-alkyl
moiety could enhance the α-proton acidity (Figure 4).
Figure 6 - Time course for α-proton exchange for thioesters of α-phenylpropionic acid as
monitored by 1H NMR. Ht = α- proton signal intensity at each time point. H0 = initial α-
proton signal intensity.
Table 2- Rates of R-Proton Exchange for R-Phenylpropionate Thioesters (1, X = Ph)
The allyl thioester showed slightly enhanced α-proton acidity relative to the ethyl thioester.
The benzyl and phenyl thioesters showed further enhanced rates while the propargyl and
trifluoroethyl thioesters exhibited rates of α-proton exchange enhanced approximately 5-fold
and 20-fold relative to the ethyl thioester. The propargyl and trifluoroethyl thioesters appeared
sufficiently acidic to allow racemization-coupled resolution over the course of a few days.
The relative acidity of the thioesters in Table can be correlated with the group
electronegativity values of the functional group attached to the thiomethylene group. While
the ethyl thioester 1h has a methyl group attached to this methylene, the allyl 1i and benzyl 1j
thioesters have the more electronegative vinyl and phenyl groups, respectively. This greater
electronegativity and the resulting enhanced acidity of 1i,j relative to the ethyl thioester 1h is
attributed to the greater S-character of the orbitals used in the bond to the thiomethylene
carbon. The propargyl thioester 1l, having the more electronegative alkynyl group, and the
trifluoroethyl thioester 1m, having the very electronegative trifluoromethyl group, exhibit
even greater acidity. The phenyl thioester 1k has the phenyl group attached directly to the
thioester sulfur atom with no intervening methylene group. However, the kinetic acidity of
this thioester was only slightly enhanced over that of the benzyl thioester. So, phenyl
thioesters are thus probably less attractive substrates for dynamic resolution than the others.
Thiophenol and allyl and benzyl thiols are commercially available and are very inexpensive.
Trifluoroethanethiol is somewhat expensive while propargyl thiol is to our knowledge not
commercially available but is readily prepared from the inexpensive propargyl chloride. The
results of R-proton exchange studies suggest that thioesters of these thiols may be valuable
substrates for dynamic enzymatic resolution of acids for which the simple ethyl thioesters are
not sufficiently acidic.
2.2.2 Thioester as Substrates for Hydrolytic Enzyme
In order to achieve enzymatic resolution of a thioester, an enzyme must be identified which

catalyses hydrolysis of the thioester substrate and which exhibits sufficient enantioselectivity
for the desired stereoisomer. In this view, Drueckhammer, due to the fact that the ability of
common hydrolytic enzymes to hydrolyze thioester substrates had not been well documented,
investigated the hydrolysis of thioester carried out by many enzymes commonly used in
enzymatic resolution of oxoester (Scheme 24). As showed in table 3 only pig liver esterese
was a very poor catalyst for hydrolysis of the thioester.
Scheme 24 - Hydrolysis of Ethyl butyrate and Ethyl Thiobutyrate with various enzyme
Table 3 - Rates of Enzymatic Hydrolysis of Ethyl Butyrate and Ethyl Thiobutyrate
(µmol/(min*g of enzyme))
2.2.3 Demonstration of Dynamic Enzymatic Resolution of Thioesters
As consequence of the promising results the Drueckhammer proposed some DKR of

thioesters. For this further demonstration 1l and 1m were selected as substrate. A survey of
several enzymes identified subtilisin Carlsberg as having moderate enantioselectivity in
hydrolysis of the propargyl thioester of R-phenyl propionate.
Hydrolysis of the propargyl thioester 1l under racemizing conditions taken to 95% completion
gave product in 80%, instead the trifluoroethyl thioester, which has a greater rate of
racemization, gave product in 83% ee upon hydrolysis to 97% completion under racemizing
conditions.
Table 4 - Resolution of 1l,m with Subtilisin Carlsberg under Nonracemizing and Racemizing
Conditions
In addition, also the DKR of dichlorophenoxypropionate thioester was investigated: as a

result of its lower racemization rate, it posed further challenges due to their susceptibility to
nonenzymatic hydrolysis. This enhanced rate of nonenzymatic hydrolysis is attributed to the
inductive effect of the dichlorophenoxy group, which is much greater than the inductive effect
of the phenyl group. So, the applicability of a successful DKR depends by the structure of the
substrate. Remembering that the nonenzymatic hydrolysis precluded efficient enzymatic
dynamic resolution by hydrolysis.
2.2.4 Conclusion
The work reported by Drueckhammer paved the way to DKR based on the use of thioesters as
substrates. It also demonstrated that the choice of thiol mojety can have large effect on the α-
proton acidity of thioester and that many thioesters investigated can be sufficiently able to
perform a good dynamic resolution. Thiophenol has been identified as one of the most
interesting substrate. Instead, regarding the hydrolytic enzyme, the high activity and
enantioselectivity in oxoester hydrolysis does not guarantee high activity and
enantioselectivity in hydrolysis of a thioester of the same acid. In the end, subtilisin Carlsberg
has been identified as advantageous biocatalyst.
2.3 Previous work43
Starting from the important results obtained by Drueckhammer, in our previous work was
investigated the possibility to apply a DKR based on thioesters to produce amino acids. Also
in this case, several factors regarding the general applicability of this procedure were
unknown, including the acidity of the α-proton of thioesters of amino acids, and the influence
played by several factors like the amino acids class, the hydrolytic enzyme, the thioester
moiety, the protective group on α-NH2.
So, the α-proton exchange rate of different classes of amino acid thioester were measured.
Initially were studied aryl amino acid thioesters and after non-α-aryl-substituted and aliphatic
amino acid thioesters. Kinetics of racemization were performed by 1H-NMR exchange
experiments in which 20 mg of aryl-substrate was dissolved in 500 μL of DMSO-d6 and 100
μL of CD3OD and 0.5 eq. of TOA was added at 31°C. No exchange was observed in the
absence of TOA and control experiments with the corresponding oxoesters and acids showed
no measureable proton exchange under similar conditions. The presence of the protective
group doesn’t impede the racemization.
O O
R
S R
H S
HN O trioctyl amine /DMSO
HN D O
CD3OD, T=304°K
O
O
1 R = Ph 4 R = 4-Cl-Ph 5 R = 4-F-Ph
6 R = 2-Cl-Ph 7 R = 2-F-Ph 8 R = 2-thienyl
2 = control N-Boc-Phgly-OEt
Scheme 25 - General System to Measure α-Proton Exchange rate by 1H NMR (Previous

Work)
Comparison of racemization rates of the group of rings substituted phenyl-glycines allows
quantifying the effect of electron withdrawing groups present in the molecules. The data
reported in Figure 7 are explained taking in account steric and electronic effects. The thienyl
group is the most electron withdrawing group, followed by the benzene group substituted in
the para-position with a halogen, then the phenyl group itself, and at the end the less efficient
substituent seems to be the phenyl with a halogen in the orto-position.
100,00
 proton exchange rate (%)
90,00
80,00
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
0 20 40 60
time (minutes)
80 100 120 140
Nboc-Phegly-SEt (1) Nboc-4 Cl-PheGly-SEt (4)

Nboc-2 Cl-PheGly-SEt (6) Nboc-2 F-PheGly-SEt (7)
Nboc-4F-PheGly-SEt (5) NBoc-2-thienylGly-SEt (8)
Nboc-Phegly-OEt (2)
Figure 7 - Proton Exchange Rate of Thioester of α-Aryl-Amino Acids

In order to extend the applicability of the base catalyzed dynamic kinetic resolution set of
different substrates with defined structural differences (Scheme 26). In additional, from these
further experiments were evaluated the effect of the nature of the thioester (ethyl vs. benzyl)
and the impact of the presence (or absence) and position of an aromatic ring in the N-Boc
amino acid derivatives.
The success of the DKR method depends from the relatively high acidity of the α-proton
which is ensured by the aromatic ring on the α-carbon. In non α-aryl- substituted amino
thioester the acidity of the α-H is lower and therefore their racemization is more difficult:
TOA is not strong enough to promote racemization. pKa of thioesters of amino acid
derivatives increases markedly from α-aryl-substituted compounds to aliphatic one. Then,
from a survey of several non-nucleophilic organic bases was identified diazobycycloundecene
(DBU) as the most effective base for a DKR system.
The reactions were carried out in the same apparatus and in the same condition, exception for
the substitution of TOA with DBU. Also in this case no exchange was observed in absence of
the base and the corresponding oxoesters showed no measurable proton exchange under
similar condition. The results are reported in figure. The presence of an aromatic group in β-
or γ- position and/or benzyl group in the thioester moiety increases the proton exchange rate;
a similar effect could be predicted by considerations based on electronic effects or
stabilization energy. Instead aliphatic amino acid thioesters are not so influenced by the
presence of an aromatic group in the thioester side.
O
O
R1 R2
S R1 R2
H DBU /DMSO S
HN O D
CD3OD, T=304°K HN O
O
O
9 : R1 = CH2Ph, R2=CH3CH2 18 : R1 = CH2Ph, R2 = CH2Ph
10 : R1 = CH2CH2Ph, R2 = CH3CH2 19 : R1 = CH2CH2Ph, R2 = CH2Ph
12 : R1 = , R2 = CH3CH2 20 : R1 = , R2 = CH2Ph
Scheme 26 - System Used to Measure α-Proton Exchange Rate of Aryl and Aliphatic Amino
Acid Thioesters
Figure 8 - Proton Exchange Rates of Thioesters of non-α-aryl-substituted-amino amino acids
2.3.1 DKR of α-aryl Amino Acid Thioesters
In order to highlight the improvement of the novel DKR system, it was tested only to PheGly
derivatives thioesteres since the present of an aromatic has been identified as important
feature.
Racemization
O O O
water/MTBE
R pH-Stat: pH7.5 R R
S S OH
Alcalase, 36°C
HN O
(0,5 eq trioctylamine)
HN O + HN O
O O O
1 R = Ph 4 R = 4-Cl-Ph 5 R = 4-F-Ph
6 R = 2-Cl-Ph 7 R = 2-F-Ph 8 R = 2-thienyl
Scheme 27 - DKR of α-Aryl Amino Acid Thioesters

The DKR of Compounds 1, 4-8 were performed in biphasic system (water-MTBE) in
presence of Alcalase 2.5L DX from Novozymes (50 U/mL) and TOA (base), the reaction time
course was followed on a pH-stat apparatus (Scheme 27). At the end of the reaction the water
phase was separated from the organic layer and the acidic product was recovered by
extraction. Enantiomeric excess was evaluated with chiral HPLC analysis. The results are
summarized in table. It’s important to stress that the reaction system includes an organic
hydrophobic solvent (MTBE) and water. In MTBE the substrate is solubilized togheter with
the organic base needed for the racemization while the aqueous phase kept at constant pH the
reaction system and it solubilises the enzyme. Moreover, the water is where final product is
accumulated. An important limitation of this system is that can be applied only to substrate
with high α-proton acidity that can be racemized by TOA.
Table 5 summarized the dynamic kinetic resolution of compounds 1, 4-8
Substrate R =
O Conversion % TOA e.e.
R
S Time
HN O (pH-stat) equivalents (L)-N-Boc-aa-OH
O
1 Phenyl- >95% 0.5 48 h >99%
6 2-Cl-Phenyl- >95% 1 96 h >99%
4 4-Cl-Phenyl- >95% 0.5 36 h >99%
7 2-F-Phenyl- >95% 1 12 h >99%
5 4-F-Phenyl- >95% 0.5 20 h >99%
8 2-Thienyl- >95% 0.5 5h >99%
2.3.1 DKR of aryl and aliphatic Amino Acid Thioesters
To extend the method just described to a larger group of compounds must be studied the
racemization conditions of a variety of non-α-aryl-α-substituted amino acids thioesters. These
substrates require not only a different racemizing agent but also different choice of solvent
and form of enzyme to achieve a successful DKR.
As reported in scheme 28, the base identified for aryl and aliphatic amino acid thioesters was
DBU, which unfortunately cannot be used as racemizing agent in water because is so strong
that, at pH=7, it is completely protonated. The solution to this problem was based on the
minimization of water: a system consisting in an organic solvent, compatible with the enzyme
(no denaturation, no inhibition) able to solubilize both the thioester and the base, and
containing the stoichiometric amount of water needed for the reaction. Initially, due to the
absence of water, a subtilisin immobilized on solid ceramic support and supplied by
Novozymes© was chosen among those commercially available and also 2-propanol and tert-
butanol was chosen as solvents to minimize the possibility of enzymatic transesterification.
This novel system presented good results but to further improve this procedure CLEA© was
chosen as biocatalyst. The acronym means: Cross-linked Enzyme Aggregates. This is a
particular enzyme preparation, where the protein is cross-linked with glutaraldehyde and
successively precipitated from the solution giving a powder retaining most of the activity.
(Fig. 6.2) CLEA© is more expensive respect to the previously employed enzyme but it is
easily recyclable showing improved mechanical stability. With the form of the enzyme, the
solvent was also changed due to the fact that in 2-propanol/CLEA© system transesterification
was observed.
Figure 9 - CLEA
Then, the DKR of aryl and aliphatic amino acids was carried out in tert-butanol with CLEA©
as biocatalyst and DBU as racemizing agent (Scheme 28). The L- enantiomer excesses of all
thioesters are in all cases excellent (98-99%).
Racemization
O O O
20 ml tert-Butanol
R S R S + R OH
sub.CLEA, DBU
HN T=37°C HN HN
boc boc boc
Aromatic Aliphatic
11-R= CH3CH2 24-R= CH3CH2
9-R= Ph 22-R= Ph
10-R= CH2Ph 23-R= CH2Ph 12-R= CH3CH2CH2 25-R= CH3CH2CH2
13-R= 26-R=
Scheme 28 - DKR of Aryl and Aliphatic Amino Acid Thioester with CLEA and DBU
Table 6 - Results of DKR with CLEA
Thioester R t Conv% yield% e.e.(L)% αD
9 CH2Ph 12 >99 >98 >99 34,8
10 CH2CH2Ph 40 >99 >94 >98,5 48,1
12 CH3CH2 24 >99 >98,5 >99 24,8
12 CH3CH2CH2 48 98 91 99 23,4
13 110 99 95,8 >99 25,87
3 Synthesis of enantiopure aliphatic amino acids
3.1 introduction
The DKR applied directly to aliphatic amino acid thioester is not an easy task. A possibility
investigated in the previous work, to achieve the synthesis of enantiopure aliphatic amino
acids through DKR, was based on reaction engineering. In fact, the reaction conditions were
modified to permit the racemization of aliphatic amino acid thioesters that due to the lower α-
proton acidity needed a strong base as DBU. Moreover, an expensive biocatalyst was used.
In this work a new route has been investigated, based on substrate engineering. The idea is to
design a thioester substrate with high α-proton acidity, that provide optimal DKR, and also
easily convertible in an aliphatic amino acid. Starting from the amino acid thioester
investigated in the previous, 2-NBoc-thienylglycine thioester was identified as possible
candidate. The latter is the amino acid thioester with the highest α-proton exchange rate
investigated in the previous work and moreover in literature are reported many procedures for
the desulfurisation of a thienyl ring44.
The general procedure can be split in three main parts: first the thienylglycine thioester is
transformed into one of the two enantiomers through a DKR, then the protection is removed
and finally the resulting substituted thienylglycine is reduced into the final desired aliphatic
amino acid (Scheme 29).
Scheme 29 - General Procedure to produce Aliphatic α-Amino Acids
3.2 Preparation of Substrates
The selected amino acid thioesters were reported in Figure 10. Each of them was prepared
from the corresponding aldehyde, subsequently their amino group was protected with Boc2O
and then they were converted into thioesters.
Figure 10 - Selected Substrates
3.2.1 Production of D,L-2-ThienylGlicyne
D,L 2-Thienil-Glycine was produced from the corresponding aldehyde via Strecker synthesis.
Scheme 30 - Production of D,L-2-ThGly

First the aldehyde reacts with NH3 and potassium cyanide in methanol at room temperature.
Initially the carbonyl oxygen of the aldehyde is protonated, followed by nucleophilic attack of
ammonia. Then water molecule is eliminated and there is the formation of an iminium ion
intermediate. Now the cyanide ion reacts with iminium ion intermediate producing the
aminonitrile.
At this point benzaldehyde was added with toluene and a solution 1N of NaOH. At the end of
the reaction, part of the solvent was evaporated and then were added water and ethyl acetate.
The product was in the organic layer and so it was separated and concentrated.
The remaining solid was solved in acetone and was added a solution of HCl 12 N. The
hydrochloric acid reacts forming corresponding amino amide that precipitates. The solution
was filtered and the solid was washed with acetone and let to dry.
In the end the amino amide produced was added at a solution of THF and NaOH 4 M. The
reaction was heated to reflux until the complete formation of the D,L Thienyl-Glycine. Then
the reaction was left to cool; the formation of two phases was observed. The amino acid was
present in the water phase, which was separated from the organic phase. Into the water phase
was added hydrochloric acid until pH 5 and it was let in the refrigerator until crystallization of
the amino acid. In alternative, the amino acid can be recovered with the use of ion-exchange
resin.
This procedure was also applied at all the other substrate . The aldehyde for the first three
substrates is commercially available, but for the D,L Ethyl-Thienyl-Glycine production the
aldehyde was produced from 2-Ethyl-Thiophene (scheme 31 ).
Scheme 31 - Production of Ethyl-Thienyl Aldehyde
This reaction was conducted in N2 atmosphere. In the flask were added 2-Ethyl-Thiophene,
buthyl lithium and TMEDA all dissolved in hexane. At the end of the reaction the flask was
cooled at – 40° C with a bath of dry ice and ethanol and THF and DMF were added.
Then the reaction mixture was let to stir at room temperature. In the end the solution was put
in a becker containing ice, HCl 6 M and ethyl acetate. When the ice was melted the two
phases was separated. The organic phase containing the aldehyde was finally concentrated.
3.2.2 Production of D,L NBoc Thienyl-Glycine
In general for a hydrolysis the amino group must be protected in order to avoid side-reactions
such as ring-closure with the formation of diketopiperazines. So, to avoid possible by-product
the amino group was protected with tert-butyloxycarbonyl group.
Scheme 32 - Protection Reaction
The D,L 2-Thienyl-Glicyne react with Di-tert-butyl dicarbonate in a double liquid phase
system, NaOH solution and tert-butanol. To speed up the reaction was added a drop of N,N-
Dimethylmethanamide. In the end D,L NBoc-2-Thienyl-Glicyne can be easily isolated with a
series of extraction and then it can be concentrated.
This procedure was used for all the starting amino acids. The effect of the protective group on
the racemisation during the biotransformation was also checked in the previous work. (chapter
2.3).
3.2.3 Production of D,L NBoc Thinyl-Glycine thioester
Scheme 33 - Production of D,L-2-NBoc-ThGly Thioester

Thioesters of a protecting amino acid can be prepared by condensation of thiols and
carboxylic acids in the presence of dehydrating agents. In this case, D,L NBoc-2-Thienyl-
Glicyne react with Ethanethiol in the presence of DCC as dehydrating agents. The reaction
was carried out in CH2Cl2 at low temperature in an ice bath. To speed up the reaction was also
added DMAP. The final product was purified through column chromatography with a small
loss in yield.
This procedure was used for all the protecting amino acids. The reactants were purchased
from.
3.3 Dynamic Kinetic Resolution of D,L NBoc Thienyl-Glycine-Thioester
Mediated by Solution of Subtilisin Carlsberg
Scheme 34 - DKR of NBoc-2-ThGlySEt

The DKR of thioesters were performed in biphasic system (water-MTBE) in presence of
Alcalase 2.5L DX from Novozymes (50 U/mL) and TOA (base), the reaction time course was
followed on a pH-stat apparatus at pH 9. At the reaction mixture was also added Na2B4O7 as
buffer to limit excessive pH oscillations. The reaction was carried out in a jacketed reactor
heated at 40 °C. At the end of the reaction the water phase was separated from the organic
layer and the acidic product was recovered by extraction. Enantiomeric excess was evaluated
with chiral HPLC analysis.
Scheme 35 - Racemization mechanism

Remembering that TOA favour the α-hydrogen atom for the continuous racemization. This
procedure was used for all the thioesters previously produced. The results are reported in
table.:
Table 7 - Results of DKR of ThienylGlycine Thioesters
Substates Yield Time ee
NBoc-2-ThGlyOSEt >99% 6h >97%
NBoc-3-ThGlyOSEt >99% 11 h >97%
NBoc-Me-2- >99% 24 h >99%

ThGlyOSEt
NBoc-Et-2-ThGlyOSEt >99% 22 h >99%
Table 8 - Measured Optical Power of ThienylGlycines
Substrates α sper (°) αD α specifico
NBoc-2-ThGly 0.569 100.7 104
NBoc-3-ThGly 0.343 84.7 87
NBoc-Me-2- 0.434 98.63 99

ThGly
NBoc-Et-2-ThGly 0.080 18.6 18

3.4 Deprotection of Enantiomerically Pure Amino Acids
Deprotection of NBoc-Thienyl-glycine from the biotransformation can be performed in

different ways.
Scheme 36 - Deprotection Reaction

In this work we used:
‐ Hydrochloric acid in dioxane

‐ Trifluoracetic acid in CH2Cl2
Both the methods are well known in the literature and usually employed for deprotection of
tert-butyl carbamates45. These procedures have been checked on the D,L NBoc-Thienyl-
Glycine with good result. The problem was that the produced amino acids are not always so
easily isolated. To overcome at this problem the pure amino acid can be recovered with the
use of or ionic exchange resin or, after concentration, or through precipitation caused by the
use of solvent as diethyl ether or acetone. The first possibility ensures high yield and the
second yield about 80%. In particular, the precipitation with solvent worked well with the first
three amino acids.
Also in these reactions enantiomeric excess was evaluated with chiral HPLC analysis. For the
HPLC analysis the derivatization of the products was used then they reacts with OPA-TBC to
became more easily recognisable. So, a small quantity of the product was solved in HCl 0.1 N
and 25 μL of it was added to 175 μL of borate buffer with 50 μL of OPA-TBC.
The use of hydrochloric acid in dioxane was already reported with very good results46. The
ability of TFA solved in CH2Cl2 to eliminate the Boc group was checked on the pure
enantiomers NBoc Me-Thienyl-Glycine and NBoc Et-Thienyl-Glycine with an enantiomeric
excess in both the case of ˃90%.
3.5 Production of aliphatic amino acids by reduction with RaNi
For the reduction of the thiophen ring wasn’t used RaNi© and H2 as reported in literature44
because it was always accompanied by some racemization. Then was tried another way.
Scheme 37 - Reduction of Thiophen Ring

The reduction was performed in water solution in which RaNi© was used as catalyst and
hydrazine as source of hydrogen for the hydrogenation of thiophene group. It is a transfer
hydrogenation mechanism similar to the one reported by Mebane et al., in which RaNi shifts
the hydrogen of 2-propanol to a ketone producing acetone and an alcohol47. The reaction was
heated until reflux.
This procedure was used for all the enantiomerically pure amino acid and the enantiomeric
excess was evaluated with chiral HPLC analysis. Also in this case the derivatization of the
products with OPATBC was used.
The enantiopure nor-Leucine and iso-Leucine was obtained with yield >90%, ee>99% in 2-3
h. While from the reduction of Et-2-ThGly, octanoic amino acid was obtained with yield
>99% and only one peak was observed from HPLC analysis. The product was also confirmed
by 1H-NMR analysis. Instead from the reduction of Me-2-Th-Gly the enantiopure heptanoic
amino acid was obtained but with low yield as confirmed by HPLC and 1H-NMR analysis. On
this last substrate further investigations must be done, due to the very good results obtained
from the other substrates.
3.6 Conclusion & Prospects
In this work a new route to produce aliphatic amino acid has been developed consisting in the
coupling of a DKR and a reduction of a thiophen ring.
The simultaneous presence of Thioester and thiophene as functional group were found
fundamental to carry out this synthetic path. In particular, both increase the acidity of α-
hydrogen that permit the continuous racemisation during the biotransformation while
thiophene group can be design to produce the wanted amino acids. From the experimental
data can be notice that, regarding the biotransformation, more the thiophen ring is substituted
and more is the enantiomeric excess, according with the concept that its functional group
stabilize the thioester deprotonated.
The quantitative yield and high enantiomeric excess, at least 97%, for all the amino acid
produced make this a very promising way but there is space for some upgrade. For now the
use of subtilisin for DKR limit the production at only one of the two enantiomer so can be
interesting find an enzyme for yield the other enantiomer. Another attractive possibility is the
reduction of the thiophen ring directly on the Boc-protected amino acid due to the fact that
amino acids usually are used in protected form so this can increase the value of the final
product.
This final aspect was investigated through four different procedure:
 reduction with sodium borohydride catalysed by NiCl2

 reduction with sodium borohydride catalysed by MoO3
 reduction with H2 catalysed by RaNi
 reduction with isopropanol catalysed by RaNi at reflux
The first two don’t work while the other work but with a yield of ~50 %. From a kinetic study
with HPLC the formation of an intermediate has been observed in the first 30 min so the
cause of the low yield can be the generation of a complex between RaNi and the protected
amino thioesters. From an analysis at the mass spectroscopy the result confirms this
hypothesis in fact the result show a peak of xxx that indicate the presence of a complex. A
possible configuration of the complex is. Then more experiment must be made.
Another aspect that can be improved is the way to produce the substrates because NaCN is an
expensive and toxic compound.
4 Experimental Section and Analytical Data
4.1 Procedures for the production of enantiopure nor-Leucine
4.1.1 Production of D,L-2-Thienylglycine
O
NH2
S O NH
NH4Cl, NaCN 2
PhCHO
NC
NH3,MeOH NaOH
H S N
S
Chemical Formula:
Chemical Formula: C6H6N2S
C5H4OS
Ph
Chemical Formula:
C13H12N2OS
Scheme 38 - Synthesis of D,L-2-ThGly (1)

To a 250 mL flask equipped with a magnetic stirring bar was added Potassium cyanide (1,92
g, 39,1 mmol , 1,10 eq.) and 20 mL of methanol. To this solution was also added Ammonium
chloride, Thiophene-2-carbox aldehyde (4 g, 35,6 mmol, 1.00 eq.), 10 mL NH3 (30%) and
other 20 mL of methanol. The reaction mixture was left to stir at room temperature until it has
become reddish-brown. After 4 hours was added benzaldehyde (3,77 g, 35,6 mmol, 1 eq.), 50
mL of toluene and 36 mL of NaOH 1 N (1 eq.). The reaction was left stir for a night.
The day after a large part of the solvent was evaporated and then was added 60 mL of water
and 60 mL of Ethyl acetate. The solution was transferred in a separatory funnel and the water
solution was discarded (and clean). Instead the organic solution was dried with sodium solfate
and then concentrated under vacuum.
The remaining solid was mixed with 80mL of acetone and it was transferred in 250 mL flask
with a magnetic stirring bar. To the solution was added 4 mL of HCl 12 N (1,3 eq.) and it was
left to stir for 2 hours. Precipitation of a light solid was observed. The mixture was filtered
and the solid was washed with acetone and it was left to dry.
1
H NMR (400 MHz, DMSO-d) δppm 5.25-5.42 (d,1H) 7.02-7.07 (m,1H) 7.30-7.31 (d,1H)
7.58-7.59 (d,1H) 8.18 (s,1H) 8.83-8.92 (t,3H).
13
C NMR (100 MHz, DMSO-d) δppm 51.04, 127.05, 128.78, 128.98, 129.37, 134.46, 135.58,
157.76, 158.12, 158.50, 168.49, 169.10.

The solid was transferred in a 100 mL flask, equipped with a magnetic stirring bar, with 40
mL of NaOH 4 N and 20 mL of THF. The reaction was started and was heated to reflux for a
night. The complete consumption of the starting material was checked by TLC (eluent
“ammine”).
Then, the reaction micture was left to cool at room temperature. The formation of two phase
was observed (the organic phase is brown). The two phase was separated; the product is in
the water phase. The water phase was washed also with THF and it was filtered. To the
remaining solution was added HCl 12 N until pH 5,5. Then the mixture was left to cool in the
refrigerator for a night. The day after the mixture was filtered and the solid was washed with
THF.
Through crystallization the yield obtained was 62 %. To obtain yield >90% can be used ionic
exchange resin (amberlite IRA-400(OH)) but first the amino acid was dissolved in water with
pH > 7. When the amino acid was totally adsorbed by the resin, it was put in a column and
was washed with water. To recover the amino acid, a solution of formic acid was put in the
column until total desorbtion of the product. Then the solution was concentrated under
vacuum and the amino acid isolated. The resin was regenerated with a solution of NaOH 1M.
1
H NMR (400 MHz, DMSO-d) δppm 5.08-5.43 (s,1H) 7.05-7.07 (m,1H) 7.29-7.30 (d,1H)
7.57-7.59 (m,1H) 8.94 (s,3H).
13
C NMR (100 MHz, DMSO-d) δppm 169.35, 159.49, 159.10, 158.72, 158.34, 129.27,
128.35, 127.63, 119.93, 117.06, 114.19, 111.33.
4.1.2 Production of D,L-NBoc-2-Thienylglycine
Scheme 41 - Synthesis of D,L-NBoc-2-ThGly

To a mechanical stirred solution of water (15 mL) was dissolved NaOH (0,23 g, 5,7 mmol,
1,1 eq.). After that the amino acid and 10 mL of tert-butanol were added to the same solution.
To the reaction mixture was dropped very slowly Boc2O dissolved in tert-butanol over 1
hour. In the end a drop of N,N-Dimethylmethanamide was added to the mixture as catalyst to
speed up the reaction. The reaction was left to stir for some hours. The completion of the
reaction was checked by TLC (eluent “acidi”).
The reaction was exstracted two times with exane and then to the water remaining phase was
added HCl 12 N until Ph 2. The water solution was extracted 3 times with ethyl acetate (the
product migrates to organic phase). The organic phases was gathered an then it was dried with
sodium solfate and concentrated under vacuum.
To the remaing product was added 50 mL of exane and the mixture was put in the fridge.
After a night formation of crystal was observed. In the end they was filtered and was washed
with hexane.
Yield 89%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.97 - 1.57 (m, 9 H) 5.34 –
5.55(d,1H)6.89 (dd, J=5.14, 3.42 Hz, 1 H) 7.04(s,1H) 7.7.10 - 7.22 (m, 1 H) 7.61(s,1H)
10.60(s,1H)
13
C NMR (100 MHz, CHLOROFORM-d) δppm 174.36, 172.98, 156.86, 155.24, 140.63,
128.78, 127.51, 126.05, 125.39, 82.46, 81.06, 54.56, 53.48, 28.53, 28.35.
4.1.3 Production of D,L-NBoc-2-Thienylglycine Thioester
Scheme 42 - Synthesi of D,L NBoc-2-ThGly

To a well stirred solution of CH2Cl2 (15mL) were added the substratum ( 500 mg, 1,943
mmol, 1 eq.), ethanethiol ( 362 mg, 5,830 mmol, 3 eq) and DMAP (23,75 mg, 0,194 mmol,
0,10 eq.). The mixture was cooled by an ice bath and after 10 min DCC (441 mg, 2,137
mmol, 1,1 eq.) dissolved in CH2Cl2 was added dropwise. After 30 min the reaction was left to
cool at room temperature. After 2 hours was checked the state of the reaction, to consume
totally the substratum other DCC was added (0,3 eq.). The end of the reaction was checked by
TLC with eluent “acidi”.
The reaction mixture was filtered through porous baffle and then the remainig solution was
concentrated under vacuum. In the end 40 mL of diethyl ether was added to the product and it
was filtered and the was concentrated under vacuum.
The residue was left to solidify and in the end 10 mL of exane was added to obtain a white
solid.
Yield 82%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.07 - 1.26 (m, 3 H)1.36 – 1.38
(s, 9H) 2.72 - 2.94 (m, 2 H)5.42 – 5.47(s,2H) 6.89-7.01(d,1H) 7.01(s,1H) 7.16 - 7.24 (m, 1 H)
13
C NMR (100 MHz, CHLOROFORM-d) δ 197.92, 128.96, 127.04, 126.11, 28.26, 23.66,
14.29
4.1.4 Enzymatic hydrolysis of D,L-NBoc-2-Thienylglycine Thioester
Boc Boc
NH NH
Alcalase, TOA
S S pH 9, 45°C S OH
water/MTBE
O O
Chemical Formula: Chemical Formula: C11H15NO4S

C13H19NO3S2
Scheme 43 - Enzymatic Hydrolysis of D,L NBoc-2-ThGly Thioester

In a 40 mL jacketed reactor heated at 40 °C, equipped with a magnetic stirring bar , were
poured Na2B4O7·10H2O (12,65 mg, 33,2 mmol, 0,10 eq.) and 10 mL of water. To the
solution were added trioctylamine (58,67 mg, 72,34 μL, 0,165 mmol, 0,50 eq.) and 5 mL of
MTBE. Subsequently 5 mL of enzyme alcalase was added. To the mixture was added NaOH
0,1 M until pH by using 718 STAT titrino. Then was added the substratum (100 mg, 0,332
mmol, 1 eq). The pH of reaction was mantained at 9,00 by using 718 STAT titrino.
The reaction was left to stir and heated until the total consumption of starting reactant
(checked by monitoring the performance of pH and added volume recorded by 718 STAT
titrino).
After the reaction mixture was extracted 3 times with AcOEt. Then to the water phase was
added HCl 6 N until pH 2. In the end the mixture was extracted 3 times with AcOEt and then,
the organic phase was dried with NaSO4 and was concetrated under vacuum.
Yield >99%, ee >97%.

4.1.5 Deprotection of enantiopure NBoc-2-Thienylglycine
Scheme 44 - Deprotection Reaction of NBoc-2-ThGly

To a flask were added the substrate and a solution of TFA dissolved in CH2Cl2 (50 μL of
TFA each 1 mL of CH2Cl2). The reaction was left to stir at room temperature. The
completion of the reaction was checked by TLC (eluent “acidi”). At the end of the reaction
the product was concentrated with vacuum and then it was precipitated with the use of
acetone. In the end the amino acid was filtered and recovered. Yield >80 %.
The enantiomeric excess was checked with HPLC. For the analysis the amino acid was solved
in a solution of HCl 0.1N and then it was derivatized with OPA TBC in a solution of borate
buffer (25 μL of product in HCl, 50 μL of OPA TBC, 175 μL of buffer).
4.1.6 Reduction of enantiopure 2-Thienylglycine
Schema 45 - Reduction of 2-ThGly

To a well stirred water solution were added 2ThGly (40 mg, 0,3 mmol ,1 eq) and hydrazine
(40 mg, 0,8 mol, 3 eq.). Subsequently was added 1 mL of RaNi (50% water solution). The
reaction was left to stirr and reflux (50°C). The total consumption of starting substratum was
checked by TLC with eluente AA.
In the end the reaction was filtered to separate RaNi catalyst and then, to the remaining
solution was added an ionic exchanger resin (amberlite IRA-400(OH)) enough to adsorb all
the product of the reduction. After a column was packed with this resin. Fistly, the resin was
washed with water until neutral pH. After the product was eluted with a solution 0,2 M of
formic acid until acid pH. Then the obtained solution was concentrated by a rotavapor
apparatus.
To regenerate the resin, after use formic acid, was washed firstly with water, secondly with
NaOH 0,1 M (until basic pH) and thirdly with water.
Yield 99%. 1H NMR (400 MHz, METHANOL-d + TFA-d) δ ppm 0.83-0.89 (m, 3H) 1.28-
1.35 (m, 4H) 1.78-1.84 (m, 2H) 3.82-3.85 (t, 1H).
4.2 Procedures for the production of Isoleucina/Alloleucina
4.2.1 Production of D,L-3-Thienylglycine

In a 250 mL flask equipped with a magnetic stirring bar was added Potassium cyanide (1,92
g, 39,1 mmol , 1,10 eq.) and 20 mL of methanol. To this solution was also added Ammonium
become reddish-brown.
After 4 hours was added benzaldehyde (3,77 g, 35,6 mmol, 1 eq.), 50 mL of toluene and 36
mL of NaOH 1 N (1 eq.). The reaction was left stir for a night.
NH2 NH2
O O
N HCl aq NH3+
Ph
S Cl-
S
Chemical Formula: C13H12N2OS Chemical Formula: C6H9ClN2OS

Through crystallization the yield obtained was of 60 %. To obtain yield >90% can be used
ionic exchange resin (amberlite IRA-400(OH)) but first the amino acid was dissolved in water
with pH > 7. When the amino acid was totally adsorbed by the resin, it was put in a column
and was washed with water. To recover the amino acid, a solution of formic acid was put in
the column until total desorbtion of the product. Then the solution was concentrated under
1
H NMR (400 MHz, DMSO-d) δppm 4.94-5.08 (d,1H) 7.31-7.32 (d,1H) 7.54-7.59 (m,1H)
7.71-7.72 (t,1H) 8.17 (s,1H) 8.80-8.84 (t, 5H)
13
C NMR (100 MHz, DMSO-d) δppm 51.34, 124.78, 126.66, 128.89, 128.49, 134.66, 168.56.
“ammime”).
was observed (the organic phase is brown). The two phases was separated; the product is in
remaining solution was added HCl 12 N until Ph 5,5. Then the mixture was left to cool in the
THF.
1
H NMR (400 MHz, DMSO-d) δppm 5.11-5.23 (s,1H) 7.21-7.22 (d,1H) 7.58-7.60 (t,1H) 7.69
(s,1H) 8.80-8.84 (d,3H).
13
C NMR (100 MHz, DMSO-d) δppm 169.68, 159.41, 159.03, 158.66, 158.28, 133.40,
129.68, 129.24, 127.76, 127.14, 126.17, 119.95, 117.08, 114.21, 111.34.
4.2.2 Production of D,L-NBoc-3-Thienylglycine
Scheme 49 - Synthesis of D,L-NBoc-3-ThGly

To the reaction mixture was dropped very slowly Boc2O dissolved in tert-butanol over 1 hour.
In the end a drop of N,N-Dimethylmethanamide was added to the mixture as catalyst to speed
up the reaction.
The reaction was left to stir for some hours. The completion of the reaction was checked by
TLC (eluent “acidi”).
product migrates to organic phase). The organic phases were gathered an then it was dried
with sodium solfate and concentrated under vacuum.
After a night formation of crystal was observed. In the end they were filtered and was washed
with exane.
Yield 84%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.05 - 1.58 (m, 9 H)5.12 –
5.49(t,1H) 7.14(s,1H) 7.30 - 7.48 (m, 2 H)7.65(s,1H)10.99(s,1H)
13
125.92, 123.22, 122.69, 81.83, 80.59, 54.69, 53.45, 28.27, 28.05.
4.2.3 Production of D,L-NBoc-3-Thienylglycine Thioester
Scheme 50 - Synthesi of D,L NBoc-3-ThGly

To a well stirred solution of CH2Cl2 (15mL) were added the substratum (500 mg, 1,943 mmol,
1 eq.), ethanethiol ( 362 mg, 5,830 mmol, 3 eq) and DMAP (23,75 mg, 0,194 mmol, 0,10 eq.).
The mixture was cooled by a ice bath and after 10 min DCC (441 mg, 2,137 mmol, 1,1 eq.)
dissolved in CH2Cl2 was added dropwise. After 30 min the reaction was left to cool at room
temperature. After 2 hours was checked the state of the reaction, to consume totally the
substratum other DCC was added (0,3 eq.). The end of the reaction was checked by TLC with
eluent “acidi”.
solid.
Yield 79%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.05 - 1.26 (m, 3 H)
1.36–1.38 (s, 9H) 2.70 - 2.93 (m, 2 H) 5.37 – 5.43(s,1H) 6.99 – 7.01(d,1H) 7.19 - 7.26(m, 2H)
13
C NMR (100 MHz, CHLOROFORM-d) δ 198.70, 129.00, 126.74, 126.25, 123,56, 60.25,
28.32, 23.54, 14.38.
Scheme 51 - Enzymatic Hydrolysis of D,L NBoc-3-ThGly Thioester

poured Na2B4O7·10H2O (12,65 mg, 33,2 mmol, 0,10 eq.) and 10 mL of water. To the solution
were added trioctylamine (58,67 mg, 72,34 μL, 0,165 mmol, 0,50 eq.) and 5 mL of MTBE.
Subsequently 5 mL of enzyme alcalase was added. To the mixture was added NaOH 0,1 M
until pH by using 718 STAT titrino. Then was added the substratum (100 mg, 0,332 mmol, 1
eq). The pH of reaction was mantained at 9,00 by using 718 STAT titrino.
titrino).
Yield >99%, ee >97%.
4.2.5 Deprotection of enantiopure NBoc-3-Thienylglycine
Scheme 52 - Deprotection Reaction of NBoc-3-ThGly

the product was concentrated with vacuum and then it was precipitated with the use of
diethyl-ether. In the end the amino acid was filtered and recovered. Yield >80 %.
in a solution of HCl 0.1N and then it was derivatized with OPA TBC in presence of borate
4.2.6 Reduction of enantiopure 3-Thienylglycine

Scheme 53 - Reduction of 3-ThGly
To a well stirred water solution were added 3ThGly (40 mg, 0,3 mmol ,1 eq) and hydrazine
In the end the reaction was filtered to separate RaNi catalyst and then, to the remaining
solution was added an ionic exchanger resin (amberlite IRA-400(OH)) enough to adsorb all
the product of the reduction. After a column was packed with this resin. Fistly, the resin was
washed with water until neutral pH. After the product was eluted with a solution 0,2 M of
formic acid until acid pH. Then the obtained solution was concentrated by a rotavapor
apparatus.
Yield 99%. 1H NMR (400 MHz, METHANOL-d + TFA-d) δ ppm 0.28-0.36 (m, 6H) 0.40-
0.51 (m, 3H) 0.58-0.69 (m, 2H) 0.85-0.89 (m, 1H) 3.23-3.32 (m,1H).
4.3 Procedures for the production of Heptanoic Amino Acid
4.3.1 Production of D,L-Me-2-Thienylglycine
Scheme 54 - Synthesis of D,L-Me-2-ThGly (1)

g, 39,1 mmol, 1,10 eq.) and 20 mL of methanol. To this solution was also added Ammonium
chloride, Thiophene-2-carbox aldehyde (4 g, 35,6 mmol, 1.00 eq.) , 10 mL NH3 (30%) and

1
H NMR (400 MHz, DMSO-d) δppm 2.41-2.42 (d, 3H) 5.13-5.26 (t,1H) 6.72-6.75 (m, 1H)
7.06-7.09 (m, 1H) 8.80-9.05 (m, 5H).
13
158.51, 168.49.
“ammime”).
was observed (the organic phase is brown). The two phases were separated; the product is in
remaining solution was added HCl 12 N until Ph 5,5. Then the mixture was left to cool in the
THF.
Through crystallization the yield obtained was of 58%. To obtain yield >90% can be used
ionic exchange resin (amberlite IRA-400(OH)) but first the amino acid was dissolved in water
with pH > 7. When the amino acid was totally adsorbed by the resin, it was put in a column
and was washed with water. To recover the amino acid, a solution of formic acid was put in
the column until total desorbtion of the product. Then the solution was concentrated under
1
H NMR (400 MHz, METHANOL-d) δppm 2.47 (d,3H) 5.26 (s,1H) 6.74-6.75 (m,1H) 7.05-
7.05 (d,1H) 7.46-7.48 (m, 3H).
13
C NMR (100 MHz, METHANOL-d) δppm 170.24, 161.01, 160.62, 160.22, 159.82, 159.45,
159.03, 144.48, 132.11, 130.66, 130.51, 129.30, 126.94, 121.09, 118.24, 117.62, 115.40,
114.79, 112.55, 111.97, 57.91, 53.25, 15.25.
4.3.2 Production of D,L-NBoc-Me-2-Thienylglycine
Scheme 57 - Synthesis of D,L-NBoc-Me-2-ThGly

In the end a drop of N,N-Dimethyl methanamide was added to the mixture as catalyst to speed
up the reaction. The reaction was left to stir for some hours. The completion of the reaction
was checked by TLC (eluent “acidi”).
The reaction was extracted two times with hexane and then to the water remaining phase was
product migrates to organic phase). The organic phases were gathered and then it was dried
with sodium sulphate and concentrated under vacuum.
To the remain product was added 50 mL of hexane and the mixture was put in the fridge.
with hexane.
Yield 87%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.02 - 1.59 (m, 9 H) 2.37(s,2H)
5.04-5.43(t,1H) 6.52 -6.53(d,1H) 6.80(d,1H) 7.30 - 7.76 (m, 1 H) 10.41(s,1H)
13
140.56, 139.81, 135,77, 128.92, 128.48, 127.22, 126.27, 125.82, 125.11, 124.82, 80.55, 54.37,
53.41, 28.24, 27.97, 15.26.
4.3.3 Production of D,L-NBoc-Me-2-Thienylglycine Thioester
Scheme 58 - Synthesi of D,L NBoc-Me-2-ThGly

1 eq.), ethanethiol ( 343 mg, 5,528 mmol, 3 eq) and DMAP (22,5 mg, 0,184 mmol, 0,10 eq.).
The mixture was cooled by an ice bath and after 10 min DCC (418,2 mg, 2,027 mmol, 1,1
eq.) dissolved in CH2Cl2 was added dropwise. After 30 min the reaction was left to cool at
room temperature. After 2 hours was checked the state of the reaction, to consume totally the
eluent “acidi”.
solid.
Yield 84%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.06 - 1.27 (m, 3 H) 1.36 – 1.38
(S,9H) 2.37 (s,1H) 2.69 - 2.95 (m, 2 H) 5.32-5.47(d,1H) 6.45 - 6.62 (m, 1 H) 6.78(d,1H)
7.19(s,1H)
13
C NMR (100 MHz, CHLOROFORM-d) δ 140.96, 136.89, 129.00, 127.52, 125.16, 28.31,
23.57, 15.33, 14.34
Scheme 59 - Enzymatic Hydrolysis of D,L NBoc-Me-2-ThGly Thioester

poured Na2B4O7·10H2O (12,09 mg, 31,7 mmol, 0,10 eq.) and 10 mL of water. To the solution
were added trioctylamine (56 mg, 69,124 μL, 0,158 mmol, 0,50 eq.) and 15 mL of MTBE.
Subsequently 5 mL of enzyme alcalase was added. To the mixture was added NaOH 0,1 M
until pH by using 718 STAT titrino. Then was added the substratum (100 mg, 0,317 mmol, 1
eq.). The pH of reaction was maintained at 9,00 by using 718 STAT titrino.
titrino).
Yield >99%, ee >99%.
4.3.5 Deprotection of enantiopure NBoc-Me-2-Thienylglycine
Scheme 60 - Deprotection Reaction of NBoc-Me-2-ThGly

the product was concentrated with vacuum and then the remaining solution was first solved in
basic water and after an ionic exchanger resin (amberlite IRA-400(OH)) was added, enough
to adsorb all the product of the reduction. A column was packed with this resin. Fistly, the
resin was washed with water until neutral pH. After the product was eluted with a solution 0,2
M of formic acid until acid pH. Then the obtained solution was concentrated by a rotavapor
apparatus. Yield > 95%. To regenerate the resin, after use formic acid, was washed firstly
with water, secondly with NaOH 0,1 M (until basic pH) and thirdly with water.
4.3.6 Reduction of enantiopure Me-2-Thienylglycine
Scheme 61 - Reduction of Me-ThGly

To a well stirred water solution were added MeThGly (40 mg, 0,2 mmol ,1 eq) and hydrazine
In the end the reaction was filtered to separate RaNi catalyst with a solution of NH3 1M and
then, to the remaining solution was added an ionic exchanger resin (amberlite IRA-400(OH))
enough to adsorb all the product of the reduction. After a column was packed with this resin.
Fistly, the resin was washed with water until neutral pH. After the product was eluted with a
solution 0,2 M of formic acid until acid pH. Then the obtained solution was concentrated by a
rotavapor apparatus.
Yield 99%. 1H NMR (400 MHz, METHANOL-d + TFA-d) δ ppm 0.86-0.87 (t, 3H) 1.23-1.35
(m, 6H) 1.86-1.92 (m, 2H) 3.89-3.91 (d, 1H).
4.4 Procedures for the production of Octanoic Amino Acid
4.4.1 Production of D,L-Et-2-Thienylglycine
Scheme 62 - Synthesis of D,L Et-2-ThGly (1)

g, 39,1 mmol, 1,10 eq.) and 20 mL of methanol. To this solution was also added Ammonium

1
H NMR (400 MHz, DMSO-d) δppm 1.19-1.23 (t, 3H) 2.75-2.81 (m, 2H) 5.15-5.16 (d, 1H)
6.76-6.77 (d, 1H) 7.09-7.10(d, 1H) 8.81-8.85 (t, 5H).
13
168.49.
“ammime”). Then, the reaction micture was left to cool at room temperature. The formation
of two phase was observed (the organic phase is brown). The two phases were separated; the
product is in the water phase. The water phase was washed also with THF and it was filtered.
To the remaining solution was added HCl 12 N until Ph 5,5. Then the mixture was left to cool
in the refrigerator for a night. The day after the mixture was filtered and the solid was washed
with THF.
Through crystallization the yield obtained was 65 %. To obtain yield >90% can be used ionic
exchange resin (amberlite IRA-400(OH)) but first the amino acid was dissolved in water with
pH > 7. When the amino acid was totally adsorbed by the resin, it was put in a column and
was washed with water. To recover the amino acid, a solution of formic acid was put in the
column until total desorbtion of the product. Then the solution was concentrated under
1
H NMR (400 MHz, DMSO-d) δppm 1.20-1.24 (t, 3H) .77-2.83 (m, 2H) 5.34 (s,1H) 6.80-
6.81 (d,1H) 7.08-7.09 (d,1H) 8.87 (s, 3H).
13
22.82, 15.88.
4.4.2 Production of D,L-NBoc-Et-2-Thienylglycine
Scheme 65 - Synthesis of D,L-NBoc-Et-2-ThGly

In the end a drop of N,N-Dimethylmethanamide was added to the mixture as catalyst to speed
up the reaction. The reaction was left to stir for some hours. The completion of the reaction
was checked by TLC (eluent “acidi”).
product migrates to organic phase). The organic phases were gathered an then it was dried
with sodium solfate and concentrated under vacuum.
with exane.
Yield 85%. 1H NMR (400 MHz, CHLOROFORM-d) δppm 1.07 - 1.27 (m, 3 H) 1.27 - 1.52
(m, 9 H) 2.63 - 2.97 (m, 2 H) 5.06-5.46(d,1H) 6.57 – 6.58 (d,1H) 6.84 (d,1H) 8.01 (s,1H)
10.21(s,1H).
13
C NMR (400 MHz, CHLOROFORM-d) δppm 174.41,173.23, 156.29, 154.95, 136.48,
135.49, 128.55, 127.25, 126.10 ,123.27, 80.55, 54.52, 53.41, 28.28, 23.47, 15.78.
4.4.3 Production of D,L-NBoc-Et-2-Thienylglycine Thioester
Scheme 66 - Synthesi of D,L NBoc-Et-2-ThGly
1 eq.), ethanethiol (326 mg, 5,257 mmol, 3 eq) and DMAP (21,4 mg, 0,175 mmol, 0,10 eq.).
The mixture was cooled by an ice bath and after 10 min DCC (397,7 mg, 1,927 mmol, 1,1
eq.) dissolved in CH2Cl2 was added dropwise. After 30 min the reaction was left to cool at
room temperature. After 2 hours was checked the state of the reaction, to consume totally the
eluent “acidi”.
The residue was left to solidify and in the end 10 mL of hexane was added to obtain a white
solid.
Yield 80%. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.27 (dt, J=10.39, 7.40 Hz, 6
H)1.46 – 1.47 (s,9H) 2.73 - 2.85 (m, 2 H) 2.92 (qd, J=7.42, 1.22 Hz, 2 H) 5.43 – 5.56 (d,1H)
6.65 (dt, J=3.48, 1.07 Hz, 1 H) 6.87-6.88(d,1H)
13
C NMR (100 MHz, CHLOROFORM-d) δ ppm 198.08, 148.59, 126.41, 123.23, 60.11,
28.27, 23.61, 23.44, 15.67, 14.29.
3.4.4 Enzymatic hydrolysis of D,L-NBoc-Et-2-Thienylglycine Thioester
Scheme 67 - Enzymatic Hydrolysis of D,L NBoc-Et-2-ThGly Thioester

poured Na2B4O7·10H2O (17,65 mg, 30,4 mmol, 0,10 eq.) and 10 mL of water. To the
solution were added trioctylamine (53,7 mg, 66,2 μL, 0,152 mmol, 0,50 eq.) and 15 mL of
MTBE. Subsequently 5 mL of enzyme alcalase was added. To the mixture was added NaOH
0,1 M until pH by using 718 STAT titrino. Then was added the substratum (100 mg, 0,303
mmol, 1 eq.). The pH of reaction was mantained at 9,00 by using 718 STAT titrino.
titrino).
Yield >99%, ee >99%.
4.4.5 Deprotection of enantiopure NBoc-Et-2-Thienylglycine
Scheme 68 - Deprotection Reaction of NBoc-Et-2-ThGly

the product was concentrated with vacuum and then the remaining solution was first solved in
basic water and after an ionic exchanger resin (amberlite IRA-400(OH)) was added, enough
to adsorb all the product of the reduction. A column was packed with this resin. Fistly, the
resin was washed with water until neutral pH. After the product was eluted with a solution 0,2
M of formic acid until acid pH. Then the obtained solution was concentrated by a rotavapor
apparatus. Yield > 95%.
4.4.6 Reduction of enantiopure Et-2-Thienylglycine
Scheme 69 - Reduction of Et-2-ThGly

To a well stirred water solution were added EtThGly (40 mg, 0,2 mmol ,1 eq) and hydrazine
checked by TLC with eluent AA.
In the end the reaction was filtered to separate RaNi catalyst with a solution of NH3 1M and
then, to the remaining solution was added an ionic exchanger resin (amberlite IRA-400(OH))
enough to adsorb all the product of the reduction. After a column was packed with this resin.
Fistly, the resin was washed with water until neutral pH. After the product was eluted with a
solution 0,2 M of formic acid until acid pH. Then the obtained solution was concentrated by a
rotavapor apparatus.
Yield 99%. 1H NMR (400 MHz, METHANOL-d + TFA-d) δ ppm 0.89-0.93 (t, 3H) 1.28-1.44
(m, 8H) 1.89-1.94 (m, 2H) 3.94-3.97 (t, 1H).
4.5 Eluents Used for TLC
Eluent AA: ethyl acetate 2, acetic acid 1, water 1, isobutanol 1
Eluent “acidi”: toluene 30, acetone 10, n-BuOH 5, acetic acid 1
Eluent “ammine”: ethyl acetate 10, methanol 2, ammonia solution 0,5
4.6 Preparation OPATBC
The OPATBC solution were prepared by dissolving 10 mg of OPA (ophtaldialdehyde) and 10

mg of TBC (N-tert-butyloxycarbonyl-L-cysteine) in 1 mL of HPLC grade methanol.
4.7 HPLC Analysis of the Biotrasformation Product
The following chromatogram was conducted at T amb with th use as column Chiralcel OD
and as eluente Hexane/MTBE 8:2 + 0.1% of TFA. Flow rate = 1mL/min, at 230 nm. The
sample was dissolved in MTBE.
D,L NBoc-2-ThGly
NBoc-2-ThGly from Biotrasformation

D,L NBoc-3-ThGly
NBoc-3-ThGly from Biotrasformation

D,L NBoc-Me 2-ThGly
NBoc-Me-2-ThGly from Biotrasformation

D,L NBoc-Et-2-ThGly
NBoc-Et-2-ThGly from Biotrasformation

4.8 HPLC Analysis of the Deprotection
The following chromatogram was conducted at 35°C with th use as column Luna 5u C18(2)
and as (Acetate buffer/MeOH 95/5)/MeOH 5:5. Flow rate = 0.8 mL/min. The sample was
dissolved in HCl and it was derivatized with OPATBC, as it described in the procedures.
D,L 2-ThGly
2-ThGly from biotrasformation
D,L 3-ThGly
3-ThGly from biotrasformation
D,L Me-2-ThGly
Me-2-ThGly from biotrasformation
D,L Et-2-ThGly
Et-2-ThGly from biotrasformation

4.9 HPLC Analysis of the Reduction
The following chromatogram was conducted at 35°C with th use as column Luna 5u C18(2)
and as (Acetate buffer/MeOH 95/5)/MeOH 5:5. Flow rate = 0.8 mL/min. The sample was
dissolved in HCl and it was derivatized with OPATBC, as it described in the procedures.
D,L Nor-Leucine
Nor-Leucine from Biotrasformation
L-isoleucine and D-allo-isoleucine

D-isoleucine
Hexanoic Amino Acid from Biotrasformation

D,L Heptanoic Amino Acid
Heptanoic Amino Acid from Biotrasformation

Octanoic Amino Acid
4.10 MS Spectra
NBoc-2-ThGlySEt
Molecular Weight: 301,42 g/mol
NBoc-3-ThGlySEt
NBoc-Me-2-ThGlySEt
NBoc-Et-2-ThGlySEt
2-ThenylGlycine
3-ThienylGlycine
Me-2-ThienylGlycine
Et-2-ThienylGlycine
Nor-Leucine from Biotrasformation (negative ions)

Nor-Leucine from Biotrasformation
Isoleucine from Biotrasformation (negative ions)

Isoleucine from Biotrasformation
Hepatonoic amino acid (neg. ions)

Hepatonoic amino acid
Otanoic amino acid (neg. ions)

Octanoic amino acid
4.11 NMR Spectra
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POLITECNICO DI MILANO

Scuola di Ingegneria Industriale e dell’Informazione

Relatore: Prof. Davide Tessaro

Tesi di Laurea Magistrale di:

Tra le tante tecniche scoperte una è particolarmente interessante: la risoluzione cinetica

Questo lavoro di tesi si inserisce nell’ampliamento della risoluzione cinetica dinamica

Figura 2 - Sintesi norLecina a partire da NBoc-2-ThGlySEt

1.1 Amino Acids

Amino acids are organic compounds characterized by the simultaneous presence of

Figure 3 - Amino Acids Classification

 discovery of novel peptides and proteins

1.2 Synthesis of α-Amino Acids

1.2.1 Strecker Synthesis

Scheme 1 - Strecker Synthesis

1.2.2 Amination of α-Halogen Acids

Scheme 3 - Cahours synthesis, X=Cl or Br

Scheme 5 - Gabriel Synthesis

The well-known rearrangement of acid azides to isocyanates, with the concomitant

Scheme 6 - Curtius and Sieber Synthesis

This sequence involves the rearrangement of an alkyl- or aryl-malonylazidic acid to the

Scheme 7 - Darapsky Synthesis

OEt RX OEt OH-

H2N COOH H2NOC COOH

Scheme 8 - Huang, Lin and Li Synthesis

1.3 Synthesis of Entiomerically Pure Amino Acids

 Asymmetric synthesis or desymmetrization

Scheme 9 - Asymmetric Synthesis

Enantioconvergent process Dynamic kinetic resolution Stereoinversion

A,B = Substrate enantiomers

Scheme 11 - Deracemization Processes

 Spontaneous crystallization procedures

1.4 Asymmetric Synthesis

In the enantioselective catalysis, usually is based on a catalyst characterized by chiral ligands,

1.4.1 Non-enzymatic Catalytic Asymmetric Synthesis

Non-enzymatic catalytic asymmetric synthesis is based on the generation of chirality by the

1.4.2 Enzymatic Asymmetric Synthesis

1.5 Kinetic Resolution

Scheme 14 - Asymmetric Oxidation (b)

Scheme 15 - Kinetic Hydrolysis (d)

Scheme 16 - Acid Hydrolysis

1.5 Deracemization Process

1.5.1 Dynamic Kinetic Resolution

Scheme 17 - DKR by Hydantoinase/Racemase

1.5.2 Stereoinversion of Crixivan

Scheme 18 - Stereoinversion of Hafner and Wellner

Scheme 19 - Stereoinversion of Crixivan

 The mechanical separation of crystals possessing requisite hemihedrism

1.7.1 Crystallization Procedures

The type of crystallization may be known from the phase diagram.

In particular compounds known to crystallize as conglomerates are much easier to resolve

1.7.2 Diastereoisomeric Salts

(+A) + (D,L) B (+A)(L)B + (+A)(D)B

Scheme 20 - Diastereoisomeric Salts

Figure 5 - Phase Diagrams

1.8 Paper Chromatographic Approaches

2.2 Dynamic Enzymatic Resolution of Thioesters40

RS R' RS+ R'

Scheme 21 - Resonance of Thioester

Scheme 22 - Racemization of Thioester

2.2.1 Kinetic α-Proton Acidity of Thioesters

Scheme 23 - General System Used to Measure α-Proton Exchange Rate (Drueckhammer)

2.2.2 Thioester as Substrates for Hydrolytic Enzyme

In order to achieve enzymatic resolution of a thioester, an enzyme must be identified which