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An Efficient Synthetic Strategy towards

4-alkyl Piperidines

Master Thesis by

Valentinos Mouarrawis

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An Efficient Synthetic Strategy towards

4-Alkyl Piperidines

Master thesis by Valentinos Mouarrawis

14 February 2015

Vrije Universiteit Amsterdam

&

Universiteit van Amsterdam

(10407618)

Chemistry: Molecular Design, Synthesis and Catalysis

Supervisor:

Prof. Dr. Romano Orru

Daily Supervisor:

Gydo van der Heijden

Second Reviewer:

Dr. Chris Slootweg

Research performed at:

Synthetic & Bio-organic chemistry

Division of Organic Chemistry

Vrije Universiteit Amsterdam

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‘’Eὰν μὴ ἔλπηται ανέλπιστον οὐκ ἐξευρήσει,

ανεξερεύνητον ἐὸνκαὶ ἄπορον’’

-Iraklitos

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Summary Nowadays, organic chemists put emphasis on how environmentally friendly is the synthesis of

novel compounds due to the great importance of the environment. In the last 15 years many

developments took place towards this direction. Despite the progress there is always space

available for further developments. An attractive method to add in the synthetic chemist’s

sustainable repertoire is multicomponent reactions (MCR) methodology that surprisingly only

recently has become an important topic of research.

A step forward to MCR methodology will be the combination of more than one green synthetic

method so as to achieve even more advantages over conventional synthesis. In particular,

biocatalysis which is a powerful and ‘‘green’’ methodology for both simple and complex

transformations can be combined with MCR and serve as co-acting green synthetic step that

cooperates in an environmentally friendly manner.

In this thesis we were particularly interested in the development of an efficient and green

synthetic method towards 4-alkyl piperidines because of their great interest as inputs for the

ideal combination of enzyme-catalyzed and multicomponent reactions. These cyclic amines have

a structural motif which is present in numerous natural alkaloids and they are widely used as

building blocks in the synthesis of natural products.

Two approaches were developed for the synthesis of 4-substituted piperidines. The first method

is a three step synthetic route that includes: i) piperidone protection, ii) Wittig reaction and iii)

one-pot double bond reduction / deprotection step. However, after lack of success to meet the

criteria we set concerning the overall yields and how green is the process, we developed an

alternative approach that completely satisfies our goals. This modified strategy towards 4-alkyl

piperidines includes: i) SN2 reaction between 4-picoline anion with various alkybromides and ii)

reduction of the 4-substituted pyridine to the HCl salts of the corresponding piperidines.

A subsequent challenge that we briefly investigate includes the optimization of (i) the MAO-N

catalyzed oxidation step towards the corresponding imines in both high ee’s and conversions and

(ii) the Ugi 3 component reaction (U-3CR) that can be used to generate 4-substituted piperidyl

peptides. Furthermore, this synergistic approach can be further applied as a powerful

methodology in the synthesis of actual medicines that contain the appropriate structural

information.

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List of abbreviations

Ac acetate mp melting point ATP adenosine triphosphate

n.c

no conversion

BIOS

biology-oriented synthesis NADP

nicotinamide adenine dinucleotide phosphate phosphate bs broad signal n-Bu n-butyl

°C degrees Celsius NMR nuclear magnetic resonance

CALB

Candida Antarctica lipase B P-3CR Petasis three component reaction

CBz carboxybenzyl ppm parts per million

CHMO

cyclohexanone monooxygenases PS

protein S

CRL

Candida rugosa q quartet

d doublet R rectus d.r.

diastereomeric ratio rt room temperature

3D three dimensional S sinister

DCM dichloromethane

s singlet

de diastereomeric exces TBDMS tert-Butyldimethylsilyl

DKR dynamic kinetic resolution

TLC thin layer chromatography

DNA deoxyribonucleic acid

TMSOTf

trimethylsilyl trifluoromethanesulfonate

DOS

Diversity-oriented synthesis U-4CR Ugi four component reaction

e.g. examplia gratia (for example) vs versus

ED enzymatic desymmetrization

ee enantiomeric excess

Eq. equivalents

Et ethyl

et al. et alii (and others)

FAD

flavin adenine dinucleotide

FTIR Fourier transform infrared spectroscopy

GC gas chromatography

GDH

glucose dehydrogenase

h hour(s)

HRMS high resolution mass spectroscopy

Hz Hertz z

i.e. id est (that is)

IMCR isocynanide multicomponent reaction

KR kinetic resolution

KRED

ketoreductase

LDA lithium diisopropylamide

m multiplet

MAO-N

Monoamine oxidase N

MCR multicomponent reaction

Me methyl

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Table of Contents

Summary .......................................................................................................................................... i

List of abbreviations ........................................................................................................................ii

Table of Contents ........................................................................................................................... iv

1. Introduction ............................................................................................................................ 1

1.1 Green Chemistry ........................................................................................................................... 1

1.2 Multicomponent reactions ........................................................................................................... 4

1.3 Biocatalysis .................................................................................................................................. 10

1.4 Biocatalysis & Multicomponent Reactions ................................................................................. 21

1.5 Aim and Outline of this Thesis .................................................................................................... 26

2. Results & Discussion.............................................................................................................. 28

2.1 Synthesis of 4-substituted piperidines ........................................................................................ 28

2.1.1 Exploring the synthetic approach (i) towards 4-substituted piperidines. ........................... 28 2.1.2 Exploring the synthetic approach (ii) towards 4-substituted piperidines. .......................... 32

2.2 Monoamine Oxidase N: Biocatalytic desymmetrizations of 4-alkylpiperidines. ........................ 36

2.3 Investigation of the U-3CR towards 4-substituted piperidyl peptides. ...................................... 39

3. Conclusions ............................................................................................................................ 43

4. Future prospects.................................................................................................................... 45

4.1 Optimization and further investigations of the MAO-N catalyzed reaction. .............................. 45

4.2 Establishing U-3CR reaction as a highly diastereoselective methodology. ................................ 46

4.3 The discovery of valuable applications of the Biocatalysis/U-3CR method. ............................... 47

Acknowledgement ........................................................................................................................ 50

5. Experimental Section ............................................................................................................ 52

5.1 General remarks .......................................................................................................................... 52

5.2 Synthetic procedure (1): A synthetic strategy towards 4-substituted piperidines. .................... 52

5.2.1 Synthesis of phosphonium salts .......................................................................................... 53 5.2.2 Synthesis of CBz-protected 4-substituted piperidones ........................................................ 54 5.2.3 Synthesis of 4-alkyl piperidines ........................................................................................... 56

5.3 Synthetic procedure (2): A synthetic strategy towards 4-substituted piperidinium salts .......... 58

5.3.1 Synthesis of 4-alkylpyridines ............................................................................................... 58 5.3.2 Synthesis of 4-substituted piperidinium salts ...................................................................... 61

5.4 Synthetic procedure (3): The 3 component Ugi reaction towards 4-substituted piperidyl

peptides. .............................................................................................................................................. 64

References ..................................................................................................................................... 67

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1. Introduction

1.1 Green Chemistry Organic synthesis is the science of replicating the molecules of nature and creating others like

them in the laboratory. [1] This is of great importance for the discovery of new medicines which

has served as the driving force for the development of new ways to achieve the synthesis of any

complex compound that can have potential biological activity. Over the past two centuries

fundamental theories and reactivities have been soundly established and these days the total

synthesis of natural products with very high complexity, such as vitamin B12, [1]

(Figure 1) in the laboratory are evidence of the great success in the field of organic synthesis.

Figure 1: The remarkably complex structure of vitamin B-12

However, despite the great developments in the artificial construction of natural products, we

are facing substantial challenges in future chemical synthesis. The present state-of-the-art

synthetic approaches in obtaining natural products are highly inefficient due to the large

amounts of chemical waste. Therefore, organic chemists started to develop innovative

methodologies towards green or sustainable organic synthesis. Since the early 1990’s when the

concept of green chemistry was first formulated as the design of chemical products and processes

to reduce or eliminate the use and generation of hazardous substances, the field has received

extensive attention due to its ability to connect chemical novelty and creativity with

environmental and economic goals.[2-4] Thus, green synthesis, including feedstocks, reactions,

solvents and separations, became an important topic of research. The idea of environmentally

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friendly processes has its origins 50 years ago when Rachel Carson published Silent Spring, a book

that drew the attention to the connection between chemicals and their effects on the

environment and human health.[5] It was the birth of an environmental view in science that

captured the public’s interest and challenged the old scientific and industrial methods, and over

time, inspired significant changes in our awareness for the environment.

Since the beginning of green chemistry, it has developed into a major focus field within chemistry

and major research, education and outreach activities have been established globally. One solid

example of this development are the twelve principles of green chemistry (Table 1) which have

played a major role in promoting the subject and explaining its goals, ever since they were first

reported.[2] Specifically these principles have served as a handy assessment tool of how green a

plausible chemical route actually is.

Table 1: The twelve principles of green chemistry

1. It is better to prevent waste than to treat or clean up waste after it is formed.

2. Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.

3. Whenever practicable, synthetic methodologies should be designed to use and generate substances that possess little or no toxicity to human health and the environment.

4. Chemical products should be designed to preserve efficacy of function while reducing toxicity.

5. The use of auxiliary substances (e.g. solvents, separation agents, etc.) should be made unnecessary whenever possible and, innocuous when used.

6. Energy requirements should be recognized for their environmental and economic impact and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure.

7. A raw material or feedstock should be renewable rather than depleting wherever technically and economically practicable.

8. Unnecessary derivatization (blocking group, protection/deprotection, and temporary modification of physical/chemical processes) should be avoided whenever possible.

9. Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.

10. Chemical products should be designed so that at the end of their function they do not persist in the environment and break down into innocuous degradation products.

11. Analytical methodologies need to be developed further to allow for real-time-in-process monitoring and control prior to the formation of hazardous substances.

12. Substances and the form of a substance used in a chemical process should be chosen so as to minimize the potential for chemical accidents, including releases, explosions, and fires.

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Among others, synthetic chemists have an essential role in our society to find and develop ideal

synthetic methods that are able to maintain our standard of living without depleting the earth’s

resources. The concept of the ideal synthesis has its origins in 1975 when it was first addressed

by Hendrickson as one that [6] ˝creates a complex molecule in a sequence of only construction

reactions involving no intermediary refunctionalizations, and leading directly to the target, not

only its skeleton but also its correctly placed functionality.˝ In the 20th century the model of ideal

synthesis has been modernized and adjusted to the current needs of our society. P.A Wender et

al. describe this as the synthesis that should lead to the target molecule safely, from readily

available starting materials in one or two reaction steps, in good overall yield and using

environmentally benign reagents.[7] Thus, the environmentally friendly way of thinking

constitutes one of the factors of the ideal synthesis (Figure 2).

Figure 2: Ideal synthesis described by Wender and coworkers.

Surprisingly, a very essential methodology that was first introduced by Strecker in 1850 is

multicomponent reaction (MCR). This relatively old synthetic tool satisfies most of the criteria of

the ideal synthesis and can actively offers numerous advances towards the direction of a green

synthetic mentality. Therefore, MCR can play an important role in the expansion of the synthetic

chemist’s repertoire due to the high atom economy, efficiency, mild reaction conditions, step

economy, and compatibility with green solvents. Thus, MCRs nearly meet all the criteria of an

ideal synthesis and offer many advantages, if a compound allows to be synthesized in that way.[8]

IDEAL SYNTHESIS

Readily available starting materials

Safe

Resource effective

One pot

Environmentally friendly

Total conversion

100% yield

Simple

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Despite the unique benefits that MCR offers, there is still further space in the development of a

highly valued green process that can dramatically expand the field of organic synthesis in parallel

with the 12 principles of green chemistry. A versatile method that generates useful intermediates

and at the same time enhances the dimensions of ideal synthesis is biocatalysis. In this case,

enzymes serve as the catalyst and the key feature that establishes enzymatic transformations a

green process is that enzymes can be obtained from nature without the necessity to be

synthesized. Furthermore, enzyme-catalyzed reactions can prevent waste generation with high

stereo- and regio-selectivity, and by avoiding or reducing the use of dangerous organic reagents.

In addition, one can design safe procedures with relatively high energy efficiency by conducting

reactions under ambient temperature and pressure. Also, atom economy can be increased by

avoiding protection and deprotection steps.

The benefits of MCR and biocatalysis over conventional chemical synthesis are significant due to

the high chemical and energy efficiency. In addition, the green criteria of biocatalysis are highly

unique and in combination with MCRs would represent an important step towards ideal

synthesis. In particular these two methodologies can cooperate in a synergistic manner and add

to the development of a powerful green synthetic approach.[9]

1.2 Multicomponent reactions Nowadays, the state of the environment is becoming an international issue and organic chemists

should also adapt an ecological synthetic mentality. Alongside the development of conventional

synthetic methods, multicomponent reactions strongly meet the principles of the ideal synthesis.

The MCR methodology is defined as a one-pot reaction employing more than two starting

materials where most of the atoms of the starting materials are incorporated in the final product

(Figure 3).[10]

Figure 3: Multicomponent reaction methodology (1) versus conventional multistep synthesis (2).

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Numerous novelties have been reported in the field of MCR since its discovery in the 50s and

justifiably most of them are essentially considered as an important area of research. Specifically

this reaction methodology was first introduced in 1850 by Strecker, a reaction towards α-

aminonitriles, which are useful intermediates for the synthesis of amino acids via hydrolysis of

the nitrile. The first step is the condensation of ammonia with the aldehyde (1) for the iminium

(2) formation followed by the nucleophilic attack of the cyanide to generate the α-aminonitrile

(3). Hydrolysis forms the corresponding aminoacid (4, Scheme 1).[11]

Scheme 1: Strecker amino acid synthesis

An additional important development in the MCR field is the Mannich reaction that was first

reported in 1912.[12] It is used to convert primary or secondary amines and two carbonyl

compounds into β-amino carbonyls. The reaction mechanism starts with the formation of an

iminium ion (7). The compound with the carbonyl functional group (8) can tautomerize to the

enol form, and then attack to the iminium ion which after deprotonation provides the final β-

amino-carbonyl (also known as a Mannich base; 9) with two stereocenters (Scheme 2). The

applications of the Mannich reaction includes agro chemicals, for instance plant growth

regulators,[13] and polymer chemistry. However, the most popular use of the Mannich reaction

can be found in the pharmaceutical industry due to facile synthesis of 1, 3-amino alcohols or

Michael acceptors via the Mannich bases.[14]

Scheme 2: The Mannich reaction

Another significant MCR is the Passerini reaction which was discovered in 1921 in Florence, Italy

by Mario Passerini.[15] It is the most fundamental multi-component reaction involving

isocyanides. These compounds are chemical species with an extraordinary functional group and

scarce valence structure and reactivity. They are the only stable compounds with a formally

divalent carbon.[8] Several studies have taken place in order to grasp their electronic and

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geometric structures [16] so that their electrophilic and nucleophilic character could be explained.

Romain Ramozzi and coworkers have showed by the means of high-level valence bond

calculations that isocyanides are dominantly of the carbenic type (Figure 4), contrasting the

usually used zwitterionic character.[17] Isocyanide based MCRs are widely compatible with a range

of functional groups not taking part in the initial MCR. This category of MCRs offers the possibility

to utilize such functional groups in a secondary reaction step in order to move beyond towards

more diverse synthetic steps and improve the current complex synthesis toolbox.

Figure 4: Sugested representation of isocynides by Romain Ramozzi et al.

The classic Passerini reaction is a three-component reaction involving a carboxylic acid (12), an

aldehyde or a ketone (10) and an isocyanide (11) to form α-acyloxycarboxamide (15). Over the

years, the scope of the reaction has broadened and now it is widely involved with accessing

biologically active molecules. The mechanism of the reaction is still a subject of uncertainty and

kinetic studies have led to different mechanistic elucidations.[18,19] However, Ugi discovered that

the reaction is accelerated in aprotic solvents indicating a non-ionic mechanism.[20] Hydrogen

bonding is believed to play an important role in the formation of the presumed cyclic transition

state for this reaction and the reaction mechanism which proceeds through an intermediate (14)

and further rearranges to the final product (15) (Scheme 3).

Scheme 3: The nonionic mechanism of the Passerini reaction.

Despite the discovery of the Passerini reaction, isocyanide chemistry started to flourish in the

late 1950s when Ugi et al. discovered the first isocyanide based 4-component reaction (U-4CR)

in 1959.[21-23] This versatile reaction is a reaction between a ketone or aldehyde (16), an amine

(19), an isocyanide (17) and a carboxylic acid (18), generating α-aminoacyl amide derivatives (23).

The Ugi reaction, compared to Passerini reaction, is much more multipurpose due to both library

size and scaffolds that can be accessed. This difference can be primarily attributed to the acid

component variability and their rearrangement options, the amines structures and to the many

intramolecular variations. The protein-like small chain products of the Ugi reaction have potential

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pharmaceutical applications. Thus, it is particularly of great interest in diversity-oriented

synthesis, where e.g. libraries of compounds are created for screening purposes. The suggested

mechanism involves a prior formation of an imine by condensation of the amine with the

aldehyde, followed by formation of an iminium ion (20) by proton exchange with the carboxylic

acid. The next step is isocyanide addition to the iminium ion to form a nitrilium ion intermediate

(21), after which nucleophilic addition of the carboxylate ion takes place, generating an acylated

isoamide (22). The last step is a rearrangement of the acyl group to afford the desired product

(23,Scheme 4).

Scheme 4: Suggested mechanism of the U-4CR.

One of the most recent isocyanide based multicomponent reactions is the Orru reaction, a three-

component reaction developed in 2003.[24] It is a reaction between an amine (24), an aldehyde

(25) and α-acidic isocyanide (26), generating substituted 2-imidazolines (29). The first step of the

proposed mechanism is the imine formation followed by attack of the α-acidic isocyanide. The

formation of the intermediate (28) is believed to be promoted by traces of amine present in the

reaction that may act as a basic catalyst. The final step is a ring closure of 28 to afford the desired

product (29, Scheme 5 ).

Scheme 5: The mechanism of the Orru three-component reaction.

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During the past decades, it has become clear that the aim of state-of-the-art research is the

development of green and simplified synthetic routes that give access to molecules of high

structural complexity.[10] In this perspective, MCRs have become an emerging field of research

because of their plethoric benefits for the facile and green synthesis of small-molecule libraries.

However, one of the main limitations of this methodology is the absence of stereocontrol which,

in most cases, is neither straightforward nor generally applicable. The necessity of asymmetric

synthesis stems from the high complexity of the compounds isolated from natural products which

have diverse polycyclic ring systems and significantly high 3D structural information.

Over the years there have been several developments in the direction of stereoselective MCR

that can be divided into two main categories that include (i) the use of catalysts and (ii) the use

of chiral MCR inputs. The toolbox of organocatalysis contains many promising solutions towards

asymmetric synthesis in multicomponent reaction methodology. Catalytic asymmetric

multicomponent chemistry creates a challenge in the synthesis of optically pure compounds with

structural diversity and complexity.[25]

Two examples worth mentioning are depicted in Scheme 6. Shi-Xin Wang et al. have described

an efficient enantioselective Passerini three-component reaction (P-3CR) catalyzed by Lewis acid

catalyst (34) that generates a-acyloxyamides (33) in good to excellent enantioselectivities.[26]

Another asymmetric MCR was reported by Feng shi et al. that is considered the first catalytic

asymmetric five-component reaction using chiral phosphoric acids (39), which directly assembles

aldehydes (37), anilines (35) and β-keto esters (36) into functionalized tetrahydropyridines (38)

with the creation of five s bonds and two stereocenters in high diastereo- and

enantioselectivities.[27] In particular, this is a very attractive method towards chiral

tetrahydropyridine derivatives which are core structures in a wide range of natural products and

pharmaceuticals. Thus, the development of an efficient catalytic enantioselective MCR for the

synthesis of optically pure tetrahydropyridines is highly desirable.

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Scheme 6: Catalytic asymmetric multicomponent reactions.

Yet, the discovery of a generally applicable catalytic asymmetric Ugi reaction is a momentous

event that would open many beneficial opportunities for the future of the diverse-oriented

synthesis and it is justifiably considered as the Holy Grail in MCR chemistry.[28]

The second main approach to achieve stereocontrol in MCR methodology is the asymmetric

induction of optically pure MCR inputs. In particular, this remarkable approach provides access

to complex compounds - otherwise hard to achieve - where one stereogenic center can efficiently

control the newly formed center in a diastereoselective manner.

A representative example that uses optically active inputs is the diastereoselective Petasis 3CR,

reported by N.A Petasis in 1998.[29] It is a one-step three component reaction between an

organoboronic acid (40), an amine (41), and an R-hydroxy aldehyde (42) to give the

corresponding β-amino alcohol (43). In particular, the reaction generates only the anti-products

with de of more than 99% (Scheme 7). Interestingly the products are obtained as single

enantiomers when optically pure R-hydroxy aldehydes such as glycerinaldehyde (42) are used,

due to the absence of racemization.

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Scheme 7: Diastereoselective Petasis 3CR with chiral aldehyde.

Despite the fact that this synthetic approach is experimentally simple and it proceeds with very

high diastereoselectivity, the limited availability of the chiral starting materials create the need

to develop simplistic, efficient and sustainable methods towards these optically active inputs. In

this perspective, biocatalysis can play an important role as an efficient and sustainable strategy

in order to gain access to highly desirable optically pure materials. For this reason the future of

asymmetric multicomponent reactions via enzyme catalyzed chiral inputs can be considered a

very promising field of research.

1.3 Biocatalysis Biocatalysis is the field that uses enzymes as catalysts for the chemical transformations of organic

compounds. A number of various reactions are feasible by means of biocatalytic processes and

can be divided into six main categories depicted in Table 2.

Table 2: Classification of enzymes

Enzyme class Reaction type

Oxidoreductases Oxidation–reduction reactions

Transferases Transfer of groups

Hydrolases Hydrolysis formation of esters, amides, lactones, lactams, epoxides,

Lyases Addition–elimination reactions

Isomerases Isomerizations

Ligases Formation-cleavage of C–O, C–S, C–N,

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The first reported example of biocatalytic process goes back to 1858 when Louis Pasteur

described an enzyme-catalyzed kinetic resolution. This remarkable discovery has been

recognized as a Milestone in the field of biocatalysis due to the wide application in both academia

and industry.[30] In 1894, Emil Fischer introduced the lock and key hypothesis of stereoselective

enzyme catalysis model. In this model, only the substrate with the specific shape that is

complementary to the active site can bind and react. An additional keystone of biocatalysis was

described by Eduard Buchner who in 1897 reported the first successful cell-free fermentation of

sugar by yeast extracts, which establishes unquestionable evidence that biological

transformations do not necessarily need living cells.[31] This criterion alteration opened the door

to present-day biocatalysis containing fermentation technology for the production of both achiral

and chiral products. In 1913, Ludwig Rosenthaler described the preparation of (R)-mandelonitrile

(46) by treating benzaldehyde (44) with HCN (45) in the presence of emulsin extracted from bitter

almonds (Scheme 8).[32] This discovery can be considered as what we call today enzyme-catalyzed

asymmetric synthesis.

Scheme 8 : The first enzyme-mediated asymmetric catalysis described by Ludwig Rosenthaler.

Yet another landmark that likewise set the stage for present day research and technology is the

fundamental discovery in 1926 by James B. Summer in which enzymes are proven to be

proteins.[33] In 1965, Jacques Monod expanded the known information about enzymes by

proposing the allosteric model which describes the allosteric transitions of proteins made up of

identical subunits.[31] Last but not least, Whitesides et al. reported a remarkable asymmetric

example using an aldolase as biocatalyst in stereoselective aldol addition of ketone (48) to

aldehydes (47) for the synthesis of aldol adducts (49) in an asymmetric fashion (Scheme 9).[34]

Scheme 9: Biocatalytic asymmetric aldol addition of ketone to aldehydes.

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Despite, the rapid development of enzyme catalysis, a serious obstacle persevered until the late

1970s that is how to obtain proteins in sufficient amounts for practical applications in a wide-

ranging manner. By then, enzymes were isolated from their sources like microorganisms, fungi,

insects, plants, or mammals which were often problematic and not efficient.

The revolutionary work in 1980 completed by Paul Barg, Herbert Boyer, and Stanley Cohen was

the vital step to overcome the long-standing restriction of the development of biocatalysis in

synthetic organic chemistry. Specifically they proposed the idea of recombinant DNA

methodology, according to which an enzyme occurring in one organism can be overexpressed in

a host organism, a typical host organism are Echerichia coli and Basillus subtillikl.[35] Biocatalysis

has numerous advantages compared to chemocatalysis in organic synthesis. The major

advantage of a biocatalyst is its high selectivity. This selectivity can be chiral (stereoselectivity),

positional (regioselectivity), and functional group specific (chemoselectivity). This characteristic

is very desirable in organic synthesis as it may offer numerous benefits. Opposed to conventional

chemocatalysis, biocatalysis opens the door for using protecting group free substrates. A

representative example is an aldol reaction depicted in Scheme 10 for the asymmetric synthesis

of β-hydroxy α-amino acid. Furthermore, biocatalytic processes minimize side reactions, such as

isomerization, racemization, and rearrangement compared to the classic catalysis. In addition,

due to the last 10 years of increased emphasis on developing synthetic methods that are

environmentally friendly, biocatalysis as a process is as green as catalysis gets.

Scheme 10: Conventional multistep synthesis using a chemocatalyst vs. protecting-free one-step biocatalytic approach in an aldol reaction.

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The use of enzymes has immediate advantages in turning a chemical process green and greatly

satisfies the 12 principles of green chemistry. The mild reaction conditions, the high purity

products in one step, the low energy requirements, and both biodegradability and

biocompatibility of enzymes establishes bio-based routes an attractive sustainable tool towards

novel and complex synthesis. Biocatalysis, using either enzyme technology or whole cells, has

won numerous awards according the US Environmental Protection Agency (Table 3).[36]

Table 3: Green Chemistry Challenge Awards in biocatalysis over the past ten years

Product Technology Complany Year

Succinic acid as chemical feedstock Fermentation BioAmber 2011

1,4-butandiol for polymers and chemical feedstock Fermentation Genomatica 2011

Sitagliptin: a pharmaceutical ingredient for treatment of type 2 diabetes

Enzyme Merck and Codexis 2010

Atorvastatin intermediate for treatment of high cholesterol

Enzyme Codexis 2006

Low trans fats and oils for human nutrition Enzyme ADM and

Novozymes 2005

Taxol for treatment of breast cancer Fermentation Bristol Myers

Squibb 2004

Despite these advantages, enzymes as catalysts in synthetic organic chemistry continued to

suffer from some limitations that cannot be overlooked. In fact, the narrow substrate scope

compared to native substrate or chemocatalysis substrates prevents their establishment as a

catalyst for a wide range of inputs. The cost of enzyme including isolation and purifications

processes is a major obstacle towards commercializing biocatalysts due to the current cost-

competitive established classic methods. Furthermore, enzymes are provided by nature in only

one enantiomeric form and it is impossible to invert the chiral induction of a given enzymatic

reaction due to the limited general methods of generating mirror-image enzymes.

Another disadvantage is the insufficient stability under operating conditions due to the general

unstable nature of enzymes when their removed from their natural conditions. Even though

enzymes are very flexible in tolerating non-natural substrates, they are practically cofactor

depended in order to be functional. The most common cofactors are depicted in Figure 5.

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Figure 5: Cofactors often applied in enzymatic organic synthesis.

Last but not least, the specificity of enzymes is not strictly limited to substrates. Frequently, the

activity of an enzyme is reduced by specific interactions with molecules termed inhibitors.

Specifically, these inhibition phenomena can occur at high substrate and/or product

concentrations and lead to low reaction rates, a factor that significantly limits the efficiency of

the enzymatic process.

Undoubtedly, the field of biocatalysis has reached a high level of sophistication through a

significant number of technological research and innovations. However, the above-mentioned

limitations in many cases dramatically prevent the revolutionary development of biocatalysis to

establish enzymatic processes a widely applicable synthetic tool. Nowadays, molecular biology

methods such as protein engineering provide the missing components to create the ideal enzyme

and to overcome these walls.[37-39] The basic goal of protein engineering is the creation of

improved versions of known enzymes in order to meet the demands in each individual case.

These improvements can include one or more properties such as, increased catalytic function

relative to the original enzyme, altered substrate specificity or stereospecificity and, increased

stability to the conditions that are required.[40] Two strategies have actively contributed to that:

(i) the so-called rational design approach and (ii) and directed evolution. [41] Rational design is the

process where enzymes are re-designed rationally at the molecular level by specifying the

sequence. It requires comprehensive understanding of structures and mechanism in order to

adjust their functions for the desired applications. Specifically, site-directed mutagenesis is used

to introduce site-specific functionalizations into the enzyme, frequently in combination with

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computational means.[42] A noteworthy example is the development by Kazlauskas and

coworkers of an esterase with higher enantioselectivity for the hydrolysis of 3-bromo-2-

methylpropionate via targeted mutagenesis.[43] Moran Brouk et al. reported the use of a

monooxygenase variant with increased substrate specificity which resulted in 190-fold higher

reaction rate for 2-phenylethanol.[44] Another attention-grabbing example of enzyme utilization

was reported by Sneha Srikrishnan et al. whereby higher catalytic efficiency obtained via site-

directed mutagenesis.[45] The mutant displayed 2.5-fold improvements in hydrolytic activity on

cellulosic substrates, while preserving thermostability. However, its wide application site-

directed mutagenesis has two major drawbacks which cannot be ignored. The high complexity of

interpreting the structure of an enzyme and the relation in an enzyme between structure and

activity is still not a trivial. Even when the target enzyme is fully characterized, distinguishing the

amino acid residues that control the catalytic activity is a challenging assignment.

Directed evolution involves the modifications of a biocatalyst via an in vitro form of Darwinian

evolution and offers a powerful method for the growth of enzymes with unique properties. The

developed enzyme can include altered substrate specificity, thermal stability and organic solvent

resistance. The major advantage of directed evolution over rational design is that novel

properties can be induced in enzymes without the need of prerequisite knowledge of enzyme

structure and/ or catalytic mechanism. There are a considerably large number of prominent

examples of directed evolution of enzymes reported in the last 10 years. M.T. Reetz and

coworkers reported an enantioselective cyclohexanone monooxygenases (CHMO) as a catalyst

for the desymmetrization of 4-hydroxycyclohexanone (50) in Baeyer–Villiger reaction whereby

the selectivity of the oxidation inverted by using the directed evolution approach (Scheme 11).[46]

Scheme 11: CHMO-Catalyzed Oxidation of 4-Hydroxycyclohexanone.

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Although, directed evolution has already proven its substantial role in inducing new functional

properties into an enzyme, there are a few major challenges in order to harness the advantages

of biocatalysis that trigger further technological research and innovation. For example, low

efficiency, tricky screening in complex mutant libraries for the desired functions, time-

consuming, and technically challenging. Unfortunately, there is not a single ideal method of

protein engineering due to the diversity of choosing’s observed from enzyme to enzyme and from

application to application. The growing attention paid to the cooperative method of rational

design and directed evolution towards the targeted randomization of certain areas of the protein,

enzymes can be designed in a more efficient manner in correlation with the desired property.

Biocatalytic steps are often used to introduce chirality in a reaction sequence towards the desired

product. The growing need of creating molecules with increased complexity (e.g large molecules

with many chiral centers) offers a broad spectrum of opportunities for this technology to be

further developed and established as a key component in the synthetic chemist’s arsenal. Over

the years, enormous efforts have been made to develop enantioselective methodologies for

simplistic preparation of enantiomerically pure compounds because of their significance in the

pharmaceutical, food and agricultural sectors. There are two main categories of stereoselective

biotransormations including, asymmetric synthesis and kinetic resolutions of racemic mixtures.

Kinetic resolution (KR) is a process to separate two enantiomers in a racemic mixture. In

particular, the idea is based on the different reaction rates of the two enantiomers causing an

enantioenrichement of the less reactive enantiomer.[47] The major drawbacks of this method are

the 50% maximum yield and its low atom economy. A yeast-mediated reduction of β-keto esters

illustrates representatively a kinetic resolution of racemic alcohols (Scheme 12).[48]

Scheme 12: Kinetic resolution of racemic alcohols.

Due to the limitations of this method a different approach has been developed called dynamic

kinetic resolution (DKR). In DKR, a racemization reaction takes place alongside to the enantiomer

formation, leading to the desired enantiomer in 100% theoretical yield. Specifically the

enantioselective reaction is slower than the racemization one and leads to formation of the

enantiomer with the lower activation energy. An interesting example of dynamic kinetic

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resolution was published by Sonia Rodriguez et al. for the asymmetric synthesis of β-hydroxy

esters by recombinant Escherichia coli (Scheme 13).[49]

Scheme 13: Dynamic kinetic resolution (DKR) of β-hydroxy esters

Asymmetric synthesis, constitutes the second main class of stereoselective biotransormations,

and implies the formation of one or more chiral centers in a substrate. One of the approaches in

the field of asymmetric synthesis is the enzymatic desymmetrization (ED) of meso or prochiral

compounds and opposed to kinetic resolution a maximum yield of 100% can be achieved. Meso

and prochiral compounds have either a planar trigonal group with two enantiotopic faces or two

enantiotopic groups. The enzymatic process is faster at one of the enantiotopic groups or faces

of the substrate resulting in high enantioselectivities. Goswami et al. reported a nice example of

an industrial application of EDs for the efficient synthesis of 51 with Candida Antarctica as a

biocatalyst (Scheme 14).[50] The yield of the reaction was 99.8% and the 1S,2R-monoester (51)

was enantiomerically pure.

.

Scheme 14: An industrial enzymatic desymmetrization (ED) process.

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Biocatalytic processes have been employed progressively towards widespread applications

especially in the environmentally benign synthesis of optically pure compounds. This is due to

the great interest stems from pharmaceutical and agrochemical industries where enantiopure

molecules are of a great importance as synthons for numerous pharmaceutically active

substances and agrochemicals. The examples depicted below are a few biocatalytic methods to

produce these highly vital synthons that illustrate the great potentials of biocatalysis in the

direction of both high enantioselectivities and conversions.

Enantiomerically enriched amines are key starting materials and intermediates for the synthesis

of a wide range of biologically active compounds. For this purpose, kinetic resolution of racemic

amine, hydrolases are the most frequently used enzymes. For example, Burkholderia plantari

lipase has been widely used for the production of enantiopure amines (54) via kinetic resolution

of racemic amines (52, Scheme 15).[51] It is worthwhile to mention the need of using an acyl donor

(53) in order to activate the carbonyl. The expected major downside of this approach is the

limitation in relation to the reaction yield of the kinetic resolution, which cannot surpass 50 %.

Scheme 15: Kinetic resolution of racemic amines with Burkholderia plantari lipase

The yield limitations of kinetic resolution can be overtaken by asymmetric synthesis approach

which is more preferred due to the maximum yield of 100%. An efficient approach to shift the

equilibrium of the reaction towards the products is to remove the co-product which is generated

from the de-amination of the amine donor. Wang et al. have reported the asymmetric synthesis

of (S)-1-phenylethan-1-amine (60) using as the amine donor 3-aminocyclohexa-1,5-

dienecarboxylic acid (57) and as the enzyme several variants of ω-transaminases (Scheme 16).

The generated ketone (58) was efficiently removed by a subsequent tautomerization to 3-

hydroxybenzoic acid (59). The quantitative formation of (S)-60 (99%) in an excellent enantiomeric

excess (>99%) established this approach at least promising for the asymmetric synthesis of

amines.[52]

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Scheme 16: Asymmetric synthesis of a chiral amine with an artificial amine donor.

Optically active aldehydes and ketones are vastly valuable intermediates in the synthesis of

complex compounds with high 3D structural information. From a chemical point of view,

enantiopure α-hydroxy ketones (also called acyloin) are highly valuable building blocks for many

applications for the fine chemistry sector as well as pharmaceuticals. Due to their significance,

several biocatalytic approaches have been developed by means of different categories of

enzymes such as lyases, oxido-reductases and hydrolases. The hydrolase-catalyzed DKR of

racemates is an important example of a well-developed methodology to afford enantioenriched

α-hydroxy ketones (62). For example a two-compartment DKR processes have been reported,

involving Candida Antarctica lipase B (CALB) as biocatalyst for the enantioselective substrate

transesterification in a first compartment, and the simultaneous racemization of the residue

alcohol facilitated by Amberlyst 15 in a separated compartment (Scheme 17).[53]

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Scheme 17: Two compartments DKR of racemic acyloins (61).

Chiral alcohols play an essential role as reactive intermediates or starting materials in agro-,

pharma-, and fine chemical industries. In recent times, remarkable progress has been done

towards biocatalytic preparation of enantiopure alcohols. An attractive pathway towards the

synthesis of optically active alcohols is the biocatalytic reductions of ketones. Ma et al. developed

a novel green-by-design method for the synthesis of a key intermediate for atorvastatin which is

the active ingredient of LipitorR, an anti-cholesterol drug. The process includes a biocatalytic

reduction step of ethyl 4-chloro-3-oxo-butanoate (63) using a ketoreductase (KRED) combined

with glucose as a reductant and a NADP-dependent glucose dehydrogenase (GDH) for co-factor

regeneration.[54] Glucose is oxidized to gluconic acid and neutralized using sodium hydroxide. The

(S) ethyl-4-chloro-3-hydroxybutyrate (64) product was obtained in high yields and high

enantiomeric excess (Scheme 18).

Scheme 18: Biocatalytic reduction of ethyl 4-chloro-3-oxo-butanoate using a ketoreductase (KRED).

Chiral carboxylic acids can be derived from ester hydrolysis. Kazlauskas et al. reported a kinetic

resolution approach including a simple 2-propanol treatment that converts crude lipase from

Candida rugosa (CRL) to a form with an increased both activity and enantioselectivity by a factor

of 1.2-1.6 and 25 respectively. The chiral 2-substituted carboxylic acid (67) was obtained in

moderate yield and excellent enantioselectivity (Scheme 19).[55]

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Scheme 19: Kinetic resolution of 2-substituted carboxylic acids ester by 2-propanol treated Candida rugosa lipase (CRL).

1.4 Biocatalysis & Multicomponent Reactions As mentioned in previous sections MCRs are remarkably efficient key methodology to access

highly diverse synthesis of complex molecules due to their high atom economy, mild conditions

and step economy. Yet, the not straightforward stereochemical control and the lack of catalytic

asymmetric methods create the necessity to utilized methods towards efficient and

environmentally friendly enantioselective methods. In most of the cases asymmetry is achieved

by inducing chiral inputs and the generation of those inputs is at least a significant key step

forward to MCRs stereocontrol. The wide-ranging toolbox of the biocatalytic generation of chiral

materials opens up the opportunity to control the stereochemical outcome of MCRs by

introducing a biocatalytic step throughout the synthesis.

The synergy of biocatalysis with MCRs is a handy methodology that overcomes the limiting factor

of MCRs stereoselectivity and at the same time presents a powerful process with many

advantages concerning the environment. In addition, a biocatalytic step can be combined with a

MCR step in several ways throughout the synthetic route making this method flexible and more

widely applicable. These combinations can be: (i) kinetic resolution of a racemic MCR product,

(ii) dynamic kinetic resolution in which the optically pure product reacts in MCR and (iii)

biotransformation to generate enantiopure inputs for diastereoselective MCRs.

Despite the great individual potentials of both biocatalysis and MCRs, the synergistic approach

towards stereochemical diversity in DOS/BIOS-based library design is up till now at an early stage

of development. Nevertheless, over the last years several attractive strategies of

biocatalysis/MCR were reported sowing the great interest towards the development of powerful

and generally applicable biocatalysis/MCR/ combinations. [56-58]

A very interesting example where an enzyme-catalyzed kinetic resolution was combined with

multicomponent chemistry was reported by R. Ostaszewski in 2013. They reported a simple and

efficient methodology towards enantioenriched Ugi products that could be also applied to other

type of substrates. In particular it is a very attractive approach for obtaining enantiomerically

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pure Ugi product via enzyme mediated kinetic resolution of crude Ugi product (68) to access 1,3-

diol peptidomimetics (69) . Unfortunately the enzymatic diastereoselective acylation of hydroxyl

groups was not efficient and as a result not further pursued. On the other hand, the

enantioselective strategy was found to be very efficient and the corresponding carboxylic acid

(69) was obtained in excellent ee (Scheme 20).[59] .

Scheme 20: Enzyme mediated kinetic resolution of crude Ugi product to enantiomerically pure 1, 3-diol peptidomimetic.

Besides employing an enzymatic resolution step throughout the synthetic route, enzymes can

also be used to generate enantiomerically pure MCR adducts and therefore benefit from the

substrate-controlled diastereoselection reached through the MCR step. In addition, the

biocatalytic asymmetric synthesis of one or more MCR component would expand the input’s

diversity otherwise not easily accessible from the chiral pool, resulting in broadening the scope

of any MCR. Although the literature is still rather limited concerning this strategy, there are few

examples indicating the great potentials of this method.

One of the first examples of a MCR combined with an enzymatic desymmetrization was reported

by Larock and coworkers in 1991. They developed an efficient enzyme-desymmetrized

monoacetate (71) which was further transform to a protected diol (72) and coupled to a MCR for

the synthesis of chiral prostaglandins (75).[60] The mechanism of the MCR includes the formation

of two intermediates. The first step is the addition of the alcohol moiety in 72 to the palladium-

complex vinyl ether to afford 73. The formed complex undergoes two olefin insertions; an

intramolecular one (faster) that gives 74 and an intermolecular one that involves the unsaturated

ketone and generates the final product (75) (Scheme 21). With regard to the stereochemistry, all

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the stereogenic centers are controlled, apart from the acetal’s one and therefore an epimeric

mixture is obtained.

Scheme 21: A 3-CR approach to prostaglandins, starting from desymmetrized diol.

Diastereoselection in isocyanide-based multicomponent reactions (IMCR) and particularly in the

Ugi reaction is very tricky. Chiral amines are the only inputs that gave good results concerning

diastereoselectivity. Though, intramolecular versions are more potentially active towards good

to excellent diastereoselectivities due to the sterics imposed by cyclic transition states. [61-63]

A representative study that highlights this approach for the synthesis of pharmacologically

relevant polyfunctionalized pyrrolidine systems (79) was described by V.Cerulli et al. in 2012.[64]

In the event, enantiomerically pure cyclic imine precursors (77), synthesized by Amano PS lipase

followed by a fully diastereoselective Ugi-Joullie reaction which gave 79, in a diastereoselective

ratio of >99:1 (Scheme 22).

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Scheme 22: Diastereoselective Ugi reaction using chiral cyclic imine synthesized by Amano PS lipase.

Another attention-grabbing, two-step approach including the asymmetric enzyme-catalyzed

synthesis of enantiopure pyrrolines (81) combined with multicomponent chemistry was reported

by Turner and Orru.[65,66] They developed a desymmetrization method of a series of monocyclic,

bicyclic, and tricyclic meso pyrrolidines (80) by means of monoamine oxidase N (MAO-N) from

Aspergillus Niger optimized by directed evolution. What makes the biocatalytic step interesting

is the use of whole cells, thus isolation of the enzyme was not necessary. Furthermore, due to

the use of O2 as the stoichiometric oxidant, the addition of catalytic flavin adenine dinucleotide

(FAD) cofactor was needless, making this method relatively cost-effective. The MAO-N-produced

pyrrolines were used as chiral inputs in Ugi 3CR for the synthesis of 3,4-disubstituted propyl

peptides (82) with good to excellent diastereoselectivity in favor of the trans product (Scheme

23).[67] This development clearly showed in a very representative way how powerful the

combination of biocatalysis and MCR can be in the synthesis of molecules with high structural

and three dimensional information. The mild conditions, simple experiment procedures,

excellent yields, d.r and ee values also illustrate how do they synergistically facilitate the synthesis

of complex molecules in an environmentally friendly manner.

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Scheme 23: MAO-N desymmetrization combined with U-3CR.

Most importantly, these propyl peptides were proved to hold great potentials as useful

intermediates for the efficient synthesis of molecules of highly structural complexity that can

found applications in medicinal chemistry. In this direction, a very short and efficient synthesis

of an important hepatitis C NS3 protease inhibitor, Telaprevir (IncivekTM) was developed starting

from simple biocatalytically produced pyrrolines (83). This application can be considered without

doubt as the best example of the cooperation between MCR and biocatalysis that gave access to

an actual pharmaceutical drug (84, Scheme 24).[68]

Scheme 24: Synthesis of hepatitis C NS3 protease inhibitor Telaprevir (IncivekTM).

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In addition, the same group further expand this study towards the development of a novel Ugi

and Pictet-Spengler-type cyclization for the synthesis of polycyclic 2,5-diketopiperazines (88).[69]

This strategy constitutes the first application of MCR chemistry to generate 5-membered ring-

fused diketopiperazines establishing these compounds interesting targets for medicinal

investigations. In the event, the use of α-oxoacid (86) and an isocyanide (87) containing an

electron-rich arene promotes a subsequent MCR Pictet–Spengler (PS) reaction just by treating 88

with trimethylsilyl triflate (TMSOTf). The desired compounds (89) were obtained in moderate to

very good yields and diastereomeric ratios.

Scheme 25: Alkaloid-type compounds by the MAO-N, Joullie-Ugi and PS sequence.

1.5 Aim and Outline of this Thesis Being inspired by the work done on the MAO-N and Ugi sequence for the synthesis of 3,4-

disubstituted propyl peptides we turned our focus on developing an analogous synthetic

approach to gain access to 4-substituted piperidyl peptides. Our strategy involved the adjustment

of the method applied on the meso pyrrolidines in order to develop a green and efficient

methodology that contains all the advantages of the combination between MCR and biocatalysis.

Unfortunately, 4-substituted piperidines which are the suitable inputs of the MAO-N catalyzed

step are not commercially available and the incorporation of an additional synthetic route

towards the synthesis of these highly valuable meso 4-alkylpiperidines was more than necessary.

In fact, we wanted to develop a synthetic route that stays on the pathway of green chemistry and

can be well-combined with the follow up chemistry in an environmentally friendly manner so as

to establish an overall green methodology that includes the synthesis of MAO-N inputs,

biocatalysis and MCR chemistry. For this reason this project primarily involves the development

of an efficient and green synthetic strategy towards 4-substituted piperidines (90), which

constitute promising inputs in a MAO-N catalyzed reaction to generate optically pure 4-alkyl-

2,3,4,5-tetrahydropyridines. The first approach to synthesize these chiral 4-substituted

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piperidines (A) included a Wittig reaction of a protected piperidone (92), followed by a one-pot

double bond reduction/deprotection step of 91 to afford 90. However, due to our continuous

effort to establish methods that stay on the path of green chemistry, we developed a different

and more efficient approach (B) that involves the one-pot 4-picoline (96) deprotonation step /SN2

nucleophilic substitution with various alkylbromides, followed by a low hydrogen pressure

pyridine reduction step to afford 94 (Scheme 26).

Scheme 26: Retrosynthetic analysis of the two developed synthetic pathways towards 4-substituted piperidines.

The second goal of this thesis involves the investigation of how enantioselective the biocatalytic

oxidation of 4-substituted piperidines is towards optically pure 4-alkyl-2,3,4,5-

tetrahydropyridines by means of MAO-N. The last part aims to describe how efficient the

combination of enzymatically produced imines with U-3CR is by investigating the stereocontrol

provided by the biocatalytically generated stereogenic center over the additional stereocenter

formed during the Ugi reaction (diastereoselectivity).

The following chapters describe all the implemented experiments in this thesis including the two

synthetic strategies to synthesize 4-substituted piperidines. In addition, we illustrate the initial

screening results related to the enantioselectivities of different 4-alkyl piperidines towards the

corresponding optically active imines by using MAO-N as the enzyme. Lastly, we focus on using

the enzymatically produced imines in a U-3CR and illustrating the observed diastereoselectivities.

Next, we describe the conclusion of this research thesis, followed by some future prospects.

Lastly, detailed experimental procedures and data are described.

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2. Results & Discussion

2.1 Synthesis of 4-substituted piperidines: As mentioned in chapter 1, a powerful synthetic approach so as to satisfy most of the criteria of

ideal synthesis would be the combination of two efficient and at the same time environmentally

friendly methodologies. Biocatalysis and multicomponent chemistry present a real example of

such a methodology that works synergistically towards ideal synthesis. Nevertheless, the need to

expand the substrate scope of Biocatalysis-MCR methodology described by our group has

triggered us to develop an efficient and short synthetic strategy to generate a library of 4-

substituted piperidines that can be used as inputs for a MAO-N catalyzed reaction. In this section,

the two attempted approaches are described concerning the synthesis of 4-substituted

piperidines.

2.1.1 Exploring the synthetic approach (i) towards 4-substituted piperidines. Our initial strategy for the synthesis of 4-substituted piperidines includes a sequence of 3 steps.

The first step is the protection of 4-piperidone monohydrate hydrochloride (97) using

carboxybenzyl (CBz) group, a carbamate which is often used as an amine protecting group in

organic synthesis.[70] This amine protection step is highly essential in this synthetic route because

the free form of 4-piperidone is not stable due the existing of an amine and a ketone in the same

molecule that will lead to the formation of iminium. The Cbz protective group was chosen in

order to facilitate a one-pot reaction that includes the reduction of the double bond and the

deprotection of the amine in the last step of our synthetic route by using palladium on activated

carbon. In a total reaction time of 10h, full conversion was obtained. The N-Cbz protected product

98 obtained pure as a yellow liquid in an excellent yield of 99% (Scheme 27).

Scheme 27: N-CBz protection of 4-piperidone.

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The following more complex step involves a Wittig reaction between a ketone and various alkyl

triphenylphosphonium ylides. In the event, we first synthesized the phosphonium salts, followed by

addition of n-butyl lithium (n-BuLi) so as to furnish the corresponding ylide. The compound 98 was

reacted with a number of different alkyl triphenylphosphonium ylides in order to generate a library

of 4-alkylene substituted N-CBz protected piperidines. Despite of the commercial availability of

various Wittig reagents most of them have been synthesized by us, so as to increase the cost-

effectiveness of the corresponding method. This has been done according to literature procedure by

reacting triphenylphoshine with different bromoalkanes in order to generate the desired Wittig

reagents.[70,71] Unfortunately, this SN2 nucleophilic substitution reaction was only plausible in the

case of using primary alkylhalides which was rationalized by the effects of steric hindrance present

in the secondary ones(Table 4). After all the efforts – i.e. by increasing the reaction temperature

and/or employing a halogenalkane bearing a better leaving group such as iodine – to overcome this

barrier, the conversions remained fairly low.

Table 4: Substrate scope of the Wittig reagent synthesis.[a]

[a] Reaction conditions: alkyl bromide 99 (1 eq) triphenylphoshine (1 eq) were mixed in dry toluene under N2 atmosphere, 10 h at 120

°C. Yields refer to isolated material. [b] Methylbromide (3 eq).

At this point, the Wittig reaction could be investigated by using different phosphonium salts. In the

event, a Wittig reaction between a series of different phosphonium salts and the N-protected 4-

piperidone was studied in order to get access to the corresponding N-protected 4-alkenylpiperidines

that can be further transformed to the desired products. We initially performed the Wittig reaction

by utilizing 1.5 equiv of (ethyl) triphenylphosphonium bromide in THF and 1.4 equiv of n-butyl lithium

at -78°C. After column purification this procedure led to moderate to good yields, also containing

some unreacted material. Under the conditions mentioned above, a series of different primary

phosphonium salts reacted with the N-protected piperidone, affording the desired product in

moderate to good yields (Table 5). The next step was to investigate how efficient the Wittig reaction

is by employing several secondary phosphonium salts.

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To our disappointment, 1H NMR indicated no conversion to the desired product. We believe that the

reason behind that, is the sterically hindered proton present in the secondary alkyl

triphenylphosphonium salts, making problematic the approach of n-BuLi so as to furnish the ylide.

An additional reason when the ylide was furnished in low yields can be the difficulty of the ylide to

attack the ketone due to the effects of steric hindrance present in the secondary ylide.

Table 5: Substrate scope of the Wittig reaction.[a]

[a] Reaction conditions: (alkyl) triphenylphosphonium bromide 102 (1.5 eq) and n-BuLi (1.4 eq) were mixed in dry THF under N2

atmosphere, 1h at -78 °C. Upon completion CBz-protected piperidone (1 eq) was added at -78 °C and stirred, 12h at -78 °C to r.t. Yields

refer to isolated material after column chromatography. [b] (alkyl) triphenylphosphonium bromide 102 (1.3 eq) and n-BuLi (1.2 eq).

Despite our unsuccessful attempts on improving the Wittig reaction to furnish better than moderate

yields we moved to the last step of this approach in order to generate an overview of this synthetic

methodology towards 4-substituted piperidines. This step involved a one-pot double bond

reduction/deprotection reaction. Specifically, catalytic transfer hydrogenation using 10% Pd/C,

reduced carbon-carbon double bond and cleaved the carbamate in a one-pot process. A series of

compounds (105a-d) were isolated after stirring for 16 h, under 1 bar of hydrogen pressure at room

temperature in excellent yields ranging between 89-97 % (Table 6). Despite that, the previous steps

were not so efficient and green, this one-pot reduction/deprotection step showed to be highly

efficient due to the simple experiment procedure, mild conditions and excellent yields in all of the

substrates.

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Table 6: Substrate scope of the one-pot reduction/deprotection step.[a]

[a] Reaction conditions: Compound 104 (1 eq) and 10% Pd/C (0.1 eq) were mixed in methanol under N2 atmosphere. The mixture was

stirred under hydrogen atmosphere, 16 h at r.t. Yields refer to isolated material.

At this stage of the research, we were highly questioning the accessibility of these 4-substituted

piperidines through this approach due to several limitations as far as diversity of the created library

and the overall yields are concerned. In addition, the use of hazardous solvents such as toluene and

the low atom economy (Wittig reaction) are substantial factors that decrease the value of this

method as a green process. For these reasons this approach became a less attractive synthetic route

towards the generation of a representative library of 4-alkyl piperidines.

Since, the initial synthetic effort towards the desired product was not completed successfully due to

the above-mentioned reasons we turned our attention to an alternative and more environmentally

benign approach. The first stage of this alternative route development was to predetermine the

desired features that they can beneficently be combined with the low environmental impact of

Biocatalysis-MCR sequence. As mentioned in chapter one, the green criteria of this synergistic

strategy are highly unique and in combination with an efficient synthetic route towards 4-alkyl

piperidines would represent an important step towards ideal synthesis. For these reasons our major

concern was to increase yields, atom economy, step economy and the use of less hazardous solvents.

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2.1.2 Exploring the synthetic approach (ii) towards 4-substituted piperidines. After facing the problems mentioned in the previous section of the initial synthetic approach, we

were set to move forward with an alternative strategy towards 4-substituted piperidines. In the

event, we developed a 2-step synthetic route that includes an SN2 substitution of 4-picoline with

different alkylhalides, followed by pyridine reduction.

The first step is a one-pot two-stage procedure that involves SN2 substitution of 4-picoline anion by

a series of different alkylbromides. This procedure was performed, by first deprotonating the methyl

group of 4-picoline by addition of lithium diisopropylamide (LDA) at -78°C. The 4-picoline anion was

furnished by using 1.2 equiv. of LDA which was freshly made in the same pot by reacting n-BuLi with

LDA. The required reaction time for the deprotonation step is approximately one hour and it can be

observed by the formation of a characteristic deep red color.

Next, a diverse library of 4-alkyl pyridines was generated by adding a series of different alkyl

bromides (1 equiv.) This SN2 nucleophilic substitution reaction has a remarkably broad substrate

scope and many alkylbromides could be used ranging from short alkyl chains to longer and more

complex ones such as pentyl, isopropyl, tert-butyl, cyclohexylmethyl and cyclopropylmethyl analogs.

The best yields (89-97%) were obtained with the linear straight-chain alkyl substrates (108a-c) and

decreased (36-76%) in the branched-chain ones (108d-f). (Table 7)

In addition to our efforts to further broaden the substrate scope of this reaction we employed

dibromo alkanes as electrophiles, for a secondary cyclization step towards the synthesis of cyclic

analogs. This procedure was performed, by using 2 equiv. of LDA so as to drive a second

deprotonation step, furnish the deprotonated species and complete the intramolecular cyclization.

Despite our efforts, the yields remained very low due to the formation of the double alkylated

product prior to the intramolecular ring closing step and/or the formation of the elimination

products. In spite of the formation of the above-mentioned side-products, purification by the means

of column chromatography led successfully to isolation of 108g and 108h in a yield of 25% and 18%

respectively. (Table 7).

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Table 7: Substrate scope of the 4-alkyl pyridines synthesis.[a]

[a] Reaction conditions: 4-picoline 106 (1 eq) and LDA (1.2 eq) were mixed in dry THF under N2 atmosphere, 1h at -78 °C. Upon

completion alkyl bromide 107 (1 eq) was added at -78 °C and stirred, 12h at -78 °C to r.t. Yields refer to isolated material after column

chromatography. [b] LDA (2 eq). Dibromoalkanes (1eq.)

After having established a library of several 4-substituted pyridines we turned our attention to

develop an efficient and environmentally friendly reduction step. Although the catalytic reduction of

pyridine and pyridine derivatives has been thoroughly described in the literature, it usually involves

harsh reaction conditions such as high pressures and/or temperatures.[72-74]

We therefore started our investigations by exploring an alternative pathway towards the reduction

of these aromatic heterocycles. In the event, we developed a PtO2-catalyzed hydrogenation process

that involves an in situ generation of pyridinium salt prior to the reduction event. This salt formation

lowers the activation energy towards the reduced product making the reduction process plausible in

milder reaction conditions. As a result, pyridinium salts are in every instance more readily reduced

than pyridine and the desired 4-substituted piperidines were obtained as HCl salts in very good to

excellent yields (Table 8).

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Table 8: PtO2-catalyzed hydrogenation of various 4-substituted pyridines.[a]

[a] Reaction conditions: 4-substituted pyridine 109 (1 eq), Hydrogen chloride / ethanol solution ( 5 eq, 1.25 N) and platinum oxide

(0.05 eq.) were mixed in methanol. The mixture stirred under 1 bar of hydrogen atmosphere, 36h at r.t. Yields refer to isolated

material.

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Delighted by this result, we set out to further investigate how the pyridinium ring influences the

reduction of an aromatic system attached to it at the para position. Surprisingly, we were able to

fully reduce the commercially available 4-phenyl pyridine (111) to 4-cyclohexyl piperidinium salt

(112) under the same conditions mentioned above (Scheme 28). A possible explanation to this is the

in situ generation of pyridinium lowers the activation energy towards the reduced product, thus the

catalytic hydrogenation of the phenyl group is plausible under the same conditions via the effect of

conjugation with the pyridinium salt. For these reasons this process represents a powerful and

environmentally friendly reduction tool that can be widely applied as a green methodology for the

catalytic hydrogenation of pyridine derivatives with excellent yields.

Scheme 28: PtO2-catalyzed hydrogenation of 4-phenyl pyridine.

To summarize, we have developed a very short and efficient synthesis of 4-substituted piperidines

which present great potentials as inputs of enzyme-catalyzed desymmetrizations. We initial set

several criteria from an environmentally point of view for this synthetic route so as to develop an

attractive and powerful combination with the follow up enzymatic and MCR chemistry. Efficient

access to libraries of 4-substituted piperidines is essential for further development of the synergistic

approach of Biocatalysis and MCR. A powerful synthesis of these alkyl piperidines can readily expand

the dimensions of Biocatalysis-MCR methodology and can efficiently allow the rapid generation of

complex and structurally diverse molecules.

Despite the discouraging results of the initial approach, we developed an efficient synthetic route

towards the generation of the desired library that includes numerous advantages compared to our

initial efforts. In this respect, the lack of using hazardous solvents, elevated temperatures and high

pressures make our methodology attractive from an environmentally point of view. In addition, the

second tactic involves fewer synthetic steps, higher yields and it allows the in situ formation of

piperidinium salts which are stable, odorless, water-soluble and off-white solid compounds that can

be stored at r.t.

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2.2 Monoamine Oxidase N: Biocatalytic desymmetrizations of 4-

alkylpiperidines. Enzymes proficient of oxidizing amines are abundant in organisms such as bacteria, yeasts, plans and

mammals and can be divided into two main categories, flavoprotein and qiunoprotein. The

mechanism of the qiunoprotein amine oxidases has been well described and occurs via the formation

of an enzyme-substrate covalent adduct.[75] On the other hand, amine oxidation by flavoproteins

(monoamine oxidase, amino acid oxidases and polyamine oxidases) is still debatable.[76,77] Despite

the controversy over the mechanism of flavoproteins as amine oxidizing enzymes, MAO-N presents

a versatile biocatalyst that has been extensively reported in literature. Turner and cowers were the

pioneers that have been thoroughly investigated the use of enzymes capable to oxidize amines and

significantly contribute to the design and the development of MAO-N.[78,79] Many application have

been reported by the same group and two nice representative applications are the following: (i)

deracemization of primary, secondary and tertiary amines using mutant amine oxidases in

combination with ammonia borane,[78-80] and (ii) Meso-pyrrolidines used as substrates for amine

oxidation by MAO-N D5 towards 1-pyrrolines.[81] These highly valuable applications in parallel with

the relatively high stereoselectivity and the cofactor independency establish MAO-N one of the

leading biocatalysts for amine oxidation.

Stimulated from the inspiring work of Turner and Orru on the oxidation of meso-pyrrolidines using

MAO-N D5 we turned our focus on the desymmetrization of 4-substituted piperidines which can lead

to analogous conversions and enantioselectivities. Since we developed an efficient synthetic strategy

to gain access to libraries of 4-substituted piperidines we wanted to investigate the effectiveness of

MAO-N, by incorporating it in our synthetic strategy. The aim of this section was to perform initial

set of screening reactions of different substrates and assess the trend of both conversions and

enantioselectivities towards optically active imines. In the event, we performed several enzymatic

reactions using different mutations of MAO-N (D5 and D9) so as to enzymatically desymmetrize

prochiral 4-alkyl piperidines and generate a library of optically active imines.

The MAO-N enzyme screening process included the use of freshly prepared MAO-N pellet cells of

two different mutations in order to assured that the activity of the enzyme has not been decreased.

Interestingly, the entries 1 and 6 (R= methyl and tert-butyl) furnished the highest enantioselectivities

accompanied with the highest conversions. In particular, the mutant D5 gave 100% conversion to

the entries 1 and 6 with enantioselectivities of 82% and 93% respectively. Furthermore, the

enantioselectivity observed concerning the D9 variant was 96% which is the best achieved, however

combined with 50 % conversion. Despite this promising results, it is hard to estimate any trend how

the R group of 4 substituted piperidines influence the enantioselectivities.

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Table 9: MAO-N enzyme screening results.[a]

Entry Substrate Enzyme Conversion (%) ee (%)

1

MAO-N D5 MAO-N D9

100 1

82 n.c

2

MAO-N D5 MAO-N D9

100 30

8 93

3

MAO-N D5 MAO-N D9

100 70

8 83

4

MAO-N D5 MAO-N D9

50 50

16 >85

5

MAO-N D5 MAO-N D9

50 50

20 80

6

MAO-N D5 MAO-N D9

100 50

93 96

7

MAO-N D5 MAO-N D9

100 20

16 53

8

MAO-N D5 MAO-N D9

4 40

5 92

9

MAO-N D5 MAO-N D9

13 50

5 85

[a] The enzyme-screening reactions were done by group of Prof. Nicholas Turner from the University of Manchester as part of a joint

collaboration on this project.

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Unfortunately, at room temperature these cyclic imines are prone to trimerization reactions that

lead to trimmers (115) formation (Scheme 29). This can lead to several problems in the GC-analysis

that imposes a few difficulties to identify the characteristic peaks of the chiral imine. In addition, we

faced various implications concerning the structural elucidation by means of NMR. For these reasons

we had to incorporate a derivatization step in our synthetic approach so as to generate additional

characteristic peaks and be able to characterize and determined the ee of the desired products.

Scheme 29: Trimmer formation proposed mechanism.

Particularly, addition of acetic anhydride (116) to these imines gives the corresponding acetylated

analogs (117) that can be straightforwardly visualized by proton NMR (Scheme 30).

Scheme 30: Imine derivatization towards acetylated analogs.

To our delight, the excellent conversions in combination with the promisingly high

enantioselectivities stimulate further optimization of the corresponding enzymatic process so as to

reach both excellent conversions and ee’s. What is highly valuable about these optically active imines

is the prospective use as inputs in a highly diastereoselective MCR.

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2.3 Investigation of the U-3CR towards 4-substituted piperidyl peptides. As previously emphasized, we focused on the development of two green approaches that can

cooperate in a synergistic manner towards the synthesis of complex molecules. In the last two

sections we described an environmentally friendly method to generate chiral imines that can be used

as optically active inputs for the last MCR step that facilitates the one-pot formation of highly valued

molecules in a diastereoselective manner. Even though, the biocatalytic desymmetrization is at the

early stages of research, the initial screening results are very promising towards excellent ee’s and

full conversions. In this section, we primarily turned our focus on the investigation of Ugi-type 3CR

between different isocyanides, carboxylic acids and the generated chiral imines. In addition we

subsequently explored how diastereoselective is the above-mentioned multicomponent reaction

racemic imines. As mentioned in the introduction our group discovered a very diastereoselective Ugi-

3CR with 1-pyrrolines for the synthesis of enantioenriched propyl peptides (Scheme 31). The high

selectivity is due to the bulk of the R-groups and increases with the size of the R-groups.[66]

Scheme 31: Diastereoselective U-3CR towards substituted propyl peptides.

After this remarkable discovery we focused on applying the same principles towards 4-substituted

piperidyl peptides (119) by incorporating racemic six-membered ring imines (118) to MCR step.

(Scheme 32).

Scheme 32: Diastereoselective U-3CR towards 4-alkylpiperidyl peptides.

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In our case we envisioned that the newly formed stereocenter can be controlled by the para

substituted R-groups on the imine six-membered ring. In particular, the suggested mechanism of the

Ugi reaction involves nucleophilic attack of the isocyanide (121) to the iminium ion (123) that is

formed by proton exchanged with the carboxylic acid (122). Subsequently, an acylated isoamide

(125) is formed by the nucleophilic addition of the carboxylate ion to the nitrilium anion (124). The

last step is a rearrangement, driven by the formation of the amide bond relocating the acyl group

from the oxygen to the nitrogen to afford the desired product (126) (Scheme 33).

Scheme 33: Suggested mechanism of the U-3CR.

Interestingly, the stereochemistry of the newly formed chiral center is set by the nucleophilic

addition of the isocyanide to the iminium anion making the U-3CR as a highly diastereoselective

methodology. In particular, the isocyanide addition can in principle take place either from the top or

the bottom face of the unsaturated six-membered ring which in each case provides different

stereochemical outcome. In detail, attack from the top face leads to the formation of an unstable

twist boat with high-energy transition state (128) which is less favorable than the chair conformation

transition state (127) that is formed when the attach takes place from the bottom face (Scheme

34).[70]

Scheme 34: Top face (ii) versus bottom face (i) isocyanide addition.

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In the event, we started our investigations with the reaction between methyl substituted imine (129),

two different isocyanides (130) and four different acids (131, Table 10). The methyl analog was

selected as the components of the model reaction and we only screen the substrate tolerance in

relation to different acid and isocyanides. In analogy with the optimized reaction conditions reported

by our group in 2010 towards the synthesis of substituted propyl peptides via U-3CR we applied the

same conditions for the synthesis of 4-substituted piperidyl peptides (132). Our primary goal was to

evaluate how efficient is the corresponding reaction in relation to the yields and subsequently

establish a representative result that indicates how diastereoselective this method is based on GC

analysis. Pleasingly all the examined substrates underwent clean conversion to the desired Ugi

product in moderate to good yields (35 - 70%). Benzoic acid analog (132c) is an exception in the trend

and further purification is needed by the means of column chromatography resulting in low yield of

35 %. A plausible explanation why 132c shows such a considerable lower yield compared to the other

Ugi products is the isolated polymer-like side product which suggests oligomerization or

polymerization reactions. In addition, is known that imines are prone to trimerization reactions

suggesting that the unconverted material form a trimmer prior to the MCR step.

Table 10: U-3CR with various carboxylic acids.[a]

[a] Reaction conditions: Imine 129 (1 eq), isocyanide 130 ( 1.33) and carboxylic acid 131 (1.33 eq.) were mixed in DCM. The mixture

stirred for 48h at room temperature. Yields refer to isolated material. [b] Purified by column chromatography.

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The next aim of the last section was to qualitative establish a representative example in relation to

the diastereoselectivity of this reaction and subsequently trigger future optimizations in order to

develop a fully diastereoselective U-3CR methodology towards 4-substituted piperidyl peptides. In

the event, we performed several GC measurements by analyzing our synthetically produced Ugi

products (Scheme 35). Based on GC analysis, we were delightful to discover that the generated

piperidyl peptides 132a-d show high d.r’s. To illustrate these high d.r’s, we chose 132a as an example

that representatively shows the high value of this method to synthesize complex molecules in an

asymmetric fashion. The compound 132a was generated in one-pot by the addition of 4-methyl-

2,3,4,5-tetrahydropyridine, tert-butyl isocyanide and acidic acid in DCM.

Scheme 35: Diastereoselectivity of the U-3CR based on GC analysis of U-3CR between 126, acidic acid and tert-butyl isocyanide. Peaks (1) and (2) show the two diastereomers.

Based on the GC analysis, illustrated in Scheme 35 we can presumably suggest that the minor peak

(2) is the minor diastereomer which indicates that the peak (1) is the major diastereomer. If this is

the case, the minor formation of the cis- product (134) clearly shows that the U-3CR towards 4-

substituted piperidyl peptides takes place in diastereoselective manner due to the formation of the

trans-product (133) as the major diastereomer. Despite this promising results, further analysis is

necessary to be done in order to clearly prove the major formation of the one diastereomer.

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3. Conclusions. The primary aim of this research was to develop an efficient synthetic method towards the synthesis

of 4-substituted piperidines. In this research we first synthesized the alkyl piperidines through a

sequence of 3 steps and overall yields ranging between 21-57%, depending on the R-group.

Unfortunately, this approach became a less suitable synthetic route towards the generation of a

representative library of 4-alkyl piperidines due to the limited diversity of the created library, the low

overall yields and the lack to meet the principles of green chemistry. After this setback, a relatively

greener and more efficient synthetic pathway was developed towards the desired product from the

commercially available 4-picoline, in overall yields of 36-94% over 2 steps. This strategy includes

numerous advantages compared to the previous one, such as fewer synthetic steps and higher yields,

and it can be considered as a more environmentally friendly process.

After establishing the above-mentioned green synthetic route towards these highly valuable inputs,

an initial set of enzyme-catalyzed desymmetrization reactions were implemented by using MAO-N

(D5 and D9) as the enzyme. These investigations were performed by the research group led by Prof.

Nicholas Turner at the University of Manchester as part of a joint collaboration on this project. It

turned out that most of the entries converted to the corresponding imine in good to very good

enantioselectivities which in some cases reached ee’s that are greater than 90%.

The incorporation of these imines in U-3CR is of utmost importance so as to develop a powerful

methodology to gain access to structurally complex products. In this perspective, the MCR step

showed, in an initial stage of research, to be very efficient and the chosen substrates convert to the

desired Ugi product in moderate to good yields (35 - 70%) and excellent diastereoselectivities.

Nevertheless, further investigations should be done to investigate the diastereoselectivity and to

establish representative library of these 4-substituted piperidyl peptides.

The further development and the optimization of this synergistic process that includes the synthesis

of 4-substituted piperidines/ MAO-N catalyzed desymmetrization/U-3CR can give access in an

environmentally friendly manner to a highly diverse library of compounds with high molecular

complexity (Scheme 36). The most remarkable potentials about this method is that is green and

efficient and it can find many applications in to future developments in drug discovery.

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Scheme 36: The synergistic strategy towards 4-alkylpiperidyl peptides.

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4. Future prospects. Taking everything into account this thesis research has led to the development of an innovative

and green synthetic strategy towards 4-substituted piperidines. It further illustrated how these

highly valued inputs can incorporate in an enzyme catalyzed step for the synthesis of optically

active MCR inputs that can give access to highly complex molecular structures. However, there is

always space available for supplementary things that can be improved or studied. The main focus

of follow-up studies should involve the optimization of the biocatalytic and MCR step towards

higher yields, enantioselectivities and diastereoselectivities. In addition, further investigations

towards the desymmetrization of more diverse substrates that can be further functionalized in

follow up synthetic steps in order to gain access to molecules with higher structural complexity.

Lastly, the need to discover valuable applications in the design of pharmaceutically interesting

compounds is more than necessary so as to establish our method as a highly applicable approach

in medicinally oriented synthesis.

4.1 Optimization and further investigations of the MAO-N catalyzed

reaction. Despite the preliminary investigations done on the biocatalytic step for the generation of

optically pure imines, there is still plenty of space available towards higher conversions and ee’s.

In particular, the reaction conditions can be further optimized to fully convert the meso 4-

substituted piperidines to the corresponding optically active imines. Since, the reported

conversions in chapter 2 are the outcome of a reaction time of 24h, prolonging that time can

complete the reaction and fully convert the starting materials to the desired product. In addition,

a more extensive substrate screening can be done in order to obtain valuable insights about the

factors that influence the selectivity of MAO-N, and develop through the directed evolution

process different MAO-N variants with higher enantioselectivities. These insights can be further

combined with different protein engineering techniques such as rational design and/or in silico

studies in the direction to develop a diverse library of enantioselective MAO-N variants, suitable

for different and most exotic inputs. These inputs can be further functionalized in subsequent

steps such as several couplings and azide transformation reactions. The only limiting factor with

respect to the diversity of these promising inputs is the need to be MCR inert in order not to

interfere in the following U-3CR step (Figure 6).

Figure 6: Proposed inputs for MAO-N desymmetrization.

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4.2 Establishing U-3CR reaction as a highly diastereoselective

methodology. Developing asymmetric MCR methods is of high importance; however, uncertainty always

remains how to efficiently control the newly formed chiral center during the process. For that

reason, inducing chirality through optically active starting materials is an alternative and robust

approach. Despite the initial studies taken place in this thesis research in respect to

diastereoselectivities, there are several investigations to be done in the direction of developing

an efficient and more diverse diastereoselective methodology. Our investigations involved

several GC measurements by analyzing our synthetically produced 4-methyl piperidyl peptides.

Despite the promising results, these MCR products were synthesized by using racemic imines

instead of the enzymatically enantioenriched ones. For that reason it would be most ideal if the

4-substituted piperidyl peptides could be diastereoselectively synthesized in an MCR of the

enzymatically produced optically active imines. Next to this, a diverse library of chiral MCR

products can be generated by inducing the MAO-N produced optically active imines in U-3CR step

(Figure 7). Subsequently, the relation between the R groups of the chiral imines and the

diastereoselectivity observed in the final product can be investigated by means of gas

chromatography and obtain valuable insights about the factors that enhance the

diastereoselectivity.

Figure 7: Proposed U-3CR library.

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4.3 The discovery of valuable applications of the Biocatalysis/U-3CR

method. The development of a versatile and green synthetic methodology was of utmost importance in

this research thesis in order to access highly molecular structures which are otherwise hard to

achieve. However, the need to find applications in the design of pharmaceutically interesting

compounds is highly important and necessary to establish our method as a highly applicable

synthetic tool. The structural motif of the U-3CR products can be found in several medicines, thus

this can be used to either synthesize derivatives of the actual medicine and/or used as an

alternative green approach to produce the actual medicine. A very nice example is Argatropan

(153) which is an antithrombin agent and it is used for the treatment of thrombosis in patients

with heparin-induced thrombocytopenia.[82] The core-structure of Argatropan is a 4-substituted

piperidyl peptide and the synthetic route could be facilitated by an MCR step combined with a

short supplementary synthesis (Scheme 37). The suggested approach is very interesting to

explore, however, there are a few uncertainties that need to be investigated. In particular, the

use of an isocyanide (151) that can be converted to the free acid is highly important and it is a

project that is currently under investigations in our group.

Scheme 37: Argatropan proposed synthetic route.

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A few other pharmaceutically interested target molecules that their synthesis can be simplified

by MCR step are Boceprevir (154), Asunaprevir (155) and Faldaprevir (156, Figure 8). These drugs

contain the structural information that is required in order to synthesize several derivatives of

them via U-3CR mediated step. Nevertheless, subsequent investigations should be done to

develop the entire synthetic route towards these medicines and if this methodology proved to

be efficient, it would provide a novel asymmetric procedure for these types of molecules.

Figure 8: Actual medicines containing the structural information of U-3CR product.

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Acknowledgement

After 9 months of research and successfully achieving the goal of my thesis project, I would like

to thank several important people that essentially contribute to complete my internship. First of

all, I would like to thank prof. Romano Orru and dr. Eelco Ruijter for giving me the opportunity to

do my Master internship in the synthetic organic chemistry group. Most importantly, I would like

to thank Gydo van der Heijden for all his help and daily supervision that definitely helped me to

develop myself as a synthetic chemist and of course for the good times in the lab. Without this

guidance this project would undoubtedly not have been this fruitful. Also, I would like to thank

Veronica Estévez for her helpful advices and solutions to my questions. Dr. Chris Slootweg is

thanked as well for being my second reviewer. Last but not least, I would like to thank the whole

synthetic organic group for the good times in the labs and during the well-organized and nice

“borrels”. I really appreciate all the help and knowledge that I gain in the lab and I can definitely

recognize a big improvement in my synthetic organic skills.

Valentinos Mouarrawis

14 February 2015

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5. Experimental Section

5.1 General remarks: Unless indicated otherwise, all reagents and solvents were purchased from Sigma Aldrich or

Acros Organics and used without any further purification. Diisopropylamine was stored in a

Schlenk flask under nitrogen atmosphere. Cyclohexane was distilled prior to use. Anhydrous THF

and Toluene were obtained by distillation using appropriate drying agents prior to use. Reactions

requiring anhydrous conditions were performed in vacuum flame – dried glassware under a

nitrogen atmosphere. Air and moisture sensitive liquids were transferred via syringe into the

reaction flasks through rubber septa. Flash chromatography was performed on silica gel (particle

size 40 - 63 μm, pore diameter 60 Å). Analytical TLC was performed on Merck Kieselgel 60F254

aluminum plates which were visualized using potassium permanganate /Δ, para – anisaldehyde/

Δ and UV light (254 nm). The 1H and 13C NMR spectra were recorded on a 400 MHz and 500 MHz

Bruker Avance spectrometer. Chemical shifts are quoted (δ) in parts per million (ppm),

referenced to the residual solvent resonance. Coupling constants are quoted in Herz (Hz).

Multiplicity is assigned as s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet

and bs for broad signal. Mass spectrometry analysis was performed on a Bruker micrOTOF-Q

instrument. Infrared spectra were recorder on a Shimadzu FTIR-8400s spectrometer and

wavelengths are reported in cm -1. Melting points were recorded on a Buchi M-565 melting point

instrument. GC-FID analysis was performed on Agilent 6850 GC.

5.2 Synthetic procedure (1): A synthetic strategy towards 4-substituted

piperidines.

Benzyl 4-oxopiperidine-1-carboxylate (1): 4-Piperidone monohydrate

hydrochloride (19.96 g, 130 mmol, 1.1 eq.) was dissolved in a mixture of THF/

water (400 mL, 1:1). K2CO3 (20, 44 g, 147.95 mmol, 2.5 eq.) was added and the

resulting mixture stirred for 20 min at 0 °C. Benzy chloroformate (8.44 mL, 59.18

mmol, 1 eq.) was added dropwise and the mixture allowed to warm to rt and

stirred for 12h. The reaction solution was quenched with NH4Cl aq. and the water layer was

extracted with ethyl acetate (3x). The combined organic layers were washed with brine, dried

over anhydrous Na2SO4. The volatiles were removed by means of a rotary evaporator yielding a

yellow liquid. Yield 46: 13.8 g, 59.18 mmol, 98 %. ; 1H-NMR (500 MHz, Chloroform-d) δ 7.45 (dd,

J = 24.6, 4.5 Hz, 5H), 5.14 (s, 2H), 3.53 (t, 4H), 2.19 (d, J = 39.1 Hz, 4H), ppm; 13C-NMR (500 MHz,

CDCl3) δ 155.14 (C), 136.87 (C), 134.82 (C), 128.35 (CH) , 127.81 (CH), 127.71 (CH), 66.89 (CH2),

35.48 (CH2), 27.58 (CH2) ppm; IR (neat): νmax (cm-1) = 2867, 1700, 1400, 1230, 1115, 730 ; HRMS

(ESI): m/z calculated for C13H15NO3 (M+H) = 233.1052, found = 234.1066

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5.2.1 Synthesis of phosphonium salts:

General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with

alkyl bromide (60 mmol, 1 eq.) in anhydrous toluene (30ml). Triphenylphosphine (60 mmol, 1

eq.) was added and the reaction mixture refluxed for 10h. The resulting mixture was cooled to

room temperature and the precipitate was filtered, washed with toluene, and concentrated in

vacuo to afford a white solid.

(n-Ethyl)triphenylphosphonium bromide (2):

Prepared from bromoethane (13.5 mL, 180 mmol, 3.00 eq.) and triphenylphosphine

(15.74 g , 60 mmol, 1 eq.) according to the general procedure. The title compound

was isolated as a white solid. Yield 66: 15 g, 40 mmol. 67 %. m.p.: 203 – 206 °C; 1H

NMR (500 MHz, Chloroform-d): δ 7.9 – 7.86 (m, 6H), 7.79 (t, J = 7.6 Hz, 3H), 7.72 (t,

J = 8.1 Hz, 6H), 3.76 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H) ppm; ; 13C-NMR (500 MHz, Chloroform-d): δ

135(CH), 133.7(CH), 130.4(CH), 118.9 (C), 23 (CH2), 15 (CH3) ppm; IR (neat): νmax (cm-1) = 2847,

1431, 1113 746, 730, 690, 518, 490; HRMS (ESI): m/z calculated for C20H20BrP (M+H) = 370.0486,

found = 291.1303

(n-Propyl)triphenylphosphonium bromide (3):

Prepared from bromopropane (5.45 mL, 60 mmol, 1,00 eq.) and

triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general

procedure. The title compound was isolated as a white solid. Yield 33: 15 g, 39

mmol, 65 %. m.p.: 220-223 1H NMR (500 MHz, Chloroform-d): δ 7.88– 7.83 (m, 6H), 7.81 (t, J =

7.6 Hz, 3H), 7.72 (t, J = 8.1 Hz, 6H), 3.78 (m, 2H), 2.04 (m, 2H), 1.58 (t, J = 7 Hz, 3H) ppm; ; 13C-

NMR (500 MHz, Chloroform-d): δ 138(CH), 134 (CH), 130(CH), 118 (C), 26 (CH2), 21 (CH2), 18 (CH3)

ppm; IR (neat): νmax (cm-1) = 2834, 1430, 1122 745, 735, 678, 593 565,, 572, 440; HRMS (ESI):

m/z calculated for C21H22BrP (M+H) = 384. 0642, found = 305.1344

(n-butyl)triphenylphosphonium bromide (4):

Prepared from bromobutane (6.48 mL, 60 mmol, 1,00 eq.) and

triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general procedure.

The title compound was isolated as a white solid. Yield 36: 20.2 g, 50 mmol, 84 %.

m.p.: 225-227 1H NMR (500 MHz, Chloroform-d): δ 7.93 – 7.89 (m, 6H), 7.81 (t, J

= 7.7 Hz, 3H), 7.76 (t, J = 8.3 Hz, 6H), 3.86 (m, 2H), 1. 63 (m, 4H), 1.12 (t, J = 6.9 Hz, 3H) ppm; ; 13C-

NMR (500 MHz, Chloroform-d): δ 136(CH), 133.9(CH), 131.4(CH), 119.2 (C), 24 (CH2), 22 (CH2),

19.8 (CH2), 11 (CH3) ppm; IR (neat): νmax (cm-1) = 2849, 1428, 1119 744, 722, 680, 605, 580, 466;

HRMS (ESI): m/z calculated for C22H24BrP (M+H) = 398.0799, found = 319.1199

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(Isobutyl)triphenylphosphonium bromide (5):

Prepared from 1-bromo-2-methylpropane (6.52 mL, 60 mmol, 1.00 eq.) and

triphenylphosphine (15.74 g.60 mmol, 1 eq.) according to the general procedure.

The title compound was isolated as a white solid. Yield 47: 50.88 g, 14.7 mmol,

24.5 %. m.p: 194-197 °C; 1H NMR (500 MHz, Chloroform-d): δ 7.89 – 7.81 (m, 6H),

7.77 (t, J = 7.7 Hz, 3H), 7.69 (t, J = 8.0 Hz, 6H), 3.70 (dd, J = 13.3, 6.2 Hz, 2H), 2.08 – 2.02 (m,1H),

1.04(d, J = 6.7 Hz, 6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 134.9(CH), 133.5(CH),

130.2(CH), 118.6 (C), 30.3 (CH2), 24.5 (CH), 24.2 (CH3) ppm; IR (neat): νmax (cm-1) = 2850, 1434,

1105, 748, 690, 528, 490; HRMS (ESI): m/z calculated for C22H24BrP (M+H) 398.0799, found =

319.1609

(Isopropyl)triphenylphosphonium bromide (6):

Prepared from 2-bromopropane (5.6 mL, 60 mmol, 1.00 eq.) and

triphenylphosphine (15.74 g ,60 mmol, 1 eq.) according to the general procedure.

The title compound was isolated as a white solid. Yield 38: 2.31g, 6 mmmol, 10%,

m.p.: 212-214 °C; 1H NMR (500 MHz, Chloroform-d): δ 7.89 – 7.81 (m, 6H), 7.77 (t, J = 7.7 Hz, 3H),

7.69 (t, J = 8.0 Hz, 6H), 3.70 (dd, J = 13.3, 6.2 Hz, 2H), 2.08 – 2.02 (m,1H), 1.04(d, J = 6.7 Hz, 6H)

ppm; 13C-NMR (500 MHz, Chloroform-d): δ 134.9(CH), 133.5(CH), 130.2(CH), 118.6 (C), 30.3 (CH2),

24.5 (CH), 24.2 (CH3) ppm; IR (neat): νmax (cm-1) = 2850, 1434, 1105, 748, 690, 528, 490; HRMS

(ESI): m/z calculated for C22H24BrP (M+H) 384.0642, found = 308.1466

5.2.2 Synthesis of CBz-protected 4-substituted piperidones:

General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with

phosphonium salt (1.5 eq.) in anhydrous THF and cooled to -78 °C. N-butyl lithium (1.6 N in

hexanes, 1.4 eq.) was added dropwise and the resulting solution was stirred for 1 hour. The

temperature was raised to 0 °C and the mixture stirred for 1 hour. Piperidone ( 1 eq.) was added

dropwise over a period of 20 min at - 78°C and stirred for 12h at room temperature. The reaction

mixture was then quenched with NH4Cl aq. The whole mixture was extracted with ethyl acetate

(3x), and the combined organic layers were washed with brine, dried over Na2SO4 and

concentrated under reduced pressure. The residue was purified by column chromatography on

silica gel using the eluent indicated below.

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Benzyl 4-ethylidenepiperidine-1-carboxylate (7):

Prepared from (n-Ethyl)triphenylphosphonium bromide (8.75 g, 30 mmol, 1.5

eq.) in THF (150ml), n-Butyl lithium (17.5 mL, 28 mmol, 1.4 eq.) and benzyl 4-

oxocyclohexane-1-carboxylate (4.66 g, 20 mmol, 1.00 eq.) according to the

general procedure. Column chromatography [Ethyl acetate/cyclohexane

(1:10)] gave a yellow liquid. Yield 69: 2.94 g, 12 mmol, 60%. TLC

(cyclohexane/Ethyl acetate, 10 : 1 v/v): Rf = 0.28; 1H NMR (500 MHz,

Chloroform-d): δ 7.42 – 7.28 (m, 5H), 5.29 (t, J = 7.0, 6.4 Hz, 1H), 5.15 (s, 2H), 3.47 (t, J = 6.2 Hz,

4H), 2.19 (d, J = 41.1 Hz, 4H), 1.60 (d, J = 6.7 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ

155.15 (C), 136.78 (C), 134.82 (C), 128.35 (CH), 127.81, (CH), 127.71 (CH), 118.17 (CH), 66.89

(CH2), 45.66 (CH2), 44.63 (CH2), 12.54 (CH3) ppm; IR (neat): νmax (cm-1) = 2860, 1693, 1425, 1250,

1110, 1014, 401; HRMS (ESI): m/z calculated for C15H19NO2 (M+H) 245.1416, found = 246.1477

Benzyl 4-propylidenepiperidine-1-carboxylate (8):

Prepared from (n-propyll)triphenylphosphonium bromide (12 g, 31.14 mmol, 1.5

eq.) in THF (150ml), n-Butyl lithium (18.17 mL, 29 mmol, 1.4 eq.) and benzyl 4-

oxocyclohexane-1-carboxylate (4.84 g, 20.8 mmol, 1,00 eq.) according to the

general procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)]

gave a yellow liquid. Yield 41: 3.27 g, 12.6 mmol, 61 %. TLC (cyclohexane/Ethyl

acetate, 10 : 1 v/v): Rf = 0.29; 1H NMR (500 MHz, Chloroform-d): δ 7.34 (dd, J =

24.2, 4.1 Hz, 5H), 5.23 (t, J = 6.7 Hz, 1H), 5.15 (s, 2H), 3.51 – 3.39 (m, 4H), 2.18 (d, J = 37.3 Hz, 4H),

2.01 (q, J = 7.1 Hz, 2H), 0.95 (t, J = 7.5 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ

155.14(C), 136.79 (C), 133.60 (C), 128.35 (CH), 127.81 (CH) 127.71 (CH), 126.13 (CH), 66.90 (CH2),

45.74(CH2), 44.87 (CH2), 20.26 (CH2), 14.53 (CH3) ppm; IR (neat): νmax (cm-1) = 2900, 1695, 1425,

1220, 1112, 1095, 985, 696; HRMS (ESI): m/z calculated for C16H21NO2 (M+H) 259.1572, found =

260.1634

Benzyl 4-propylidenepiperidine-1-carboxylate (9):

Prepared from (n-butyll)triphenylphosphonium bromide (20 g, 50 mmol, 1.3 eq.) in

THF (200ml), n-Butyl lithium (28.84 mL, 46.2 mmol, 1.2 eq.) and benzyl 4-

oxocyclohexane-1-carboxylate (8.97 g, 38.5 mmol, 1 eq.) according to the general

procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)] gave a

yellow liquid. Yield 44: 5.77 g, 21.12 mmol, 55 %. TLC (cyclohexane/Ethyl acetate,

10:1 v/v): Rf = 0.31; 1H NMR (500 MHz, Chloroform-d): δ 7.34 (dd, J = 24.0, 4.2 Hz,

5H), 5.23 (t, J = 7.1 Hz, 1H), 5.15 (s, 2H), 3.51 – 3.43 (m, 4H), 2.19 (d, J = 32.5 Hz, 4H), 1.98 (q, J =

7.0 Hz, 2H), 1.35 (h, J = 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-

d): δ 155.40 (C), 137.06 (C), 134.60 (C), 128.62 (CH), 128.08 (CH) 127.98 (CH), 124.54 (CH), 67.16

(CH2), 46.07(CH2), 45.15 (CH2), 29.28 (CH2), 23.20 (CH2), 13.89 (CH3) ppm; IR (neat): νmax (cm-1)

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56 | P a g e

= 2956, 1697, 1425, 1217, 1113, 1099, 696, ; HRMS (ESI): m/z calculated for C17H23NO2 (M+H)

273.1729, found = 274.1790

Benzyl 4-propylidenepiperidine-1-carboxylate (10):

Prepared from (Isobutyl)triphenylphosphonium bromide (10 g, 25 mmol, 1.3 eq.)

in THF (150ml), n-Butyl lithium (14.45 mL, 23.12 mmol, 1.2 eq.) and benzyl 4-

oxocyclohexane-1-carboxylate (4.5 g, 19.26 mmol, 1 eq.) according to the general

procedure. Column chromatography [Ethyl acetate/cyclohexane (1:10)] gave a

yellow liquid. Yield 53: 1.23 g, 4.5 mmol, 23.4 %. TLC (cyclohexane/Ethyl acetate,

10:1 v/v): Rf = 0.34; 1H NMR (500 MHz, Chloroform-d): δ 7.30 – 7.26 (m, 5H), 5.9 (t, J = 1.6 Hz,

1H), 6.77 (s, 2H), 4.39 – 4.36 (m, 4H), 4.08 (d, J = 29.5 Hz, 4H), 2.54-2.47 (m, 1H), 1.23 (d, J = 6.8,

6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 156 (C), 138 (C), 136 (C), 128.76 (CH), 128.18 (CH)

129.098 (CH), 124.34 (CH), 68 (CH2), 43.6 (CH2), 28.09 (CH2), 28.3 (CH), 23.98 (CH3) 23.34 (CH2)

ppm; IR (neat): νmax (cm-1) = 2956, 1697, 1425, 1210, 1114, 1101, 696; HRMS (ESI): m/z

calculated for C17H23NO2 (M+H) 273.1729, found = 274.1801

5.2.3 Synthesis of 4-alkyl piperidines:

General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with a

solution of CBz-protected piperidone (1 eq.) in methanol and 10 % Pd/C (0.1 eq.). The flask was

evacuated and flushed with H2 (3X) using a balloon. The suspension was stirred at room

temperature under hydrogen atmosphere for 16 h. Upon completion, the suspension was filtered

through celite. The celite was washed with MeOH (3x) and concentrated in vacuo to afford an oil.

4-ethylpiperidine (11):

Prepared from benzyl 4-ethylidenepiperidine-1-carboxylate (2.5 g, 10.2 mmol, 1 eq.) in

MeOH (50mL) and 10 % Pd/C (1.08 g, 1.02 mmol, 0.1 eq.) according to the general

procedure. The title compound was isolated as yellow oil. Yield 70: 1.04 g, 9.19 mmol,

90 %.1H NMR (500 MHz, Chloroform-d): δ 3.06 (d, J = 11.9 Hz, 1H), 2.94 (d, J = 11.4 Hz,

1H), 2.56 (t, J = 12.0 Hz, 1H), 2.36 (s, 1H), 1.88 (t, J = 11.1 Hz, 1H), 1.65 (t, J = 15.6 Hz, 2H),

1.30 – 1.01 (m, 5H), 0.92 – 0.82 (t, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 53.01

(CH2), 47.08 (CH2), 38.31 (CH), 32.40 (CH2), 11.47 (CH3) ppm; IR (neat): νmax (cm-1) = 2986, 2918,

2850, 1461, 1446, 466, 493; HRMS (ESI): m/z calculated for C7H15N (M+H) 113.1204, found

=114.1279

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4-propylpiperidine (12):

Prepared from benzyl 4-propylidenepiperidine-1-carboxylate (3 g, 11.56 mmol, 1 eq.) in

MeOH (50mL) and 10 % Pd/C (1.23 g, 1.16 mmol, 0.1 eq.) according to the general

procedure. The title compound was isolated as yellow oil. Yield 42: 1.31 g, 10.28 mmol,

89 %. 1H NMR (500 MHz, Chloroform-d): δ 3.41 (d, J = 12.5 Hz, 1H), 3.09 (s,1H) 3.02 (d, J

= 10.4 Hz, 1H), 2.80 (t, J = 12.5 Hz, 1H), 2.06 (s, 1H), 1.83 (d, J = 13.6 Hz, 1H), 1.69 - 1.40

(m, 3H), 1.37 – 1.11 (m, 5H), 0.92 – 0.78 (m, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 52.68

(CH2), 44.58 (CH2), 38.34 (CH2), 34.31 (CH), 29.40 (CH2), 14.45 (CH3) ppm; IR (neat): νmax (cm-1)

= 2954, 2893, 2846, 1648, 1452, 442; HRMS (ESI): m/z calculated for C8H17N (M+H) 127.1361 ,

found = 128.1433

4-butylpiperidine (13):

Prepared from benzyl 4-butylidenepiperidine-1-carboxylate (5 g, 18.3 mmol, 1 eq.) in

MeOH (70mL) and 10 % Pd/C (1.95 g, 1.83 mmol, 0.1 eq.) according to the general

procedure. The title compound was isolated as yellow oil. Yield 49: 2.47 g, 17.5 mmol,

95 %. 1H NMR (500 MHz, Chloroform-d): δ 3.07 (d, J = 12.2 Hz, 2H), 2.77 – 2.50 (m, 3H),

1.67 (d, J = 12.9 Hz, 2H), 1.38 – 1.16 (m, 7H), 1.10 (q, J = 14.7, 13.7 Hz, 2H), 0.87 (d, J =

6.4 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 46.74 (CH2), 36.97(CH2), 36.19

(CH), 33.43 (CH2), 28.87 (CH2), 23.02 (CH2), 14.20 (CH3) ppm; IR (neat): νmax (cm-1) = 2916,

2852, 1670, 1444, 493, 468 ; HRMS (ESI): m/z calculated for C9H19N (M+H) 141.1517, found =

145.1593

4-isobutylpiperidine (14):

Prepared from benzyl 4-butylidenepiperidine-1-carboxylate (5 g, 18.3 mmol, 1 eq.) in

MeOH (50mL) and 10 % Pd/C (1.95 g, 1.83 mmol, 0.1 eq.) according to the general

procedure. The title compound was isolated as yellow oil. Yield 49: 2.47 g, 17.5 mmol,

95 %. 1H NMR (500 MHz, Chloroform-d): δ 3.65 (d, 2H), 2.74-2.61 (m, 2H), 1.72-1.54

(m, 3H), 1.42-1.48(m, 1H,), 1.11-0.98 (m, 4H), 0.86 (d, J = 6.4 Hz, 6H). ppm; 13C-NMR

(500 MHz, Chloroform-d): δ 45.9 (CH2), 33.4 (CH2), 32.3 (CH2), 28.4 (CH), 24.4 (CH3),

22.7 (CH) ppm; IR (neat): νmax (cm-1) = 2954, 2901, 2898, 1670, 1458, 476, 435 ; HRMS (ESI):

m/z calculated for C9H19N (M+H) 141.1517, found = 142.1665

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5.3 Synthetic procedure (2): A synthetic strategy towards 4-substituted

piperidinium salts:

5.3.1 Synthesis of 4-alkylpyridines:

General procedure: A flame-dried round-bottom flask under N2 atmosphere was charged with a

solution of diisopropylamine in THF. The reaction mixture was cooled to −78°C and a solution of

n-butyllithium (1.6 M in hexanes) was added dropwise over a period of 20 min. The solution was

stirred at −78°C for 10 min, warmed to 0°C and stirred for 20 min. Subsequently, a solution of 4-

picoline in THF was added using a syringe over a period of 20 min at -78 °C. The resulting mixture

was stirred at –78°C for 20 min, warmed to -40 and stirred for 30 min. A solution of alkyl bromide

in THF was added dropwise over a period of 20 min at -78 °C. Afterwards, the reaction mixture

was stirred for 1 h at -78 °C. The temperature was allowed to reach room temperature while

stirring overnight. Subsequently, the reaction solution was quenched with NH4Cl aq. and washed

with water. The water layer was extracted with ethyl acetate (3x) and the combined organic

layers were washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The

crude product was purified by column chromatography on silica gel using the eluent indicated

below.

4-propylpyridine (15):

Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl

amide (2.93 ml, 21.6 mmol, 1.2 eq) and bromoethane (1.34 ml, 18 mmol, 1 eq) according

to the general procedure. Column chromatography [Ethyl acetate/cyclohexane (1:3)]

gave a yellow liquid. Yield 127: 2.10 g, 17.33 mmol, 95 %. TLC (Ethyl acetate/

Cyclohexane/, 1:3 v/v): Rf = 0.29; 1H NMR (500 MHz, Chloroform-d): δ 8.47 (d, J = 4.8 Hz,

2H), 7.09 (d, J = 4.8 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 1.65 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H)

ppm; 13C-NMR (500 MHz, Chloroform-d): δ 151.44 (C), 149.58 (CH), 123.93 (CH), 37.21 (CH2),

23.43 (CH2), 13.65 (CH3) ppm; IR (neat): νmax (cm-1) = 2960, 2933, 2871, 1600, 1413, 794, 626,

445; HRMS (ESI): m/z calculated for C8H11N (M+H) , 121.0891 found = 122. 0969.

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4-butylpyridine (16):

Prepared from 4-picoline (3.21 ml, 33 mmol, 1 eq) in THF (50 ml), lithium diisopropyl

amide (5.37 ml, 39.6 mmol, 1.2 eq) and bromopropane (2.98 ml, 33 mmol, 1 eq)

according to the general procedure. The title compound was isolated as an orange

liquid. Yield 127: 4.33 g, 32.02 mmol, 97 %. 1H NMR (500 MHz, Chloroform-d): δ 8.46

(d, J = 5.1 Hz, 2H), 7.09 (d, J = 5.0 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 1.60 (p, J = 7.6 Hz, 2H),

1.35 (h, J = 7.4 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H ppm; 13C-NMR (500 MHz, Chloroform-d): δ 151.83

(C), 149.73 (CH), 124.03 (CH), 35.05 (CH2), 32.54 (CH2), 22.37 (CH2), 13.97 (CH3). ppm; IR (neat):

νmax (cm-1) = 2965, 2931, 2860, 1600, 1413, 630, 582, 526, 501; HRMS (ESI): m/z calculated for

C9H13N (M+H) 135.1048, found = 136.1121.

4-Pentylpyridine (17):

Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (40 ml), lithium diisopropyl

amide (2.40 ml, 17.76 mmol, 1.2 eq) and bromopropane (1.6 ml, 14.8 mmol, 1 eq)

according to the general procedure. Column chromatography [Ethyl

acetate/cyclohexane (1:5)] gave a yellow liquid. Yield 127: 1.94 g, 13.05 mmol, 89%.

TLC (Ethyl acetate/ Cyclohexane/, 1:4 v/v): Rf = 0.3; 1H NMR (500 MHz, Chloroform-

d): δ 8.49 (s, 2H), 7.10 (d, J = 3.5 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 1.62 (p, J = 7.5 Hz,

2H), 1.32 (q, J = 11.0 Hz, 4H), 0.89 (t, J = 6.8 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ

151.87 (C), 149.75 (CH), 124.10 (CH), 35.34 (CH2), 31.47 (CH2), 30.12 (CH2), 22.58 (CH2), 14.11

(CH3) ppm; IR (neat): νmax (cm-1) = 2965, 2927, 2858, 1600, 1413, 630, 536, 503, 496; HRMS

(ESI): m/z calculated for C10H15N (M+H) 149.1204, found = 150.1278.

4-Isobutylpyridine (18):

Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (40 ml), lithium diisopropyl

amide (2.40 ml, 17.76 mmol, 1.2 eq) and 2-bromopropane (1.39 ml, 14.8 mmol, 1 eq)

according to the general procedure. Column chromatography [Ethyl

acetate/cyclohexane (1:3)] gave a yellow liquid. Yield 127: 1.52 g, 11.24 mmol, 76 %.

TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.26; 1H NMR (500 MHz, Chloroform-

d): δ 8.48 (d, J = 4.7 Hz, 2H), 7.07 (d, J = 4.9 Hz, 2H), 2.46 (d, J = 7.2 Hz, 2H), 1.89 (dq, J = 13.8, 6.8

Hz, 1H), 0.91 (d, J = 6.6 Hz, 6H) ppm;13C-NMR (500 MHz, Chloroform-d): δ 150.64 (C), 149.68

(CH), 124.70 (CH), 44.79 (CH2), 29.70 (CH), 22.41 (CH3) ppm; IR (neat): νmax (cm-1) = 2965, 2925,

2898, 1602, 1413, 630, 595, 536, 490;HRMS (ESI): m/z calculated for C9H13N (M+H) 135.1048,

found = 136.1121.

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4-(cyclohexylmethyl)pyridine (19):

Prepared from 4-picoline (1.44 ml, 14.8 mmol, 1 eq) in THF (30 ml), lithium

diisopropyl amide (2.40 ml, 17.76 mmol, 1.2 eq) and bromocyclohexane (1.88 ml,

14.8 mmol, 1 eq) according to the general procedure. Column chromatography

[Ethyl acetate/cyclohexane (1:5)] gave a yellow liquid. Yield 127: 1.15 g, 6.6 mmol,

44%. TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.32;1H NMR (500 MHz,

Chloroform-d): δ 8.46 (d, J = 4.8 Hz, 2H), 7.05 (d, J = 4.9 Hz, 2H), 2.46 (d, J = 7.1 Hz,

2H), 1.73 – 1.59 (m, 5H), 1.58 – 1.48 (m, 1H), 1.26 – 1.08 (m, 3H), 0.93 (q, J = 11.9 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 150.37 (C), 149.58 (CH), 124.76 (CH), 43.49 (CH2), 39.14

(CH), 33.12 (CH2), 26.47 (CH2), 26.27 (CH2) ppm; IR (neat): νmax (cm-1) = 2923, 2850, 1600, 1448,

1413, 804, 592,499; HRMS (ESI): m/z calculated for C12H17N (M+H) 175.1361, found = 176.1438.

4-(cyclopropylmethyl)pyridine (20):

Prepared from 4-picoline (3.21 ml, 33 mmol, 1 eq) in THF (50 ml), lithium diisopropyl

amide (5.37 ml, 39.6 mmol, 1.2 eq) and bromocyclopropane (2.64 ml, 33 mmol, 1 eq)

according to the general procedure. Column chromatography [Ethyl

acetate/cyclohexane (1:2)] gave an orange liquid. Yield 103: 1.57 g, 11.8 mmol, 36%.

TLC (Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.3; 1H NMR (500 MHz, Chloroform-

d): δ 8.49 (d, J = 4.9 Hz, 2H), 7.19 (d, J = 4.9 Hz, 2H), 2.53 (d, J = 6.9 Hz, 2H), 0.98 (dt, J

= 12.5, 5.3 Hz, 1H), 0.57 (d, J = 7.7 Hz, 2H), 0.21 (d, J = 4.7 Hz, 2H) ppm; 13C-NMR (500 MHz,

Chloroform-d): δ 151.20 (C), 149.69 (CH), 123.92 (CH), 39.69 (CH2), 10.83 (CH), 4.86 (CH2) ppm;

IR (neat): νmax (cm-1) = 2999, 1601, 1415, 815, 588 ,495, 487, 418, 406; HRMS (ESI): m/z

calculated for C9H11N (M+H) 133.0891, found =134.0969.

4-cyclohexylpyridine (21):

Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl

amide (4.88 ml, 36 mmol, 2 eq) and 1,5-dibromopentane (2.45 ml, 18 mmol, 1 eq)

according to the general procedure. Column chromatography [Ethyl

acetate/cyclohexane (1:2)] gave a yellow liquid. Yield 125: 0.7 g, 4.34 mmol, 25%. TLC

(Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.34; 1H NMR (500 MHz, Chloroform-d): δ

8.47 (d, J = 4.7 Hz, 2H), 7.11 (d, J = 4.8 Hz, 2H), 2.48 (s, 1H), 1.85 (s, 4H), 1.75 (d, J = 12.5

Hz, 1H), 1.40 (q, J = 11.3, 10.1 Hz, 4H), 1.31 – 1.18 (m, 1H) ppm; 13C-NMR (500 MHz, Chloroform-

d): δ 156.64 (C), 149.85 (CH), 122.46 (CH), 43.92 (CH), 33.62 (CH2), 33.50 (CH2), 26.64 (CH2) ppm;

IR (neat): νmax (cm-1) = 2923, 2850, 1595, 1448, 811, 622 ,555, 493, 420; HRMS (ESI): m/z

calculated for C11H15N (M+H) 161.1204, found =162.1278.

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4-cyclopentylpyridine (22):

Prepared from 4-picoline (1.75 ml, 18 mmol, 1 eq) in THF (40 ml), lithium diisopropyl

amide (4.88 ml, 36 mmol, 2 eq) and 1, 4-dibromobutane (1.83 ml, 18 mmol, 1 eq)

according to the general procedure. Column chromatography [Ethyl

acetate/cyclohexane (1:2)] gave a yellow liquid. Yield 128: 0.477 g, 3.24 mmol, 18%. TLC

(Ethyl acetate/ Cyclohexane/, 1:3 v/v): Rf = 0.33; 1H NMR (500 MHz, Chloroform-d): δ

8.47 (d, J = 4.7 Hz, 2H), 7.14 (d, J = 4.8 Hz, 2H), 2.97 (p, J = 8.6 Hz, 1H), 2.12 – 2.00 (m,

2H), 1.84 – 1.77 (m, 2H), 1.74 – 1.66 (m, 2H), 1.58 (p, J = 9.3 Hz, 2H) ppm; 13C-NMR (500 MHz,

Chloroform-d): δ 155.69 (C), 149.72 (CH), 122.76 (CH), 45.24 (CH), 34.04 (CH2), 25.63 (CH2) ppm;

IR (neat): νmax (cm-1) = 2948, 2916, 2867, 1596, 1409, 813, 630 ,545, 493; HRMS (ESI): m/z

calculated for C10H13N (M+H) 147.1048, found =148.1120.

5.3.2 Synthesis of 4-substituted piperidinium salts:

General procedure: To a solution of 4-alkylpyridine in ethanol in a 2-neck round bottom flask,

HCl/methanol (1.25 N) and platinum oxide were respectively added. Subsequently, the flask was

evacuated and flushed with H2 (3X) using a balloon. The mixture was stirred at room temperature

under hydrogen atmosphere for 36 h. Upon completion, the reaction mixture filtered through

Celite with thorough washing (EtOAc) and concentrated in vacuo to afford a solid.

4-ethylpiperidinium chloride (23):

Prepared from 4-ethylpyridine (0.57 ml, 5 mmol, 1 eq) in ethanol (30 ml), HCl/methanol

(20 ml, 25 mmol, 5 eq) and platinum oxide (0.057 g, 0.25 mmol, 0.05 eq) according to the

general procedure. The title compound was isolated as an off-white solid Yield 123: 0.735

g, 4.91 mmol, 98%. m.p.: 170 – 176.1 °C; 1H NMR (500 MHz, Chloroform-d): δ 9.37 (d, J =

158.3 Hz, 2H), 3.45 (d, J = 8.5 Hz, 2H), 2.83 (s, 2H), 1.88 (d, J = 13.2 Hz, 2H), 1.59 (d, J = 11.1

Hz, 2H), 1.46 – 1.25 (m, 3H), 0.88 (t, J = 7.0 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-

d): δ 44.30 (CH2) , 35.91 (CH2), 28.59 (CH) , 28.57 (CH2), 11.10 (CH3) ppm; IR (neat): νmax (cm-1)

= 2973, 2794, 2727, 2497, 1456, 1396, 993, 574, 441; HRMS (ESI): m/z calculated for C7H16ClN

(M+H) 149.0971, found = 148.1113.

4-propylpiperidinium chloride (24):

Prepared from 4-propylpyridine (1 g, 8.26 mmol, 1 eq) in ethanol (50 ml), HCl/methanol

(33.04 ml, 41.3 mmol, 5 eq) and platinum oxide (0.096 g, 0.42 mmol, 0.05 eq) according

to the general procedure. The title compound was isolated as an off-white solid. Yield

133: 1.34 g, 8.19 mmol, 99%. m.p.: 189 – 196.8 °C; 1H NMR (500 MHz, Chloroform-d): δ

9.35 (d, J = 149.9 Hz, 2H), 3.47 (d, J = 8.3 Hz, 2H), 2.87 (s, 2H), 1.93 (d, J = 12.9 Hz, 2H),

1.64 (d, J = 11.4 Hz, 2H), 1.46 – 1.28 (m, 3H), 1.13 (m,2H), 0.84 (t, J = 6.9 Hz, 3H) ppm;

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13C-NMR (500 MHz, Chloroform-d): δ 44.37 (CH2) , 35.98 (CH2), 28.69 (CH) , 28.64 (CH2), 20.36

(CH2) 11.14 (CH3) ppm; IR (neat): νmax (cm-1) = 2941, 2923, 2796, 2718, 1456, 574; HRMS (ESI):

m/z calculated for C8H18ClN (M+H) 163.1128, found = 156.1740

4-butylpiperidinium chloride (25):

Prepared from 4-butylpyridine (1.35 g, 10 mmol, 1 eq) in ethanol (55 ml),

HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05 eq)

according to the general procedure. The title compound was isolated as an off-white

solid. Yield 116: 1.65 g, 9.26 mmol, 93%. m.p.: 180 – 193 °C; 1H NMR (500 MHz,

Chloroform-d): δ 9.37 (d, J = 151.5 Hz, 2H), 3.44 (d, J = 11.3 Hz, 2H), 2.82 (d, J = 10.3

Hz, 2H), 1.85 (d, J = 13.6 Hz, 2H), 1.59 (q, J = 12.2, 11.6 Hz, 2H), 1.45 (s, 1H), 1.26 (s,

6H), 0.85 (t, J = 6.7 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.21 (CH2), 35.47 (CH2),

34.20 (CH), 28.88 (CH2), 28.67 (CH2), 22.76 (CH2), 14.09 (CH3) ppm; IR (neat): νmax (cm-1) = 2947,

2920, 2844, 2792, 2769, 2719, 1448, 576 ; HRMS (ESI): m/z calculated for C9H20ClN (M+H)

177.1244, found = 176.1421

4-pentylpiperidinium chloride (26):

Prepared from 4-pentylpyridine (1 g, 6.7 mmol, 1 eq) in ethanol (35 ml),

HCl/methanol (27ml, 33.5 mmol, 5 eq) and platinum oxide (0.08 g, 0.335 mmol, 0.05

eq) according to the general procedure. The title compound was isolated as an off-

white solid. Yield 134: 1.16 g, 6.05 mmol, 90%. m.p.: 178 –191 °C; 1H NMR (500

MHz, Chloroform-d): δ 9.39 (d, J = 160.0 Hz, 2H), 3.45 (d, J = 8.9 Hz, 2H), 2.91 – 2.73

(m, 2H), 1.87 (d, J = 13.0 Hz, 2H), 1.60 (d, J = 11.3 Hz, 2H), 1.47 (s, 1H), 1.26 (d, J =

14.6 Hz, 8H), 0.86 (t, J = 6.6 Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.28 (CH2), 35.76

(CH2), 34.26 (CH), 31.91 (CH2), 28.94 (CH2), 26.17 (CH2), 22.65 (CH2), 14.14 (CH3) ppm; IR (neat):

νmax (cm-1) = 2945, 2910 2846, 2792, 2767, 2734, 1448, 1074, 576, 437 ; HRMS (ESI): m/z

calculated for C10H22ClN (M+H) 191.1441, found = 200.1999

4-(isobutyl)piperidinium chloride (27):

Prepared from 4-isobutylpyridine (1.03 g, 7.61 mmol, 1 eq) in ethanol (38 ml),

HCl/methanol (30.44ml, 38.05 mmol, 5 eq) and platinum oxide (0.09 g, 0.381 mmol,

0.05 eq) according to the general procedure. The title compound was isolated as an

off-white solid. Yield 131: 1 g, 7.03 mmol, 92%. m.p.: 175 – 195 °C; 1H NMR (500

MHz, Chloroform-d): δ 9.41 (d, J = 161.8 Hz, 2H), 3.46 (d, J = 9.9 Hz, 2H), 2.84 (s, 2H),

1.85 (d, J = 9.6 Hz, 2H), 1.61 (dd, J = 17.0, 10.0 Hz, 4H), 1.17 (s, 2H), 0.86 (d, J = 6.2 Hz,

6H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 45.11 (CH2), 44.28 (CH2), 31.84 (CH), 29.07 (CH2),

24.52 (CH), 22.75 (CH3) ppm; IR (neat): νmax (cm-1) = 2890, 2796, 2769, 2736, 1448, 595, 499;

HRMS (ESI): m/z calculated for C9H20ClN (M+H) 177.1284, found = 176.1423

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4-(tert-butyl)piperidinium chloride (28):

Prepared from 4-(tert-butyl)pyridine (1.35 g, 10 mmol, 1 eq) in ethanol (55 ml),

HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05 eq)

according to the general procedure. The title compound was isolated as an off-white

solid. Yield 130: 1.73 g, 9.73 mmol, 98%. m.p.: 247 – 293 °C; 1H NMR (500 MHz,

Chloroform-d): δ 9.39 (d, J = 150.6 Hz, 2H), 3.53 (d, J = 12.2 Hz, 2H), 2.79 (q, J = 11.9 Hz,

2H), 1.85 (d, J = 13.8 Hz, 2H), 1.70 (q, J = 13.0 Hz, 2H), 1.21 (t, J = 12.0 Hz, 1H), 0.87 (s,

9H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.95 (CH), 44.85 (CH2), 32.42 (C), 27.15 (CH3),

24.02 (CH2) ppm; IR (neat): νmax (cm-1) = 2960, 2837, 2798, 2777, 2761, 2744, 2719, 1448, 1396,

1361, 1078, 568, 509; HRMS (ESI): m/z calculated for C9H20ClN (M+H) 177.1284, found = 176.1424

4-(cyclohexylmethyl)piperidinium chloride (29):

Prepared from 4-(cyclohexylmethyl)pyridine (0.75 g, 4.8 mmol, 1 eq) in ethanol (30

ml), HCl/methanol (19.2 ml, 24 mmol, 5 eq) and platinum oxide (0.055 g, 0.24

mmol, 0.05 eq) according to the general procedure. The title compound was

isolated as an off-white solid. Yield 93: 0.68 g, 3.73 mmol, 78 %. m.p.: 267 – 283

°C; 1H NMR (500 MHz, Chloroform-d): δ 9.40 (d, J = 164.6 Hz, 2H), 3.59 – 3.37 (m,

2H), 2.93 – 2.73 (m, 2H), 1.85 (d, J = 8.7 Hz, 2H), 1.65 (q, J = 18.2 Hz, 8H), 1.36 – 1.06

(m, 6H), 0.85 (q, J = 11.0 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.31

(CH2), 43.66 (CH2), 34.11 (CH), 33.56 (CH2), 31.09 (CH), 29.22 (CH2), 26.66 (CH2), 26.37 (CH2) ppm;

IR (neat): νmax (cm-1) = 2945, 2849, 2821, 2785, 2763, 2729, , 1446, 1074, 950, 597; HRMS (ESI):

m/z calculated for C12H24ClN (M+H) 217.1597, found = 216.1896

4-(cyclopropylmethyl)piperidinium chloride (30):

Prepared from 4-(propylmethyl)pyridine (1.33 g, 10 mmol, 1 eq) in ethanol (55 ml),

HCl/methanol (40 ml, 50 mmol, 5 eq) and platinum oxide (0.114 g, 0.5 mmol, 0.05

eq) according to the general procedure. The title compound was isolated as an off-

white solid. Yield 115: 1.52 g, 8.65 mmol, 86 %. m.p.: 170 – 174.8 °C; 1H NMR (500

MHz, Chloroform-d): δ 9.40 (d, J = 163.6 Hz, 2H), 3.47 (d, J = 10.5 Hz, 2H), 2.96 –

2.78 (m, 2H), 1.96 (d, J = 9.9 Hz, 2H), 1.63 (s, 3H), 1.21 (s, 2H), 0.63 (s, 1H), 0.43 (d,

J = 7.6 Hz, 2H), 0.33 (d, J = 4.2 Hz, 2H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.31 (CH2),

40.86 (CH2), 35.23 (CH), 28.95 (CH2), 8.37 (CH), 4.62 (CH2) ppm; IR (neat): νmax (cm-1) = 2939,

2839, 2794, 2769, 2733, 2700, 1591, 1454, 1074, 1010, 819, 590, 497; HRMS (ESI): m/z calculated

for C9H18ClN (M+H) 175.1128, found = 174.1424

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4-cyclohexylpiperidinium chloride (31):

Prepared from 4-cyclohexylpyridine (0.776 g, 5 mmol, 1 eq) in ethanol (30 ml),

HCl/methanol (20 ml, 25 mmol, 5 eq) and platinum oxide (0.057 g, 0.25 mmol, 0.05 eq)

according to the general procedure. The title compound was isolated as an off-white

solid. Yield 129: 0.91 g, 4.5 mmol, 89 %. m.p.: 180 – 222 °C; 1H NMR (500 MHz,

Chloroform-d): δ 9.43 (d, J = 163 Hz, 2H), 3.47 (d, J = 9.8 Hz, 2H), 2.78 (m, 2H), 1.88 –

1.63 (m, 10H), 1.26 – 1.20 (m, 4H), 1.22 (q, J = 10.3 Hz, 2H) ppm; 13C-NMR (500 MHz,

Chloroform-d): δ 44.97 (CH), 44.86 (CH2), 36.23 (CH), 29.34 (CH2), 27.32 (CH2) 13.65

(CH2), 11.65 (CH2) ppm; IR (neat): νmax (cm-1) = 2964, 2848, 2794, 2777, 2727, 1450, 541; HRMS

(ESI): m/z calculated for C11H22ClN (M+H) 203.1441, found = 202.1745

4-cyclopentylpiperidinium chloride (32):

Prepared from 4-cyclopentylpyridine (0.2 g, 1.36 mmol, 1 eq) in ethanol (10 ml),

HCl/methanol (5.44 ml, 6.8 mmol, 5 eq) and platinum oxide (0.015 g, 0.07 mmol, 0.05

eq) according to the general procedure. The title compound was isolated as an off-

white solid. Yield 136: 0.247 g, 1.3 mmol, 96 %. m.p.: 259 – 298 °C; 1H NMR (500

MHz, Chloroform-d): δ 9.29 (d, J = 213.0 Hz, 2H), 3.49 (s, 2H), 2.85 (s, 2H), 2.04 – 1.44

(m, 11H), ), 1.27 (m, 1H) 1.09 (s, 2H).ppm; 13C-NMR (500 MHz, Chloroform-d): δ 44.88

(CH), 44.77 (CH2), 36.22 (CH), 29.45 (CH2), 27.55 (CH2), 11.65 (CH2) ppm; IR (neat): νmax (cm-1)

= 2966, 2850, 2792, 2765, 2738, 2704, 2495, 1589, 1444, 1076, 522; HRMS (ESI): m/z calculated

for C10H20ClN (M+H) 189.1284, found = 188.1588

5.4 Synthetic procedure (3): The 3 component Ugi reaction towards 4-

substituted piperidyl peptides.

General procedure: Imine was dissolved in 9 ml of DCM together with the substituent addition

of isocyanide and carboxylic acid. The mixture was stirred at r.t for 48 h and upon completion 9

ml of DCM were added. The resulting mixture was washed with Na2CO3 (2x12ml), dried over

Na2SO4, filtered and concentrated in vacuo.

1-acetyl-N-(tert-butyl)-4-methylpiperidine-2-carboxamide (33):

Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq), tert-

butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and acetic acid (0.077 g, 1.33 mmol,

1.33 eq) in DCM (9 ml) according to the general procedure. The title compound

was isolated as yellow solid. Yield 109: 0.177 g, 0.74 mmol, 74%. m.p.: 85.4 - 91

°C; 1H NMR (500 MHz, Chloroform-d): δ 5.96 (s, 1H), 5.13 (d, J = 5.8 Hz, 1H), 3.71

(d, J = 13.6 Hz, 1H), 3.13 (t, J = 13.4 Hz, 1H), 2.28 – 2.07 (m, 3H), 1.62 (s, 4H), 1.33 (d, J = 20.4 Hz,

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9H), 0.92 (d, J = 6.6 Hz, 3H). ppm; 13C-NMR (500 MHz, Chloroform-d): δ 171.04 (C), 170.58(C),

52.88 (C), 44.72 (CH), 40.00 (CH2) 34.31 (CH2) , 33.93 (CH2), 29.15 (CH3), 27.14 (CH), 22.43 (CH3),

22.20 (CH3) ppm; IR (neat): νmax (cm-1) = 2988.42, 1683.74, 1558.38, 1541.02, 1458.08, 1419.51;

HRMS (ESI): m/z calculated for C13H24N2O2 (M+H) 240.1838, found = 240.1911

1-acetyl-N-isopropyl-4-methylpiperidine-2-carboxamide (34):

Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),

isopropyl isocyanide (0.13 ml, 1.33 mmol, 1.33 eq) and acetic acid (0.077 g, 1.33

mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. The title

compound was isolated as yellow oil. Yield 110: 0.116 g, 0.52 mmol, 52%. 1H NMR

(500 MHz, Chloroform-d): δ 5.90 (d, J = 7.9 Hz, 1H), 5.17 (d, J = 5.9 Hz, 1H), 4.01

(dt, J = 13.8, 6.9 Hz, 1H), 3.71 (d, J = 13.7 Hz, 1H), 3.13 (t, J = 13.4 Hz, 1H), 2.27 – 2.03 (m, 4H),

2.03 – 1.87 (m, 1H), 1.66 (d, J = 13.4 Hz, 1H), 1.10 (qd, J = 14.2, 12.9, 6.1 Hz, 7H), 0.92 (d, J = 6.5

Hz, 3H) ppm; 13C-NMR (500 MHz, Chloroform-d): δ 170.83 (C), 170.07 (C), 52.20 (CH), 44.46 (CH2),

41.39 (CH), 34.02 (CH2), 33.73 (CH2), 26.91 (CH), 22.72 (CH3) 22.15 (CH3), 21.95 (CH3) ppm; IR

(neat): νmax (cm-1) = 2925.81, 1697.33, 1533.30, 1429.15 1365.51, 981.70; HRMS (ESI): m/z

calculated for C18H26N2O2 (M+H) 226.1681, found = 227.1751

1-benzoyl-N-(tert-butyl)-4-methylpiperidine-2-carboxamide (35):

Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),

tert-butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and benzoic acid (0.17 g,

1.33 mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. Column

chromatography [Ethyl acetate/cyclohexane (1:3)] gave a white solid. Yield

111: 0.24 g, 0.35 mmol, 35%. m.p.: 81 – 89.5 °C; 1H NMR (500 MHz,

Chloroform-d): δ 7.42 (q, J = 7.9 Hz, 5H), 6.42 (s, 1H), 5.19 (d, J = 5.5 Hz, 1H),

3.68 (d, J = 13.7 Hz, 1H), 3.03 (t, J = 13.4 Hz, 1H), 2.26 (d, J = 14.1 Hz, 1H), 1.65 (s, 1H), 1.36 (s,

9H), 1.27 – 1.04 (m, 2H), 0.96 (d, J = 6.6 Hz, 3H). ppm; 13C-NMR (500 MHz, Chloroform-d): δ 172.15

(C), 170.05 (C), 135.31 (C), 130.17 (CH), 128.58 (CH), 126.96 (CH), 53.31 (CH), 50.97 (C), 45.87

(CH2), 34.00 (CH2), 33.47 (CH2), 28.79 (CH3), 26.94 (CH), 22.05 (CH3) ppm; IR (neat): νmax (cm-1)

= 2954.33, 1672.17, 1630.23, 1533.30, 1450.37, 1413.72, 1321.15, 1207.36; HRMS (ESI): m/z

calculated for C12H22N2O2 (M+H) 302.1994, found = 303.2064.

N-(tert-butyl)-4-methyl-1-pivaloylpiperidine-2-carboxamide (36):

Prepared from 4-methyl-2,3,4,5-tetrahydropyridine (0.097 g, 1 mmol, 1 eq),

tert-butyl isocyanide (0.15 ml, 1.33 mmol, 1.33 eq) and pivalic acid (0.14 g, 1.33

mmol, 1.33 eq) in DCM (9 ml) according to the general procedure. The title

compound was isolated as white solid. Yield 112: 0.20 g, 0.67 mmol, 67 %. m.p.:

88 – 99.3 °C; 1H NMR (500 MHz, Chloroform-d): δ 6.05 (s, 1H), 5.07 (d, J = 5.4

Hz, 1H), 4.15 (d, J = 13.7 Hz, 1H), 2.91 (t, J = 13.4 Hz, 1H), 2.35 – 2.13 (m, 1H),

1.62 (d, J = 13.1 Hz, 1H), 1.29 (s, 18H), 1.11 – 0.98 (m, 2H), 0.90 (d, J = 6.5 Hz, 3H) ppm; 13C-NMR

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(500 MHz, Chloroform-d): δ 178.28 (C), 170.45 (C), 50.98 (C), 44.44 (C), 39.00 (CH2), 33.93 (CH2),

33.63 (CH2), 31.05 (CH) 28.87 (CH3), 28.32 (CH3), 27.15 (CH), 22.15 (CH3) ppm; IR (neat): νmax

(cm-1) = 2867.33, 1687.21, 1602.74, 1514.02, 1473.51, 1458.08, 1363.58; HRMS (ESI): m/z

calculated for C16H30N2O2 (M+H) 282.2307, found = 305.2198.

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