asymmetric synthesis of c-glycosylated amino acids

Asymmetric Synthesis of C-glycosylated Amino Acids

- Incorporation in Collagen Glycopeptides and Evaluation

in a Model for Rheumatoid Arthritis

by

TOMAS GUSTAFSSON

Akademisk avhandling Som med tillstånd av rektorsämbetet vid Umeå universitet för erhållande av Filosofie Doktorsexamen vid Teknisk–Naturvetenskapliga fakulteten, framlägges till offentlig granskning vid Kemiska institutionen, Umeå universitet, sal KB3B1, KBC, fredagen den 25 november 2005, kl. 13.00. Fakultetsopponent: Prof. Peter Somfai, Organisk Kemi, Kungliga Tekniska Högskolan, Stockholm

COPYRIGHT © 2005 TOMAS GUSTAFSSON ISBN: 91-7305-975-7

PRINTED IN SWEDEN BY VMC–KBC UMEÅ UNIVERSITY, UMEÅ 2005

Title Asymmetric Synthesis of C-glycosylated Amino Acids - Incorporation in Collagen Glycopeptides and Evaluation in a Model for Rheumatoid Arthritis Author Tomas Gustafsson, Department of Chemistry, Organic Chemistry, Umeå University, SE – 901 87, Umeå, Sweden. Abstract

This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition.

The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of p-methoxybenzylidene acetals was obtained.

The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis.

The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide Keywords

Total synthesis, stereoselective synthesis, chiral glycine templates, Evan’s alkylation, asymmetric hydrogenation, α-amino acid synthesis, C-glycoside, rheumatoid arthritis, MHC, T cell.

ISBN: 91-7305-975-7

CCoonntteennttss 11.. LLiisstt ooff PPaappeerrss __________________________________________________________________________________________________________ 77

22.. LLiisstt ooff AAbbbbrreevviiaattiioonnss ______________________________________________________________________________________________ 99

33.. IInnttrroodduuccttiioonn____________________________________________________________________________________________________________ 1111

3.1. T cell response to antigens _____________________________________11

3.2. Rheumatoid arthritis and collagen induced arthritis __________________12

3.3. Objectives of the Thesis _______________________________________16

44.. CC--GGllyyccoossiiddeess__________________________________________________________________________________________________________ 1177

4.1. Carbohydrates vs C-glycosides __________________________________17

4.2. Synthetic strategies of C-glycosides ______________________________18 4.2.1. Anomeric nucleophiles ___________________________________18 4.2.2. Anomeric electrophiles ___________________________________19 4.2.3. Anomeric radicals _______________________________________22 4.2.4. Miscellaneous methods ___________________________________22

55.. AAssyymmmmeettrriicc ssyynntthheessiiss ooff αα--aammiinnoo aacciiddss ________________________________________________________________ 2255

5.1. Introduction _________________________________________________25

5.2. Synthesis of α-Amino acids ____________________________________25 5.2.1. Route A: Asymmetric Strecker synthesis _____________________26 5.2.2. Route B: Asymmetric hydrogenation ________________________28 5.2.3. Route C: Asymmetric carbon-carbon bond formation____________29 5.2.4. Route D: Asymmetric carbon-nitrogen bond formation __________32

66.. SSyynntthheessiiss ooff hhyyddrrooxxyyllyyssiinnee ____________________________________________________________________________________ 3355

6.1. Introduction _________________________________________________35

6.2. Retrosynthetic analysis ________________________________________35

6.3. Synthesis of the electrophilic constituent __________________________36

6.4. Enantioselective alkylation of a glycine template ____________________39

6.5. Investigation of the reductive opening of p-methoxybenzylidene acetals. _42

6.6. En route to galactose hydroxylysine C-glycoside ____________________46

77.. SSyynntthheessiiss ooff CC--GGaallaaccaattoossyyllaatteedd hhyyddrrooxxyynnoorrvvaalliinnee ________________________________________________ 4477

7.1. Introduction _________________________________________________47

7.2. Retrosynthetic analysis ________________________________________47

7.3. Synthesis of C-linked galactose on hydroxynorvaline ________________48

7.4. Incorporation of building bock into epitope CII(256-270) _____________51

7.5. En route to galactose hydroxylysine C-glycoside ____________________52

88.. SSyynntthheessiiss ooff aa ββ--DD--GGaallaaccttoossyyll tthhrreeoonniinnee CC--ggllyyccoossiiddee __________________________________________ 5555

8.1. Introduction ________________________________________________ 55

8.2. Synthesis: strategy and execution________________________________ 55

8.3. En route to galactose hydroxylysine C-glycoside ___________________ 59

99.. SSyynntthheessiiss ooff ggaallaaccttoossee hhyyddrrooxxyyllyyssiinnee CC--ggllyyccoossiiddee ________________________________________________ 6611

9.1. Introduction ________________________________________________ 61

9.2. Synthetic strategy ____________________________________________ 61

9.3. Assembly of the components ___________________________________ 62

9.4. The key asymmetric hydrogenation ______________________________ 63

9.5. Completing the synthetic route__________________________________ 67

9.6. Peptide synthesis ____________________________________________ 68

1100.. IImmmmuunnoollooggiiccaall eevvaalluuaattiioonn ______________________________________________________________________________________ 7711

10.1. Introduction ______________________________________________ 71

10.2. T cell response assays ______________________________________ 72

10.3. Results __________________________________________________ 72

1111.. CCoonncclluuddiinngg rreemmaarrkkss ________________________________________________________________________________________________ 7777

1122.. AAcckknnoowwlleeddggeemmeennttss ________________________________________________________________________________________________ 7799

1133.. RReeffeerreenncceess ______________________________________________________________________________________________________________ 8833


⎯ 7 ⎯

11.. LLiisstt ooff PPaappeerrss I Tomas Gustafsson, Magnus Schou, Fredrik Almqvist and Jan

Kihlberg; A total synthesis of hydroxylysine in protectedform and investigations of the reductive opening of p-methoxybenzylidene acetals. Journal of Organic Chemistry, 2004, 69(25), 8694-870.

II Eric Wellner, Tomas Gustafsson, Rikard Holmdahl and Jan

Kihlberg; Synthesis of a C-glycoside analogue of β-D-galactosyl hydroxynorvaline and its use in immunological studies. ChemBioChem, 2000, 1(4) 272-280

III Tomas Gustafsson, Maria Saxin and Jan Kihlberg; Synthesis of a

C-glycoside analogue of β-D -galactosyl threonine. Journal of Organic Chemistry, 2003, 68 (6), 2506-2509

IV Tomas Gustafsson, Mattias Hedenström and Jan Kihlberg;

Synthesis of a C-glycoside analogue of β-D-galactosyl hydroxylysine and incorporation in a glycopeptide from type II collagen Submitted to Journal of Organic Chemistry.

Reprinted with kind permission from the publishers.


⎯ 8 ⎯


⎯ 9 ⎯

22.. LLiisstt ooff AAbbbbrreevviiaattiioonnss

Abbreviation Meaning Ac Acetate APC Antigen Presenting Cell Aq. Aqueous Bn Benzyl Boc tert-Butoxycarbonyl BuLi Butyllithium CAN Cerium ammonium nitrate Cbz Benzyloxycarbonyl CIA Collagen Induced Arthritis CII Type II collagen CPT Chiral phase transfer DBAD di-tert-Butylazodicarboxylate DEAD Diethylazadicarbyxylate DIC 1,3-diisopropyl carbodiimide DMAP 4-N,N-Dimethylaminopyridine DMF Dimethylformamide DMS Dimethylsulfide DMSO Dimethyl sulfoxide DPPA Diphenylphosphoryl azide Fmoc 9-Fluoenylmethoxycarbonyl FmocOSu 9H-Fluorenylmethyloxycarbonyl succinate? Gal Galactose Glc Glucose HATU 1-Hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole KHMDS Potassium hexamethyldisilazane LDA Lithium diisopropyl amide LHMDS Lithium hexamethyldisilazane LN Lithium Naphtalide MHC Major Histacompatibility Complex Ms Methanesulfonyl NaHMDS Sodium hexamethyldisilazane NBS N-Bromo succinimide OSu Succinate OxCl Oxalyl chloride


⎯ 10 ⎯

Pd-C Palladium on charcoal Pg Protective group Piv Pivaloyl, trimethylacetyl PMB p-Methoxybenzyl PMP p-Methoxyphenyl RA Rheumatoid Arthritis Rt Room temperature SEM 2-(Trimethylsilyl)ethoxymethyl TBA, Q Tetrabutyl ammonium TBDMS tert-Butyldimethylsilyl TEA Triethyl amine TES Triethylsilyl Tf Triflate, Trifluoromethylsulfonate THF Tetrahydrofuran TIPS Triisopropylsilyl TMGA Tetramethyl guanidinium azide TMS Trimethylsilyl Tol Tolyl Trisyl Triisopropylbenzenesulfonyl Ts p-Toluenesulfonyl


⎯ 11 ⎯

33.. IInnttrroodduuccttiioonn

33..11.. TT cceellll rreessppoonnssee ttoo aannttiiggeennss11

HE IMMUNE SYSTEM CONSTITUTES A defence against infectious agents by distinguishing foreign pathogens and eliminating

them from the body. One part of the immune system, called the innate or non-adaptive immune system, acts through macrophages and neutrophils that engulf material and degrade it. This immune response is rapid but coarse, and can be evaded by infectious organisms. Moreover, the innate immune system does not allow for a memory of pathogens to be formed. Another part of the defence system is the adaptive immune system that elicits a response that is highly specific for a certain pathogen. It also includes a memory effect where the immune system remembers pathogenic antigens, thereby making future responses quicker and more effective. The adaptive immune system operates through lymphocytes in two types of responses: (1) B lymphocytes, or B cells, produce antibodies that bind to toxins and other soluble antigens, thereby neutralizing them. Antibodies also facilitate uptake, degradation and elimination of microorganisms. (2) B cells require help from CD4+ T cells, which also activate phagocytic cells to eliminate pathogens. Last but not least, other T cells (CD8+) act by destroying cells presenting antigen.

T cells identify their targets by recognizing peptide fragments that are presented on a cell surface. These peptides originate from proteins that are degraded and attached to so called major histacompatibility complex (MHC) molecules. MHC molecules transport antigens to the cell surface and present them to circulating T cells. There are two types of MHC molecules, class I and class II. The former are present on all cells and present peptides derived from proteins endogenous to the cell. Hence MHC I molecules can display fragments originating from viral proteins or from transformed cells, e.g. cancer cells. MHC II molecules are generally only found on specialized cells, i.e. antigen presenting cells (APCs), such

T


⎯ 12 ⎯

as macrophages, dendritic cells and B cells. These cells internalize proteins; both extracellular proteins and proteins attached to the cell surface, digeste them into shorter peptides and transport these to the surface of the antigen presenting cell bound by MHC II molecules. It is important to note that not only foreign peptides and peptides from pathogens are presented by MHC molecules. The vast majority of the MHC-peptide complexes on a cell surface are from normal endogenous cellular proteins.

The response of a T cell depends on both the type of T cell and the peptide-MHC complex. Cytotoxic CD8+ T cells recognize peptides bound by MHC I molecules and, if stimulated, kill the presenting cell by releasing cytotoxic compounds. In contrast, peptides bound to MHC II molecules are identified by inflammatory T cells or helper T cells (CD4+). If these are activated they release soluble proteins (cytokines) that serves as signal substances to other cells. Macrophages are activated and B cells start to multiply and produce antibodies. For the immune system to function properly, T lymphocytes need to be able to distinguish between self and non-self antigens. For this purpose T cells are tested during maturation in the thymus. T cells that display too weak affinity for self MHC molecules or those that are stimulated by self peptides bound by MHC molecules, are deleted. The role of this system is to ensure that T cells become tolerant to self antigens. However, all mechanisms have a risk of breakdown. Lymphocytes that escape the selection process can cause an auto-reaction, i.e. the immune system attacks the body’s own tissue or organs. Breaking of tolerance in this, or other ways, is believed to constitute one step in development of autoimmune diseases.2, 3

33..22.. RRhheeuummaattooiidd aarrtthhrriittiiss aanndd ccoollllaaggeenn iinndduucceedd

aarrtthhrriittiiss

HEUMATOID ARTHRITIS (RA) IS AN example of a disease that is regarded to be autoimmune. In RA, joint cartilage is primarily

destroyed, eventually leading to bone erosion. Although the process leading to RA is not fully understood, development of disease has been associated with certain MHC class II molecules called DR4 (HLA-DR4)

R


⎯ 13 ⎯

and DR1 (HLA-DR1). This implies that T cells are involved in the development of RA. The antigen responsible for initiating RA is unknown, but different constituents of cartilage4-6 or pathogens7 have been proposed as the culprits. One possible candidate is collagen type II (CII), which is the major protein component in cartilage.4 Immunization of certain strains of mice with CII from rat leads to an inflammatory respons called collagen induced arthritis (CIA), where symptoms similar to those of RA, i.e. erythema and swelling of peripheral joints, are displayed.8 In mice, CIA is associated with particular MHC II molecules, Aq (H-2Aq) and Ar (H-2Ar). Today murine CIA is the most frequently used animal model of RA, and recently a humanized version has been developed where transgenic mice expressing the human MHC II molecule DR4 are used.9 It has been previously shown that a panel of 29 T cell hybridomas, obtained from immunization of mice with type II collagen, recognize an immunodominant T cell epitope located within a polypeptide fragment of CII. For Aq MHC II, the CII epitope that is recognized by T cells was first identified as CII256-270.10 Recently the minimal epitope has been assigned to be CII260-267.11 The epitope for DR4 is located between CII259-273, but it is shifted four amino acids towards the C-terminus.

However, even though all of the T cells hybridomas recognized type II collagen and fragments containing CII256-270 after antigen processing, only 6 out of 29 responded to the synthetic peptide CII256-270 (Figure 3.1).12 It was later discovered that lysine is position 264 (Lys264) in native CII could be post-translationally hydroxylated and subsequently glycosylated. This hydroxylysine (Hyl264), and glycosylated derivatives thereof, are important features of the epitope. It was also found that a few disaccharide-peptide analogues could trigger T cells to respond.13, 14


⎯ 14 ⎯

O

NH

O

OHHO

HOOH

O

H2N

264

O

NH

O

OHHO

HOOH

O

264

O

NH

O

OHHO

OOH

O

H2N

264

OHO

HOHO HO

O

NH

O

OHHO

OOH

O

264

OHO

HOHO HO

Hyl264

Lys264

Gal-Hyl264 Gal-Hnv264 GlcGal-Hyl264 GlcGal-Hnv264

H-Gly256-Glu-Pro-Gly259-Ile-Ala-Gly-Phe-AA264-Gly-Glu-Gln-Gly-Pro-Lys270-Gly-Lu-Thr273-OH

CII256-270: AA = Hyl: The first identified epitope associated with 2-Aq CII260-267, AA = Gal-Hyl: Minimal epitope (2-Aq)CII259-273, AA = Gal-Hyl: Epitope associated with HLA-DR4

Figure 3.1. Summary of the recognition pattern for 29 different T cell hybridomas toward post-translationally modified peptides. Shown at the bottom are the sequences for the different epitopes discussed.

It is believed that the glycosyl moiety is directed outwards from the MHC molecule so as to allow interactions with the T cell receptor. The peptide-MHC complex is stabilized by anchoring certain pivotal amino acid residues of the peptide in hydrophobic pockets in MHC II. The anchor points for binding of CII256-270 to Aq are Ile260 and Phe263,15 and the corresponding residue for DR4 is Phe263.16, 17

In an attempt to probe the interactions between the peptide-MHC complex and the T cell receptor, a series of analogues were synthesized. Analogues of the peptide where hydroxylysine lacking the


⎯ 15 ⎯

aminomethylene-group in δ-position, called hydroxynorvaline, could in three instances still be recognized.18 In addition, deoxygenated galactose derivatives have been synthesized attached to hydroxylysine and incorporated in CII256-270 or CII259-273.19, 20 For most hybridomas interactions of the T cell receptor with the hydroxyl groups in the 2- and 4-position, and to a lesser extent the 3-position, on galactose were important for recognition (Figure 3.2).

H-Gly-Glu-Pro-Gly Ala-Gly Gly-Glu-Gln-Gly-Pro-Lys-Gly-Lu-Thr-OHHN

O

NH

HN N

H

O

H-2Aq H-2Aq HLA-DR4

HN

O

O

O

OHHO

HO OH

CD4+ T cell

Required for recognition

Weaker binding site

MHC molecule

Figure 3.2. T cell interactions with the galactose moiety (top) and anchoring positions for murine and human MHCII (bottom). The T cell interactions denoted with the arrows are general and summarize the behaviour of most hybridomas.


⎯ 16 ⎯

33..33.. OObbjjeeccttiivveess ooff tthhee TThheessiiss

O GAIN FURTHER INSIGHTS IN the T cell specificity, more glycopeptide analogues need to be synthesized. The 2-, 3-, 4-

and 6-deoxygenated galactose derivatives on hydroxylysine, along with the 4-methoxy and 4-fluoro galactose analogues, have already been prepared, incorporated in the epitope and tested for rocognition by T cells. Other analogues could include C-glycosides, where the anomeric oxygen is replaced by a methylene group. C-Glycosides have the added benefit of being much more stable towards chemical and enzymatic degradation. If glycopeptides at some point will arise as drug candidates suitable for vaccination, stability towards degradation is essential. Therefore, the main goal of this study was to develop a synthetic route to the C-glycoside analogue of galactosylated hydroxylysine. When obtained, this building block was to be incorporated in a peptide for evaluation in immunological studies of collagen induced arthritis. The C-glycoside of galactosylated hydroxylysine constitutes the most complex amino acid C-glycoside chosen as target to date since the synthesis involves construction of three stereogenic centra in a target with complex functionalization. To achieve this goal, two other C-glycosides, i.e. the C-glycosides of galactosylated hydroxynorvaline and threonine, as well as hydroxylysine itself, were chosen as targets for model studies.

OBnO

BnO

OBn

BnO

OH

O

N3

OTBDMSBocHN CO2H

NHFmocO

BnO

BnO

OBn

BnO

NHBocOH

O

OOH

HO

OH

OH

NHFmocOH

OBocHNGalactose hydroxylysine C-glycoside

Galactose hydroxynorvaline C-glycoside Galactose threonine C-glycoside 5-Hydroxylysine

Figure 3.3. The target molecules of this study.

T


⎯ 17 ⎯

44.. CC--GGllyyccoossiiddeess

44..11.. CCaarrbboohhyyddrraatteess vvss CC--ggllyyccoossiiddeess

ARBOHYDRATES HAVE GENERATED INTEREST in different fields of research during a long time. Of course, their value as

nutrition and structural elements cannot be ignored. In cell biology and medicine, carbohydrates and glycoconjugates display a great variety of functions including acting as cell signatures, regulating cell differentiation and mediating cell adhesion. A large number of synthetic chemists have focused their efforts on carbohydrates, attracted by the synthetic challenges they bring as well as the abundant applications. There seems to be an endless number of biological targets that depend on glyco-conjugates. However, theraputic agents containing a carbohydrate motif suffer from a number of drawbacks. These include poor uptake after oral administration, as well as rapid metabolism and excretion; the latter being due to the high polarity and inherent lability of the glycosidic bond. As other acetal linkages it can easily be degraded under chemical or enzymatic conditions. To utilize the possibility of drugs based on carbohydrates to the fullest, this and other problems need to be resolved. One way to overcome the instability of the glycosidic bond is to exchange the exocyclic oxygen atom for a methylene group, thereby making a C-glycosidic analogue or a C-glycoside.21, 22 C-Glycosides can be regarded as isosters of O-, S- and N-glycosides with high stability toward severe conditions, while most properties of the original glycoside is retaianed. Their bond lengths, van der Waals surfaces and conformations are almost identical.23-25 Even though there are many findings that support that C-glycosides compare well with O-glycosides, there are some indications of unique conformations of C-glycosides, not found in O-glycosides.26, 27 Of course, by the exchange of an oxygen atom by a carbon atom, there is no anomeric effect, no possibility for hydrogen bonding and the dipole moment is reduced. Still, the most pronounced

C


⎯ 18 ⎯

distinction between O- and C-linked carbohydrates lies within the chemical reactivity and, in many cases, the difficulty in preparation of C-glycosides.

44..22.. SSyynntthheettiicc ssttrraatteeggiieess ooff CC--ggllyyccoossiiddeess

UMEROUS METHODS HAVE BEEN USED to synthesize C-glycosides. However, a few main strategies can be discerned:

(1) use of the anomeric centre as a nucleophilic species, (2) use of the anomeric centre as an electrophilic species, (3) anomeric radicals, and (4) miscellaneous methods. These methods will be discussed briefly in this chapter.

44..22..11.. AAnnoommeerriicc nnuucclleeoopphhiilleess

NUMBER OF METHODS CAN be used for reductive lithiation of the anomeric centre, thereby allowing it to react with an

electrophilic species to form the C-C bond of the C-glycoside.

O O

Li

O

E

O

Cl

O

SnBu3

O

Li

O

ELN, Bu3SnCl

orBu3SnLi

HCl

tBuLi

BuLi

LN

"E "

"E "

"E " = aldehydes, activated alkylhalides

retains stereochemistry

α-selective

Figure 4.1. Different ways to prepare C-glycosides from a lithiated anomeric position (LN = Lithium naphtalide).

N

A


⎯ 19 ⎯

Glycals can be lithiated directly with tert-butyl lithium and reacted with electrophiles such as aldehydes or activated alkyl halides, e.g. MeI, allyl halides, and benzyl halides (Figure 4.1).28 2-Deoxy derivatives can be generated by treating a glycal with HCl to give glycosyl chlorides that can be lithiated either directly or via the stannane.29 Direct lithiation always provides the α-anomer, while lithiation via the stannane retains the stereochemistry of the stannane.30

O

X

LDAO

XLi

X = NO2, CO2R, SR, SOR, SO2R

Figure 4.2. Activated anomeric lithiations. Electron withdrawing groups at the anomeric centre will facilitate deprotonation. (Figure 4.2).31-33 The α/β-selectivity in the substitution reaction will depend on the electrophiles, but can generally be said to favour β-substitution.

O

SO2Pyr

SmI2

R R´

O O

SmIII

O

HOR

R´

retains stereochemistry Figure 4.3. Reductive samaration of sulfones.

Recently, samariumII iodide has been used together with glycosides sulfonated at the anomeric position to form a nucleophilic SmIII species that can react with aldehydes or ketones in situ (Figure 4.3).34

44..22..22.. AAnnoommeerriicc eelleeccttrroopphhiilleess

OSSIBLY SACCHARIDES WITH ELECTROPHILIC ANOMERIC positions are even more common than nucleophilic anomeric

species in C-glycoside forming reactions. Any activating group, may it be a halide, acetate, alcoholate or a lactone, can in principle be used to make

P


⎯ 20 ⎯

the anomeric position electrophilic, analogous to methods used for synthesis of O-glycosides. As mentioned earlier, C-glycosides lack the anomeric effect, leaving the stereoelectronic effects as the main influence on the stereochemical outcome of the reaction. As a consequence of this, the α-isomers are often more accessible than their β-counterparts. In O-glycosidations the stereochemical outcome is to a great extent directed by neighbouring group participation. This effect is far less pronounced in formation of C-glycosides.

O

X

OSiMe3

Lewis Acid

X = OH, OR, OAc, F, Cl

α-selective

Figure 4.4. C-Glycosylations with allyl silanes. Allyl silanes (Figure 4.4) in combination with Lewis acids can be used for C-C bond formation for a variety of electron withdrawing groups.35-37 In spite of being somewhat solvent dependent, the stereochemical outcome usually favours formation of the α-isomer.

O

Br

O

RO

O

ORNaH

O

OR

O

OOR

Figure 4.5. Activated enolates are useful nucleophiles in reactions with glycosyl bromides. Figure 4.5 illustrates that it is possible to achieve β-selectivity in formation of C-glycosides.38 This is due to the SN2 character of the reaction and the fact that α-glycosyl bromides are easily attainable.


⎯ 21 ⎯

O

O

O

HO

β-selectiveMgBr

n

n = 0,1n

O

BrAcO

via 1,2-anhydro glycoside

Figure 4.6. 1,2-Anhydro sugars as anomeric electrophiles. The SN2-type reaction can also be utilized to give β-linked C-glycosides after opening of 1,2-anhydro glycosides, usually formed by oxidation of glycals (Figure 4.6).39 This reaction leaves the hydroxy group in the 2-position unprotected.

O

O

O

OHR

O

R

Et3SiH,BF3

β-selectiveRLi

RMgBr

Figure 4.7. Organometallic reagents readily add to lactones. One of the most frequently used reaction types to form β-isomers of C-glycosides are Grignard and organolithium additions to glycolactones.40-42 The reaction results in an intermediate hemiacetal that can be reduced to the corresponding β-C-glycoside with triethylsilane and boron trifluoride etherate.

O

OH

Ph3POR

OO

O

OR

α,β mixture, equilibrates toβ-anomer in basic solution

Figure 4.8. Addition of Wittig-type reagents to a hemiacetal. Reagents belonging to the Wittig family can add to the masked aldehyde of a hemiacetal. The reaction is limited to activated Wittig43 or Horner-Emmons reagents.44 Both isomers at the anomeric position can be formed


⎯ 22 ⎯

but the destabilized α-isomer equilibrates to the β-isomer in basic solution.

44..22..33.. AAnnoommeerriicc rraaddiiccaallss

NOMERIC RADICALS CAN BE GENERATED from halides, methylthiothiocarbonates, nitro glycosides, thiophenols and

phenylselenium glycosides. The radical can be quenched either with tributyltin hydride or another radical acceptor, such as acrylonitrile. Both isomers are accessible, but the selectivity depends on radical stability and can be hard to direct. Radical reactions can also be plagued by rearrangements, especially when neighbouring hydroxyl groups are protected with acetyl groups.

O

O2N R

O

R CN

CN

Bu3SnH

CN

Bu3SnHO

Br

OCN

O O

O

Bu3SnH Figure 4.9. Different radical couplings to form C-glycosides. Figure 4.9 shows a few examples of C-glycosides prepared from anomeric radicals.45, 46

44..22..44.. MMiisscceellllaanneeoouuss mmeetthhooddss

FEW EXAMPLES OF METHODS not obviously belonging to any of those discussed above will be briefly summarized here.

A

A


⎯ 23 ⎯

OAcORZnI, BF3 Et2O

O

Ror RZnI, PdCl2

Figure 4.10. Organozinc couplings.

Organozinc reagents can be coupled to acetate protected glycals (Figure 4.10).47 The resulting 2,3-glycal can be oxidized with osmium tetraoxide to the corresponding glycoside, usually producing C-mannosides.

OOTBDMSO

O

OMe

O

Heat

Figure 4.11. Claisen rearrangements.

Acetates can be transformed into silylenol ethers which then undergo a Claisen rearrangement to give a C-glycoside.48 Again, 2,3-glycals are formed, which often require oxidation tfor use as carbohydrate analogues.


⎯ 24 ⎯


⎯ 25 ⎯

55.. AAssyymmmmeettrriicc ssyynntthheessiiss ooff αα--aammiinnoo aacciiddss

55..11.. IInnttrroodduuccttiioonn

VEN THOUGH THE NUMBER OF COMMONLY occuring amino acids in biological systems are as low as 20, over 500 diverse α-

amino acids have been found in nature.49 Just as carbohydrates, α-amino acids are also one of the main types of building blocks found in all living beings. Nature, in its ingenuity is able to produce practically all endogenous substances from the biogenic precursor acetic acid. In comparison, only crude synthetic methods are available to man in general and organic chemists in particular, even though the progress that has been made the last 50 years is truly staggering. Due to their importance in biological systems and their usefulness as a source of chirality in organic synthesis, a considerable interest has been given to asymmetric synthesis of α-amino acids, natural and unnatural alike.

55..22.. SSyynntthheessiiss ooff αα--AAmmiinnoo aacciiddss

HIS CHAPTER WILL SERVE AS a short introduction to the field of amino acid synthesis, exemplifying some traditional and some

more recent approaches. Figure 5.1 shows four general methods to generate the stereogenic centrum at the α-position of amino acids. In brief, the different routes rely on carbon-carbon (routes A and C), carbon-hydrogen (B) and carbon-nitrogen (D) bond forming reactions.

E

T


⎯ 26 ⎯

NHR1

ROR2

O

A B

C D

O

HR NR1

R3

NR1

ROR2

OOR2

O

or

R1NH2, HCN

+ +

chiral cat.

NHR1

ROR2

O

NHR1

ROR2

O

Figure 5.1. The four main strategies for synthesis of α-amino acids presented in this chapter.

55..22..11.. RRoouuttee AA:: AAssyymmmmeettrriicc SSttrreecckkeerr ssyynntthheessiiss

VER 150 YEARS AGO STRECKER PUBLISHED his results on the synthesis of α-amino nitriles in a three component reaction

that now carries his name.50 Treatment of aldehydes with ammonia and hydrogen cyanide result in amino nitriles that can be hydrolyzed to amino acids. At the time of the discovery of the reaction the concept of chirality was not well recognized and the original reaction was consequently achiral. Recently, great efforts have been devoted to finding a stereoselective version of the reaction. These efforts have resulted in two major strategies; formation of an intermediate chiral imine or use of chiral ligands (Schemes 5.1 and 5.2).

In the first example (Scheme 5.1), an amino acid reacts with phenylalanine amide to form a chiral imine, which is attacked by cyanide.51 When using phenylalanine amide, this reaction produces two equilibrating products of which one precipitates and the product is obtained as a single diastereomer. Percipitation of one diastereomer may not be applicable to every substrate, but in the case of non-crystalline products chromatography may be used for purification. After

O


⎯ 27 ⎯

hydrogenation and hydrolysis the amino acid is obtained in an excellent enantiomeric excess. In the second example, Enders’ SAMP forms a hydrazone that undergoes cyanation. The amino acid is then released through a sequence involving protection, oxidation and hydrolysis.52

O

H1)

Ph

CONH2H2N

2) NaCN, AcOH

Ph

CONH2HN

CN

NH2

CO2H

1), H2SO42) H2/Pd-C

3) HCl (6 M)

(93%, >99:1 dr) (73%, >98% ee)

N

HAlk

N

OMeTiCl4, TMSCN

NH

CNAlk

N

OMe

(75-93%, 88-91% de)

NH2

CO2HAlk

3 steps

(29-53%, 94-97% ee) Scheme 5.1. Asymmetric Strecker synthesis via chiral imines.

Two out of a multitude of chiral ligands that can be used for the asymmetric Strecker synthesis are shown in Scheme 5.2.53 Lipton and co-workers used a diketopiperazine as a catalyst that gave good yields and selectivities for imines derived from aromatic aldehydes.54 Kobayashi et. al. used a more elaborate zirconium-catalyst containing no less than three binaphtyls.55 The aldehydes that can be used with this catalytic system can be of aliphatic or aromatic nature and usually give yields higher than 80%. After methylation of the phenolic hydroxyl group, hydrolysis can be performed either to the ester or amide. Removal of the aromatic group with cerium ammonium nitrate (CAN) liberates the free amine that can be used for recrystallization to giva a hydrochloride salt which affords an enantiomerically pure α-amino acid ester.


⎯ 28 ⎯

Ph

PhNAr

HCN

HNNHPh HN

O

O

NH

NH2

Ph

PhNH

Ar

CN

(71-97%, 10-99% ee)

O

HR

(5 mol%)

Br

Br

OO

ZrBr

Br

dimer

HN

CNROH

(76-100%, 84-94% ee)

(1-5 mol%)

MeMe

OHH2N

HCN

O

Scheme 5.2. Ligands for induction of chirality in the Strecker synthesis.

55..22..22.. RRoouuttee BB:: AAssyymmmmeettrriicc hhyyddrrooggeennaattiioonn

SYMMETRIC CATALYSIS PLAYS AN INCREASINGLY important role in organic synthesis. Even though the catalytic transition

metals are the same today as in the past, great achievements have been made in the development of chiral ligands. In the synthesis of α-amino acids through asymmetric hydrogenation the most frequently used metal is rhodium and most ligands are based upon the coordinating ability of phosphorous. To a great extent, the mechanistic details that allowed for this ligand development have to be credited to the work of Halpern56 and Brown.57

A


⎯ 29 ⎯

O

O

PPh2

PPh2

P

P

PhPh

OMe

OMe

P P

R

RR

R

P P

R

RR

R

DIOP DIPAMP BPE DuPHOS

R1

R2

R3 NHAcOMe

O R1

R2

R3 NHAcOMe

O[Et-DuPHOS-Rh]+

H2

Regioselectivity >98%Stereoselectivity >98%

Scheme 5.3. Some common ligands for Rh-catalysts and an example of an asymmetric hydrogenation using DuPHOS-Rh.

A few of the most frequently used ligands for rhodium catalysts are exemplified in Scheme 5.3. DIOP was one of the earliest ligands, developed by Kagan.58 DIPAMP, which is chiral at phosphorous, has found industrial applications in the synthesis of L-DOPA.59 However, the ligands encountered most frequently in the literature today are those developed by Burk, i.e. the BPE and DuPHOS-ligands.60 These catalytic systems are used under very mild conditions with high selectivity and tolerate a great deal of functionalities in the substrate.

55..22..33.. RRoouuttee CC:: AAssyymmmmeettrriicc ccaarrbboonn--ccaarrbboonn bboonndd

ffoorrmmaattiioonn

LYCINE TEMPLATES HAVE A DOMINANT role for the synthesis of α-amino acids and chirality can be introduced through two

major strategies. Either glycine is coupled to a chiral auxilary, or a chiral catalyst can be used. The number of chiral auxilaries that can be attached

G


⎯ 30 ⎯

to glycine to induce chirality is constantly growing. The templates can act as nucleophiles, electrophiles or through radical mechanisms.61

NN

MeO

OMe

BuLi

RXN

NMeO

OMeR

O

OHR

NH2Hydrolysis

CO2Me

or O

OHNH2

MeO2C

or

N

ONH2Ph

OH

LDA, LiClRX N

ONH2Ph

OH R

NaOH

O

OHR

NH2

(a)

(b)

Scheme 5.4. The chiral glycine templates developed by Schöllkopf (a) and Myers (b).

Two examples of chiral glycine templates are shown in Scheme 5.4; (a) Schöllkopf’s bis-lactim ether62-64 and (b) Myers psuedoephedrine glycinamide.65 They both give very good stereoselectivity but require harsh conditions, both in the alkylation and the hydrolysis step. The electrophiles generally useful in these reactions are limited to activated alkyl halides (allyl, benzyl, methyl, ethyl, some longer alkyls) and α,β-unsaturated esters in the case of Schöllkopfs bis-lactim ether (a).

The morpholinone templates developed by Williams66 (Scheme 5.5) are commercially available in both enantiomeric forms, protected either as t-butyl or benzyl carbamates. These templates are used either as nucleophilic66 or electrophilic67 species. The enolate can be formed with a strong base, such as LHMDS or NaHMDS and reacted with an alkyl halide as shown in route (a). As often is the case with this type of alkylations, the use of activated alkyl halides is hugely beneficial for the reaction. The diphenylethylene auxiliary can be removed with medium pressure hydrogenation over palladium or by using Birch conditions.


⎯ 31 ⎯

RNO

O

PhPh

R = Cbz, Boc

NaHMDS

R1X

RNO

O

PhPh

R1

O

OHR1

NHR

O

OHR1

NH2Pd-C,

H2

or Li, NH3

orNBS

RNO

O

PhPh

Br

RNO

O

PhPh

R1

ZnCl2

R1M

(a)

(b)

R1M = Organozinc, Organocuprate Silyl enol ether, Allyl silane

Scheme 5.5. Two strategies to form α-amino acids using William’s glycine templates.

Bromination (b) of the same glycine template with NBS in refluxing CCl4 produces an electrophilic glycine equivalent. Activation with ZnCl2 followed by addition of an organometallic species, such as organocuprates, allyl silanes or silyl enol ethers, leads to the same type of precursor for α-amino acids as in route (a).

Lygo68 and Corey69 have developed a chiral phase-transfer catalytic system that promotes alkylations of the diphenyl Schiff base of glycine t-butyl ester. The chiral catalyst is based upon the cinchona alkaloids (Scheme 5.6). With all the benefits from a catalytic reaction it allows for alkylation of the protected glycine, most often with activated alkyl halides, but also with α,β-unsaturated esters.


⎯ 32 ⎯

N

N O

Br

PTC =

NPh

Ph

Ot-Bu

ON

Ph

Ph

Ot-Bu

O

R

PTC, CsOH

RX

RX = MeI, EtI, AllylBr, BnBr, etc

> 70% yield> 95% ee

Scheme 5.6. Asymmetric alkylation using a cinchonidinium based phase transfer catalyst.

55..22..44.. RRoouuttee DD:: AAssyymmmmeettrriicc ccaarrbboonn--nniittrrooggeenn bboonndd

ffoorrmmaattiioonn

LYCINE TEMPLATES HAVE A COMMON DRAWBACK in alkylations (C-C-bond formation) since activated alkyl halides are often

required for the reaction to proceed. This may be avoided by instead making an amination (C-N-bond formation). To introduce an amine in α-position to a carboxylic acid derivative either an electrophilic amine-equivalent, or a good leaving group that can be substituted by a nucleophilic nitrogen, is required There are several ways to accomplish this in a stereoselective fashion, three of which will be presented here.

Evans pioneered this research area with the development of the amination of chiral oxazolidinones.70 Two complementary approaches to the stereoselective amination are presented in Scheme 5.7. In the first example trisylazide is used in an azido transfer reaction with a potassium carboximide enolate. The same type of reaction can be carried out by using di-tert-butyl azodicarboxylate (DBAD) as electrophile but that leaves a Boc-protected hydrazone that has to be deprotected (TFA) and reduced (Raney nickel, 500 psi H2). In contrast, the oxazolidinone auxiliary can be removed under mild basic conditions with LiOOH, formed in situ from LiOH and H2O2.

G


⎯ 33 ⎯

O

ONR

Bn

O O

ONR

Bn

O

N3

SN3

O O

Trisyl azide =

1) KHMDS, Trisyl azide 2) AcOH, AcOK

1) Bu2BOTf, TEA2) NBS

O

ONR

Bn

O

Br

O

ONR

Bn

O

N3

TMGA TMGA =

N

NNH

HN3

Scheme 5.7. Azidation of Evans’ oxazolidionone through direct azide transfer or via the bromide.

Bromination of the oxazolidinone enolate, formed with a Lewis acid and amine base, generates another intermediate suitable for azido substitution (Scheme 5.7). It has been found that tetramethyl guanidinium azide (TMGA) is the best choice of nucleophile for this reaction, providing a clean inversion to the corresponding azide.

O

R CCl3RZnX

O

CCl3Cl

O

R H

Cl3C1)

2)Oxidation

N BO

Ph Ph

BuO

BHO

OH

R CCl3

NaOH

NaN3 O

OHR

N3

+

Scheme 5.8. Rearrangement of chiral α-trichloromethyl alcohols to amino acids with NaN3.

Another aesthetically appealing method for synthesis of amino

acids, developed by Corey et. al., utilizes the asymmetric CBS reduction (catecholborane- oxazaborolidine, Scheme 5.8). α-Trichloromethyl-ketones can be conveniently formed either through reaction with


⎯ 34 ⎯

organozinc compounds and trichloroacetic acid chloride, or by oxidation of the product ovtained be reaction of an aldehyde and trichloromethyl anion. These ketones are then reduced to the corresponding chiral alcohol with the CBS reduction, treatment of which with NaOH/NaN3 results in formation of α-azido acids. In this reaction the alcohol is first deprotonated and forms a dichloroepoxid, which is then attacked by the azide nucleophile, opening the epoxide. This results in an intermediate acid chloride that is hydrolyzed to the corresponding azido acid.

O

HR

L-proline

NN OR1

OR1O

O

O

HR

NNH

OO OR1

R1O

O

OHR

NH2

OHR

NH2

or

Scheme 5.9. Proline-catalyzed asymmetric synthesis of α-amino acids or α-amino alcohols

Two research groups have independently developed the catalytic asymmetric amination of aldehydes, using proline as catalyst (Scheme 5.9).71, 72 Azodicarboxylates are used as electrophilic amine equivalents, resulting in formation of carbamate protected hydrazones that then have to be reduced to amines. A drawback with obtaining aldehydes as products is that they are prone to epimerization. The aldehydes are therefore often directly reduced to amino alcohols or oxidized to amino acids.


⎯ 35 ⎯

66.. SSyynntthheessiiss ooff hhyyddrrooxxyyllyyssiinnee


YDROXYLYSINE, A CENTRAL ELEMENT IN our studies of glyco-peptides containing glycosylated hydroxylysines, has

previously been synthesized by other research groups. The syntheses are is of wide variety, ranging from a racemic synthesis73 to synthesis of several stereoisomers followed by separation,74, 75 and several relatively recent reports of the synthesis of pure stereoisomers.76-81 Even though hydroxylysine now is commercially available, it is both expensive and requires protective group manipulation to be useful in synthesis. These manipulations have proven to be rather cumbersome, and new efficient synthetic routes to hydroxylysine protected suitably for further manipulations are still of some interest.13, 18 More importantly, the method for preparation of hydroxylysine presented in this chapter can be regarded as a model system for the preparation of galactose-hydroxylysine C-glycoside.

66..22.. RReettrroossyynntthheettiicc aannaallyyssiiss

HE IDEA BEHIND THE SYNTHESIS of hydroxylysine was to utilize a nucleophilic, chiral glycine template for introduction of the

α-amino acid stereogenic centrum (cf Scheme 6.1 and Chapter 5.2.3). The chiral R-malic acid (1) would provide the ε-stereogenic centrum and would be attached to the glycine template by alkylation. Before connecting these two synthons, a series of manipulations including differentiation between three hydroxyl groups, a selective acetal ring opening and several functional group conversions was realized.

H

T


⎯ 36 ⎯

OH

OHO

O

OH

IN3

OPg

O

R*PgN

BocHNOH

OH

O

NHFmoc

+

1

2

Scheme 6.1. Retrosynthetic analysis of protected hydroxylysine 2.

66..33.. SSyynntthheessiiss ooff tthhee eelleeccttrroopphhiilliicc ccoonnssttiittuueenntt

ALIC ACID WAS FIRST REDUCED with borane-dimethylsulfide complex to triol 3 (Scheme 6.2). The 1,3-diol was then

protected as an acetal using p-methoxybenzaldehyde and pyridinium toluene-4-sulfonate (PPTS). The unprotected primary alcohol was converted into a leaving group with p-toluenesulfonyl chloride (TsCl) and then substituted for an azide to give 6 in 76% yield from 1. A key step of the synthesis was then regioselective opening of the acetal. The intention was to open the acetal in a manner to leave a terminal unprotected hydroxy group and the other oxygen atom protected as its p-methoxy benzyl ether (PMB), 8 in figure 6.1. There are several reports of acetal openings in a selective manner.82-84 Use of DIBAL is reported to give an oxygen-aluminate complex to the sterically least hindered oxygen (i.e. the oxygen denoted as 1 in Figure 6.1). After complexation, reductive opening will occur, leaving a free primary alcohol and the secondary alcohol protected as a PMB-ether, i.e. formation of 8 was expected from 6. When this was attempted on substrate 6 the selectivity was reversed, yielding 8 as sole product in 60% yield. Using borane and a Lewis acid is supposed to have the same outcome as with DIBAL, but again the selectivity was reversed, i.e. BH3 · DMS and Bu2BOTf gave only secondary alcohol 7. With other Lewis acids the reduction either failed or resulted in reduction of the azide to an amine.

M


⎯ 37 ⎯

OH

OHO

O

OH

OHHO

OH

OHO

O

OMe

OTsO

O

PMP

ON3

O

PMP

BH3 DMS, B(OMe)3

THF, rt

p-methoxybenz-aldehyde, PPTS

CH2Cl2, reflux, 89%

TsCl, TEA, DMAP

CH2Cl2, rt, 98%

NaN3

DMF, 55 ºC, 88%

13

4

5

6 Scheme 6.2. Synthesis of acetal 7 from R-malic acid 1. Instead our attention was turned to a method frequently utilized in carbohydrate chemistry for regioselective opening of 4,6-benzylidene acetals (e.g. 9).85-87 This method relies on activation of the acetal with a Lewis acid, complexing to one of the two oxygen atoms, followed by reduction with NaCNBH3. The idea here is that if a small Lewis acid (such as a proton from TFA) is used, the most basic oxygen atom, i.e. no. 4 in Figure 6.1,

ON3

O

PMP

12

OPMBN3

OH OHN3

PMBO

OO

O

PMP

OMe

BnOBnO

6

4

OPMBO

OH

OMe

BnOBnO

OHO

OPMB

OMe

BnOBnO

67 8

910 11 Figure 6.1. Schematics for the regioselectivity of the opening of acetals 6 and 9.


⎯ 38 ⎯

attacks the Lewis acid and reductive opening gives a free secondary alcohol (10).87 With a larger Lewis acid, where trimethylsilyl chloride (TMSCl) is most commonly used, steric repulsion favors kinetic complexation (oxygen atom no. 6). After reduction this results in the reversed regioselectivity as with a proton, i.e. leaving a free primary alcohol (11). For 6 the expected product in the presence of TMSCl would then be 8. When the reductive opening of acetal 6 was attempted with NaCNBH3 and TMSCl, it was found that as with DIBAL, the expected selectivity was reversed giving primary alcohol 7 as the only product (entry 1, Table 6.1).

ON3

O

PMP

OPMBN3

ORSilyl chloride (2 eq), NaCNBH3 (2 eq)

7a-dMeCN, rt

6 Entry Silyl

chloride 7a R = H

7b-d R = SiR3

1 TMSCl 93% - 2 TESCl 62% 7b, R = TES, 15% 3 TBDMSCl 8% 7c, R = TBDMS, 89% 4 TBDPSCl 60% 7d, R = TBDPS, 30% 5 TIPSCl 81% - 6 TFA 85% - 7 - - - Table 6.1. Investigation of reductive acetal opening with silyl chlorides and NaCNBH3.

In an attempt to reverse the selectivity, the steric bulk of the silyl chloride was increased. Thus, TESCl was used as Lewis acid but the same product as before was formed in 62% yield. In addition, some silylation of the alcohol occurred to yield 7b (15%). With TBDMSCl (entry 3), silylation was even more pronounced and TBDMS ether 7c could be isolated in 89% yield along with 8% of alcohol 8a. With even larger silyl groups (TBDPSCl and TIPSCl), 7 was still the favored regioisomer, but silylation of the product appeared to be more sluggish (entries 4 and 5). In a control experiment where TFA was employed as Lewis acid product 7a was again formed. Without added Lewis acid no reaction occurred, demonstrating that the reduction requires activation of the acetal and that the unexpected selectivity was not due to reductive opening of


⎯ 39 ⎯

nonchelated substrate. When the reduction with TBDMSCl as Lewis acid was tested in DMF as solvent, complete silylation was obtained, but in a lower yield than with MeCN (72%). Another interesting observation was that if less than 2 equivalents of TBDMSCl in MeCN was used, the degree of silylation was affected. This, and a more thorough examination of the selectivity in the reductive opening of acetals is discussed later (chapter 6.5). Even though the opening of the benzylidene acetal gave opposite selectivity than the one expected, this could be exploited in the synthetic strategy. A TBDMS ether can be utilized instead of having the secondary alcohol protected as a PMB ether. TBDMS ethers and PMB ethers can be deprotected in an orthoganal manner and will not cause any further strategic problems.

OPMBN3

OTBDMS

OHN3

OTBDMS

DDQ

MeCN/H2O, 0 ºC

OMsN3

OTBDMS

MsCl, TEA, DMAP

CH2Cl2, 0 ºC

NaI

acetone, reflux(76% from 7c)

IN3

OTBDMS

7c 12

13 14 Scheme 6.3. Accordingly, primary alcohol 12 was liberated by oxidation of the PMB-ether with DDQ. The hydroxyl groups were then activated as a leaving group by conversion into a mesylate and then substituted for an iodide (Scheme 6.3, 76% yield over three steps). Iodide 14 is a suitable electrophile for alkylation with a glycine template.

66..44.. EEnnaannttiioosseelleeccttiivvee aallkkyyllaattiioonn ooff aa ggllyycciinnee tteemmppllaattee

S DISCUSSED IN CHAPTER 5.1.3 SEVERAL glycine templates and methods that have been developed for enantioselective

alkylation with electrophiles. The template first examined by us was Myer’s template based on pseudoephedrine-glycine amide (15, Figure 6.2).65 Deprotonation of the template was done with LDA or BuLi in THF at 0 °C with LiCl as a complexing additive to give a dianion.

A


⎯ 40 ⎯

Unfortunately, treatment of the dianion with iodide 2 did not provide any product.

Instead the method relying on catalytic chiral phase transfer (CPT) developed by Lygo and Corey was investigated.68, 69, 88, 89 The catalyst is based on the cinchonidinium alkaloid and acts as a phase transfer mediator at temperatures as low as -78 °C in CH2Cl2 with CsOH⋅H2O as base. Disappointingly, alkylation of benzophenone imine glycine t-butyl ester according to the method frequently cited, 69 resulted in no detectable product formation, whereby this method was discarded. Both methods discussed above were aimed at giving a product that could be deprotected without affecting the azide; the azide being a small group would make glycosylation of azide-protected hydroxylysine interesting. Since none of these methods could be employed, attention was directed at the morpholinone template of Williams et. al (17).66, 90

OtBu

ON

Ph

PhN

ONH2Ph

OH

CbzNO

O

PhPh

N

N OBr

+15

1617

Figure 6.2. Glycine templates for enantioselective alkylation: Myers (top left), CPT-glycine (right), Williams (bottom left). Williams’s glycine template has previously been used by van der Nieuwendijk et. al. in the synthesis of hydroxylysine.81 The template is available in both enantiomeric forms with the amine protected as its tert-butyl or benzyl carbamate. As our objective was to prepare enantiopure hydroxylysine suitably protected for Fmoc solid-phase synthesis, the Cbz-protected morpholinone would be more convenient even though higher yields are often reported in the alkylation step with the Boc-analogue.81, 91 However, for iodide 14 the reaction with 17 proceeded smoothly to give alkylated product 18 in 94% yield (Scheme 6.4). Only one of the two


⎯ 41 ⎯

possible diastereomers could be detected by 1H NMR spectroscopy and HPLC. The stereochemical outcome of this alkylation is well established in the literature and can also be confirmed from the 1H NMR spectrum of the product. The methine protons of the diphenylethylene bridge of an anti isomer displays a larger ∆δ value (typically 0.9-1.1 ppm) than for the corresponding syn isomer (∆δ < 0.7 ppm).66 For 18 the ∆δ value for the two methine protons was found to be 0.89 ppm, strongly indicating that the anti

OTBDMSN3

OCbzN

PhPh

O

OTBDMSBocHN CO2H

NHFmoc

IN3

OTBDMS

17, NaHMDS,15-crown-5

THF, -78 to -20 ºC,94%

OTBDMSH2N

OCbzN

PhPh

O

PPh3

THF/H2O, 130 ºC

OTBDMSBocHN

OCbzN

PhPh

O

Boc2O, TEA

THF, rt, 85%

1) Pd-C, H2 (65 psi) THF/H2O, rt

2) FmocOSu, Na2CO3 acetone/H2O, rt, 77%

14 18

19 20

2 Scheme 6.4. The final steps towards hydroxylysine. product 18 had been formed as expected. After the successful alkylation, some protective group transformations remain to complete the synthesis of hydroxylysine. First azide 18 was reduced with triphenylphosphine to give amine 19, which was protected with a Boc group (85% over two steps). The Cbz group of the α-amine, and the diphenylethylene bridge of the morpholinone, were simultaneously removed by hydrogenation over Pd-C and H2-atmosphere (65 psi). Fmoc-protection of the free amine gave the desired protected (2S,5R)-5-hydroxylysine 3 in 77% yield.


⎯ 42 ⎯

66..55.. IInnvveessttiiggaattiioonn ooff tthhee rreedduuccttiivvee ooppeenniinngg ooff pp--

mmeetthhooxxyybbeennzzyylliiddeennee aacceettaallss..

FEW UNEXPECTED OBSERVATIONS WERE made during the initial attempts to reductively open benzylidene acetal 7 (cf. Chapter

6.3 and Figure 6.1). First, opening occurred with opposite selectivity than was expected. Second, use of less than 2 equivalents of TBDMSCl resulted in a more incomplete silylation of the intermediate alcohol. In order to understand these observations some additional experiments were performed. The regioselectivity of the reductive opening of the p-methoxy-benzylidene acetal is determined by the chelation of the Lewis acid. A thermodynamic attack would favor chelation of the more electronrich oxygen atom (no. 2 in Sceme 6.2). However, with increased steric bulk of the Lewis acid chelation to the more accessible oxygen atom (no. 1) would be favored. To study the competion of these two effects further, a few analogues of 6 were synthesized and investigated in the acetal-opening reaction (Scheme 6.5). These analogues were: a methoxy analogue (22), the small methoxy group allowing chelation to O-2 but being less electronwithdrawing than an azide; an acetate (25) with similar size as the methoxy group but with a stronger electronwithdrawing effect;92 and a methyl analogue (28), which is smaller in size and has no possibility for chelation. It was observed that the methoxy analogue gave reversed selectivity in the reductive opening as compared to azide 6. The ratio between the two products 23:24 was ~1:3, as compared to 1:0 (7:21) for azide 6. Reductive opening of the acetate analogue gave an even more distinct selectivity differance (~1:6). For the methyl analogue, no selectivity between the two alternate openings could be discerned (~1:1). Most unexpectedly, the secondary alcohol 29 resisted silylation while for the related secondary alcohols 23 and 26 silylation was predominant. Thus it seems that the methoxy and acetate groups exert a greater steric hindrance than the azide, and/or make the secondary oxygen atom more electron poor through inductive effects. Both of these effects should contribute to direct the chelating silyl chloride towards the primary oxygen atom.

A


⎯ 43 ⎯

22

25

OR

OPMBN3

OPMB

ORN3

OR

OPMBMeO

OR

OPMBAcO

OPMB

ORMeO

OPMB

ORAcO

21a R = H, 0% b R = TBDMS, 0%

24a R = H, 9% b R = TBDMS, 55%

27a R = H, 5% b R = TBDMS, 60%

7a R = H, 8% c R = TBDMS, 89%

23a R = H, 3% b R = TBDMS, 21%

26a R = H, 12% b R = TBDMS, 0%

O ON3

PMP

O OMeO

PMP

O OAcO

PMP

+

+

+

28

OR

OPMB

OPMB

OR

30a R = H, 7%b R = TBDMS, 37%

29a R = H, 37% b R = TBDMS, 0%

O O

PMP

+

6

Scheme 6.5. Reductive opening of some analogues of benzylidene acetal 6.

However, it may be argued that the azide should act in the same manner as the methoxy and acetate groups. In an attempt to explain the difference between them, it was speculated that the azide (in addition to the secondary oxygen atom) might form a chelate to silyl chloride, thereby directing it to the more sterically crowded position. In order to establish if this was the case some NMR studies were undertaken. First, two experiments were performed where azide 6 and methoxy analogue 22 were dissolved in MeCN-d3 and titrated with TBDMSCl (0.5-2 eq). No alterations of the 1H NMR or 13C NMR spectra could however be detected. Instead, a 15N-isotope enriched azide was prepared from tosylate 5 and mono-15N labeled sodium azide, and a 15N NMR spectrum was recorded. The spectrum showed two singlets of approximately equal intensity separated by 36.7 ppm (Figure 6.3, top). The signal most downfield was assumed to originate from the nitrogen atom attached to the methylene group in 6. When 1 equivalent of TBDMSCl was added to 15N-enriched azide 6 in MeCN-d3 another resonance appeared slightly


⎯ 44 ⎯

upfield of the signal from the nitrogen bound to the methylene carbon (Figure 6.3, bottom). After this spectrum had been recorded, a 1H NMR spectrum was recorded which revealed that the sample was still intact and that no decomposition had occured. The new 15N resonance indicates that chelation between TBDMSCl and the azido group occurs. This chelation effect might even activate silyl chloride further for silylation of the intermediate alcohols formed from 6, 22 and 25, which could explain why the secondary alcohol of the methyl analogue (29) resisted silylation.

Figure 6.3. 15N NMR spectra of azide 15N-6 (top) and 15N-6 + 1 eq TBDMSCl (bottom). When less than 2 equivalents of TBDMSCl was used in the reductive opening of 6, silylation of the secondary alcohol was incomplete. Thus, with 1 equivalent no silylation was observed. With 1.5 equivalents equal amounts of silylated and nonsilylated product was formed, whereas use of 2 equivalents gave almost complete silylation (>10:1). The same trend was observed with oxygen analogues 22 and 25. Seemingly, 1 equivalent of the silyl chloride is consumed during the reaction. A proposed mechanistic explanation, both for the unexpected regoselectivity and the consumption of 1 equivalent of silyl chloride, is shown in Scheme 6.6.


⎯ 45 ⎯

O O

OMe

SiCl

BH H

NC H

O O

N3

OMe

SiCl

BH

NCH

O

OPMBN3

Si

ClBHCN

H

O

OPMBN3

BCN

H O

OPMBN3

Si

SiCl

31 32

33 34

6

R3SiH

N3

Scheme 6.6. Tentativemechanism for reductive opening of 6. Chelation of the secondary oxygen atom and the azide with silyl chloride enables reductive opening of the acetal. The intermediate alkoxide then has two Lewis acids in close proximity; silyl chloride and cyanoborane. It reacts with the stronger of the two Lewis acids, the cyanoborane, to form borate 32. This borate is activated as a reducing agent as compared to NaCNBH3. This allows the silyl chloride to be reduced to a silane by the activated borohydride, while being inert to NaCNBH3. This would explain the consumption of one equivalent of silyl chloride. Additional amounts of silyl chloride react with boron ester 33, forming an O-Si bond that is thermodynamically more stabile than an O-B bond. It should be stressed that this mechanism remains speculative. However, in support of the mechanism, the silanes corresponding to TESCl and TBDMSCl (triethyl silane and dimethyl-t-butyl silane, respectively) were detected in the reaction mixture when analysed by GC (TESH and TBDMSH as internal references), one-dimensional 1H NMR spectroscopy and COSY experiments. Integration of the 1H NMR spectra showed that the silanes were formed in approximately equimolar amounts as products 7b and 7c.


⎯ 46 ⎯

66..66.. EEnn rroouuttee ttoo ggaallaaccttoossee hhyyddrrooxxyyllyyssiinnee CC--ggllyyccoossiiddee

HEN ALKYLATION WITH WILLIAMS’S GLYCINE template proved successful in the synthesis of hydroxylysine, this

approach was applied immediately to the synthesis of the C-glycosidic analogue of galactosylated hydroxylysine (Scheme 6.6). The electrophile B required for alkylation was prepared from alkene A in two steps (for a more detailed discussion of the synthesis of some of the compounds in Scheme 6.6, see Chapter 8 and 9).

OBnO

BnO

OBn

BnOTBDMSO

NaHMDS, morpholinone 17

THF, -78 to -20 ºC,22%

OBnO

BnO

OBn

BnOTBDMSO

I

OBnO

BnO

OBn

BnOTBDMSO

OCbzN

PhPh

O

i) Pd-C, H2 (75 psi), AcOH

ii) FmocOSu, Na2CO3, acetone/H2O

OAcO

AcO

OAc

OAcTBDMSO

NHFmoc

O

OH

iii) Ac2O, pyridine rt, 0%

A B

C D

Scheme 6.6. Attempts to apply the Williams glycine template strategy in the synthesis of the C-galactosyl-hydroxylysine analogue D.

Alkylation of Wiliams glycine template with iodide B was more difficult than with iodide 14 in the synthesis of hydroxylysine (22% yield compared to 94% yield). However, the major setback was when the reaction sequence following the alkylation was attempted. Hydrogenation of C was very sluggish and a very polar and unwieldy product was formed. Direct Fmoc protection followed by acetylation of the hydroxyl groups did not provide any product, even though repeated attempts were made. Therefore, this route to C-galactosylated hydroxylysine was abandoned.

W


⎯ 47 ⎯

77.. SSyynntthheessiiss ooff CC--GGaallaaccaattoossyyllaatteedd

hhyyddrrooxxyynnoorrvvaalliinnee


HREE OUT OF THE 29 T CELL hybridomas discussed in Chapter 3 recognize the epitope CII256-270 carrying a galactosylated

hydroxynorvaline in position 264 (Hnv264).93, 94 Synthesis of this amino acid requires introduction only of the α-stereogenic centrum in addition to the β-C-glycoside. Therefore it makes a good first target before pursuing efforts to synthesize more complex C-glycosylated amino acid isosteres.

77..22.. RReettrroossyynntthheettiicc aannaallyyssiiss

HE RETROSYNTHETIC ANALYSIS OF C-GALACTOSYLATED hydroxynorvaline is shown in Figure 7.1. It was decided to use

a well established methodology for synthesis of the β-C-glycoside moiety based on Grignard attack on a glycolactone (cf. 4.2.2).95, 96 The α-stereogenic centrum was then to be introduced by coupling of chiral Garner’s aldehyde (39) in a Wittig reaction.97-99 After deprotection of the amino alcohol moiety and oxidation to amino acid, the building block may be incorporated in a C-glycopeptide using a mixed Fmoc/Boc strategy.

T

T


⎯ 48 ⎯

OBnO

BnO

OBn

BnO O

OBnO

BnO

OBn

BnO

OBnO

BnO

OBn

BnOPPh3 I

BocNO

O

H

+

OBnO

BnO

OBn

BnO

NHBocOH

O

BrMg+

35 3637

38 39

40

Figure 7.1. Retrosynthetic analysis of β-C-galactosylated hydroxynorvaline.

77..33.. SSyynntthheessiiss ooff CC--lliinnkkeedd ggaallaaccttoossee oonn

hhyyddrrooxxyynnoorrvvaalliinnee

HE LACTONE 35 THAT CONSTITUTES THE electrophile used in the synthesis of all C-glycosides discussed in the following

chapters was synthesized in five steps from galactose pentaacetate (Scheme 7.1).100 First, thioglycoside 42 was prepared by reaction with boron trifluoride etherate and thiocresol, and the acetates were removed with NaOMe in methanol. The protecting groups of the galactose hydroxyl groups will have to endure a wide variety of reaction conditions in the subsequent synthetic steps; consequently, a reasonably inert protecting group had to be chosen. Therefore, the hydroxyl groups were protected as benzyl ethers, using sodium hydride and benzyl bromide in DMF. The anomeric thiocresol group was removed with NBS and the resulting hemiacetal oxidized under Swern conditions to give lactone 35.

T


⎯ 49 ⎯

OAcO

AcO

OAc

AcOOAc

OAcO

AcO

OAc

AcOSTol

OHO

HO

OH

HOSTol

OBnO

BnO

OBn

BnOSTol

OBnO

BnO

OBn

BnO OH

OBnO

BnO

OBn

BnO O

BF3 OEt2TolSH, CH2Cl2

rt, 79%

NaOMeMeOH, rt

NBS, TEAMeCN/H2O, rt,

82%

OxCl, DMSO, TEA

THF, -78 ºC, 99%

NaH, BnBrDMF, 0 ºC to rt,

89%

41 42

43 44

45 35 Scheme 7.1. Preparation of lactone 35. The C-C bond of the C-glycoside was formed in a two step reaction: attack on lactone 35 with homoallylmagnesium bromide followed by reduction of the intermediate hemiacetal with boron trifluoride and triethylsilane (Scheme 7.2). The reaction is highly stereoselective and only formation of the β-isomer 37 (the alkyl chain in an equatorial position) was observed. The stereochemical outcome is well established from previous publications,96 in addition the coupling constant between H-1 and H-2 of galactose confirmaed formation of the β-isomer (J1,2 = 9.1 Hz, for the α-isomer a coupling constant J1,2 of approx. 5.8 Hz is expected).


⎯ 50 ⎯

OBnO

BnO

OBn

BnO O THF, -78 ºCO

BnO

BnO

OBn

BnO OH

BrMg BF3 OEt2, Et3SiH

MeCN, -40 ºC, 81%

OBnO

BnO

OBn

BnO

O3, NaBH4,

MeOH/CH2Cl2,-78 ºC to rt, 91%

OBnO

BnO

OBn

BnOOH

OBnO

BnO

OBn

BnOI

OBnO

BnO

OBn

BnOPPh3 I

PPh3, Imidazol, I2Toluene, rt, 88% 120 ºC, 88%

PPh3

35

37 46

47 38 Scheme 7.2. Synthesis of phosphonium salt 38 required for the Wittig reaction.

Ozonolytic cleavage of the double bond in 37 with reduction of the intermediate ozonide gave alcohol 46 in 91% yield. Alcohol 46 was converted to iodide 47 using the conditions of Garegg et. al., i.e. iodine, imidazol and triphenylphosphine in toluene.101 Phosphonium salt 38 was prepared under solvent free conditions in a melt of 47 and 5 equivalents of triphenylphosphine at 120 °C. The resulting salt could be purified by trituration with toluene and diethyl ether or, for the best result albeit less convenient, by column chromatography on silica.

Garner’s aldehyde (39) containing the α-amino acid stereogenic centrum was synthesized in four steps from L-serine methyl ester.98 In brief, serine methyl ester was N-protected with Boc2O and TEA, then N,O-cyclized to the oxazolidine with 2,2-dimethoxypropane and boron trifluoride etherate. LiAlH4 reduction followed by Swern oxidation gave aldehyde 39 in 61% total yield. The aldehyde was then coupled directly with the ylide obtained from phosphonium salt 38 by deprotonation with potassium hexamethyldisilazane (KHMDS) in THF (Scheme 7.3).102 The resulting alkene was isolated as a mixture of isomers, with the expected Z-isomer as the major component (93:7 Z:E). The alkene was hydrogenated using Pearlman’s catalyst (Pd(OH)2 on charcoal) in EtOAc/MeOH,103 after attempts of reduction either with in situ generated diimide or H2 and Pd-C had failed.


⎯ 51 ⎯

OBnO

BnO

OBn

BnOPPh3 I

BocNO

O

H

+KHMDS

THF, -45 ºC to rt,71%

OBnO

BnO

OBn

BnOBocN

O

Pd(OH)2/C, H2

EtOAc/MeOH, rt, 81%

OBnO

BnO

OBn

BnO

BocNO

OBnO

BnO

OBn

BnO

NHBocOH

O

CrO3

H2SO4/acetone0 ºC to rt, 66%

38 39

4849

40

Scheme 7.3. Introduction of the α-stereogenic centrum and further transformations to give C-linked galactose on hydroxynorvaline 40.

The synthesis of building block 40 was completed by concomitant cleavage of the oxazolidine ring and oxidation of the resulting alcohol under Jones oxidation conditions (CrO3, H2SO4 (aq.), acetone). Interestingly, the otherwise acid labile Boc-group remains intact under these seemingly strongly acidic conditions.

77..44.. IInnccoorrppoorraattiioonn ooff bbuuiillddiinngg bboocckk iinnttoo eeppiittooppee

CCIIII((225566--227700))

HE BUILDING BLOCK WAS INCORPORATED in a peptide corresponding to the CII(256-270) epitope.104 The synthesis

was performed on a Merrifield resin, using a combined Boc and Fmoc-strategy. This eliminates the need for protective group manipulations of building block 40, the peptide could be cleaved under conditions that deblock the galactose hydroxyl groups which carried benzyl protecting groups. The Boc-protected Nα-groups of Lys270 (preloaded on resin), Glu266 and Gal-CH2-Hnv264 were deprotected with 25% TFA in CH2Cl2. All other amino acids carried Nα-Fmoc protecting groups, which were

T


⎯ 52 ⎯

deprotected with 20% piperidine in DMF. The peptide was cleaved from the resin by treatment with TESOTf in TFA, using thioanisole as cation scavenger.105 After reversed phase HPLC purification and lyophilization, the peptide could be isolated in 19% overall yield. The results of the ensuing immunological studies are discussed in Chapter 10.


HE STRATEGY DISCUSSED ABOVE WAS also applied in attempted synthesis of galactose hydroxylysine C-glycoside (Scheme

7.4). Alcohol 50 was oxidized under Jones conditions and Evans oxazolidinone was coupled via the pivaloyl anhydride. Stereoselective alkylation of oxazolidinone 51 with trimethylsilylethoxymethyl iodide (SEMI, formed in situ from SEMCl and tetrabutylammonium iodide) proceeded in 72% yield. Next, reduction of 52 with LiAlH4 and substitution of the alcohol to iodide 53 gave the precursor for phosphonium salt 54 required for the Wittig reaction. Using the same conditions as earlier (PPh3, 120 °C (melt)) gave the desired product 54 in comparable yields to 38 obtained before, even though the reaction was complete after heating for 20 h instead of 2 h for iodide 47 (Scheme 7.2). This indicates that the TMSEtOCH2 side chain exerts some steric hindrance in this substitution. When the Wittig reaction was attempted, no reaction could be observed, whereas for analogue 38 (Scheme 7.2), reaction was complete in less than 2 h. Other bases (LDA, LHMDS) and different reaction conditions (temperature and solvents) were investigated without success. Consequently, this seemingly simple route was abandoned.

T


⎯ 53 ⎯

OBnO

BnO

OBn

BnOOH

OBnO

BnO

OBn

BnON

O

O

O

i-Pr

OBnO

BnO

OBn

BnON

O

O

O

i-Pr

LDA, TBAITMS O Cl

THF, -78 ºC,72%

O

TMS

OBnO

BnO

OBn

BnOI

TMSEtO

PPh3

120 ºC, 79%

OBnO

BnO

OBn

BnOPPh3 I

TMSEtO

KHMDS, Garner's aldehyde (39)

THF, -45 ºC to rt,0%

OBnO

BnO

OBn

BnO

BocNO

TMSEtO

46 50

5152

53 54 Scheme 7.4. Attempts to apply the Garner’s aldehyde strategy in the synthesis of C-galactosylated hydroxylysine.


⎯ 54 ⎯


⎯ 55 ⎯

88.. SSyynntthheessiiss ooff aa ββ--DD--GGaallaaccttoossyyll tthhrreeoonniinnee CC--

ggllyyccoossiiddee


HE SYNTHESES OF C-LINKED ANALOGUES of naturally occuring glycosylated amino acids that can be found in the literature are

mainly those of glycosylated serine106-109 and asparagine.97, 110, 111 Thus previous studies have targeted amino acids without any stereocentra besides the α-position of the amino acid moiety. However, a great assortment of glycoproteins where the saccharide is linked to amino acids having two stereocentra is also found in Nature. In mucines found on epithelial cells, threonine is glycosylated with α-D-N-acetylglucosamine.112 Nuclear pore proteins, transcriptional factors and cytoskeletal proteins carry β-D-N-acetyl-glucosamine moieties on threonine.113 Hydroxylysine, a constituent of collagen that has been discussed in chapter 3, is often glycosylated with β-D-galactose.114 In this chapter, synthesis of the C-glycoside analogue of β-D-galactosylated threonine is described. This constitutes the first synthesis of a C-glycoside of a naturally occuring amino acid carrying more than one stereocentra in the amino acid moiety. Galactosylated threonine residues are found in the cuticle of the deep-sea hydrothermal vent worm Riftia pachyptila.115

88..22.. SSyynntthheessiiss:: ssttrraatteeggyy aanndd eexxeeccuuttiioonn

HE STEREOGENIC CENTRE OF THE anomeric methylene linkage in C-galactosylated hydroxylysine was established according to

the method used in the preperation of galactosylated hydroxynorvaline C-glycoside (Chapter 7.3).100 The two remaining stereogenic centra, i.e. the methylgroup in β-position and the α-amino acid functionality, were both

T

T


⎯ 56 ⎯

envisioned to be achieved through the use of Evan’s chiral oxazolidinone methodology.70, 116 The synthesis started by oxidation of alcohol 46 under Jones conditions to provide carboxylic acid 55 in near quantitative yield (Scheme 8.1). Activation of carboxylic acid 55 as a mixed anhydride with triethylamine and pivaloyl chloride, followed by substituted with the lithium salt of (R)-4-benzyloxazolidin-2-one gave carboximide 56. This was deprotonated with LDA and then treated with allyl iodide at -78 °C to afford 57. Only one of the two possible diastereomers was observed by 1H NMR spectroscopy, indicationg a selectivity of >99:1; analytical HPLC in two different systems also showed only one peak. The carboximide was reduced with LiEt3BH since LiAlH4 was surprisingly sluggish and plagued with some formation of aldehyde, which resisted reduction even with additional equivalents of LiAlH4. Deoxygenation of the resulting alcohol was then to be attempted after converting it to a leaving group.

OBnO

BnO

OBn

BnOOH

46

OBnO

BnO

OBn

BnOOH

55

O

OBnO

BnO

OBn

BnO

56

O

ON

O

Bn

OBnO

BnO

OBn

BnO

57

O

ON

O

Bn

CrO3

H2SO4/acetonert, 99%

1) PivCl, TEA, THF, -78 ºC2) BuLi, (R)-4-benzyloxazolidin-2-one, THF, -78 ºC, 70%

LDA, allyliodide

THF, -40 to -20 ºC, 62%

LiEt3BH

THF, rt, 94%

OBnO

BnO

OBn

BnO

58 OH

OBnO

BnO

OBn

BnO

59 I

PPh3, Imidazol, I2Toluene, rt, 90%

Scheme 8.1. Establishment of the stereogenic centrum at β-position in the threonine moiety. The tosylate formed from alcohol 58 proved unreactive towards reduction with LiAlH4 or LiEt3BH. Instead iodide 59 was prepared from 58 in 90% yield by treatment with iodine, triphenylphosphine and imidazol.101


⎯ 57 ⎯

OBnO

BnO

OBn

BnO

59 I

LiEt3BH

THF, rt, 96%

OBnO

BnO

OBn

BnO

60

O3, NaBH4

MeOH/CH2Cl2,-78 ºC to rt

OBnO

BnO

OBn

BnO

OH

OBnO

BnO

OBn

BnO

OH

61

62O

CrO3

H2SO4/acetonert, 93%

1) PivCl, TEA, THF, -78 ºC2) BuLi, (R)-4-benzyloxazolidin-2-one, THF, -78 ºC, 79%

OBnO

BnO

OBn

BnO64

O

N O

O

Bn

1) PivCl, TEA, THF, -78 ºC2) BuLi, (S)-4-benzyloxazolidin-2-one, THF, -78 ºC, 87%

OBnO

BnO

OBn

BnO63

O

N O

O

Bn

Scheme 8.2. Synthesis of two carboximides for introduction of the α-stereogenic centre.

Iodide 59 was reduced to methyl analogue 60 with LiEt3BH in a satisfying 96% yield. The alkene terminus of 60 was cleaved to the alcohol by treatment with ozone followed by NaBH4, and alcohol 61 was then directly oxidized to carboxylic acid 62. This was coupled with both (S)- and (R)-4-benzyloxazolidin-2-one via the mixed pivaloyl anhydride. Both diastereomers were prepared to be able to make use of either of the two available strategies for introduction of the α-stereogenic centre; i.e. alkylation with an electrophilic nitrogen atom (trisyl azide) or bromination of the carboximide followed by substitution with azide anion (Scheme 8.3).70


⎯ 58 ⎯

OBnO

BnO

OBn

BnO64

O

N O

O

Bn

OBnO

BnO

OBn

BnO63

O

N O

O

Bn

OBnO

BnO

OBn

BnO65

O

N O

O

BnN3

1) LHMDS or KHMDS2) Trisyl-N3

THF, -78 º, 0%

3) AcOH

1) DIPEA, Bu2BOTf, CH2Cl2, -78 º to rt

2) NBS, CH2Cl2, -78 º, 57%

OBnO

BnO

OBn

BnO66

O

N O

O

BnBr

TMGA

CH2Cl2, rt, 97%

OBnO

BnO

OBn

BnO67

O

OHN3

OBnO

BnO

OBn

BnO65

O

N O

O

BnN3 LiOH

H2O/THF, rt, 97%

Scheme 8.3. Conclusion of the synthesis of β-D- galactose threonine C-glycoside. Direct azidation of carboximide 63 was first attempted. Thus, the lithium or potassium enolate of 63 was formed at -78 °C and treated with trisyl azide. The reaction was quenched with acetic acid, but no product could be observed. Instead the boron enolate of R-isomer 64 was brominated with NBS. The resulting bromide 66 was substituted with tetramethylguanidinium azide,117 the nucleophile that has shown most merit in this type of reaction.70 It was found that the bromination gave a mixture of diastereomers in the ratio of 93:7 as observed by 1H NMR spectroscopy. This mixture could easily be seperated by column chromatography on silica gel, but for conveniance, the seperation was done with azide 65. Both 65 and 66 decomposed on silica or alumina gel, but seperation of azide 65 gave a higher total yield than if the seperation was done with bromide 66. The yield for these two steps including separation of the diastereomers was a moderate 57%. The synthesis was completed by hydrolysis of the chiral auxilary with LiOH in THF and water which gave target 67 in 97% yield. To ensure that no epimerization had occurred, either at the nucleophilic substitution of bromide 66 or during hydrolysis, bromide 66 was completely epimerized by with tetrabutylammonium bromide refluxing in THF. This mixture was then converted into a mixture of 67 and epi-67 by treatment with TMGA and subsequent hydrolysis. Two sets


⎯ 59 ⎯

of signals could be distinguished by 1H NMR spectroscopy and reverse-phase HPLC showed two clearly separated peaks. When comparing these spectra and chromatograms to those obtained from the synthesis, it could be concluded that no epimerization had occurred for 67. Galactosylated threonine C-glycoside 67 is suitably protected for direct use in solid-phase peptide synthesis. It has been demonstrated that α-azido acids can be incorporated in peptides without epimerization or other side reactions.118 Azides can also be reduced to the corresponding amines on solid-support with great ease.118, 119 However, as later studies (cf chapter 9) have shown, benzyl ethers are far from optimal as protecting groups for hydroxyl functionalities. Even though there are several successful examples of simultanous cleavage from the solid suport and deblocking of benzyl ethers,42, 105, 120 it is our experince that the deprotection conditions are not sufficiently reliable to be the foundation for an elaborate synthetic scheme.


LTHOUGH THE SYNTHESIS OF galactosylated threonine C-glycoside was successful, the method was never adapted for

the synthesis of galactose hydroxylysin C-glycoside. This is in part because of the slightly dissapointing selectivity in the introduction of the bromide/azide (93:7), and in part because other attractive synthetic routes had surfaced during this work (see Chapters 6 and 9). A very brief feasible synthetic route to galactosylated hydroxylysine C-glycoside is depicted in Scheme 8.4. The oxazolidinone would first be allylated, then reduced and the resulting alcohol TBDMS-protected (these synthetic steps will be discussed in Chapter 9.2, Scheme 9.1). Cleavage of the alkene to aldehyde E followed by chain extension via a Wittig reaction would provide F. The double bond would be hydrogenated, a new Evan’s oxazolidinone introduced after hydrolysis of the ester and the TBDMS ether would be replaced with a phtalimide (NPht) via a Mitsunobu reaction. All that is remaining of the synthesis is to introduce the α-azido group according to the strategy in Scheme 8.3 and some protective group manipulation.

A


⎯ 60 ⎯

OBnO

BnO

OBn

BnO

O

ON

O

Bn

OBnO

BnO

OBn

BnO

O

ON

O

Bn

LDA,

OBnO

BnO

OBn

BnO

allyl iodide

TBDMSO

OBnO

BnO

OBn

BnOCHO

TBDMSO

OBnO

BnO

OBn

BnOTBDMSO

OEt

O

Ph3P=CHCO2Et

OBnO

BnO

OBn

BnOPhtN

O

N O

O

BnO

BnO

BnO

OBn

BnOPhtN

O

N O

O

BnN3

6768

70 E

F

G H Scheme 8.4. Suggested synthetic route to galactose hydroxylysine C-glycoside using the α-amine strategy described in this Chapter.

The synthesis to galactose threonine C-glycoside described in this

chapter may not be the most straightforward one. The methyl sidechain could have been introduced to the carboximide with methyl iodide. However, one of the key achievements made during the synthesis was the novel use of the carbonyl carbon of the carboximide as the aminomethylene sidechain of hydroxylysine. The major drwbacks in utilizing Evan’s oxazolidinone alkylation method are that it requires activated electrophiles and that functionalization of the electrophile often hinders the reaction. With the method presented herein these drawbacks can to a certain degree be avoided, even though all limitations are not circumvented.


⎯ 61 ⎯

99.. SSyynntthheessiiss ooff ggaallaaccttoossee hhyyddrrooxxyyllyyssiinnee CC--

ggllyyccoossiiddee


ITH THE SYNTHESIS OF TWO galactosylated amino acid C-glycosides and the synthesis of hydroxylysine completed, the

main objective of this study, i.e. the synthesis of galactosylated hydroxylysine C-glycoside, still remained. As described in some of the preceding chapters several synthetic routes had been attempted but proven unsuitable. This target is more complex than the C-glycoside of galactose hydroxynorvaline due to the additional stereogenic centre. It is also more complex than the C-glycoside of galactose threonine since the functionalization is more intricate and because there is a need for a carbon chain elongation. Compared to the synthesis of hydroxylysine, the side chain of galactose hydroxylysine C-glycoside has a lot more steric bulk than that of hydroxylysine which makes alkylations more difficult. Considering all these factors the synthesis turned out to be a challenge.

99..22.. SSyynntthheettiicc ssttrraatteeggyy

HE SYNTHETIC STRATEGY WAS REQUIRED to take three stereogenic centra in consideration (Figure 9.1). Establishment

of the stereogenic centrum at the anomeric position followed the procedure described earlier (Chapters 7 and 8), as did the aminomethylene stereogenic centrum in ε-position (Chapter 8). An asymmetric hydrogenation of an enamide was decided to be used for the α-position. For the asymmetric hydrogenation there are several catalytic systems available, but the one most frequently encountered in the

W

T


⎯ 62 ⎯

litarature is the one developed by Burk et al.60 This rhodium(I) based catalyst carries chiral phosphine ligands (DuPHOS or BPE) and reduces enamide carboxylate esters to α-amino acids with high chemo- and stereoselectivity. It also tolerates many functional groups in the substrate.

OOR

RO

OR

RONHPg

OR´

O

NHPg

OOR

RO

OR

RO OBrMg+

NHPgOR´

O

H2, asymmetric catalyst

+

O

ON

O

Bn

+ LDA, allyl iodide

Figure 9.1. Establishing the required stereogenic centra.

99..33.. AAsssseemmbbllyy ooff tthhee ccoommppoonneennttss

HE SYNTHESIS BEGAN FROM carboxylic acid 55, synthesized as described previously (Chapter 8).121 The lithium salt of (S)-4-

benzyloxazolidin-2-one was coupled to 55 activated as a mixed pivaloylanhydride to give carboximide 67 in 88% yield (Scheme 9.1). Deprotonation with NaHMDS followed by addition of allyl iodide afforded 68 as a single diastereomer as determined from 1H NMR spectroscopy and normal-phase HPLC. This product was treated with LiAlH4 and the resulting alcohol 69 was directly silylated with TBDMSOTf with collidine as base. Direct silylation both facilitated purification and improved the yield (82% over two steps). Next the carbon chain was to be elongated by one methylene group and then functionalized as an enamide methyl carboxylate. This was done by sequentially cleaving the alkene moiety to an aldehyde with

T


⎯ 63 ⎯

ozone and triphenylphosphine on polystyrene to give 71, then by coupling this aldehyde to N-Cbz glycine methyl ester via an Wadsworth-Emmons reaction. This gave a mixture of the two alkene isomers in a ratio of 98:2 (Z:E) that could be separated by flash column chromatograhphy. However, when using Burk’s catalytic system stereoselectivity is not affected by Z/E mixture to any significant extent and synthesis was continued with the isomeric mixture.

OBnO

BnO

OBn

BnOOH

55

OO

BnO

BnO

OBn

BnO

67

O

ON

O

Bn

OBnO

BnO

OBn

BnO

68

O

ON

O

Bn

1) PivCl, TEA, THF, -78 ºC2) BuLi, (S)-4-benzyloxazolidin-2-one, THF, -78 ºC, 88%

LDA, allyl iodide

THF, -40 to -20 ºC, 70%

diethyl ether, rtO

BnO

BnO

OBn

BnO

69 OH

OBnO

BnO

OBn

BnO70 OTBDMS

TBDMSOTf, collidine

CH2Cl2, rt, 82%

LiAlH4

OBnO

BnO

OBn

BnO

71

O

TBDMSO

OBnO

BnO

OBn

BnO

72TBDMSO

H

NHCbzOMe

O

O3, PS-PPh3

MeOH/CH2Cl2,-78 ºC, 96%

(MeO)2P(O)CH(NHCbz)CO2Me

DBU, CH2Cl2, rt, 92%

Scheme 9.1. Introduction of the ε–stereogenic centrum and the enamide ester functionality.

99..44.. TThhee kkeeyy aassyymmmmeettrriicc hhyyddrrooggeennaattiioonn

URK’S CATALYST IS QUITE TOLERANT to reaction conditions and functionalization of the substrate and the conditions normally

found in the literature are often very similar. The catalyst most frequently employed is the Rh-DuPHOS+ (Rh-Bis(phospholano)benzene) but use of

B


⎯ 64 ⎯

the more flexible ligand BPE (Bis(phospholano)ethane) can sometimes be advantageous. Degassed methanol, 2-5 mol% catalyst, hydrogen at 50-70 psi (can be as low as 15 psi) and 24 h reaction time at room temperature can be considered as standard conditions. The selectivity is excellent for most substrates, i.e. in the range of 97-99.9 % ee, while the selectivity for sterically hindered substrates can sometimes be lower (< 96 % ee). With enamide 72 in hand there are several parameters to investigate to achieve good selectivity in the reduction. Different substrates can be explored, such as the TBDMS ether, the corresponding alcohol, azide or Boc protected amine (72-75, Figure 9.1). According to the theory behind the catalytic systems, the selectivity can be improved by decreasing the H2 pressure and by raising the temperature.122

OBnO

BnO

OBn

BnOTBDMSO

NHCbzOMe

O

OBnO

BnO

OBn

BnOHO

NHCbzOMe

O

OBnO

BnO

OBn

BnON3

NHCbzOMe

O

OBnO

BnO

OBn

BnOBocHN

NHCbzOMe

O

72 73

74 75 Figure 9.1. Substrates that were envisioned to be investigated in the asymmetric hydrogenation. First the standard conditions were tested. Thus, enamide 72 was dissolved in degassed methanol under nitrogen and 5 mol% catalyst; [(S,S)-(COD)-Et-DuPHOS-Rh(I)]OTf, henceforth referred to as Rh-Duphos or Burk’s catalyst, was added. The system was put under vacuum and then aggitated under an atmosphere of hydrogen gas (75 psi) for 72 h to give 74% of product. After removal of the catalyst by filtration through silica gel, the product was still contaminated with startingmaterial 72 (appr. 10%) and the undisered R-isomer (S:R 4:1).123 When the reaction was attempted at atmospheric pressure of hydrogen gas, no reaction could be detected. From these experiments it could be concluded that both the rate of the reaction and the selectivity were unusually poor. These


⎯ 65 ⎯

somewhat disappointing results warranted a more thorough investigation of the parameters of the asymmetric reduction, starting with the steric hinderance from the side chain (Table 9.1, entries 1-4). First, the TBDMS group in 72 was removed by treatment with TBAF to give alcohol 73 along with byproduct 76 (Figure 9.2), formed by intramolecular 1,4-conjugated nucleophilic attack of the intermediate alkoxide ion. However, formation of this byproduct could be avoided by deprotection with TFA:H2O. When a Mitsunobu reaction to substitute the hydroxy group in 73 for an azido group was attempted the annulated product 77 (Figure 9.2) was formed. Similar cyclizations have been reported earlier for related systems,124-126 and it seems that the intramolecular reaction proceeds much faster than the azide-substitution. All other attempts to introduce an azide were unsuccessful and therefore analogues 74 and 75 could not be synthesized for investigating the selectivity.

OBnO

BnO

OBn

BnO O

NHCbzOMe

O76

OBnO

BnO

OBn

BnONCbz OMe

O

77 Figure 9.2. Byproducts formed during synthesis.

When using the standard conditions for reduction with alcohol 73 as substrate, the reaction rate was in the same magnitude as for TBDMS-ether 72 and the selectivity decreased to S:R 7:4. Therefore, instead of decreasing the steric bulk as with 73, the larger TIPS group was introduced. The selectivity was however almost identical with the TIPS ether (Table 9.1, entry 3) as with TBDMS ether 72. A somewhat lower selectivity was obtained with the pivaloate ester (entry 4). It thus appears that, the steric bulk of the side chain is of less consequence for the stereoselectivity than the steric bulk of the galactose moiety. As mentioned above, the selectivity is theorized to improve with reduced hydrogen pressure and increased temperature. Since performing the reaction at atmospheric pressure had already been found to be detrimental to the conversion (entry 1), it was decided to increase the temperature in following experiments.


⎯ 66 ⎯

Table 9.1. Varying the reaction conditions to improve stereo selectivity.

OBnO OBn

BnOBnO

OR

NHCbz

CO2MeO

BnO OBn

BnOBnO

OR

NHCbz

CO2Me

(COD)Rh(S,S)-Et-DuPHOS OTf (2 mol%)

Solvent, P (H2), Temp

For the conveniance of controlling the temperature and pressure a

microwave oven was used for the hydrogenations. After mixing substrate 72, catalyst (2 mol%) and methanol the reaction vessel was put under vacuum and filled with hydrogen gas (atmospheric pressure, approx. 15 psi). Heating at 100 °C for 10 min resulted in 25% conversion of starting material yielding product with very high de (>99% according to 1H NMR spectroscopy). The reaction was restarted and run for a total of 40 min with complete conversion of starting material and excellent selectiviy. Unfortunately, it turned out that different batches of catalyst had different thermal stability and when the experiment was repeated with another batch of catalyst less than 5% conversion was obtained. Therefore the reaction temperature was lowered to 70 °C, which resulted in lower selectivity (11:1, entry 6). Temperatures between 100 and 70 °C showed a trend of lower selectivity with lower temperature, and lower conversion (requiring further addition of catalyst) with higher temperature. However, when 2-propanol was used as solvent the selectivity trend was reversed; at 80 °C the selectivity was 36:1 and at 70 °C as high as > 99:1 (entries 7 and 9). Furthermore, the stability of the catalyst seemed independent of temperture in the interval 70-90 °C in 2-propanol. These results were reproducable and gave 78 in a satisfactory 95% yield.

selectivity Entry R Solvent P (psi) Temp (2S) (2R) 1 TBDMS MeOH 75 rt 4 1 2 H MeOH 75 rt 7 4 3 TIPS MeOH 75 rt 4 1 4 Piv MeOH 75 rt 5 2 5 TBDMS MeOH 15 100 °C >99 1 6 TBDMS MeOH 15 70 °C 11 1 7 TBDMS i-PrOH 15 80 °C 36 1 8 TBDMS i-PrOH 15 75 °C 75 1 9 TBDMS i-PrOH 15 70 °C >99 1


⎯ 67 ⎯

99..55.. CCoommpplleettiinngg tthhee ssyynntthheettiicc rroouuttee

HAT REMAINS OF THE SYNTHESIS, after succeeding with introduction of the third stereogenic centrum, is to

substitute the oxygen atom in the sidechain for a nitrogen containing functional group, and to exchange the protective groups of the building block to make it suitable for peptide synthesis. First the hydroxy group in 79 was to be substituted for an azide. This step had caused some problems earlier for closely related substrates; attempts to introduce an azide via a Mitsunobu reaction with NaN3, LiN3 or HN3 had failed. In addition, conversion of the alcohol into an iodide or a mesylate/tosylate followed by tratment with NaN3 had failed. Most distressing, the Mitsunobu reaction attempted on enamide 73 had resulted in an annulation reaction with the Cbz protected α-enamide (cf. 77, Figure 9.2). As was indicated by 1H NMR spectroscopy, the electronic environment for the α-amine protons of the reduced analogue, e.g. 78, differs greatly from that of the enamide.proton (∆δ approx. 1.8 ppm). With this difference it was anticipated that the chemoselectivity may be altered. Gratifyingly, alcohol 79 was successfully substituted to give azide 80 via a Mitsunobu reaction with diphenylphosphoryl azide (DPPA) without any ringclosure from the α-amine (93% yield).

W


⎯ 68 ⎯

OBnO

BnO

OBn

BnOTBDMSO

NHCbzOMe

O

78

OBnO

BnO

OBn

BnOOH

NHCbzOMe

O

79

OBnO

BnO

OBn

BnON3

NHCbzOMe

O

80

OBnO

BnO

OBn

BnONHBoc

NHCbzOMe

O

81

OBnO

BnO

OBn

BnONHBoc

NHCbzOH

O

82

OOH

HO

OH

OHNHBoc

NHFmocOH

O

TBAFTHF, rt, 94%

PPh3, DEAD, DPPATHF, rt, 93%

1) PPh3, THF/H2O, 130 ºC

2) Boc2O, TEA, THF, rt, 97%

LiOH

THF/H2O, rt

1) Pd-C, H2 (75 psi), AcOH, rt

2) FmocOSu, Na2CO3, MeCN, rt, 56%

84 Scheme 9.2. Completing the synthesis of building block 84. The azido group was then reduced to an amine with triphenylphosphine at 130 °C and directly Boc protected to give 81 in near quantitative yield. Hydrolysis of the methyl ester and complete hydrogenolysis of all benzylic groups, followed by Fmoc protection of the α-amine provided building block 84 in 56% yield. The overall yieldfor the 17 steps from galactose pentaacetate was 18%, reflecting an average yield of 90%.

99..66.. PPeeppttiiddee ssyynntthheessiiss

O ALLOW IMMUNOLOGICAL STUDIES OF CIA, building block 84 was incorporated in a glycopeptide, corresponding to CII259-

273 (discussed in Chapter 3.2). The peptide was synthesised on solid phase according to the Fmoc-strategy and the amino acids were coupled

T


⎯ 69 ⎯

from the C-terminus.13, 93, 127 The first portion of the peptide (amino acids 273-265) was coupled using HBTU and diisopropylethylamine for activation of amino acids and piperidine to deblock the α-amino groups. Builing block 84 was coupled with HBTU and HOBt in the presence of DIPEA as base.128 Even though the saccharide moiety is preferably protected in glycopeptide synthesis, protection is not required. However, care has to be taken to avoid ester formation between the free hydroxyl groups and activated amino acids. An often employed method for activating amino acids that suppresses ester formation is pentafluorophenyl ester-activation.129, 130 Therefore, the remaining amino acids (263-259) were coupled as their pentafluorophenyl esters prepared by treating the carboxylic acid with DIC and pentafluorophenol in DMF. Finally, the peptide was cleaved from the resin with TFA/H2O and the standard cation scavenger cocktail. Due to some problems with a contaminated analytic column and some mistakes during reverse phase HPLC purification, the yield for the entire peptide synthesis sequence was as low as 7% but provided enough material for immunological studies (see Chapter 10).


⎯ 70 ⎯


⎯ 71 ⎯

1100.. IImmmmuunnoollooggiiccaall eevvaalluuaattiioonn


HE TWO C-GLYCOSYLATED PEPTIDES SYNTHESIZED in Chapters 7 and 9 (Figure 10.1) were evaluated for recognition by T cell

hybridomas obtained in CIA. At the outset of the project, we had established that modification of galactasylated hydroxylysine 264 did not influence binding to the Aq MHC molecule,15 but wanted to study if such a modification would give a modulated T cell response. If this turned out to be the case, this type of glycopeptides may aid in the longterm goal of inducing tolerance towards RA. Studies in models for autoimmune disease such as experimental encephalomyelitis131 have shown that slight structural modifications of MHC-restricted peptides may give altered peptide ligands,132 (APLs) which can induce tolerance, that is, break autoimmunity.

Gly259-Ile-Ala-Gly-Phe Gly-Glu-Gln-Gly-Pro-Lys-Gly-Glu-Thr273NH O

OHO OH

HOOH

H2N

Gly256-Glu-Hyp-Gly-Ile-Ala-Gly-Phe Gly-Glu-Gln-Gly-Pro-Lys270NH O

OHO OH

HOOH

85

86 Figure 10.1. C-Glycosylated peptides 85 and 86 were used in immunological studies. Their synthesis has been described in Chapters 7 and 9, respectively.

T


⎯ 72 ⎯

1100..22.. TT cceellll rreessppoonnssee aassssaayyss

HE RESPONSE OF THE T CELL hybridomas was measuered as secretion of the cytokine interleukin 2 (IL-2). Cytokines are

soluble proteins which signal to other cells, thereby activating T cells to mount an immunoresponse and to proliferate, consequently measurment of IL-2 secretion reflects the magnitude of a key step in the immunoresponse. Two different assays were used to study the IL-2 response generated by peptides 85 and 86. They were based either on the T cell clone CTLL or on an antibody cytokine assay, as described in brief below.

In the first assay T cell hybridomas obtained from mice were co-cultured with spleen cells that express Aq class II MHC molecules and peptide antigen 85 for 24 h. After freezing the supernatant to kill transferred T cells, it was thawed and CTLL T cells, which are dependant upon IL-2 to grow were added. CTLL proliferation, induced by the IL-2, was then quantified by incorporation of 3H-thymidine in the CTLL cells.

For peptide 86, immuno plates coated with IL-2 specific capture antibody (JES6-IA12) and blocked with BSA to avoid non-specific binding, were treated with the thawed supernatant and incubated for 2 h. To measure the amount of bound IL-2, a biotinylated antibody (JES6-5H4) which also binds to IL-2 was added. Quantification was done by incubating with streptavidin-Eu3+, where bound Eu3+ was released through an enhancer solution. The amount of Eu3+, which is proportional to the amounts of IL-2 produced, was detected by a fluorometric assay.

1100..33.. RReessuullttss

EPTIDES 85 AND 86 WERE evaluated in the assays mentioned above for some selected T cell hybridomas. For peptide 85, all

three hybridomas previously found94 to recognize the glycopeptide CII256-270 GalHnv264 (cf. Fig. 3.1) were examined. For peptide 86, 5 hybridomas were selected from 4 different groups that have been distinguished from the 20 hybridomas that specifically respond to the glycopeptide CII256-270 GalHyl264.18 Hybridomas within a group have been shown todepend on the same hydroxyl groups in the galactose

T

P


⎯ 73 ⎯

moiety for their response, as revealed in a previous study with deoxygalactose analogues.19, 20 Both positive and negative controls have been included in the present study of T cell recognition. For peptide 85 the controls were CII256-270 GalHyl264 and CII256-270 GalHyl264(both positive) and non-glycosylated CII256-270 (negative), and for peptide 86 the corresponding positive and negative controls were CII259-273 GalHyl264 and non-glycosylated CII256-270, respectively. As can be seen in Figure 10.2, two of the hybridomas (HM1R.1 (a) and HM1R.2 (b)) required a 10-20 fold higher concentration of C-glycoside peptide 85 to give the same response as the native O-linked glycopeptide CII256-270 GalHyl264 used as positive control. Hybridoma HDC.1 (Figure 10.2 c) did not show any stimulation at the concentrations of 85 that were tested. However, this hybridoma is known to give weaker response than the other two, and it might be stimulated at higher concentrations of 85.

85

CII (positive control)

CII256-270 (negative control)

CII256-270 GalHyl264 (positive control)

CII256-270 GalHnv264 (positive control)

Figure 10.2. Evaluation of T cell recogition of C-linked glycopeptide 85.


⎯ 74 ⎯

The same trend could be seen for C-glycoside peptide 86, even if the evaluation ends at too low concentrations to be completely conclusive. For hybridomas from groups 2 and 3 (Figure 10.3, b-d) the response is approximatley 10 times weaker than for the reference glycopeptide used as positive control. The hybridoma from group 1 that was tested failed to be stimulated by 86, while hybridoma HM1R.1 from group 4 was stimulated just as well as the positive control.

Figure 10.3. Evaluation of T cell recognition of C-linked glycopeptide 86.


⎯ 75 ⎯

T cells are known to be highly specific in their recognition of complexes between MHC molecules and peptide antigens. Simply modifying the structure of the contact surface between the T cell receptor and antigen, e.g. by altering the side chains of the amino acids, can have a dramatic influence on the response of the T cell, ranging from induction of selective stimulatory functions to completely turning of the fuctional capacity of the cell.132, 133 As mentioned above, the selective activation of T cells by such APLs may result in induction of anergy, i.e. a reduced ability of the T cell to respond to a subsequent exposure to the antigen. It can also lead to T cell antagonism, defined as down-modulation of agonist-induced T cell proliferation when both agonist and APL are simultaneously presented to the T cell. The immunological studies presented in this chapter suggest that APLs, such as 85 and 86, could be used in the immunotherapy of autoimmune disease.


⎯ 76 ⎯


⎯ 77 ⎯

1111.. CCoonncclluuddiinngg rreemmaarrkkss VEN THOUGH THE OVERALL THEME of this thesis deals with biological applications within models for rheumatoid arthritis,

the main effort has of course been on the development of synthetic routes. With the vast number of synthetic methods available to chemists today, the step from idea to actually executing the synthesis can at the outset seem trivial. However, as most people fascinated with synthetic organic chemistry have discovered, synthetic methods that are applicable to any functionalized intermediate are few are far between. Therefore it is still of great importance to put synthetic methods to the test and, even more importantly, develop new methods to further expand the synthetic repertoire available to chemists. Below is a summary of the results and experiences obtained from the four different studies reported in this thesis. In the first study a new synthetic route to hydroxylysine was devised and executed. The original plan was to utilize an azide as a protecting group for the ε-amino group and to introduce the side chain in enantiomerically pure form by alkylation of Myers pseudoephedrine glycinamide. However, when this alkylation failed some other glycine templates were evaluated and it was found that William’s morpholinone was successful. During this study an unexpected selectivity in a reductive opening of a p-methoxybenzylidene acetal was discovered. After experimentation with the reaction conditions a tentative explanation for the selectivity could be provided, which in turn lead to new mechanistic insights in reductive opening of benzylidene acetals. In this particular case it seems that the azido group can chelate to the silyl chloride used as Lewis acid in the reaction, thereby directing the regioselectivity in the opening of the acetal. It also appears that under the reaction conditions employed NaCNBH3 can reduce silyl chlorides to the corresponding silane, after activation of the borane. In the second study the C-glycoside analogue of β-D-galactosyl hydroxynorvaline was synthesized. The key steps of the synthesis were a stereoselective Grignard reaction with homoallylmagnesium bromide on galactopyranosyl lactone, and a Wittig reaction to couple Garner’s

E


⎯ 78 ⎯

aldehyde to the functionalized galactose moiety. This amino acid was then incorporated in a glycopeptide that previously was found to stimulate T cells in a model for rheumatoid arthritis. In immunlogical studies it was found that T cells still responded to the C-linked glycopeptide, but that 10-20 fold higher concentrations were required to achieve the same response as for the native, O-linked glycopeptide. The third study constitutes synthesis of another C-glycoside analogue of a glycosylated amino acid, galactosyl threonine. In this case, the anomeric configuration of the C-glycoside was established using the Grignard reaction mentioned above. The stereogenic centra in the threonine moiety were introduced via an alkylation and a bromination of Evan’s chiral oxazolidinone. When published, this C-glycoside was the most complex C-glycosylated analogue of a naturally occuring glycosylated amino acid reported in literature. The fourth study, i.e. the synthesis of the C-glycoside of galactosylated hydroxylysine, was the main goal of the thesis, for which the three earlier studies also served as model syntheses. Several routes were attempted and found unsuitable. The route that finally was found to be successful was based on an Evan’s alkylation and an asymmetric hydrogenation. The asymmetric hydrogenation at first provided poor selectivities and poor conversion, but after performing the reaction under higher hydrogen gas pressure and with microwave assited heating, both the selectivity and yields were excellent. To the best of our knowledge, this is the first example of an asymmetric hydrogenation heated by micrwave irradiation. The C-glycosylated hydroxylysine was incorporated in a peptide closely related to that of the secnd study. Preliminary immunlogical studies with this peptide suggested a similar behaviour as in the study of C-galactosylated hydroxynorvaline, i.e. the T cell hybridomas required approximately 10 fold higher concentrations of the peptide analogue to induce equal stimulation as with the native, O-linked glycopeptide. However, one of the tested hybridomas gave T cell responses of the same magnitude as the native O-glycoside analogue.


⎯ 79 ⎯

1122.. AAcckknnoowwlleeddggeemmeennttss Många är de som varit till ovärderlig hjälp unde de senaste åren. Jag kanske inte alltid visar min uppskattning till vardags, men skulle här vilja utsträcka min tacksamhet till er. Först och främst vill jag på varmast möjliga sätt tacka Jan Kihlberg, som erbjudit allt man kan önska från en handledare, och dessutom lite extra utöver det. Hård när det behövs, mer medgörlig när det tillåts, på det hela taget en rävig strateg som är väldigt bra att ha sin hörna. Viktigast av allt, ett tack för din vänskap och ditt stöd i svåra stunder. Anders Berg, som med sitt engagemang som gymnasielärare gjorde kemin ännu intressantare. Utan det så skulle jag garanterat inte ha slagit mig in på den här banan. Roland Gustafsson, en mentor utan dess like för en novis på undervisning. Jag uppskattar att du utan (visad) tvekan sagt: ”Prova, det kommer fler stundenter att prova något annat på om det inte skule funka” när man frågar hur man bäst ska göra för att lära ut. Med vördnad; tack. Fredrik Almqvist, reserven, med mer entusiasm än vad tiden egentligen tillåter. Jag väntar fortfarande på att någon gång få höra ”Det kommer aldrig att fungera”. Erik Wellner, min gamla övervakare och den bästa medarbetare man kan hoppas på. Med två nya strategier om dagen så kändes det sällan hopplöst, det fanns ju alltid något nytt att testa. Jag hoppas vi får samarbeta igen vid något senare tillfälle. David Blomberg, min broder i studier, min broder på labbet, min broder på fiskestranden. Lotta Holm, min Lotta, för allt du gjort för mig under de här åren. Jag har svårt att tro att samma glädje att gå till jobbet hade infunnit sig utan dig. Veronica Åberg, för att du så ofta påminner om perspektiven när jag fastnar i detaljerna. Och för att filtret verkar tåla det mesta.


⎯ 80 ⎯

Andreas Larsson, för de oräkneliga timmar med högkvalitativt arbete jag undvikit tack vare dig. Och för ditt förarbete som liger till grund för den ultimata måltiden på Max (9 bitars, green garlic, smält ost). Nu har vi valt varsin halva av världen att jobba på, ett drag som kanske höjer vår produktivitet och sänker våra kolestorol värden. Stina Saitton, min forne rumskamrat, för ditt tillbakalutade, ibland sviktande men aldrig brutna, lugn och envishet som inspirerar. Tack även för korekktur lsäning av min avhandling. Axel, för att du avdramatiserat det här med den stora fasa som kallas ”barn”. Björn Holm, som under flera år pålitligt fanns som sällskap påp jobbet. Var vi inte där samtidigt, så möttes vi i dörren (”One bastard goes in, another bastard comes out”). Jon Gabrielsson, för alla timmar vi spenderat i ”kön” till Fantastisk Grill, en syssla med oviss utgång som kräver starkare nerver än man kan tro. Mattias Hedenström, som jagar 47:or när jag jagar 57:or. Det är väl alltid bra att ha någon som är förkrossande mycket bättre att driva på en. Fast jag har inga ambitioner att uppnå din kompetens vad gäller NMR, jag får se till att du finns till hands istället. David Bundle, for accepting me in his group in Edmonton for some months, even though I failed in a spectacular fashion with the project you gave me, the visit in your group was a real boost Thanks to all the people in the lab for making my stay enjoyable. In particular, thanks to Scott McGavin and Nolan Erickson for the fishing trips. I will never forget Sauer Lake, mostly because I can´t get the smell out. No, really, do you think hypochlorite would work? Och Hanna Pettersson, min med-Svensk, som hade den goda smaken att vara på universitetet när jag kom. Kjell Öberg, som även du handlett och försökt få pli på mig. Jag har försökt spegla din iver för NLO och IR, även om mina applikationer för ivern varit något annorlunda. Per Lind, som jag stod och slet tillsamans med på Bertil-tiden för att framställa kraftigt färgade, totalt olösliga föreningar. Men ”de löser sig nog efterhand” som det gamla ordspråket/sarkasmen löd…


⎯ 81 ⎯

Hans Emtenäs, min gamle vapendragare, det är ett under att labbet klarade sig från vår oömma behandling med bara några sotmärken. Carina Sandberg, Pelle Lundholm, Ann-Helén Waara och Bert Larsson, som får avdelningen att fungera på ett mirakulöst sätt, det märks en markant skillnad när någon av er är borta.

Mina gamla rumskamrater, Kjell, Vit-Per, Stina, Anna, David, Nils och Jon (Två dagar? Tre?).

Alla på Organisk kemi, för att ni gjort det roligt och minnesvärt att gå till jobbet. Det är även roligt att se att när de flesta ljus är släckta i kemihuset, så brukar det alltid lysa på just en våning.

En hel hoper med begåvade exjobbare som gjort sitt bästa för att lösa de problem jag inte klarat av att lösa själv. Jag vill också rikta ett tack till alla studenter som passerat förbi under åren, det har varit ett frustrerande nöje att bli distraherad från labbet och sina egna missöden för att få bevittna era motgångar och utveckling som organiska kemister. Cecilia Ericsson, min vän på distans numera, som jag läst med ända sedan C-kursen (tror jag, kommer du ihåg?). Någon gång kanske vi kommer att befinna oss i samma stad, eller åtminstone samma land. Alla fiskefantaster på avdelningen, det skulle behövas ett appendix för att räkna upp er alla. Ett särskilt tack till er som ibland får mindre fisk än mig. Alla Aikido-människor, som visat att begreppen "att vara nöjd och glad" och "att bli dängd ner i en brottarmatta, gärna ganska hårt" inte alls är oförenliga. Varför känns det som om jag kommer att få stå till svars för de där orden? Johan och Mikaela (och gänget), även om vårt umgänge blivit väldigt försummat nu på slutet. En gång i tiden hade jag energi nog att både jobba och ha ett socialt liv. Nuförtiden orkar jag inget utav det. Tack till min bror Jonas, far Leif, mor Maude och även Gunnar för att ni underlättar min tillvaro på alla möjliga sätt. Det finns garanterat en hel del människor jag glömt bort att nämna här. Men det är bara här, i verkligheten kommer jag ihåg er. Tack!


⎯ 82 ⎯


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1133.. RReeffeerreenncceess 1. Roitt, I.; Brostoff, J.; Male, D., Immunology. 5th ed.; Mosby

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asymmetric synthesis of c-glycosylated amino acids

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