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Synthetic studies of natural compounds and their

analogues

by

Li-Kuan Yeh

A thesis presented in partial fulfilm ent of the requirem ents for the Doctor of Philosophy degree

of the University of London

C hristopher Ingold Laboratories, D epartm ent of Chem istry, University College, London.

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ProQuest Number: 10105605

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Acknowledgem ents

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Contents

Acknowlegem ent 3

Contents 4

PART 1. The total synthesis of the cyclic hexadepsipeptide portion on the antibiotic A83586C.

Abstract of part 1 9

Abbreviations 10

Charpter 1: Introduction

1.1. General introduction to the the azinothricin fam ily of depsipeptide antibiotics and the biological background of A83586C. 12 1.2. Degradation studies on the Azinothricin fam ily of Antibiotics.

The reduction and hydrolysis of Verucopeptin. 14

1.3. Techniques of peptide synthesis. 15

1.3.1. Biochem ical methods of protein synthesis. 15 1.3.2. The chem ical m ethods for peptide synthesis. 16

1.4. Protecting groups for peptide synthesis. 19

1.5. Studies on the m echanism s of reactions used to remove

protecting groups. 21

1.5.1. Activation of carbonyl group by the inductive effect

of an adjacent substitutent. 22

1.5.2. Fragm entation via the E l cB elimination. 25 1.5.3. Acid or base catalyzed 1,6-elim ination. 26 1.5.4. Electrophilic addition on the enamine moiety. 28

1.5.5. Intram olecular cyclization. 29

1.5.6. Cleavage m ediated by neighbouring-group participation. 30 1.5.7. Hydrolysis of acetal-type intermediates. 31

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1.6. Side-reactions in peptide synthesis. 32 1.6.1. N-acyl-N’-a-am inoacylhydrazine-rearrangem ent. 33 1.6.2. The transform ation between the lactone bond and the peptide bond

by slow 0 ,N -a cyl shift in an actinom ycin-related depsipeptide. 34 1.7. Strategies for the total synthesis of depsipeptides. 36 1.7.1. The total synthesis of the antitum or FR -901,228. 36 1.7.2. The total synthesis of the antibiotic L-156,602. 39

1.8. Synthetic studies of antibiotic A83586C. 43

Chapter 2: Resulting and Discussion

2 .1. Retrosynthetic analysis of hexapeptide 1 21; The “ 1+4+1” fragm ent

condensation strategy. 46

2.2. Synthetic study of the “ 1+4+1” fragm ent condensation. 47

2.3. R etrosynthetic analysis of hexapeptide 112: The “ 1 +2+3” fragm ent

condensation strategy. 51

2.4. Synthetic study of the “ 1+2+3” fragm ent condensation. 52

2.5. Retrosynthetic analysis of hexapeptide 154: The “3+3” fragm ent

condensation strategy. 58

2 .6 . Synthetic study of the “3+3” fragm ent condensation. 59

2.7. Retrosynthetic analysis of hexapeptide 154: The “ 1+2+3” fragm ent

condensation strategy. 64

2.8. Synthetic study of the “ 1+2+3” fragm ent condensation. 65 2.9. Preparation of Alkyl N^-Z-(3S)-Piperazate. 66

2.10. Preparation of N-Fm oc-N-M e-(R)-alanine. 70

2.11. Preparation of 0-TBS-(2F?,3S)-threonine methyl ester. 72 2.12. Preparation of N -O Bn-(S)-alanine f-butyl ester. 73

2.13. Preparation of N -F m oc-0-TBS -(2S ,3S)-leucine. 75

Chapter 3: Experimental

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3.1. Reagents and Apparatus 3.2.-3.6 . Experimental Details

78 79

References 102

PART 2. Melatonin agonist and antigonist derived from the structures base on 6H-isoindolo[2,1-a]indole, 5H,6H-indolo[2,1-a]isoquinoiine and 5,6,7-trihydrobenzo[3,4]cyclohept[2,1-a]indole.

Abstract of part 2 Abbreviations

110

111

Chapter 4: Introduction

I. Biological section.

4.0. Melatonin: General background. 114

4.1. Regulation of Melatonin biosynthesis. 115

4.2. Biosynthesis and metabolism of melatonin. 119

4.3. Melatonin binding sites. 122

4.4. The classification of high affinity and low affinity m elatonin

receptors. 124

4.5. Melatonin agonists. 127

4.6. Melatonin antagonists. 129

4.7. Mapping the melatonin receptor. 130

4.8. Objectives. 134

II. Synthetic section.

4.9. Retrosynthesis of tetracyclic-m elatonin analogues. 138

4.10. Synthetic m ethods for the preparation of indole analogues. 139

4.11. Synthetic m ethods for the preparation of

5-substituted-(indol-3-yl)-ethanam ines. 144

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tetracyclic-tryptam ine. 147

4.13. Synthetic methods for the preparation of phenylalkanylhalide. 150

Chapter 5: Results and Discussion

5.1. Synthesis of indole analogues. 152

5.2. Synthesis of analogues of tryptam ine. 160

5.3. Synthesis of analogues of N -alkanoyl-tryptam ine. 164

5.4. Synthesis of analogues of N-alkanoyl-2-(2-substituted-6H -isoindolo-

[2,1 -a]indol-1 1 -yl)ethanam ine. 166

5.5. Attem pted preparation of N alkanoyl2(10substituted5H ,6H

-indolo[2,1 -a]isoquinolinyl-12-yl)ethanam ine. 172

5.6. Synthesis of N-alkanoyl

2-(11-substituted-5,6,7-trihydrobenzo-[3,4]cyclohept[2,1 -a]indol-13-yl)ethanam ine. 174

Chapter 6: Biological assays.

I. Introduction.

6 .1. Binding affinity of melatonin analogues. 180

6 .2 . Membrane preparation. 181

6.3. M em brane binding assays. 181

6.4. Data analysis. 182

6.5. Growing cells containing Meha and Mehb receptors. 182

6.5.1. Cultures of COS-7 cells. 182

6.5.2. Havesting of transfected cells 183

6.5.3. Radioligand binding studies. 183

6.5.4. Culture of NIH-3T3 cells and harvesting of transfected cells. 184

6.5.5. Radioligand binding studies. 184

6.2. Biological activity of melatonin analogues 184

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Chapter 7: Experimental

Contents 193

7.1.-7.10. Experimental Details. 199

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PART 1. The total synthesis of the cyclic hexadepsipeptide portion

on the antibiotic A83586C.

The abstract of part 1.

A83586C, a novel cyclodepsipeptide isolated from Streptom yces Kamatakensis,

exhibits antitum our properties in vitro against a CCRF-CEM human T-cell Leukaem ia line (ICso = 0.0135 pg/mL).

The strategy for synthesis of the peptide portion of A83586C can, in principle, be divided into three parts. First, the asym m etric synthesis of 3S- or 3F?-piperazic acids via the Evans-Vederas alkylation procedure. Second, the form ation of the linear hexapeptide. Third, the intram olecular coupling to form the cyclic depsipeptide.

The problem of N ,0-acyl shifts during the cyclization of depsipeptides occurs when serine or threonine residues are present. It was therefore decided to form the lactone bond between (2 S,3S)-3-hydroxyleucine and threonine in the last step. Three types of fragm ent condensations were em ployed in an attem pt to synthesis the linear hexapeptide. The successful synthesis used a “ 1+2-I-3” condensation procedure.

Synthesis of the linear hexapeptide was achieved by coupling specific am ino acids using DOC or BOP-CI. 4-M ethyl-2-E-pentenic acid was used inplace of

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Abbreviations

AcOH Ala All Alloc atm. Az Bio Bn Boo BOP-CI BOP reagent

n-Bu t-Bu GDI CH2CI2 Dabco DOC DEAD Dim DMF DMSO DMAP DMPU DPM Dts eqival. EtsN Et20 Fmoc

Acetic acid Alanine Allyl Allyloxycarbonyl Atm osphere 4-Azido-m ethyloxybenzyloxycarbonyl 5-Benzisoxazolylm ethyloxycarbonyl Benzyl fert-Butyloxycarbonyl

Bis(2-oxo-3-oxazolidiny!)-phosphinic chloride (B enzotriazol-l-yloxytris(di-m ethylam ino)- phosphonium hexafluorophosphate) n-Butyl

te rf-Butyl

1,1-C arbonyl-diim idazole Dichlorom ethane

1.4-D iazabicyclo[2,2,2]octane D icyclohexylcarbodiim ide Diethyl azodicarboxylate

5.5-D im ethyl-3-oxocyclohexen-1-yl Dim ethylform am ide

Dim ethylsulfoxide 4-Dim ethylam inopyridine

1,3-Dim ethyl-3,4,5,6-tetrahydro-2(1 H)-pyrimidinone Diphenylm ethyl

N "-dithia-succinoyl equivalent

Triethylam ine Diethyl ether

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Gly Glycine

His Histidine

HOBT 1-Hydroxybenzotriazole hydrate

HPLC High perform ence liquid chrom atography

Leu Leucine

Maq M ethylanthra-quinone

MCPBA m -C hloroperoxybenzoic acid

mol Mole

m.p. melting point

Ms Mesyl (m ethanesulfonyl)

Mtp S-m ethyl-4-thiophenol

MBS N -bromosuccinimide

Pet 2-(2-Pyridyl)ethyl

Ph Phenyl

Pip Piperazic acid

n-Pr n-Propyl

/-Pr /so-Propyl

Pro Proline

PyoG 2-(2-Pyridyl)ethyloxylcarbonyl

Sar Sarine

TBS terf-Butyldimethylsilyl

Tee 2,2,2-T richoroethanol

TFA Trifluoroacetic acid

Tfa Trifluoroacetyl

THF Tetrahydrofuran

TLC Thin layer chrom atography

TMS Trimethylsilyl

Ts Tosyl (p-toluenesulfonyl)

Troc 2 ,2 ,2-Trichloroethoxylcarbonyl group

Trt Triphenylm ethyl

Try Tryosine

Z Benzyloxycarbonyl

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C h a p te r: 1 In tro d u c tio n

1.1. G en eral in tro d u c tio n to th e a zin o th ric in fa m ily of d e p s ip e p tid e a n tib io tic s and th e b io lo g ical b a c k g ro u n d of A 8 3 5 8 6 C .

Azinothricin, the first antibiotic of this fam ily, was originally identified by Maehr and his c o w o rk e rs \ This antibiotic contains a 19-m embered cyclic hexadepsipeptide and a lipophilic side chain. The rest of the azinothricin family, which contained similar peptide sequences, were discovered and identified a few years later. These antibiotics are A83586C^, citropeptin^, L-156,602^, variapeptin^ and verucopeptin®’^. Among these six antibiotics, A83586C and citropeptin have similar lipophilic side chains to azinothricin, as shown in Fig. 1. The structures of azinothricin and A83586C were determ ined by single crystal X-ray analysis^'^.

Fig. 1. The structure of azinothricin, A83586C and citropeptin.

,.Me HO

Me OH H

Me

NH NH Me

Me

HO.

Me

HN

(1) Azinothricin, Ri=R2=Et, R3=CH20Me, R4=Me.

(2) A83586C, Ri=R3=R4=Me, R2=Et.

(3) Citropeptin, Ri=R2=Me, R3=CH20Me, R4=APr.

W ithin these six antibiotics, L-156,602 (4)"^ and variapeptin (5)® have similar cyclic depsihexapeptides and both antibiotics have the same lipophilic side chain, as showed in

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Fig. 2. The structure of L-156,602 and variapeptin.

Me OH

Me 'Me OH

NH NH Me

Me HO,

^Me

HN

(4) L-156,602, Ri=Me, R2=H.

(5) Variapeptin, Ri=CH20H, R2=CH2Ph.

The stereochem istry of verucopeptin (6 ) has not been determ ined by a single crystal • 6,7

X-ray analysis and is not certain ’ .

Fig. 3. The structure of verucopeptin.

OH

,OMe

OH Y " ^

Me Me Me Me

r ^ N H NH Me

HO

O O .

,Me Me,

o " W

I

HN— / O (6) Verucopeptin

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-0.06 ng/m L against Staphylococcus, but it did not show activity against G ram -negative bacteria. Smitka and coworkers^ showed that A83586C was inactive at doses of 4.2 mg/kg and was lethal at doses of 9.3 mg/kg in a protection test vs. Staphylococcus aureus in mice. A83586C also displayed an effect in retarding the growth of a CCRF-CEM human T- cell leukaem ia-line with an IC50 of 0.0135 mg/mL®.

1.2. D eg ra d atio n s tu d ies on the A zin o th ric in fa m ily of A n tib io tic s . T h e red u ctio n and h y d ro lys is of V e ru c o p e p tin .

.OMe OH

Me Me OMe

OH OH

Me

Me Me Me

OH

NH NH Me

HO

Me Me Me

Me NH NH Me

HO

Me Me

Me HO

^Me Me^

Me

HN

.OMe

HN OH

I.IN N a O H /

100°C /

1 hour / 2- CH2N2

Me O H f' Me

HO Me Me NH Me OMe OH OOgMe OMe

OH Me Me

Me OHp

HO

Me OH " Me

HO Me Me

Me Me NH Me

NH Me

Me MeOgO Me

OH Me

Me

Scheme 1

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{S ch e m e gave two products which were separated by reversed phase column chrom atography. The negative mass spectrum of both of these materials had a molecular ion peak at m/e 897 (M-H) and the compounds are presum ably diastereom ers about the starred positions. NMR experim ents provided some inform ation to assign structures to these com pounds. The double bond was assum ed to be stable under the reduction conditions. The resulting reduced mixture was treated with 1 N NaOH and then with diazom ethane to give the three degradation products 8, 9 and 10. The production of com pounds 8 and 9 revealed that a retro-aldol reaction had occurred at the 3- hydroxyleucine fragm ent of 7.

The structure of the cyclic depsihexapeptide of verucopeptin (6 ) is related to those of azinothricin (1), A83586C (2), citropeptin (3), and L-156,602 (4) which contain piperazic acid, 3-hydroxyleucine, N-hydroxyglycine and N-hydroxyalanine. Hence, these degradation studies are useful for suggesting targets in the synthetic studies of these depsipeptides.

1.3. T e c h n iq u e s of p ep tid e s y n th esis.

Peptide synthesis continues to be an im portant scientific activity since it provides com pounds that can be used in a widely range of biological investigations. Proteins can be isolated from natural sources but the purified natural com pounds can often only be obtained in low yield. Consequently, one of the major problem s is to obtain sufficient quantities of the peptide or protein in a pure state. In order to solve this problem, scientists have developed a num ber of methods which can roughly be divided into biochemical m ethods and chem ical methods, the latter involving either solid phase® or solution protein synthesis.

1.3.1. Biochem ical m ethods of protein synthesis.

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of the most im portant methods. In this method, the plasmid has to be introduced into the bacterium (e.g. Escherichia coii) which can now produce the desired protein or peptide. Plasmids are rings of DNA which seem to have evolved as a m echanism by which bacteria can transfer genetic material from one cell to another. Simply mixing plasmid DNA with a bacterial culture is sufficient to promote the uptake of the plasmid by a small percentage of the cells. Methods have been developed for increasing the efficiency of this uptake. Inside the cell, plasmid DNA is replicated by E. coii DNA polymerase. Although the E. coii

chrom osom es are replicated only once (just before cell division), plasmid DNA can be synthesized in 50 - 100 copies per cell. A culture of transform ed cells can be grown and then hydrolyzed, and the plasmid DNA extracted and purified. The cloned DNA will give the “natural” peptide or protein. The DNA can be m odified to give a “non-natural” peptide or protein. This is a valuable method to determ ine which amino acids cannot be substituted by others if biological activity is to be retained.

There is one method leading to an efficient production of new polypeptides in E.

coii. This method involves the preparation of a pure gene sequence fo r the polypeptide of interest. The desired DNA can be obtained by chem ical synthesis or as a natural nucleic acid sequence. DNA obtained in this fashion is mixed with different DNA sequences. This mixture of DNA is inserted into plasm ids and used to transform E. coii. W hen plated out on an agar dish, individual bacterial cells divide on the plate to form colonies. Some of these colonies contain plasm ids that can be shown to contain the desired inserted DNA. This cloning process allows identification of a cell line containing the desired DNA, as well as am plification of this DNA through cell division and extraction of the plasmid. Once the DNA is obtained, it can be altered further in vitro and the product repurified and am plified by a repeat of the cloning process.

1.3.2. The chemical m ethods for peptide synthesis.

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peptide fragm ents in the coupling steps, the synthesis of large peptides is usually undertaken by solid-phase synthesis.

The basic idea of solid-phase peptide synthesis is to “grow” the peptide chain on an insoluble polymer support. Hence, any unreacted, soluble reagent can be easily removed by filtration and washing. The extension of the peptide on the resin proceeds by system atic cycles involving addition of the amino acid and then removal of the protecting groups. This simple and speedy method usually give the desired large peptide in high yield. Once the chain of the peptides are prepared, the crude peptides have to be cleaved from the support under conditions that are minimally destructive tow ards sensitive residues in the sequence. Subsequently, these crude peptides have to be purified and characterized.

The strategy of the solid-phase peptide synthesis involves the amino acids as building blocks and sequentially addition of these blocks follow ing the C N direction, the C terminal residue having been fixed to the resin. Fig. 4 outlines the method adopted for solid-phase peptide synthesis.

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Y

DcL2chncnt o f protected peptide segment from resin, and purification

/ w X

Y Y _

\ / H f \ i / V c o o H h,n 4 / I I T T i- n X

Y Y

Segment coupling on a solid support

Y Y Y

— f \ f \ j | : c j \ j -C O O H H :N — \ y

X XYK

i

TiyXY^

support

Y Y Y Y

Fig. 4. Schem atic representation of convergent solid-phase peptide s y n th e s is ".

The synthesis of peptide in solution does not follow the same procedure as that for solid-phase peptide synthesis, which utilized resin supports linked via the C"-term inal or N "-term inal of the am ino acid followed by one-directional chain extension. The extension of the peptide chain in the solution method can take place in both directions. Consequently, the selection of protecting groups for the am ino acid functions must be carried out with great forethought. In the case of the depsipeptides, the presence of the lactone function makes the selection of protecting groups even more difficult because of the possible cleavage of the lactone ring.

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W hatever the chem ical method adopted, the first stage of peptide synthesis involves the selection of protecting groups for nonreacting functions of the various peptide segm ents. In order to form an unam biguous peptide bond the reacting segm ents must be protected at all functional groups except those which are required for the coupling.

For peptide synthesis in solution the functional groups of the amino acid side chains must be protected with the groups which are stable to the repeated treatm ents necessary for both for removal of the N" am ino protecting group and for peptide coupling of the growing peptide chain. These kinds of protecting groups are usually called ‘perm anent’ protecting groups. The protecting groups of the N“ am ino and of C" acid functions are normally referred to as a ‘tem porary’ protecting groups (Fig. 5).

3. .

-a. N "-protecting group (“tem porary” )

b. Side-chain protecting groups (“perm anent” ) c. C“ -protecting group (“tem porary” )

Fig. 5. Protection schem e for peptide synthesis

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The residue His-Phe of jasplakinolide can be used to illustrate a typical protecting scheme^®'^®, as shown in Fig. Different types of protecting groups are removed by different reagents, and are chosen so that they can be removed in any order in the presence of other types. The “perm anent” benzyloxycarbonyl protecting group (Z) is removed by hydrogenolysis using palladium on charcoal under a hydrogen atm osphere. The “tem porary” f-butyloxyl carbonyl group (Boc) at the secondary N-terminal of the peptide is removed by trifluoroacetic acid (TFA) in CH2CI2 and the methyl ester is saponified with lithium hydroxide.

NH

_ /

LION Me

Me

Boc

TFA

Pd/C

Z

Fig. 6. His-Tyr protected residue in Jasplakinolide synthesis.

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F m o c

TFA

handle

NH

-TFA Me ( Me

TFA-FBu

Me Me Me

Me

Fig. 7. One of the com m only used deprotecting schem es for solid-phase synthesis, based on graduated acidolysis to remove perm anent protecting group, f-butanol group, and the supporter^.

For the synthesis of linear or cyclic depsipeptides, the hydroxyl group side chain of the am ino acid residue is often coupled with the another residue as a “ perm anent” protecting group (Fig. 8). In the synthesis of luzopeptin^®, N-Boc-leucine is introduced as the “perm anent” protecting group for the hydroxyl function of the serine residue. The Boc group is then removed after cyclization of the depsipeptide.

Me^ Me

Me O

M e A j i

M e ^ O lactone bond

Zn, AcOH Boc

Tee

Z

Fig. 8. A protecting schem e for depsieptide synthesis em ployed in the synthesis of luzopeptin.

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The orthogonal strategy in which two or more independent classes of protecting groups are removed by different chemical m echanism s was first described by Barany Merrifield^®’^^. The “tem porary” protecting groups must be com pletely cleaved under conditions which do not affect either the perm anent protecting groups, including the anchoring linkage, or sensitive sites in the peptide itself. These groups can be removed in any order and in the presence of the other types of protecting groups (e.g. Boc, Fmoc, Alloc). A num ber of special classes of protecting groups have been developed to protect a variety of functional groups of am ino acids and described by such terms as gradative deprotection^®''^^, dual d ep ro tectio n ^\ two-step deprotection, and safety-catch protection^®'"'^. These protecting groups are also nam ed m ultidetachable protecting groups^^''^°'^^ and protected protecting groups'^'^’'^^. Unfortunately, the majority of these terms have not yet been precisely defined.

Multistep deprotection involves at least two steps with two different m echanism s for cleavage of the protecting group in the overall deprotection process. In this section the deprotecting m echanism s of these special protecting group are discussed and illustrated by some examples.

1.5.1 Activation of carbonyl group by the inductive effect of an adjacent substitutent.

H

11

R = PhCH2, Ph, Bu, t-Bu

O '

O

N'.A A

O H

H2O

RSO2' H3N -A A -1- CO2

12

Scheme 2

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the thiocarbam ic acid S,S-dioxide ester 12. Nucleophilic attack by w ater on the carbonyl

46-49

group then leads to removal of the protecting group ’ {S ch e m e 2).

Nu

ox

SCH; AA.

Y

O Mtp O

H2N-AA'

AA -C O N H -AA'

Scheme 3

The same type of mechanism is also em ployed to removed the S-m ethyl-4- thiophenol protecting group (Mtp)^° {S ch e m e 3). Mtp, a carboxyl protecting group, is stable to the acid conditions used to removed the Boc, Z and f-Bu protecting groups. Mtp can be sm oothly converted to the active 4-m ethylsulfonyl ester 14 by oxidation of the sulfur atom using MCPBA/dioxane^^^^. Nucleophilic attack then leads to loss of the protecting group. The Mtp group is also used in solid-phase peptide synthesis^^'^"^ and the synthesis of the linear^^’^^ and cyclic peptides^'^. Its application is restricted in that the oxidation step cannot be used when the peptide contains sensitive amino acids such as tryptophan, cysteine, cystine and methionine.

O

A.

HN. deprotect.^ 1 O NH. NaNOg

A A " X --- ^ A A ^ N ^ ^

---15 "

X = Z, Boc, Trt

16 H

MBS

O

AA-COgH -<

AA N = N H

17

Scheme 4

o

X

MCI ' A A ^ N = N ^ N '

18

HgN-AA'

A A -C O N H -AA'

56

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protected with Boc^^, Trt^® groups. These protected protecting groups are removed to give the free hydrazide 16. The hydrazide 16 is then converted to the diimide 17 by oxidation using N-brom osuccinim ide (MBS). Hydrolysis of the diimide 17 then gives the free acid'^V A second method involves preparation of the azide 18 by treating the free hydrazide 16 with NaN0 2 . The azide 18 can then be used to couple the C-terminal of the peptide with the another am ino acid or peptide.

In a similar way, the phenacyloxy (Pao) ester can initially be treated with hydrogen cyanide in the presence of KCN to give a strongly electrophilic interm ediate which can be easily cleaved by the incoming amino acid. One practical exam ple is illustrated in S chem e 5. Com pound 19 is treated with KCN to give interm ediate 20 which is then converted into an active vinyl ester 21 by E2 elimination. The active vinyl ester 21 is coupled with glycine methyl ester to give the corresponding peptides^^.

AA

Pao

HCN, KCN

DMF, 12h

©CN

O

A

ON

AA

OH

20

AA

Nu

Gly-OMe

AA-CO-Gly-OMe

Scheme 5

(26)

into 24. The 2-methyl vinyl ester of 25 is easily cleaved under acidic conditions to give the free acid.

PPh3 P h g P ^^'^P P h g

9 H Rh

AA O

23

(PPh3)3RhCI

Ph3P

H -R h

- [(PPh3)2RhCI]

25 26

Scheme 6

1.5.2. Fragm entation via the E lc B elim ination.

The mechanism of these deprotecting reactions involves the fragm entation of the protecting group by an E lc B process. The E lc B m echanism occurs when factors favoring the carbanion character in an E2 transition state exceed a certain limit. p-Elimination occurs sm oothly under weakly alkaline conditions provided the leaving group is situated in a position (3 to a sulfonyl or other strongly electron-w ithdraw ing group. This deprotecting m echanism was first reported by Crane and Rydon®\ Alkylthioethyloxycarbonyl®^ and arylthioethyloxycarbonyl®^’^^ groups are easily cleaved by a reaction involving this mechanism to give the free acid, as illustrated in S ch e m e 7.

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o

+ < ^ ^ S 02R

0' 28 SO2R

O R'

I

yA q- +

o R' .

j f A X _b h

Y ""R

r, H H

29

Y = AA-, AA-NH-

R = Me, Ph, 4-Me-Ph, 4-N0 2-Ph

Scheme 7

In the second method, 27 is converted into the sulfonium salt 29 by N-aikyiation®^. Elimination by a-hydrogen abstraction from 29 under basic condition occurs readily under the influence of the sulfonium salt with the form ation of the vinyl sulfide {S ch e m e 7).

Unfortunately, the N-protected amino acid [3-methylthioethyl esters has to refluxed with excess of iodom ethane to generate the sulfonium salts. The form ation of the sulfonium iodides is sometimes accom panied by side reactions. This method cannot be used for peptides containing cysteine, m ethionine or tryptam ine. These sulfonium ester can be removed conveniently and selectively from the Z, Trt and Tfa protected derivatives by treatm ent with alkali (pH 10 - 10.5).

p-Elimination is not limited to sulfonyl or sulfonium -containing groups. The 2-(2- thyloxylcarbonyl (Pyoc)®^ and Î

to be cleaved by the E lc B mechanism.

pyridyl)ethyloxylcarbonyl (Pyoc)^^ and 2-(2 -pyridyl)ethyl (Pet)^®'®^ groups were also found

1.5.3. Acid or base catalyzed 1,6-elimination.

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ethylanthra-quinone^^^ (Maq) and 4-azido-m ethyloxybenzyloxycarbonyl (AZ) use different deprotecting procedures which are discussed here.

N - A A

30

Bic

+ a 'N - A A

O'

O

NEts

H

CH3CN

A A

H

pH 7

H :B

V_y

31

- H 2 N - A A + C02+

NaO

32

Scheme 8

The 5-benzisoxazolylm ethyloxycarbonyl (Bic) group can be cleaved in a two step sequence consisting of treatm ent with an aprotic base (NEta) in a dipolar aprotic solvent, follow ed by solvolysis in w ater at pH 7 {S chem e s f^ . In the first step, the benzisoxazole ring is opened by the base to give the benzyl alcohol 31 which then undergoes a 1,6- elim ination in the protic solvent to give CO 2, 32 and the deprotected amine. The Bic group is stable to trifluoroacetic acid which can deprotect the Boc group, but is unstable to HBr/AcOH and hydrogenolysis, conditions which are used to remove the Z group.

AA red AA

[H]

34

Maq

O 35

(29)

The next protecting group, m ethylanthraquinone (Maq), is stable to acids, owing to the electron-withdrawing nature of the carbonyl groups in the parent quinone^^^. The Maq ester can be converted to the hydroquinone 34 by reduction with hydrogen. Intermediate 34 is then treated with sodium dithionite in dioxane/w ater to give the free acid and 35

{S ch e m e 9).

The 4-azidom ethyloxybenzyloxycarbonyl group can be cleaved in the presence of both Z and Boc groups. The removal of the AZ group requires reduction by SnCl2 and then acid or base catalyzed 1,6-elimination. In the first step, the azide group is reduced by SnCl2 to give the free amine 37 which loses m ethimide to give the benzylalcohol 38. Intermediate 38 is then treated with TsOH in EtOH to give the desired amine {S ch e m e 10).

O O

U Ï

O N—AA SnCl2 ^

^ MeOH ^ H HgN O

AZ 36 37

HN=CH2 O

; f " o ' ^ - A A TsOH ^ E,OH

38

S c h e m e 10

1.5.4. Electrophilic addition on the enam ine moiety.

(30)

second method^'^ the enam ine portion is treated with NaN02 in acetic acid to give iminium oxime derivative 42, which is readily hydrolyzed to give the free amine {S ch e m e 11).

Me. Me

N -A A

Me, Me Brp Br Br

N—AA

■ Me. Me

NaNO?

AcOH

N -A A

Me, Me

H?0 © ®

► Br H3N-AA +

H2N-AA +

Br Br

Me Me

Scheme 11

1.5.5. Intram olecular cyclization.

This type of amine protection has been applied when sm ooth deprotection under neutral conditions is desirable. The cleavage mechanism involving intramolecular cyclization to give a heterocyclic and the deprotected peptide. The form ation of a five- or six-m em bered ring thus provides the driving force for the final cleavage step.

■Cys

N' N— / H

4 4

X = Boc, Z

-Me S

N-H

base

NHg'Me S— NH' Me

^ N H

4 5 4 6

- C y s + hN

SH

O

A

N' -Me

(31)

This deprotection strategy has been applied to the (3-(N-acyl-N-methylaminoethyl)- carbam oylcysteine derivative^^ 44, which can be converted to the salt 45 under acid condition with the removal of either Z or Boc protecting groups. The salt 45 is then neutralized to give the (3-aminoethylcarbamoyl derivative 46, which then spontaneously cyclizes to give 1 -m ethyl-2-im idazolidone 47 and the thiol containing peptide {S chem e

12) .

1.5.6. Cleavage mediated by neighbouring-group participation.

The 2-bromoethanol^^'^^ group has been used in peptide synthesis for a long time, Zn in AcOH being used for its removal. A two-step removal procedure can also be used. The 2-brom oethyl ester 48 is treated with NEts to give the hydrophilic choline ester 49,

which is very stable to acid but can be cleaved at pH 10 w ithout racem ization {S chem e 13).

O

A,

AA O

,Br-NM03

AA

O

A

48

O

49

® pH 10

^NMes dioxane

b

P

©

Me M e J /M 6q

-► AA-CO2H + HO

A A ^ A ^

HO 50 51

Scheme 13

(32)

N -A A

N -A A H2

Pt02

53 52

I NH -AA

H2O

HO NH-AA

55

2 AA-NH2 +

S c h e m e 14

1.5.7. Hydrolysis of acetal-type intermediates.

H - '

54

NH2 ° y K ^

56 O

Pep'-CONH Pep'-CONH

CONH-Pep CONH-Pep

or Pd/h2

NH-X 57 NH2 58

X = Z, Boc

S c h e m e 15

Pep'-COHisNH-Pep

W eygand et al. have designed the 2,2,2-trifluoro-1-acylam inoethyl group for the protection of the amino groups in peptide synthesis. This group has been shown to be particularly suitable for N '^-protection of histidine-containing peptides, as illustrated in structure 57. Due to the electron-withdrawing effect of the trifluorom ethyl group, the basicity of the imidazole moiety is diminished. The NH-Z or NH-Boc function can be converted to NH2 by hydrogenation or acidification. The resulting aminal derivative 58 is then readily hydrolyzed to 2,2,2-trifluoroacetaldehyde hydrate, am m onia and the histidine derivative {S ch e m e 15).

(33)

Qp op Op

Photosensitive benzyloxycarbonyl ' or diphenylm ethyioxycarbony! groups can be cleaved with light of wavelength longer than 320 nm. The key deprotection step is a photoinduced internal oxidation-reduction reaction of an arom atic nitro com pound with a C-H bond in the ortho-position^'^. The process of deprotecting occurs by initial intram olecular hydrogen abstraction by the excited nitro group. This is followed by an electron-redistribution process to the aci-nitro form, which rearranges to the nitroso product. The resulting hemi-acetal moiety 62 is sm oothly hydrolyzed into the o- nitrosocarbonyl compound {S ch e m e 16).

A A ^ O

-H

Me 59

O

A

( o Me'^OH

62

hv

>320 nm AA_

AA-CO2H +

O Me

O Me

63

S c h e m e 16

1.6. S id e -re a c tio n s in p ep tid e s yn th esis.

(34)

1.6.1. N-acyl-N ’-a-am inoacylhydrazine-rearrangem ent.

.89-91

In the last ten years, other types ' of tem plates have been used for intramolecular

92

peptide synthesis. Among these templates, tetrahydrophthalazine with a rigid conform ation was employed in an attem pt to overcom e the disadvantage of rotation inherent in the hydrazine structure®^.

NH

®NH2 64 Cl®

Z-NH-CHRI-CO2H

BOP, DIEA

CH2CI2

N -C O -C H R I-N H Z Fmoc-NH-CHR2-COCI

NH

65

DIEA, CH2CI2

N -C O -C H R 1-NHZ N -C O -C H R 2-NH-Fmoc

66

40% aq., M02NHFrHF

(1/2, v/v)

1.5% AcOH

THF

N -C 0 -C H R 1-NHZ

N~C0“ CHR2-NH2 67

NH

N-CO -CHR2-NH-CO-CHR1-NHZ

69

Scheme 17

The hydrochloride salt of tetrahydrophthalazine 64 was m ono-acylated with an N-Z protected am ino acid using BOP-CI^'^ to give 65. The m ono-substituted derivative 65 was treated with the N-Fmoc protected amino acid chlorides^^'^^ of either alanine or glycine in the presence of diisopropylethylam ine to give the desired di-substituted derivatives 66. This method was not successful for the coupling of /so-leucine or valine with 65.

(35)

other chain gave the interm ediate 68 which then undergoes ring cleavage and proton transfer to 69.

1.6.2. The transform ation between the lactone bond and the peptide bond by slow 0 ,N -acyl shift in an actinom ycin-related depsipeptide.

N ,0-A cyl shifts in serine or threonine residues of peptides have usually been used as an approach to specific cleavage of peptide linkages or found as a side reaction during m anipulations under acidic conditions. Rearrangem ents were observed with silk fibroin,

97

which when treated with sulfuric acid showed 0 ,N -a cyl shifts in the serine residues . O ther studies of sulfuric acid mediated rearrangem ents, involving sim ple serine peptides , polyserine®®’^^ and clupeine^°°, have been reported. Liquid and BFs/HCOOH^®^ also effect N ,0 -a cyl shifts in dipeptides containing C-terminal serine or threonine residues.

O

O

OMe 4N HCI

71

li

o A

HO Me

dioxane

OMe

Me

74

Ha/Pd/C

Scheme 18

© ©

Cl HgN

72

Z-CI, NaOAc dioxane, H2O

Z OMe

Me

73

(36)

attack by the secondary alcohol of the threonine residue with subsequent cleavage of the amide bond to give 72. Compound 72 is then treated with Z-CI in the presence of NaCOa to give the all protected depsipeptide 73 {S chem e 18). By contrast, the free amine 74,

prepared from 73 by hydrogenation, readily reverted to 71 by an 0 ,N -a cyl shift. The free amine of 74 attacks the carbonyl group of lactone bond which is cleaved105

^IVIe^ /M e

Y%

o

Me O Me

Me. /M e O ^

Hp/Pd/C

Me O Me

TosOH dioxane, 80 C

slow 0 ,N-acyl shift

OH O

o

Me N Me

Me ^ 7

Scheme 19

(37)

This strategy has also been applied to the synthesis of a num ber of the cyclic peptides despite the fact that unprotected and unprotonated hydroxyl groups of serine or threonine are known to undergo a slow 0,N -acyl shift, in a m atter of hours, to give the cyclic pentapeptide from the cyclic depsipeptide. For exam ple, closely related depsipeptides have been reported as interm ediates in total the syntheses of actinomycin

and its analogues^°®'^^° and of triostin and its analogues^^^'^^^.

1.7. S tra te g ie s fo r th e to tal s y n th e s is of d e p s ip e p tid e s .

To solve the problem of the intram olecular rearrangem ent of lactone bonds, two synthetic strategies have been designed to prepare specific peptides. In the first the lactone bond is introduced before cyclization and in the second, after cyclization.

1.7.1. The total synthesis of the antitum or FR-901,228.

,116

The strategy for synthesis of the antitum or FR -901,228 (78) can, in principle, be divided into three parts involving first, the asym m etric synthesis of S-Trt-(3F?)-hydroxy m ercapto-4-heptenoic acid 79^^^'^^^; second, the form ation of the 16-mem bered cyclic depsipeptide; and third, the intram olecular oxidative thiol coupling to form a 15-membered disulfide-containing ring.

Me'

NH

NH

3F?-hydroxy mercapto 4-heptenoic acid

(38)

Synthesis of the cyclic depsipeptide was achieved by coupling one amino acid in each coupling step with the BOP reagent (benzotriazol-l-yloxytris(di-m ethylam ino)- phosphonium hexafluorophosphate)^^^. L-Threonine replaced 2-am ino-2-Z-butenoic acid as the initial amino acid. The extension of the chain of the peptide w ent in the C ^ N direction. The Fmoc group was employed to protect all the am ino functions. The carboxylic acid of L-valine was protected by form ation of the methyl ester. The Trt group was em ployed to protect the two thiol groups. Due to the steric hindrance and the low nucleophilic character of the secondary alcohol, the hydroxyl group of /.-threonine was not protected.

Me^ ^Me

BOP, DIEA OH O

Me. .Me

OMe OH

Me

OMe

1. E12NH

COgH ^ /N H

Fmoc

Fmoc',NH81

OH OMe^ ^Me

2 . BOP, DIEA

COpH

T rtS ^

A

^ NH-Pmoc

Me. .Me

OH O

V

Me OMe

1. EtgNH

O ^ ^NH

T

o

2. BOP, DIEA Fmoc

O ^ N H H o

OMe

N H -Fm oc

84

H O gC ^^N H

Me"’" ^ M e

M e. ,Me

85 O

Me Me

NH-Fmoc

86

1. TsgO

2 . D a b c o / E , , iA ^ ^

Y

NH

OMe

O.^ .,NH

O NH-Fmoc

M e^^^M e 87

S c h e m e 2 0

(39)

using the BOP reagent with diisopropylethylam ine (DIEA) as a base to give the dipeptide

82. Removal of the Fmoc group from 82 with diethylam ine follow ed by treatm ent with N- Fmoc-S-Trt-O-cysteine 83, using the BOP reagent and DIEA gave the tripeptide 84. The Fmoc group of 84 was removed by diethylamine, and the resulting free amine was then treated with N-Fmoc-D-valine 85 using the BOP reagent and DIEA to give the tetrapeptide 86. At this stage the L-threonine could be converted to 2-am ino-2-Z-butenoic acid. The hydroxyl group of threonine was tosylated and then elim inated by treatm ent with 1,4- diazabicyclo[2,2,2]octane (Dabco) and DIEA to give 87.

Me^ /Me Me. /M e

STrt

OMe OH

Me Me

NH NH

7 9a R' = H, R" = OH

7 9 b R' = OH, R" = H -|-^g

TrtS.

NH NH

BOP, DIEA 2. LiOH

NH-Fmoc NH

87 STrt

Me Me Me Me

8 8 a R' = H, R" = OH

8 8 b R' = OH, R" = H Me

EDCI, DMAP

TsOH NH

88a

TrtS

DEAD, PPhS NH

88b

NH

Me Me TrtS

Me

NH

NH

NH

Me Me

78

(40)

After removal of the Fmoc protecting group the free amine of 87 was coupled with S-Trt-(3F?)-hydroxymercapto-4-heptenoic acid 79a and the methyl ester was then hydrolyzed by LiOH give 8 8a. Under the same conditions, the free amine was coupled with 79b and then hydrolyzed to give 8 8b. A variety of conditions^^^'^^° have been investigated to effect the intram olecular cyclization of 8 8a to 89, but these methods were either unsuccessful or only went in low yield. Acid 8 8b was then treated with diethyl azodicarboxylate (DEAD) and triphenyl-phosphine to give the cyclic depsipeptide 89.

O xidation of the depsipeptide 89 with iodine in dilute MeOH solution^^^ gave FR-901,228

(78) {Scheme 21).

1.7.2. The total synthesis of the antibiotic L-156,602.

"O Me

HO.

OH

M©' I •

0 = / OH

r ^ N H NH Me

'Me Me

N Me*

O

; '

y

V^'

A

°

HO^ 1

NH o N ^ M e

H N ^ 4

^ N Z k ^ N H

Me

BnO y

+

Troc

OH

Me<•. \ Me

/ O Me

HOgC OMe 90

Me*

Me* O

-OAII HOgC

o 92

93

N - ^ O °

OAII

Scheme 22

o / — f ,,L

Alloc—N N ^ ‘ ^ O

H zn; ^

NH Me

HOgC

Me

Ô.. ^ O

r

(41)

Antibiotic L-156,602 (4) was the first member of the azinothricin fam ily to have been synthesized. The total synthesis of L-156,602^^^ involved asym m etric synthesis of the lipophilic side chain 90^^^ and cyclization of the depsipeptide. The strategy for the synthesis of the cyclic depsipeptide consisted of a “2-t-2+2” fragm ent condensation. The retrosynthetic analysis of antibiotic L-156,602 is illustrated in S che m e 22.

For the peptide synthesis, the Z protecting group was installed to protect the secondary amines of the piperazic acids and the benzyl group (Bn) was installed to protect the hydroxyl groups of the N-hydroxy-(S)-alanines. Both of these protecting groups can be removed by hydrogenation. The 2,2,2-trichloroethoxylcarbonyl group^^"^ (Troc) was installed to protect the amine of 3-hydroxy-(2S,3S)-leucine and could be removed by zinc in acetic acid after the synthesis was complete. The lactone bond was formed by the treatm ent of N-Troc threonine and N-OBn alanine. The allylo xylca rb on yf^^’^^® (Alloc) and Fmoc groups were installed as tem porary protecting groups for the am ino functions. The allyl (All) and t-butyl groups were installed as tem porary protecting groups for the carboxylic acid functions. Due to its high nucleophilicity, the coupling of glycine occurs much more readily than the amine groups of N -hydroxy-alanine and piperazic acids.

NZ •NH TMS-CI,

then Fmoc-CI 2 . 10% NaHCOs, CH2CI2.1. (C0CI)2, cat DMF.

NZ NZ BnO.

BnO (92%).

HO HO Fmoc NH M e ""

97

OAII OAII

Scheme 23

(42)

condition^^ to give the all-protected dipeptide. This was then treated with diethylam ine to remove the Fmoc protecting group and give the free amine 92.

Troc

NH Me

O.

OBn

Me Troc

NH Me

Me

Me

^Bu■0

^ OBn

Me N

tBu-O ÔH H

99

(67%)

S c h e m e 24

The lactone bond of the depsidipeptise 93 was form ed sm oothly by treatm ent of 98

and N-Troc 3-hydroxy-leucine Fbutyl ester 99 with 1,1-carbonyl-diim idazole (GDI) as a coupling reagent {S ch e m e 24).

1.10% NaHCOs 0-Bu-r

CH2CI2 0= L H

O ^ N H O N—Alloc

9 °2 H (COCI)P 'NZ HOgC

L

- —

^

V

h

N -A llo c N -A llo c 2 . CF3CO2H \

100 101

S c h e m e 2 5

Due to the low nucleophilic character and high steric hindrance of the secondary N^- am ine of piperazic acid, peptide coupling requires the other am ino acid to have a strongly activatived carbonyl group. The acid chloride 101 was prepared by treatm ent of N-alloc glycine 100 and oxalyl chloride. Treatm ent of the acid chloride 101 and N^-Z-piperazic acid Fbutyl ester 102 sm oothly gave the depeptide 94 {S ch e m e 25).

(43)

the acid chloride with oxalyl chloride and this was then treated with 93 in the presence of silver cyanide^^^ at 90 °C to give the tetradepsipeptide 103. Removal of t-butyl ester from

103 with acetic acid gave the free acid which was then converted to the acid chloride with oxalyl chloride. The acid chloride was then treated with dipeptide 92 to give the linear hexadepsipeptide 104 {S ch e m e 26).

Troc.

O

( _ ; n2

94

O

OAII

NH Me

1. (C0CI)2 hBuG Me 2 .AgCN, toluene, 90°C,

(77%) Troc^

O

o,

hBuO

NH Me

Me

0 ^ 0

BnO^

X

HN o N Me

Z N ; ^ 103

M e ''N -O B n

9 3 H

Troc

Ç

NZ NH Me

1. CF3CO2H

2.(C0CI)2, CH2CI2 /

3. AgCN, toluene, 9CP C O 0 ^ 0

Me'. / ^ T

BnO^ 1

OAII o N Me

\ o

Alloc—N N - ^ ' ^ O

Q ^ O A I I 92

H zn; ^

104

S c h e m e 2 6

(44)

occurred to give the amine which was successfully coupled to the lipophilic side chain 90 using 1-hydroxybenzotriazole hydrate (HOBt) to give 105. The Alloc and the All ester protecting groups were removed by palladium -catalyzed hydrostannolysis in one step^^®. C yclization of the resulting crude linear depsipeptide by the mixed phosphonic anhydride method gave the cyclic depsihexapeptide. Finally, hydrogenolysis of this cyclic depsihexapeptide with palladium to remove the Z and Bn groups afforded the antibiotic L- 156,602 (4).

Troc

r ^ N Z n h

BnO^ rviG'.

r NZ n h Me

y

,

-4:^ O 0 ^ , 0 —. Zn, AcOH.

O

BnO^

OAII o N" ""Me

2 . HOBt, DMF (56%

o L

Alloc— N N—

H ZN

104

O HO

Me" "I

HOgC

O Me

r ^ N Z NH

BnO

L - 4 o Ô M e '. / ^

'O ' Alio O

1. BuaSnH, (Ph3P)2PdCl2, CH2CI2, H2O.

2. [n-P rP (0)0]3, DMAP, CH2CI2.

90

OH

Me' ' • . O / ^ N H NH

O N Me

/ — ^ . ' L

Alloc—N N - ^ ’ ^ O

H

Me

105

OH'O "Me Me

Me

3. H2, MeOH, 10% Pd in charcoal.

HO

Me'

O O

NH o

Me

O HO. A

N Me

) 4

Scheme 27

1.8. S y n th e tic s tu d ie s on a n tib io tic A 83 5 8 6 C .

(45)

(2S,3S)-3-hydroxy-leucine, D-threonine, N-methyl D-alanine, N-hydroxy-L-alanine, and enantiom ers of piperazic acid. Among these amino acids, (2S,3S)-3-hydroxyleucine was prepared by a new method^^® involving Sharpless asym metric dihydroxylation (AD)^^^ of an a,[]- unsaturated ester. The synthesis of the two enantiom er piperazic acids, a key step of

130-132

which was based on the Evans-Vederas alkylation ', was accom plished by Hale et

,,Me

HO

Me Me

OH H

106

Me

NZ n h Me

Me HO,

Me BnO

OTBS Me

HN

Me OMe

Me

Me HN

Me

ZN OTBS

R = active leaving group

R', R" = protecting group 107

S c h e m e 2 8

The projected preparation of antibiotic A83586C (2) is adopted the same strategy as that for L-156,602 (4), as is illustrated in S chem e 28. The dipsipeptide portion of A83586C, with one 3-hydroxyleucine and one threonine, has two possibility sites for the 0 ,N -a cyl shift to occur. In order to avoid this shift, the form ation of the lactone bond was projected to take place in the last step of the synthesis. The TBS group was selected to protect the hydroxyl groups of 3-hydroxyleucine and threonine. The Z and Bn groups were chosen to protect the secondary amine of the N^-piperazic acids and the hydroxyl group of hydroxyalanine respectively. The strategical studies on the synthesis of the depsipeptide is discussed in the next chapter.

(46)

,134

which the pyran ring had not been form ed . Condensation between the aldehyde 109 and the a-phenyl-sulphonyl anion derived 110 gives an interm ediate which is then coupled with the aldehyde 111.

.,Me

Me

106

OPMB

Me

MeO

108

Et

P M B O ^ C H O /-BuPhsSiO.

MeO O

109

OPMB

'OHO PhSOg

OSiPhgBu-f

Me

Me

110

Me Me

111

(47)

C h a p te r 2: R e s u lts a n d D is c u s s io n

2.1. R e tro s y n th e tic a n a ly s is of h e x a p e p tid e 112: T h e “ 1 + 4 + 1 ” fra g m e n t c o n d e n s a tio n strateg y.

Studies of synthetic routes to the depsipeptide part of A83586C suggested as an initial target the linear hexapeptide 112. For the subsequent synthesis of A83586C 2, the linear hexapeptide would require the removal of the TBS group on the leucine residue and the methyl ester group of threonine, followed by intram olecular cyclization and deprotection of the cyclized depsihexapeptide. The protecting groups on the peptide need to be carefully chosen with these subsequent steps in mind.

^ N Z

F m o c I | ( j|_ | H N M e

B n O

r ^ N Z H N ^ M e ,

V

t V

- -

. ! V o

*

HO.. . . . . M e O O T B S

M e ' M e

o ' (j:02lVle N ^ " " ^ O H Ç02lVle

M e N - V ' N ^ ^ / + H g N

Z N > H

\ _ _ / O T B S 113 Ô T B S

115

112 H N . M e

M e

( I

B n O

N Z

. N . . O O g M e

Z N M e ' " ( O

117 OO2H 116

S c h e m e 30

(48)

tetrapeptide 113, N-Fm oc-3-OTBS-(2S,3S)-leuclne 114 and 3-O TB S-(2fî,3S )-threonine 115. The analysis was then continued further, with the tetrapeptide 113 being broken down to the two dipeptides 116 and 117.

2.2. S y n th e tic stu d y of th e “ 1 + 4 + 1 ” fra g m e n t c o n d e n s a tio n .

W e initially attempted to prepare the dipeptide 117 from N-Boc-N-Me-(F?)-Ala-N^-Z- (3S )-P ip-0M e 121. N -Boc-N -M ethyl-{fî)-alanine 118 and methyl N^-Z-(3S)-piperazate 120 were to be coupled using Steglich’s reagent, 4,6-diphenylthieno[3,4-d]-1,3-dioxol-2-one- 5,5-dioxide (119)^^^, as illustrated in S che m e 31.

O

BoCv.|^/Me 119

Ph Ph

Boc

2

...COzlVIe 118

120

Me

ZN

121

S c h e m e 31

(49)

o

Me Mé

QH

O

M e - 'B O C p ^

122 o " '^ 9 '

119

Me"^ "'BOC

/ ' Me

Me BOC

124

C02Me /

Z N '" ^ 120

S c h e m e 32

The coupling of the dinnethylimine active ester 128 with the secondary N amine of

120 was next investigated, as illustrated in S che m e In the first step of this process, N -Fm oc-N -M e-(fî)-alanine 125 was treated with 126 to give the interm ediate 127

which was transform ed to the salt 128. The salt 128 was then treated with 1 2 0. Unfortunately, this coupling was also unsuccessful.

FmoC\ /M e

Me O

125

H Me _

OH

c f

cr

Me

126

F m oc^^/M e

" Me

H y

127

O

Fmocx^ .Me

Me O

Fm oc..^.M e g

0 ,iCO2Me 129 M62NC(0 )H Me Cl 'Me

O H 128

120

C02Me

(50)

In order to reduce the steric hindrance, the coupling reaction cf the acid chloride

130 with 120 was investigated. Acid chloride 130 was prepared by the treatm ent of N- Fm oc-N-M e-(fî)-alanine 125 with either thionyl chloride^^® in CH2CI2 or phosphorus pentachloride^^^ in Et2 0 . The crude acid chloride 130 was then treated with 120 using N- m ethylm orpholine 131 as a base {S ch e m e 34). Unfortunately, the desired dipeptide 129

was not obtained. The reason for this unsuccessful reaction was presum ed to be that m orpholine 131 is not a strong enough base to remove the proton from 120.

M e -N O Fmocx. ^ M e

Fmocv^ .Me FmoC\ ^Me \ \ y

N I\| '' I

° Cl :n ,,CO^Me 2 f^ 'N \.'" C 0 2 M e

125 130 ZN ^

method a. SOCI2, CH2CI2. 120 129

method b. PCI5, ether.

S c h e m e 34

Two further methods for coupling the acid chloride 130 with 120 were then investigated, as illustrated in S che m e 35. In the first method, acid 125 was converted to the acid chloride 130 with thionyl chloride^^® in CH2CI2 as previously described. The crude acid chloride 130 was then stirred with 120 using sodium carbonate as a base in a mixture of H2O/CHCI3{S ch e m e 35)^^°. This method was unsuccessful and no 129 was obtained.

Fm oc^ /M e

Fmoc^ ^Me ^

1 X o Base_____,

M e ^ ^ ^ H .N . ...COgMe

Cl /.COgMe ZN

125 130 I

J

method a. SOCI2, CH2CI2; Base, 5% Na2C03 method b. Oxalyl chloride; Base, AgCN

S c h e m e 3 5

U

(51)

In the second method, the acid chloride 130 was prepared by treatm ent of the acid

125 with oxalyl chloride. The acid chloride 130 was then treated with 120 and silver cyanide in toluene at 90 °C for 2 hr. The desired dipeptide 129 was obtained in 50% yield

S che m e 35)122,141

The NMR spectrum of 129 showed a singlet at ô 3.73, indicating the presence of the methyl ester, and two broad multiplets at 5 2.85 and 2.65, which were assigned to the N-methyl group. The observation of two methyl signals presum ably reflects slow rotation on the NMR timescale about the N -C = 0 bond as deserved in other N-acyl piperazic acid compounds^^®. The IR spectrum showed a strong absorption at 1740 c m '\ which was assigned to the carbonyl stretch of the methyl ester, and another absorption at 1687 c m '\ which was assigned to the carbonyl stretch of the amide. The high resolution mass spectrum showed an M^+H peak at m/e 586.2559 (calcd. C33H36O 7N3 (M^+H), 586.2553).

F m o C \^ /M e

Me ,0

HN,Me BnO

Me .0 Me'

.N . ..COgMe

ZN

129

133

TFA

CO2H

116

'EtpNH

BnO

r ^ N Z L / N T F A

N' O

^Me

O %

Me OMe

132

S c h e m e 3 6

The route to the desired tetrapeptide 132 requires the coupling of the am ine of 117

with the acid of 116^^^. The first step was therefore the removal of the Fmoc group from

(52)

in the NMR spectrum of this product there was no absorption at 5 3.73, indicating the loss of the methyl ester. The high resolution mass spectrum show ed an M^+H peak at m/e 332.1615 (calcd. C 17H22O4N3 (M^+H), 332.1610), consistent with the cyclised com pound 133.

2.3. R etro s y n th e tic a n a ly s is of h e x a p e p tid e 112: T h e “ 1 + 2 + 3 ” fra g m e n t

c o n d e n s a tio n stra te g y .

BnO

Me'

/Fmoc

NZ HN Me

V ^ M e

o ÔTBS

-N' Q 0 C02Me

O )— f

Me^ , N ^ ' N

^ \ _ y ÔTBS

NZ NH

Fmoc HN

BnO T HO

uv N - t ^ +

Me

Me

O ÔTBS

Me

N o O Ç02M6 1 1 4

Me o W - , . î

112

FmoCs^ ,Me

Me .0

/ M e ^ I '

HN o o CC^Me

M e ^ N ^ " " N

^ ÔTBS

134

Fmoc

H + Me.

135 O O OH 136 C02Me

137 ^ ^TBS 115

S c h e m e 3 7

(53)

deprotection of the amine. It was thought that the problem of intram olecular cyclization would be much less with the tripeptide 135 than 129 because of the resulting ring size. The tripeptide 135 should be derived by coupling the dipeptide 137 with 0 -T B S -(2 fî, 3S)-theronine methyl ester 115 and removal of the Fmoc protecting group. Acid stable protecting groups, such as f-butyl ester or diphenylm ethyl ester (DPM), rather than the methyl group may be need in this procedure.

2.4. S y n th e tic s tu d y of th e “ 1 + 2 + 3 ” fra g m e n t c o n d e n s a tio n .

Acid 125 was converted to the acid chloride 130 by treatm ent with oxalyl chloride in benzene. The crude acid chloride was then treated with Fbutyl N^-Z-(3S)-piperazate 138

and silver cyanide (2 equivalents) in toluene at 90 °C to give the dipeptide 140 in ca. 80% yield {S ch e m e

Fm oC\ ^M e Fm oC \ ^Me

F m o c ^ M e i (C0CI)2, Ph CF3CO2H, toluene

H • z n' %

' ( T

V

U

: : : : z

S c h e m e 38

(54)

to the methyl carbons of the f-butyl group. The high resolution mass spectrum showed an M"+H peak at m/e 628.3028 (calcd. C36H42O7N3 (M"+H), 628.3023).

Cleavage of the f-butyl ester of 140 was successfully carried out with trifluoroacetic acid to give the desired acid 137 in 90% yield^^^. The NMR spectrum of 137 showed a broad singlet at 8 12.20, which was assigned to the acidic proton. The IR spectrum show ed a broad absorption in the range of 3800 - 2600 c m '\ which was assigned to the hydroxyl stretch of the carboxylic acid.

The diphenylm ethyl ester was also investigated as a protecting group for the carboxylic acid of N^-Z-(3S)-piperazic acid^'^^’^'^^. The crude acid chloride 130 was treated with diphenylm ethyl N^-Z-(3S)-piperazate 139 and silver cyanide (1.6 equivalents) in a toluene solution at 70 °C to give dipeptide 141 in 90% yield {S ch e m e 38). The NMR spectrum of 141 showed two multiplets at 5 2.75 and 2.57, which were assigned to the N- methyl group. In addition, com pound 141 gave a satisfactory 0 , H, and N microanalysis for C 4 5 H 4 3 O 7 N 3 .

The cleavage of the diphenylm ethyl ester group from 141^^^ occurred sm oothly with trifluoroacetic acid (12 equivalents) and phenol (2 equivalents) in CH2CI2 solution to give the desired acid 137 in 99% yield.

The coupling of dipeptide acid 137 and 0-T B S -(2R , 3S)-threonine methyl ester 115

with dicyclohexylcarbiodiim ide (DCC) (142) (1.2 equivalents), cupric chloride (143) (0.1 equivalent) and N-hydroxybenzotriazole (HOBt) (145) (2.0 equivalents) in THF occurred sm oothly to give the desired tripeptide 149 in 80% yield^"^®. The m echanism of this reaction is illustrated in S che m e 39. Dipeptide 137 is initially coupled with 142, and the lone pair of electrons on the amine of 142 then attacked cupric chloride 143 to give the interm ediate salt 144. The interm ediate salt 144 exchanged protons with 145 to give 147. The weak acid 145 was converted to its anion 146 by the donation of one proton to the amine of 144

(55)

Fm oc^ M e

'N Fmocv. .Me

146

CuCl2

H 148

Fmoc Me

Me

Me Me'

ZN

C - N

ZN

149

0TB S 147

NH Me

115

S c h e m e 39

The NMR spectrum of 149 at 110 °C showed a singlet at 5 3.55, which was assigned to the methyl ester; a singlet at 5 2.7, which was assigned to the N-methyl group; a singlet at Ô 0.85, which was assigned to the f-butyl protons of the TBS group; and two singlets at 5 0.00 and - 0.02, which were assigned to the two methyl groups of TBS. The IR spectrum showed a medium absorption at 3300 c m '\ which was assigned to the secondary amine stretch. In addition, the high resolution mass spectrum showed an M"+Na peak at m/e 823.3709 (calcd. C/oHsGOgN^SiNa (M % N a), 823.3714).

Figure

Fig. 1. The structure of azinothricin, A83586C and citropeptin.
Fig. 3. The structure of verucopeptin.
Fig. 4. Schematic representation of convergent solid-phase peptide synthesis".
Fig. 5. Protection scheme for peptide synthesis
+7

References

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