STUDIES on PEPTIDE CHEMISTRY a Thesis Submitted by DEMETRIUS SARANTAKIS for the Degree of DOCTOR of PHILOSOPHY of Imperial Colle

STUDIES ON PEPTIDE CHEMISTRY

a thesis submitted by

DEMETRIUS SARANTAKIS

for the degree of

DOCTOR OF PHILOSOPHY of THE UNIVERSITY OF LONDON

Imperial College London, S.W.7. November 1965 2

Abstract

In the first part of this thesis the methods available for N-terminal, C-terminal, and in-chain selective degradatf_c1 of peptides have been reviewed.

The selective cleavage of the C-aspartyl bond has been attempted on a model compound, N-cyclohexyl-succinamic acid via isoimide formation. A method for N-terminal analysis in which 2-fluoropyridine-N-oxide was reacted with glycine cyclohexylamide under mild conditions and the cyclohexylamine residue removed under mild conditions has been successful. A series of amidoxime and amidhydrazine derivatives of glycine cyclohexylamide have been prepared and attempts have been made to use these compounds in N-terminal analysis. In the second part, the use of 0-acyl esters of hydroxyll- amine derivatives in peptide synthesis has been reviewed. 2-Fluoropyridine-N-oxide was shown to react with acids under mild conditions to give 0-acyl--pyridones, good acylai- In/ agents.

Factors affecting the acylation rate of amines by N- substituted-N, 0-diacyl-hydroxylamines have been studied. 3 •

ACKNOWLEDGEMENTS

The author would like to thank Dr. J. K. Sutherland for his grett help and encouragement during this research and Dr. V. Tortorella for valuable discussion. Thanks are due to Professor D. H. R. Barton for the opportunity of working in his department. The author would also like to acknowledge financial support from the Greek State Scholarships Establishment. To my parents Barbara and George 4.

Index

Page Part T 5 Review 8 References 37 Theoretical 45 Experimental 74 References 97

Part II 99 Review 100 References 107 Theoretical 10i+ Experimental 127 References 147 PART I Introduction

Emil Fischer and his co-workers, in their classicR1

studies on peptides and proteins, showed that these con,, J1$1" of a-aminoacids linked by amide bonds to form polyamides the general formula:

H NCHR CO(NHCHR CO)NHCHRCOOH 2 1 3 n 2

The biologically important aminoacids are twenty one in number and differ in the nature of the side chain R. The diverse reactivity of the side chains, though a serious problem in the synthesis of polypeptides, is, on the other hand, the only means by which we can chemical. differentiate the successive units and allow selec'Ave cleavage at specific amide bonds: Much use of this principle has been made, as we will see later. The elucidation of the structure of polypeptides proteins, is a most important step to a more precise under- standing of their biological and chemical function in Naltire From an inspection of the general formula of DolyFcpbeleS

H2NCHR1CO(NHCHRnCO)nNHCH42COOH the ideal way of determining the aminoacid sequence woul,-7. be, the step by step removal of aminoacids starting from, either 7

group (N-terminal analysis) or the end bearing the free-NH2 H group (C-terminal analysib) the end bearing the free-CO2 until the complete chain has been degraded. Unfortunately at present the stepwise degradation cannot, for practical reasons, be applied to peptides containing more than thirteen or so aminoacids. This situation brings about the need for methods for the selective cleavage of a protein at a few "internal" amide bonds of aminoacids which occur in a few positions iy) the protein molecule. In this way a limited number of peptides, of a size suitable for the existing methods of sequential analysis can be obtained. The aim of this review is to describe critically the most important existing methods for: (A) N-terminal analysis (B) C-terminal analysis (C) In-chain fission. O

(A) N-TERMINAL ANALYSIS

(a) Sanger's method.

It depends upon the reaction of the free amino group the peptide, with 1-fluoro-2,4-dinitrobenzene (DNFB) undo mild alkaline conditions, forming the N-2,4-dinitropheny1 derivative of the peptide '2 . The DNP-peptide is isolated and then hydrolysed with constant boiling hydrochloric acid to give the DNP derivative of the terminal aminoacid which can be separated from the hydrolysis mixture and identified. 4 DNP-Derivatives of serine3 and aspartic acid in N-terminal position are obtained in small yields and other side groups of aminoacids react with the reagent, but satiF factory methods to overcome these difficulties have been developed and successfully applied to the elucidation of 5,6 7,8 aminoacid sequences in insulin , the corticotropins aYA other polypeptides. DNFB H2NCHR1C0(NHCHRnCO)nNHCHR2C00H NaHCO NO2 3 Hydrolysis 0 2 --NHCHR1CONHCHR CONHCHR CO H \ 1 n 2 2 with HC1 NO2 NHCHR CO H + (NH CHR CO H) + NH CHR CO H. -- 1 2 2 n 2 n 2 2 2 (b) Edman's method.

BergmanI)et al9 examined the possibility of selective removal of the N-terminal aminoacid in a peptide sequenco by condensing the free amino group of the peptide with phenyl isocyanate and cycliming the resulting carbamoyl peptide with concurrent liberation of the rest of the peptide, shorter by one aminoacid. Abderhalden and 10 Brockmann proposed a systematic method for sequential analysis but problems of insolubility make the method impractical. The most successful method at present is the so-called "Edman degradation"11 consisting of two main steps.

First coupling of the free amino group of the peptide, with phenyl isothiocyanate in slightly alkaline medium (pH = 8-9), usually pyridine-water or dioxan-water triethylamine or sodium hydroxide being added to keep the pH - 8-9. The second step is the cyclization of the phenylthio - 12 carbamyl (PTC) derivative under aqueous or anhydrous acidic conditions, to a 5-substituted-3-phenylthiohydantoin (PTH) which can be extracted into organic solvents and identified by chromatographic methods or hydrolysed to the amino acid. lo

Problems of insolubility of PTC-peptides always occur- especially with the larger peptides and a very successful procedure due to Fraenkel-Conrat consists in carrying out all the reactions on paper strips in order to increase the accessible surface of the protein13'14 . The mechanism the reaction has been discussed by Edmani5 who proposed NE Scheme 1, and recently Sheppard et al16 reported some kinetic data for the cyclization of the FTC-derivatives to phenylthiohydantoins in aqueous solution using leucyl- glycine as a model peptide, in good agreement with the proposed course of the reaction. In aqueous solution, the maximum yield of phenylthiohydantoins is obtained using a strong acid at low temperatKre for the shortest possible time, typical conditions being dissolution of the PTC-peptide in 3N-HCl at room tempera tire or at 40° for 1.5-24 hours17. SCHEME 1

pH = 8-9 PhNCS + H2 NCHR1 CONHR'

aq. or anh. H.+ PhNHCNHCHRCONHR t rapid Phenylthiocarbamyl peptide

N----CHR

PhNH-4(/ \\ CO H NR 1 3

2-anilino-4-substituted-5-thiazolinone

aq -H

S /".

PhNHCNHCHRCO H aq -H 2 \NH Phenyl-thiocarbamyl- slow C HR amino acid

Under acidic conditions all phenylthiohydantoins are unstable. Serine, threonine and cysteine phenylthiohydantoins 18 cannot be isolated after treatment of phenylthiocarbamyl derivatives with IN-HC1 at 100° for 1 hour. Phenylthiohydantoins of the above aminoacids have been prepared in aqueous solution, using conc. acids (1-5N) and low 19 temperature . The progress of the cyclization of the FTC to PTH can 20 be followed spectrophotometrically by making use of the two different absorption maxima of PTC and PTH (240 mil or lower vs. 265-270 m:µ)21. The phenylthiohydantoins of aminoacids can be extracted into ethyl acetate with the exception of those of arginine and histidine which cannot be extracted from acid solutions. The phenylthiohydantoin of histidine can be extracted from neutral solutions. The phenylthiohydantoins can be identified by paper22 23 or partition column chromatography , and recently TLC on 24 Silica gel has been employed , the great advantages of the last method being the rapidity and the extreme sensitivity. Edman in his original procedure carried out the cyclization step in anhydrous media, dry HC1 in nitromethane or acetic acid in order to avoid hydrolysis of the remaining peptide bonds, but side reactions leading to erroneous results occur readily with the relatively high temperature of the cyclization (100° C) and Konigsberg and Hill25 in a paper particularly important for its description of improved conditions for the Edman degradation, use anhydrous trifluoroacetic acid at 25° following earlier suggestions b, 26 27 Edman and Elmore and Toseland . Direct identification of phenylthiohydantoins is preferable, since those of trypt,_, phane, arginine, serine, ttareonine, cysteine and cystine ar€ unstable under the conditions used to obtain the PTC-amino acid (6N-HCl at 150° for 16 hrs.)28. In an effort to develop improved reagents other workers have proposed instead of phenyl-isothiocyanate, the use of 29 30 benzoyl-isothiocyanate , methyl-N-acetyl-dithiocarbamate , 31 32 methyl-methoxy-dithio-carbamate , carbon disulfide , and also coloured isothiocyanates such as p-phenyl-azophenyl- isothiocyanate33 and 4-dimethylamino-3,5-dinitro-phenyl- isothiocyanate34. Solubility problems leading to contamination of the terminal aminoacid with starting materials were encountered and since the above reagents do not posaNtsany significant advantage over the original Edman method, they have been little used.

Holey and Holey35 used 4-carbomethoxy-2-nitro-fluoro- benzene to form the N-(4-carbomethoxy-2-nitropheny1)-peptide and the coupling product is reduced catalytically to the o-amino compound which on acidification undergoes lactam

14 formation with removal of the terminal aminoacid as a 7- carbomethoxy-3,4-dihydro-2(IH)-quinoxalone (Scheme 2,

X . CO Me). 2

SCHEME 2. NaHCO3 in MCOH-H 0 + H NCHRCONHR' 2 2 35o

H /Pd X ' ,;--NHCHRCONHR 2

NO2

ir=\ HC1 \--NHCERCONHR'

NH 2

H NR' 1 3 ,- Co NH

In a modification of the Sanger's method, the DNP- 36 peptides are reduced catalytically to the corresponding diamino compounds and cyclised to the corresponding quinoxalone under similar conditions to those of Holey and

Holey (Scheme 2; X = NO2 or NH2). Non-specific reduction of the nitrogroup and the 15. necessity to work in the absence of oxygen is a drawback to application of the above methods and poor results have been reported when applied to complex peptides.

The selective reduction of the o-nitrogroup has been reported37 but the method has not been applied in sequential analysis. Signor et al' used 2-chloro-3,5-dinitropyridine to react with the free terminal amino group, at pH = 8.5, and room temperature. The dinitropyridyl-peptide is hydrolysed with 6N-:HC1 containing 30% HCO2H at 600, or with 90% HCO2H at 100° and the DNPyr-amino-acid is assayed by paper chromatography and optical density measurement at 340 ma. The method has obviously some advantages since the DNPyr--amino acids are bright yellow, the coupling is quantitative and relatively free of side reactions and the DNPyr- amino acids are quantitatively recovered after hydrolysis of the pc,lypeptide chains.

(d) Cyanate method.

The use of cyanate for N-terminal analysis has been reported39. The method involves six steps (i) Carbamylation of the terminal aminogroup. (ii) Separation of the carbamyl compound. (iii) Cyclization in hot 6N-HC1 for 1 hour to form 16.

a mixture of peptides, free amino acids and the hydantoin which results from the NH2- terminal residue. (iv) Isolation of the hydantoin from the mixture by ion exchange on Dowex 50-X. (v) Hydrolysis of the hydantoin. (vi) Identification and estimation of the resulting amino acids. The method is claimed to have some advantages over

Sanger's, in that incomplete reaction due to precipitation, often a problem in preparing DNP or PTC-proteins, is avoided since the reaction is carried out in 8M-urea or 4M-guanidinium chloride in which most carbamyl proteins are soluble. The excess reagent is completely decomposed to CO2 and NH3 with acetic acid. The stability of carbamyl proteins and hydantoins permits their isolation without any special precautions. Obviously since many peptide bonds are hydrolysed in the cyclization step, this prevents the sequential application of the cyanate method. This procedure is limited only by the sensitivity with which amino acids can be determined. The method was tested on Glucagon, Insulin and Ribonuclease and some synthetic peptides with satisfactory results. 17.

SCHEME 3.

KCNO + H NCHRCONHCHR CONHR -> 2 1

6N-HC1 H NCONHCHRCONHCHR CONHR 2 1 1

NH — CHR + H NCHR CO H + H NR ? CO CO 2 1 2 2 NH

dil. NaOH > H NCHRCO H 2 2

(e) Miscellaneous methods.

The labilizing effect of a methoxycarbonyl group on an adjacent peptide bond has been investigated36. Another method40 uses bromoacetate in an aqueous suspension of magnesium hydroxide to form the derivatives -COCHRN(CH2CO2H)2, which after acid hydrolysis gives the carboxymethyl derivative of the terminal amino acid in yields near 90°6. The selective N-terminal hydrolysis of simple peptides by cis- hydroxy-aquotriethylene-tetramine cobalt ions has 1 been reported , (Scheme 4). The reaction takes place in 18.

aqueous solution at 65° and pH = 7.5 in about 30 mm. In all of the reactions the product complex ions were isolated in quantitative yields by column chromatography on cellulose and characterised by infra-red spectroscopy and paper chromatography.

SCHEME 4.

[Co-triethylentetramine-(OH)(H20)]2+ + NH2CHRCONHR' > [Co-triethylentetramine-NH2CHRC00]2+

(B) C-TERMINAL ANALYSIS

There is a lack of satisfactory methods for C-terminal analysis of peptides and proteins.

(a) Hydrazinolysis42.

The method consists of treating a protein with anhydrous hydrazine at 100° C for 10 hours in a sealed tube, when the C-terminal aminoacid is liberated while all the other aminoacids are converted to the aminoacid hydrazides. The aminoacyl hydrazides are condensed with benzaldehyde to form water-insoluble benzilidene compounds, leaving the C-terminal aminoacid in the aqueous phase (Scheme 5). 19.

SCHEME 5.

N H NCHR CO(NHCHR 2 H2 1 nCO)nNHCBR2COOH

NH CHR CONHNH + NH CHR CONHNH + H NCHR COOH 2 1 2 2 n 2 2 2

PhCHO > PhCH=NCHR1 CONHN=CHPh + PhCH=NCHRnCONHN.CHPh + H2NCHR2COOH

An improved procedure43 is treatment of the hydraziriolysis products with 2,4-dinitrofluorobenzene (DNFB) to form di-DNP derivatives of the aminoacid hydrRzides and the DNP-derivative of the C-terminal aminoacid which can be readily separated using aqueous bicarbonate and identified by chromatography and estimated spectrophotometrically (Scheme 6).

SCHEME 6.

H NH2NH2 NH2CHRnCONHCHRXCONHCHRCO2

CONHNH + NH CHR CONHNH + NH CHRCO H NH2CHRn 2 2 X 2 2 2

DNFB > DNP-NHCHRnCONHNH-DNP + DNP-NHCHR F ONHNH-DNP + DNP-NH-CHRCO2H.

29.

Hydrazinolysis as an end-group analysis method suffers from the serious disadvantages of low recoveries44 and the complete loss, in a C-terminal position, of cystine, cysteine, 45 asparagine and glutamine . Methionine is converted to methionine sulfoxide, arginine to ornithine, and aspartic and glutamic acid are partially destroyed.

(b) Reductive methods.

46 Fromageot and his collaborators have reduced the free carboxyl group of the protein with LiA1H4 and identified, 47 after hydrolysis, the liberated f3-amino alcohol Reduc- tion with LiBH4, a much milder reducing agent has been 48 recommended . A stepwise degradation from the C-terminal 49 residue based on reduction was envisaged but difficulties were encountered in the reduction and rearrangement stage (Scheme 7). Methods for the isolation and estimation of ft-amino alcohols on a microsCAe have been developed50.

SCHEME 7.

esterification RCONHCHR CONHCHR CO 1 2 2H

reduction RCONHCHR 1 CONHCHR2CO2CH3

acid or acid chloride RCONHCHR1 CONHCHR2CH2OH 21.

+ reduction RCONHCHR1CO.00H2CHR2NH3

CHR CH OH RCONHCHR1CH2OH + NH2 2 2

The earliest of the C-terminal analyses methods is due 51 to Schlack and Kumpf . The method is based on the reaction of acetic anhydride and ammonium thiocyanate with an N- acylated peptide whereby a terminal thiohydantoin is formed and split off, leaving the residual N-acylpeptide for a subsequent reaction, (Scheme 8). Milder reaction conditions are possible using mixed anhydrides of thiocyanic acid52 such as (C6H50)2P0NCS. The applicability of the method to proteins is limited since proline, aspartic acid glutamic acid, serine, threonine, cystine, arginine, and lysine, cause complications when they are in C-terminal position.

SCHEME 8.

NH4NCS RCONHCHRnC ONHCHR C ONHCHR1C 02H X AC 0 2 22.

OH RCONHCHR CONHCHR CON CHR n X 1 CS Ni

Acyl thiohydantoin

RCONHCHR CONHCHR CO H HN HR n X 2 1 CS -CO NH

(d) Miscellaneous methods.

The use of aromatic carbodiimides which react with carboxylic acids has been proposed53 but difficulties in the separation stage limit its usefulness. Substractive methods have been suggested for C-terminal analysis54. The azide of the C-terminal aminoacid has been used55 to identify this amino acid and a method based on the Grignard 56 reaction has been reported .

In the last few years a new approach to sequence analysis and fragmentation of proteins has been developed. The reactivity of certain aminoacid residues, permits cleavage of peptide bonds at these sites by nonenzymatic reagents. In many cases the aminoacid residue is selectively modified prior to cleavage. Several nonenzymatic methods for selective cleavage of peptide bonds are reported in the literature. The most successful of these methods will be described in this section.

(a) N-Bromosuccinimide and brominating agents.

When brominating reagents, such as N-broMosuccinimide, N-bromoacetamide, or bromine in acidic solutioh, react with tryptophyl, tyrosyl, or histidyl residues cleavage at their carbonyl groups occurs57. The mechanism suggested58 for this cleavage involves the formation of a five membered imino-lactone ring which is readily hydrolysed with concurrent cleavage of the peptide bond (Scheme 9).

SCHEME 9.

NHCOR NHC OR1 1 NBS it I 1-Br NHR N > 0 ''.- 1\THR 2 H 2 NHCOR NHC OR 1 1 H 0 -- r' P NO/ . I

H + H H NR H2 2

24.

Two possible mechanisms are envisaged for the cleavage of tyrosyl peptides59, i.e., either hypobromite formation or bromination and subsequent displacement, (Scheme 10).

SCHEME 10. CONH-Ri Br 4.) NBS * H-0 CH,CH ---> Br- I (a) /)\ /r- - C H2 -- CH - NHC OR2 NHC OR,_, Br NBS (b) H2O

Bra Ate' NHR1 H 0 0 2 NR 0 + H2 1 A CH2— CHNHCOR2 CH-NHC OR2 Br

When a peptide or protein i3 treated with N-bromosuccinimide several amino acid residues are liable to be attacked. These include, cysteine, cystine60, methionine 62 and the liberated aminoacids . Secondary reactions with cysteines and cystines are avoided by prior oxidation with 63 performic acid to cysteic acid . Methionines are oxidised 61 by excess N-bromosuccinimide to methionine sulfone residues . 25,

The presence of formic acid in the buffer reaction medium protects the liberated aminoacid from further oxidation64. In spite of the competition between tryptophyl, tyrosyl, and histidyl residues for N-bromosuccinimide it is possible to carry out selective cleavage since tryptophyl residues react faster than tyrosyl ones57a'61 65 Witkop et al report that oxidation of a tyrosine residue is more than 200 times slower in BM-aqueous urea solution than in water alone and this difference in reactivity permits the cleavage at a tryptophyl carbonyl in a protein without cleavage of tyrosyl residues. By performing the reaction with N-bromosuccinimide at room temperature in pyridine-acetic acid-water, rupture of tryptophyl and tyrosyl linkages occurs, whereas histidyl residues are oxidised but not cleaved. The nature of the oxidation product of histidine is not known yet. After destroying the excess N-bromosuccinimide and heating the reaction mixture, cleavage of histidyl linkages occurs without any cleavage of the other peptide bonds57c. The method has been applied to some proteins with the 57a results shown in the following table- .

DETERMINATION OF THYPTOPHAN IN VARIOUS

PROTEINS BY TREATMENT WITH N-BROMOSUCCINIMIDE.

Tryptophan (io Protein Found Reported

Chymotrypsin 5.7 5.7

Chymotrypsinogen 5.7 5.6

Lysozyme 8.3 7,,1 and 9.1

Trypsinogen 3.4 3.70

Trypsin 3.3 4.5 and 1.0

(b) Methionine cleavage.

Sulfonium derivatives of methionine undergo ready displacement of the sulfur function, resulting in lactone formation. A procedure for the degradation of methionyl peptides 66 based on this observation has bean devised and recently applied to a number of methionine peptides (Scheme 11).

SCHEME 11 CH SCH 1 3 3 4-S-CH CONH 1 1 2 2 CH2 ICH CONH CH 2 CH 2 2 2 1 > CH R NHCOCHNHCOR 2 1 2 35-40° C R NHCO-CH-NHCOR 1 2

CH 2 CH 0 2 \CH 2 0 4o°c (8°6) + I H 0 I R -NH.C------CH-NHCOR 2 >R NH +CO—CH 95°C (8076) 1 2 1 2 + CH SCH CONH 3 2 2 NHCOR2

Iodoacetamide at pH-. 2.8 was found to be the most effectivL, reagent (535b peptide cleavage). A marked improvement was found on using cyanogen bromide67 in an acidic medium as the alkylating agent, (Scheme 12).

SCHEME 12

SCH + S-CN Br 1 3 CH 2 BrCN CH2 CH2 CH2 R NHCOCH.NHCOR R NHCOCHNHCOR 1 2 1 2

CH, ,CH2 / \ 0 CH2 0 CH2 + H2O I I > R NH-C CHNHC OR 2 + CO ----CHNHCOR 1 > RNH2 2

Application of this highly selective method to ribonuclease, myoglobin and collagen, has been reported68. 2g.

A new approach to the labilisation of certain peptide 69 bonds consists in the conversion of cysteine and serine units into dehydroalanine residues. This can be achieved by the conversion of the cysteine or serine residue into a derivative which possesses a "good leaving" group e.g., a sulfonium or thiodinitrophenyl group in the case of cysteine, and tosyl or phosphoryl group in the case of serine. These derivatives can be converted to the corresponding dehydroalanine derivatives by P-elimination reaction under basic conditions. Dehydroalanine residues can be cleaved by hydrolysis" or by a prior oxidation with iodine, bromine or performic acid followed by hydrolysis71. Iodine or bromine oxidation of the dehydropeptide (I) (Scheme 13) gives the compound (II) which at pH > 7 hydrolyses to give (III), and subsequently, the amide (IV) and the iodopyritvoyl amide (V) (or hydroxypyruvoyl amide (Va) from the bromine oxidation).

On oxidation of dehydropeptides with performic acid a glycol is formed which at pH > 10 is cleaved to the amide (IV) and hydroxypyruvoyl amide, (Scheme 13). 29.

SCHEME 13

X I or Br 1 R CONH-C-CONHR 2 2 R CONH-C-CONHR 1 2 1 2 CH CH X 2 2

I II X= I or Br

OH 1 R CONH-C-CONHR 1 > 1 2 > R CONH2 + 1 CH X IV XCH C0C0NHR 2 2 2 III X = I or OH V

X = I or OH

The new terminal aminoacid at the point of cleavage is released in an additional step involving oxidative hydrolysis of the pyruvoyl derivative72 to glycolic acid, carbon dioxide and the new N-terminal amino group

H 0 2 HOCH C000NHR 2 > HOCH COOH + CO + H NR 2 2 2 2 2 OH 2

In a recent paper73 the thiol group of cysteine was dinitrophenylated at pH = 5-6 and treated with sodium methoxide to give dehydroalanine. In another modification the thiol group was methylated to a dimethyl sulfonium salt and subsequently treated with bicarbonate to give the dehydroalanine derivative which is not usually isolated. The dehydroalanine peptide was cleaved by treatment with performic acid and then with alkaline hydrogen peroxide. The method was tested on several model peptides and on reduced oxytocin with success. The factors favouring the formation of dehydroalanines rather than oxazolines from 0-tosylserine have been dis- cussed74 but there are no examples of the application of this method to proteins.

(d) Glutamyl

Attempts to utilise the 7-carboxyl group of a-glutamyl peptides for specific cleavage of a peptide at a glutamyl residue, have been described75'76. The scheme involves the conversion of a-glutamyl peptides into 1-acylpyrrolid-2-ones which are then hydrolysed by dilute alkali. The cyclisation step was carried out with thionyl chloride at -20° C. Formation of acylpyrrolidone together with the imide takes place and the hydrolysis gives a mixture of a-, and 'y-glutamyl peptides at well as fission products.

SCHEME 14

R CONHCHCONHR SOC1 R 1 2 'e 1 CO-N---4- - CHCONHR2 o > I 1 CH CH COOH - 20 CO /CH 2 2 NN 2 , CH d 2 acylpyrrolidone

31.

CO \ + R CONH - CH 'NR 1 2 CH CO // CH 2 imide

R CO - CHCONHR 1 2 CHCONHR hydrolysis 2 > R COOH + CO CH 1 COCH N / 2 \ //7 2 H 2 CH2

R CONH - NR 1 2 hydrolysis > a-glutamyl peptide + CH CO 2 y-glutamyl peptide CH 2

Although a certain amount of chain fission can be brought about by this reaction under comparatively mild conditions, the course of the reaction is controlled to a large extent by the particular sequence involved. The rearrangement to y-glutamyl isomers and the relatively poor yields obtained suggest that the method is unsuitable for the selective fission of large peptides. 3 2.

(e) N 0 acyl migration.

The acid catalysed N 0 acyl migration of an N-acyl- aminoalcohol77 is the basis of attempts at selective cleavage at a serine or threonine residua, In this method the seryl peptide is treated with anhydrous acid to form the 0-peptidyl bond, which is then hydrolysed after acetylation of the newly formed aminogroup, under conditions not affecting the amide bonds (Scheme 15).

SCHEME 15

OH CH2 anhydrous H R CO-OCH R CONHCHCONHR 1 1 2 1 2 < OH - H N-CHCONHR 3 2

:cAcetic Ri COOCHn OH - CH OH _L \ G / 2 > > anhydride CH3CONHCHCONHR2 CH CONH-CH-CONHR 3 2 + R1COOH

Concentrated sulfuric acid has been used78 for this rearrangement. The use of hydrogen chloride in anhydrous methanol or nitromethane has been studied79. Methanolic solutions of hydrogen chloride or boron trifluoride were also found unsuitable8o. The reaction has been tested in

33.

a two model peptides , and attempted on some proteins82. A great number of complications were encountered, with acyl migration occurring in low yield and non-specific hydrolysis of peptide bond causing difficulties. Recently the use of 83 anhydrous hydrogen fluoride has been recommended with yields for the N 0 migration near 902b, and specific cleavage of the peptide bond occurring. The method has been tested on the A-, and B-oxidised chains of insulin and a-MSH with good results84.

(f) Prolyl peptides.

Acyl proline bonds are the only tertiary amide linkages in peptides. The special feature of the tertiary amide linkage is its J.nability to form an anion by abstraction of a proton. This makes possible its reduction in strongly basic systems to e.g. the anion (B) (Scheme 16). hydrolysis of (II) would convert it to the aminoalcohol (III), which subsequently would decompose to give an aldehyde and a secondary amine. SCHEME 16 0 21 2 e R1 H 0 OH /R 1 R--C--N R --C--N 2 > R --C --N NH 'R2 3 \ R — CHO + HNN R2 34.

An attempted specific hydrolytic cleavage of acyl proline bonds employing sodium in liquid ammonia85 has been unsuccessful but Patchornik et al has reported that lithium in methylamine88 cleaves the prolyl bond in some model peptides. Kaufmann86 in preliminary experiments has employed sodium hydrazide in anhydrous hydrazine at 0° to split selectively the one prolyl bond of the B-chain of insulin and the use of lithium aluminium hydride for the selective 87 cleavage of prolyl peptides has also been reported .

(g) Miscellaneous methods.

Cleavage at the phenylalanyl peptide bond utilising Birch reduction of the aromatic ring and oxidation of the intermediate with NBS has been reported89 Intramolecular nucleophilic attack of a y-, or b-aminogroup on a peptide, can lead to lactam formation and scission of an amide bond. N-a-Naphthylamides of L-ornithyl-glycyl- glycine, and L-2,4-diaminobutyrlglycylglycine in absolute ethanol in the presence of triethylamine at 65°, were cleaved selectively at the ornithyl peptide bond90, (Scheme 17).

35.

SCHEME 17

j NH2 H N C ONHC H C ONHCH C ONH 2 2 2

NH 2

CO H NCH CONHCH CONH 2 2 2 NH

Electrolytic cleavage of 3-nitro-tyrosylpeptides has been studied91, (Scheme 18).

SCHEME 18

H - 2e -CH - CHNHC OR I!.: 2 1 - 2H

O 2 N ‘

CH, CHNHCOR 0—00 r—=-\s, / a i 1 / H 0 I 0=-- V 2 > 0 =7,/ I\ 1 + \ 0 ---- C:7-7-NHR \ CH —CHNHC OR 2 2 1 (Not isolated).

Treatment of a threonyl peptide with phosgene, 36. cyclisation and alkaline hydrolysis of the product, causes a cleavage in yield c.a. 70,692, (Scheme 19).

SCHEME 19

CH 3 1. COC1 CHOH 2 2. Cyclisation ZNHCHMeCONHCHCOOMe C0- 0 1 ZNHCHMeCO - N CHMe CH 1 COOMe

00 --- 0 ZNHCHMeCOCH + i1 I NH CHMe NC/ 1 C OOMe 37.

REFERENCES

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Theoretical.

The lack of satisfactory methods for selective cleavage at the C-peptide bond of an aspartyl residue in peptides and proteinsl led us to examine some reactions of model compounds where intramolecular interaction between the two functional groups might be observed. The participation of amide bonds in nucleophilic displacement reactions has been demonstrated2 and optimal intramolecular interaction is observed when the resulting iminolactones are five-membered. N-Cyclohexylsuccf..namic acid (1) bears some resemblance to appartyl residue and we decided to use it as a model.

r—COOH 6x 11 (1)

It has been shown that acylation of amides occurs primarily at the oxygen atom3 and, furthermore, intramolecular 0-acylation of an amide by the activated neighbouring carboxyl group has been postulated by Stammer4 who observed the formation of carbobenzoxy-T-cyano-a-L-aminobutyric acid from carbobenzoyx-L-asparagine and dicyclohexylcarbodiimide, and noted the possibility of an isoimide intermediate

L6.

CbzNH -COX Cbz.NH C ONH 2 ;V) X NH2

where X = 0 - C = NC6H11 NHC6Hil

Isoimides have been postulated also, as intermediates in the reaction of phthaloyl chloride with ammonia to form o-cyanobenzoic acid5 in the synthesis of P-cyano estes from 6 N-unsubstituted amidic acids and in the reaction of amides with carboxylic anhydrides to form nitriles or imides7. In the light of this evidence the oxygen atom of the amide group of the carboxyl activated N-substituted-succinamic acid (2) might be expected to displace the group X by a nucleophilic attack to give the isoimide (2)

COX r- _0 NH-R NR

(2) (2)

In the isoimide (3) two positions are available for nucleophilic attack. These are the carbonyl atom (a) and the imino-carbon atom (b). 147. 0 0 OH OH COOH \ OH '0 / 0 CONHR ---1( (a) (2) MI 147-R (4) 011 (b) CO.OH 0 -- -CONHR )r 0 (5) OH OH 0 - + H NR 2 (6) 0 (7) 1COON COOH Succinic acid

In the first case (a), the: product would be the N- substituted succinamic acid while in the second case (b) two possibilities existed. The intermediate (6) could collapse either to the N-substituted succinamic acid or to succinic anhydride (7) and the amine. The succinic anhydride subsequently would hydrolyse to succinic acid. The intermediate formation of isoimide before the hydrolysis to N-substituted succinamic acid would involve

48. hydrogen exchange at the amide group and if deuterium oxide was present in the hydrolysis mixture, incorporation of deuterium into the succinamic acid obtained would be observed. In order to test this speculation, the possibility of hydrogen exchange without intermediate formation of isoimide under the mild alkaline hydrolytic conditions used during this work was tested. The methyl ester of N-cyclohexylsuccinamic acid (8)_ was dissolved in a deuterium oxide- acetonitrile-sat.NaHCO solution for 2 hours at room tem- 3 perature and the recovered substance (9) was assayed for the presence of N-D, bond, by its characteristic absorption at 2650, 2550, cm-1. Unfortunately exchange did take place and consequently the deuterium exchange test for the intermediate formation of isoimide was not valid.

,,COOCH ,,COOCH ,,COOCH3 D 0 3 D 0 2 2 Lro DO I_ N-H N-D 1 CcoH 11 c6H11 C6H11

(8) (9)

The formation of succinic acid during the hydrolysis of the carboxyl activated succinamic acid (2), is indirect 49. evidence of the intermediate formation of the isoimide pro- vided that the N-substituted succinamic acid itself is stable under the hydrolytic conditions employed. N-Cyclohexyl- succinamic acid when treated with aqueous-acetonitrile-NaHCO 3 solution, was recovered quantitatively unchanged. At this stage the need for a good procedure for sepa- rating, and estimating the succinic and N-substituted succinamic acids arose. Thin layer chromatography as described in the experimental part, was used for qualitative detection of the hydrolysis products, and a partition column prepared from silica) gel impregnated with 10% aq. H2SO4, separated succinic acid from the succinamic acids in 67 and 96% recovery respectively. We first examined the reaction of (1) with acetic anhydride expecting the mixed anhydride (10) to be formed where X graap (see formula (2)) is the acetate group and

R = C6 H11 C0.000CH 3 Co ,-CO.OH NHC6Hil

--CO (10) NHCH 11 C0.000- (1) -CONH CO 06H11 NHC61111 (n) 50.

Treatment of (1) with acetic anhydride gave a solid compound which proved to be too unstable to recrystallise for analysis, however, its infrared spectrum,, 1815, Amax 1740, and 1070, cm-1, showed that a mixed (10) or a symmetrical anhydride (11) had been formed. Succinanilic acid under the same conditions gives a crystalline compound, VIT1ax. 1815, 1745, 1090, cm-1. It was hoped that (10) on treatment with basa would undergo intramolecular cyclization and hydrolysis as out- lined previously. Any succinic acid in the hydrolysis product would indicate that the aforementioned intermediate (2) was being formed. The following basic solutions were employed for the hydrolysis. (a) Aqueous-acetonitrile-NaHCO 3 (b) Aqueous-acetonitrile-Pyridine (c) Aqueous-acetonitrile-NaOH (d) Aqueous-acetonitrile. In all cases no succinic acid was detected. In an attempt to determine the structure of the anhydride, the product from the acetic anhydride treatment of (1), was aminolysed with cyclohexylamine. If (10) is the correct structure, amides corresponding to both acid fragments (12) and (13) (and the corresponding acids) should be obtained,

51. perhaps with preponderance of the acetylamino compound. However, the symmetrical anhydride would give equal yields of (1) and (12). I_--.0CONHC 6H 11 + CH COOH --00.000CH C6H H 3 11NH2 LNHC-CO 6 11 L-CONHC 6H11 (12) r-COOH (10) + CH CONHC H --CONHC6H 11 3 6 11 (13) (1)

In the event, only (1) and (12) were found and presumably the symmetrical anhydride is formed during the evaporation of the acetic anhydride by disruption of the expected equilibrium

r-r-CO.O.COCH -c0.0c0 3 + (CH c0)° 2 I r''CO CO 3 2 NHC6H11 NHC6H11 NHC 6H 11

C6 11 2

COOH r-CONHC6H11 H L-CONHC I--CONHC 6 11 6H11

(1) (12)

We next investigated the mixed anhydride of ( ) with 8 p-toluenesulfonic acid and in the hope of generating this intermediate, (1) was treated with tosyl chloride in an aqueous-NaHCO solution, but again no succinic acid was 3 found. When the same reaction was carried out in pyridine9 a tar was obtained which on TLC showed many acidic spots. Oxalyl chloride is a convenient reagent for the preparation of sensitive acid chlorides10 so the acid (1) was reacted with this reagent in an attempt to generate the acid chloride (14) and study its hydr017S1$. A pale yellow

solid was obtained the infrared spectrum of which, A,/max, 1830, 1790, 1740, 1700, cm-1, was not in agreement with that

COOH C1C0C0C1 I - > --CO NHC6H11 (1) expected for the chloride. The absorption at 1790 cm-1 could be assigned to the -00C1 group, but the absence of NH absorption and the presence of the three other carbonyl absorptions implied that the amide group was attacked by the oxalyl chloride. Before any attempt was made to determine the structure of the product, it was treated with the basic solutions as shown below:

) )

Basic solution Ratio of succinamic to succinic acid

(a)Aqueous-acetonitrile-NaHCO 8:1 3 (b) ll ?! -Pyridine 12:1 (c) ft It -triethylamine 32:1 (d) t; tt -NaOH 12:1 (e)Aqueous-acetonitrile 20:1 (f)Pyridine Dark brown amorphous material.

In all the above cases cyclohexylsuccinamic acid (1) and succinic acid were obtained in the ratios shown, plus some other unidentified products. The action of oxalyl chloride on succinanilic acid also

gave a product with VMax' 1820, 1750, and 1700, cm-1. On alkaline hydrolysis, starting material and succinic acid were again obtained. That the amide group is attacked in all of these compounds was confirmed by treating N-cyclohexyl- acetamide (12) with oxalyl chloride. The product obtained showedV , 1810, 1740, 1660, cm max -1, but showed no absorption. for NH. The reaction of oxalyl chloride with N-mono-substituted 11 acetamide derivatives has led to some controversy. Figee first reported that the products were pyrrolidinetriones (16). 34.

R2 R R 1 1-, CHCONHR C1C0C0C1 3 2 R (15) 3 (16)

0 R_, 0 fi

R2/ R 3 (IL)

Since the product from the reaction of oxalyi chloride and acetanilide was easily hydrolysed to acetic acid and oxanilic 12 acid, Stolle and Luther considered the product to be the oxazolidinedione (17) (R1 = R2 = H, R3 = Ph). Spielman studied the ethanolysis of the reaction product from oxalyl chloride and methyl phenaceturate and assigned the oxazolidinedione structure, while Skinner and Perkins14 favoured the pyrrolidinetrione mainly from infrared data. Sheehan 15 and Corey , felt that the absorptions at 1818, 1745, and 1681, cm-1, of a series of derivatives from the reaction of oxalyl chloride with phenylacetamides, were in better agreement with the oxazolidinedione structure. They prepared the isomeric pyrrolidinetriones which were found to absorb 55. at 1780 and 1720, cm-1, but not at 1680. More recent 16 evidence from ultraviolet and NME spectra , favours the oxazolidinedione structure. In the light of the previous work, we can therefore formulate the product from the treatment of (1) with oxalyl chloride, as (18). The infra red absorption is in good agreement with the reported values15 for an oxazolidinedione, 1830, 1740, 1700, cm-1, while the 1790 cm-1, peak can be assigned to a CO-Cl group.

(18)

The hydrolysis products also are in good agreement with this structure since initial hydrolysis should give (19) which can then be hydrolysed at (a) or (b), giving succinia acid and oxamic acid in one case, and cyclohexylsuccinamic and oxalic acid in the other.

56.

COOH 1 (ad N--C6Hil -(b)

COCO2H (18) (19)

Methanolysis of the product, gave a crystalline compound, NO -1 C14H 21 6' Vmax' 1810, 1740, cm , in agreement with the expected structure (20).

r-c000H (18) Me0H 3 --CO-N-C6H11 COCOOCH 3 (20)

After the failure to obtain a succinamyl chloride, we turned our attention to the formation of a thiolester (21). Thiolesters have been prepared by treatment of an acid chloride with a mercaptan17, an anhydride with a mercaptan18 19 and by the action of mercaptides on esters . We considered the possibility of forming the thiolester from the imide (22) by nucleophilic attack of the thiol anion at the carbonyl group. •

SC,H,„-n ---CO.SC H-n 1,-CO.SC H -n ONC6H11 . I 3 > 3 7 C ONC CONHc H 0 6H11 6 11 (22) (21)

Treatment of the N-cyclohexylsuccinimide (22) with n-propyl thiol in the presence of pyridine failed to open the imide ring. Unsuccessful also was refluxing of the imide with the thiol in ethanol. However, the imide ring was opened by sodium methoxide in methanol, to the methyl ester (8), but treatment of the methyl ester (8) with the thiol in the presence of triethylamine again failed to give the desired thiolester. Mixed anhydrides have also been used to prepare thiol esters20. The mixed anhydride of (1) with ethyl chloro- carbonate was formed easily and on treatment with n-propyl thiol, the thiolester (21) was obtained in good yield. As expected the symmetrical anhydride (11) also reacts with the thiol to form the thiolester. Mild alkaline treatment of the thiolester failed to give succinic acid and only starting material was recovered. It is known that alkyl halides react with thioethers 21 to form a sulfonium salt . We envisage the following reaction scheme:

•

R + ,=c0.sc H c 3H7 3 7 L-o>, RX NHC6Hil LNH C6H1,

However, it was not possible to detect any succinic acid, when the thiolester was treated with ethyl chloroacetate or p-bromophenacylbromide or when oxidized with alkaline hydrogen peroxide. Paul and Kende22 in a study of the dehydration of maleamic acid derivatives, observed that treatment of N-butylmaleisoimide (23) with 018-water containing potassium hydroxide-018 resulted in N-butyl-maleamic acid (24) labeled at the carboxyl group only and not at the amide oxygen. This proves that hydroxyl-0i8 attack takes place only at the carbonyl carbon atom and that no nucleophilic attack takes place at C = N to give the intermediate (25). This suggested that, even if the isoimide was being formed in our reactions, it woulctnot hydrolyse in the desired way so this approach was abandoned. 0 018 KO18H 0 > _J ll H 018 NBu 2 —NHBu H0 NHBu. 0 (21) (24) (La)

59.

An attempted serine, threonine, cleavage.

The alcoholic side chain of serine or threonine suitably modified could be used for selective cleavage of a seryl-peptide at the C-amide bond next to serine. Alcohols react easily with carbon disulfide22 in alkali hydroxide to give xanthates and the anion of serine xanthate is in a favourable position to interact with the carbonyl group of the amide bond (1,6-interaction) with a possible displacement of the amine as shown below.

_.-OH 0 S CS2 S R NH/\ OH S +H2NR 1 CONHR2 R1 NH 00 0 NHR2

In order to test this scheme we prepared N-cyclohexy1-11- hydroxybutyramide (26) as a model, by treating ketene dimer with cyclohexylamine and reducing the P-ketoamide with LiBH)T.

CH =C--- 0 2 CONHC H I + H2NC6H11 > CH3C0CH2 6 11 CH2-C === 0

OH LiBH > CH3CHCH2CONHC6 H11 Treatment of (26) with carbon disulfide in aqueous 2N-NaOH gave a crystalline compound, C10H17N0, corresponding to the ap-unsaturated cyclohexylamide (27). The n.m.r. spectrum firmly establishes the structure of the amide (27). The methyl group appears as a doublet (J = 6 cps) at 8.15-°

OH CS2

CONHC6 H NaOH 11 CONHC6H11

(26) (27)

each peak being further split as a doublet (J = 2 cps) due to allylic coupling. Ha is a doublet (J = 17 cps) showing further fine splitting, while Hb is a complex multiplet. The vinyl coupling constant is consistent with it being the trans-isomer.

Obviously, dehydration of the hydroxyamide under the

alkaline conditions took place. The same i-elimination occurred under a variety of alkaline conditions causing us to abandon this approach.

N-Terminal analysis.

An oxygen or nitrogen atom when attached to oxygen or nitrogen is known to be highly nucleophilic and accordingly 61. we envisaged the following scheme for the selective splitting of an N-terminal aminoacid in a peptide.

R X R NHCH CONHR 1 --, --- 1-'-- c / 2 2 C H2NCH2CONHR2 II > II NStep...... „.... 1 N

(28)

R NH 1 '------CH Step 2 > c 2 II 1 2 + H NR N ` 2 2 -----,Y .CO

where Y = 0 or N.

In the above scheme the second step could take place either spontaneously with concurrent removal of the N-terminal aminoacid as a cyclic derivative or might be induced by the presence of a catalyst. In the first case the reagent (28) could be called a chemical peptidase since both the enzyme and the reagent would bring about the same overall change. The requirements for the reagent are rather strict and as follows: (i) The group X must be readily replaced by the terminal aminogroup.

6L.

(ii) The group Y must not replace X readily, and (iii) The group Y must be nucleophilic enough to attack the carbonyl of the amide group with displacement of the amine. 2-Fluoropyridine-N-oxide (29) prepared in this laboratory by Dr. Tortorella, from 2-fluoropyridine and trifluoroperacetic acid, seemed to us a potential reagent to fulfil all the above requirements. Glycine cyclohexylamide was used as a model peptide in all the following attempts and was synthesised as described in the second part of this thesis. The N-oxide (29) reacted readily with the glycine cyclohexylamide in aqueous sodium bicarbonate solution, and the solid obtained, C131119N302, showed infrared absorption at 3400, 3230, 3060, 1680, 1630, 1570, 1540, cm-1. Thus spontaneous cyclisation did not occur under the mild alkaline conditions of the reaction.

4NHCH CONHC H 2 6 11 ---112NCH2CONHC6Hil 0 -...,+„,;.pk„, (30) N- H NCH C00H F 2 2 ‘,/1 HC OOH 0 reflux ---1 .,,, ,, (29) I +,. ''''.-N' --,..NHCH CO H 2 2 (L) 0- 63.

Treatment of the product (30), with trifluoroacetic

acid or acetic acid did not cleave the amide bond, however refluxing with 985/o formic acid resulted in the removal of the cyclohexylamine residue and formation of the glycine-N- oxide adduct, (21), which was synthesised from (29) and glycine in aqueous sodium bicarbonate solution. The cleavage of the amide bond presumably involves intramolecular attack of the oxide oxygen atom at the carbonyl with concurrent displacement of the amine. This reaction has been further investigated by Dr. Tortorella24 and we turned to other reagents.

11 4. • E N" NH 1 I 0 O<-, CH 0 CH //CH2 / 2 2 CO H2 0 CO C OOH NHC6H11

Attempted preparation of the 2-fluoro-quinoline-N-oxide by an analogous oxidation of 2-fluoroquinoline with trifluoroperacetic acid resulted in the formation of hydroxyquinolone. Presumably the 2-fluoroquinoline-N-oxide was formed but hydrolysed to the observed product during work-up procedure. In search of other reagents fulfilling the requirements 64.

mentioned for selective splitting of the N-terminal aminoacid in a peptide, we examined the condensation of the aryl- hydroximic chlorides (32) and (22) with cyclohexylamine or glycine cyclohexylamide. Cl NHR RNH2 C---NOH > X— C ===NOH

(32) X = H

(33) X = NO2 (34) X = NO2, R = C6H11 (35) X ,H, R = CH2CONHC6H1 (36) x 1/02.. R=cii2cONHC6H:

CH ,/ 2,...,„ NH CO I ,s,„ c ,,0 ..N",,N, ,,,,- N (37) X . H (38) X = NO 2

p-Nitro-benzhydroximic chloride (33) prepared by chlorination of p-nitrobenzaldoxime25 was reacted with cyclohexylamine to give a pale yellow crystalline product,

C H N 13 17 303' and an acid insoluble solid. 65.

When (13) was reacted with glycine cyclohexylamide the pale yellow solid, C 15H201\1404, obtained, showed infrared absorption (3300, 3100 weak, 1650, 1570, 1530, cm-1)\ supporting the structure (36). An isomer, melting 20° higher was obtained on treatment of an alkaline solution of (36) with carbon dioxide. In an attempt to prepare (38) the chloride (33) was treated with sodium glycinate but the result was an un- attractive tar. From this evidence it was apparent that although we had not succeeded in cyclising (36) to (3.g.) in one step, at least we had obtained (L) and consequently we investigated conditions for cleavage, however treatment with 98/6 formic acid at room temperature or under reflux resulted in a pink mixture of many compounds (as shown on T.L.C.). The fact that the presence of nttrogroup in a compound causes problems of purification due to lower solubility in solvents led us to examine the reaction of the unsubstituted hydroximic chloride (32) with glycine cyclohexylamide. The product obtained consisted of two compounds, one insoluble in water (25% yield) (V max' 3350, 1650, 1550, cm-1) and another one soluble in water, insoluble in methylene chloride or ether (75% yield) ( max, 3350, 2750, 2650, 1670, 1600, 1560, 1530, cm-1). 66.

Suspecting that the presence of hydrochloric acid formed during the reaction caused complications during the isolation of the products, we considered the use of benzonitrile-N-oxide as a reagent which would give the desired intermediate (35) which in the most optimistic case would cyclise spontaneously and split off the cyclohexylamine. Benzonitrile-N-oxide although unstable under alkaline conditions and in concentrated solutions, reacts with amines26 to form amidoximes when prepared carefully and in dilute solution. In our case benzonitrile-N-oxide in ether reacted readily with cyclohexylamine to give the crystalline amidoxime max' 3370, 3200 broad, 1630, 1580, cm (22)' ( -1). Benzonitrile-N-oxide and glycine cyclohexylamide yielded a crystalline solid, C15H21N302, in 507'o yield \t'Imax'337° ' 3200 broad, 1640, 1540, cm-1). High resolution mass spectrometry of this compound shows the molecular ion at 275 11)/e and fragmentation supporting the structure (fl).

67,

Assignment

275 Molecular ion N II 258 c6 H5 C-NH0 114 I •

242 C6H5C=NCH2CONC6H11 NOH 176 C6H5C-NHCH.C.0 HN-- Co 161 CAIo 5 C-NH-CH 2 NHCH2 149 C6H5C=NOH

119 c6H5CNO

104 C6H5CENH

98 C6H11NH

06.

e6H11 c6H11NH2 r 0 > Ph— C =NOH

(39) H NCH C0NHC 2 2 6 H11 zCH2 NH CO NHCH2CONHC6 H11 1/ > 0 + HNCE-H Ph --C=NOH PH 2 o 11 N

Refluxing of the compound (35) in 98% formic acid, gave a pink mixture of many compounds but treatment with formic acid or trifluoroacetic acid at room temperature gave mainly starting material and some pigments. Treatment with dilute alkali resulted in a water soluble solid in small yield (Vmax, 2050, cm-1) while methanolic sodium methoxide gave starting material and some pink pigment.

From the above results it appears that although the first step in our scheme proceeds readily under very mild conditions the second step, that is the cyclisation, occurred neither spontaneously nor after treatment with acids or bases, and consequently we turned our attention to reagents of formula (40) hoping that nucleophilic attack of the nitrogen would lead to fission of the glycine cyclohexylamide.

R1.„ NHCH CONHC H C CONHC H11> C ----- 2 6 11 H2NCH2 6 II I I NN,------NHR2 NHR2

(14-o)

CH2 1 + H NC H CO 2 6 11

Ethyl tosyl-hydrazimido-carboxylate27 (41) can be prepared readily from tosylhydrazine and ethyl orthoformate and, of course, this compound has the advantage that condensation with the aminogroup givisethanol as byproduct. When (41) was treated with cyclohexylamine the white crystalline solid obtained analysed correctly for C14H21N3S02 and since it was apparent that amines displace the ethoxide group we reacted (41) with glycine cyclohexylamide. How- ever, the crystalline solid obtained (3400, 1660, 1630, 1560, cm-1), although it had a sharp melting point and on TLC appeared to be homogeneous, did not analyse correctly for the displacement product (42). From the analytical figures it was apparent that chloroform (the crystallisation solvent) was clinging tenaciously. 70.

High resolution mass spectrometry showed the molecular ion at 352 m/e and fragmentation supporting the structure (42). N.m.r. indicated the presence of the cyclohexyl and tolyl rings, and the methylene group, while infrared spectrum showed strong sulfonamine absorption (1180 cm-1).

0 NHNH CH3 0 NHN=0H.OEt 2 2 HC(OEt)3 (41)

V H2NCH2C0NHC6 H 11 \,) c1-13 - —so2NHN=CHNHC6Hii CH // 0 NHN=CHNHCH2CONHC6 11 (43) (2)

m/e Assignments

352 Molecular ion 198 H2NN=CHNHCH2CONHC6 H 11 197 C7H7S02NHNL:CH 186 2 C7H7SO NHNH2 C H 169 7 7SO2N 166 CNCH- 2CONHC 6H 11 155 NHCH2 CONHC 6 H11' C7H7S02 139 c71-17s0 71.

Treatment of (42) with dilute sodium hydroxide at room temperature leaves this product unchanged while hot alkali cleaves it to many components. Acidification of an alkaline or aqueous solution of (42) with dilute hydrochloric acid gives a crystalline solid analysing as the hydrochloride of

(42). Its mass spectrum is identical to that of (42) and n.m.r. shows that the p-tolyl and cyclohexyl rings and the methylene are present while the sulfonamide group is still present according to infrared absorption. However two facts make it unlikely that it is a simple hydrochloride derivative, firstly it has 'max' 1710 cm-1, (as opposed to 1660 cm-1 in (42)) and, more significantly, it can be recovered unchanged after dissolution in alkali and reacidi- fication with sulfuric acid. For the moment this compound has not been investigated further, but it appears that a similar type of reaction occurs in sulfuric acid. In another attempt to cleave (42) it was reduced with sodium in liquid ammonia in the hope of generating the parent hydrazone which would then cyclise. Again an anomalous crystalline product was isolated. Satisfactory analytical figures were not obtained and since n.m.r. showed the cyclohexyl ring still present this approach was abandoned.

Reaction of p-nitro-phenylhydrazine with ethyl orthoformate gave a brown solid, C9H11N303 which showed the

72. characteristic 0-ethyl n.m.r. pattern and was thus assigned structure (44), the other protons appearing at 2.9 -u (Ar-H), H 2.7 /7(Ar-H), and 3.117 ( c ==_N). Reaction of this compound with cyclohexylamine or glycine cyclohexylamide gave the same product which appears to contain two p-nitro-phenylhydrazine residues (highest m/e = 314) but is not generated by the action of triethylamine or p-nitro-phenylhydrazine on (44).

HC(OEt) 3> NHNH2 02N f NHN = CH.OEt (44)

In a related reaction the bromide (.?L) was reacted with glycine cyclohexylamide to give (46) shown to have the correct composition by mass spectrometry. The cracking pattern supported this structure, showing m/e = 297 (loss of C6H11NH2) and m/e = 243 (loss of 02NC6H4NH2 ).

Br NHCH2CONHC6 H11 PhC=NNHC H PhC=NNHC 6 4NO2-p > 6H4NO2-p (45) (46)

When (46) was treated with methanolic sodium hydroxide a pale yellow compound was obtained the composition of which 73.

was shown to be,C21H21N 503 by mass spectrometry. This apparent loss of foUihydrogens we are unable to explain at present. Obviously, some deep-seated rearrangement has occurred leading to a complete change in the chromophore. In conclusion we can say that this survey of groups for N-terminal analysis has so far yielded only one of much promise (2-fluoropyridine-N-oxide) and that in the others some anomalous reactions have been uncovered, the exact course of these reactions remains to be determined. 74.

Experimental

Melting points were determined on a Mfler block and are uncorrected. Infrared spectra were determined on a Unicam S.P. 200 spectrometer.

Nuclear magnetic resonance (n.m.r.) spectra were measured on a Varian A 60 instrument. Mass spectra were determined on a MS 9 instrument. Analyses were carried out by the staff of the micro- analytical laboratory (Imperial College). Organic solutions were dried using anhydrous magnesium or Soclium sulfate. 75.

N-Substituted succinamic acids28

The amidic acids were prepared by reaction of equimolecular amounts of the anhydride and of the amine in chloroform solution under reflux. The crude amid:racids were dissolved in dilute sodium bicarbonate and the resulting solutions were filtered and acidified. The amidic acids were obtained in 70-80% yields.

Thin layer chromatograms.

The following developing solvent-mixture was found to separate succinic acid from the N-substituted succinamic acids on a silica gel plate: Ethanol (absolute)-aqueous NH (d = 0.880)-water (80/5/15, /v).\ 3 The spraying solution was: Methyl red (100 mg) and bromothymol blue (100 mg), dissolved in ethanol (200 ml) and formalin (50 ml), and adjusted at pH = 5.2 with 0.1N--NaOH .

Partition chromatography.

35 gs. silica gel, B.D.H, was ground with 12 ml, 10,O sulfuric acid and then small portions of silica gel were added until it was a free flowing powder. This powder was slurried in 100 ml chloroform and poured into the column. The mixture of acids to be separated, dissolved in the smallest amount of chloroform, was introduced into the column, and the column was eluted with chloroform containing 2%, 4b, and 10°6, n-propanol. Aliquots of 25 ml were collected and titrated with 0.1N-NaOH with phenolphthalein as indicator. The quantity of acid present, was calculated from: mis 0-1N-NaOH x 5.9 = Succinic acid in mg., and mis 0-1N-NaOH x 19.9 = N-cyclohexylsuccinamic acid in mg.

Methyl-cyclohexylsuccinamate.

The methyl ester of cyclohexylsuccinamic acid was prepared by treating a methanolic solution of the succinamic acid with diazomethane. Crystallised from petrol (60-80°), m.p. = 101 ° 1.) max (Nujol) 3320, 1730, 1630, 1560, cm-1, (CHC13) 3450, 1730, 1660, 1530, cm-1.

Action of sodium bicarbonate on the methyl-cyclohexylsuccinamate.

Methyl cyclohexylsuccinamate (50 mg) was dissolved in an excess of aqueous-acetonitrile-sat.NaHCO solution, and 3 left to stand at room temperature for 1 hour. The mixture, thela, was acidified and extracted with chloroform. Starting material (38 mg) was obtained from the chloroform extract. j7.

Deuteration of methyl-cyclohexylsuccinamate.

Methyl cyclohexylsuccinamate (50 mg) was dissolved in an excess of D 0-acetonitrile-NaHCO (2 m1-2m1-50 mg) 2 3 siplution and left to stand at room temperature for 1 hour.

The solution was extracted with dry ether and the ether evaporated to dryness. Infrared spectra of the residue were recorded: max (CH01 ) 2650, 2550, cm-1.

Action of acetic anhydride on the N-cyclohexylsuccinamic acid. N-Cyclohexylsuccinamic acid (200 mg) was heated with an excess of acetic anhydride on a water bath for 1 hour. The excess of acetic anhydride was evapd. in vacuo, to give a pale yellow solid (165 mg) which crystallised from chloroform-petrol (60-80°) to a white solid, mp = 123-126°. (CHC1 ) 3450, 1815, 1740, 1660, 1530, 1070, cm-1. max 3 It is stable at room temperature, but on attempted recrystallisation from chloroform-petrol (60-80°) it decomposed. Attempted sublimation also failed. The above solid was also obtained by careful heating of a suspension of the succinamic acid in acetic anhydride until a clear solution resulted (about 2 min.). 73.

(a) Action of bases

Freshly prepared solid from N-cyclohexylsuccinamic acid and acetic anhydride, (100 mg) was dissolved in an

aqueous-acetonitrile-NaHCO3 (10 m1-10m1-NaHCO solid enough . 3 to saturate the solution) and after 30 min., was acidified with dil. hydrochloric acid. The acid solution was extracted with chloroform continuously overnight. From the chloroform extract only succinamic acid was obtained (60 mg). Treatment as above with (a) aqueous-acetonitrile- pyridine (2-2-1 v/v), (b) aqueous-acetonitrile-NaOH (10m1- 10ml-solid NaOH to saturate the solution), and (c) aqueous- acetonitrile, yielded in all cases cyclohexylauccinamic acid. (b) Action of cyclohexylamine Freshly prepared solid from N-cyclohexylsuccinamic acid and acetic anhydride, (200 mg) was dissolved in acetonitrile (25 ml) and treated with an excess of cyclohexylamine (1 ml). The mixture was evaporated to dryness in vacuo and the residue taken up with CHC13. The chloroform solution was extracted with aqueous-sat.NaHCO solution and 3 the organic layer separated, washed, dried and evaporated to dryness to give a white solid (112 mg) mp = 240-241°, with an infrared spectrum identical with that of 29 dicyclohexylsuccinamide . The bicarbonate layer on working up in the usual way, gave only N-cyclohexylsuccinamic acid (60 mg). 19.

Action of acetic anhydride on N-succinanilic acid.

N-Succinanilic acid (500 mg) was dissolved in acetic

anhydride (5 ml.) by gentle heating on a steam bath. The excess anhydride was distilled off in vacuo, to give a white solid (620 mg). Crystallised from chloroform-petrol (60-80°) mp = 100-105°. (CHCC1 ) 3450, 1815, 1745, max 3 1685, 1600, 1540, 1090, cm-1.

Action of oxalyl chloride on N-cyclohexylsuccinamic acids

The succinamic acid (200 mg) was treated with an excess of oxalyl chloride (1.5 ml) at 0°C for 90 mins. The excess oxalyl chloride was removed at 0° in vacuo to

give a pale yellow solid (258 mg), mp = 92-94°, V max (CHC1 1830, 1790, 1740, 1700, cm-1.

Hydrolysis of the oxalyl chloride-treatment product of

N-cyclohexylsuccinamic acid.

The pale yellow solid obtained from the reaction of N-cyelohexylsuccinamic acid with oxalyl chloride was hydrolysed with: (a) Aqueous-acetonitrile-NaHCO (1-1, with NaHCO 3 3 to saturation) (b) Aqueous-acetonitrile-pyridine (2-2-1, v/v) 80

Aqueous-acetonitrile-triethylamine (2-2-1, v) Aqueous-acetonitrile-NaOH (1-1 with solid NaOH to saturation) Aqueous-acetonitrile (1-1) Pyridine

Time of treatment at room temperature was 90 mins., after which, the hydrolysis mixture was acidified with dilute hydrochloric acid and extracted continuously overnight with chloroform. The chloroform extract was spotted on TLC on silica gel, and the succinic and succinamic acid separated by a partition column. The following results were obtained:

Ratio of succinanic Experiment TLC to succinic acid

(a) Two spots corresponding to succinamie and succinic acid plus one spot in the beginning not identified. 8 :1

(b) As in (a) 12:1 (c) As in (a) 32:1 (d) As in (a) 12:1 (e) As in (a) plus two yellow spots not identified. 20:1 (f) Dark brown acidic mixture. Many spots, one of which corresponds to succinic acid. 0110 .1111 •••• 81.

Action of methanol.

N-Cyclohexylsuccinamic acid (100 mg) was treated as usual with an excess of oxalyl chloride and the product dissolved in methanol and left to stand at room temperature overnight. A crystalline compound (30 mg) separated, m.p = 144-145°, (Nujol) 1810, 1740, cm \limax -1. (Found: C, 55,91, H, 6.95, N, 4.70, C14H21NO6 requires, C, 56.20, H, 7.00, N, 4.66, /o).

Action of oxalyl chloride on succinanilic acid.

Succinanilic acid (100 mg) was treated with an excess of oxalyl chloride at 0° for 90 min. The excess oxalyl chloride was evaporated in vacuo to give a solid (120 mg). 1max (CHC13) 1820, 1750 broad, 1700, 1600, cm-1. This solid was dissolved in an aqueous-acetonitrile-NaHCO (saturated) solution and left to stand at room temperature for 1 hour, then acidified with dilute hydrochloric acid and extracted continuously with chloroform. The chloroform extract ';as concentrated and spotted on TLC on silica gel where separated in five spots one of which was identified as succinnilic acid and another one as succinic acid. 82.

N-Cyclohexylsuccinimide.

N-Cyclohexylsuccinamic acid (150 mg) was refluxed in acetyl chloride (5 ml) for 1 hour and the excess chloride evaporated off in vacuo, to give a residue which on cooling crystallised to a white solid (90 mg). Recrystallised from water, mp = 47-48°, V max (clici ) 1760, 1690, (Nujol) 1760 3 weak, 1710, cm . Treatment of this imide with n-propyl thiol in pyridine overnight at room temperature gave starting material (imide). Starting material (imide) was obtained also when N-cyclohexylsuccinimide was refluxed with n-propylthiol in ethanol for 2 hours.

Action of sodium methoxide on N-cyclohexylsuccinimide.

N-Cyclohexylsuccinimide (60 mg) was treated with a methanolic solution of sodium methoxide (10 ml methanol and 100 mg metallic sodium) at room temperature for 3 hours. On acidification, evaporation to dryness and extraction of the residue with boiling petrol (60_800), a white solid was obtained (45 mg) (from the petrol extract) mp = 98-101°, with an infrared spectrum identical with that of methyl-N-cyclohexylsuccinamate. 83.

Preparation of n-pry-thiol-ester of N-cyclohexyl-succinami acid.

N-Cyclohexylsuccinamic acid (400 mg) was treated with one equivalent of ethyl-chloroformate (216 mg) in the presence of one equivalent of triethylamine (202 mg) in acetonitrile (10 ml). The resulting mixed anhydride was treated with n-propyl thiol (160 mg) and triethylamine (210 mg) and the mixture was left to stand for 1 hour at room temperature. The separated triethylamine salt was filtered off, and the filtrate evaporated to dryness. The residue was extracted with boiling petrol (60-80°), and the petrol extract on cooling gave a crystalline solid (300 mg) mp = 81-82°, 'Vmax (CHC13) 3450, 1660, 1530, cm-1. (Found: C, 60.56, H, 9.10, N, 5.77, c13H23Ns02 requires C, 60.70, H, 8.90, N, 5.44, °M. Treatment of the thiolester with quaternising agents ')c. ethyl bromoacetate, and p-bromophenacyl bromide in an aqueous-acetonitrile-NaHCO solution, failed to give succinic 3 acid. Succinic acid was also not detected either when the thiolester was treated with an aqueous-acetonitrile-NaHCO3 solution, or with aqueous-acetonitrile-NaHCO3-H202 30/b. 84.

Action of n-propyl thiol on the acetic anhydride-treatment product of the N-cyclohexylsuccinamic acid.

The white solid (100 mg) obtained from the treatment o the N-cyclohexylsuccinamic acid with acetic anhydride, was dissolved in acetonitrile (5 ml) and treated with n-propyl thiol (100 mg) in the presence of triethylamine (120 mg). The mixture was evaporated to dryness and the residue extracted with boiling petrol (60-80°). From the petrol extract a white solid was obtained (45 mg) mp = 80-82°, infrared spectrum identical with that of the thiolester.

N-Cyclohexyl-P-ketobutyramide.

Cyclohexylamine (5.75 ml) in 1,2-dichioroethane (45 ml) was treated with ketene dimer (b.p. = 35-40°/20 mm Hg) (3.85 ml) under stirring and cooling and the mixture was left to stand at room temperature for 15 hours. Evaporat- to dryness gave an oil which solidified when treated with petrol. Recrystallised from cyclohexane, mp = 74-75°. (Yield 90/6), V max (Nujol) 3300, 1705, 1650, 1625, 1570, cm-1.

N-Cyclohexyl-Prhydroxybutyramide.

N-Cyclohexyl-P-ketobutyramide (2.9 g) in methanol was 85.

treated with a solution of sodium borohydride 95/b (1 g) over a period of 15 min. and the mixture was stirred for 20 min. more. The methanol was evaporated in vacuo and the residue mixed with 2N-hydrochloric acid and heated on a steam-bath for 20 min. The acid solution was then extracted with ether continuously for 72 hours. On working up the ether extract in the usual way, a white solid (2.5 g) was obtained. It was crystallised from acetone-hexane, mp = 85-86°.\/max (Nujol) 3300, 1633, 1550, cm-1. (Found: C, 65.07, H, 10.59, N, 7.31, C10Hl9NO2 rcquires C, 64.83, H, 10.34, N, 7.56, 5b).

Action of carbon disulfide on N-cyclohexyl-P-hydroxy-butyraffde

A mixture of amide (520 mg) and carbon disulfide (4 ml) in 2N-aqueous sodium hydroxide (4 ml) was stirred at room temperature for 16 hours. The excess carbon disulfide was removed in vacuo at 50° C and on cooling a crystalline compound separated (100 mg) which was recrystallised from acetone-hexane, mp = 136-137°, Vmax (Nujol) 3300, 1662, 1620, 1550, cm-1. -C = 8.2-8.1 (doublet-doublet, 3H), 4.15 (doublet, 1H, a-vinylic proton), 3.15 (multiplet, 1 H, P-vinylic proton). (Found: C, 71.81, H, 10.25, N, 8.38, C10Hl7NO requires C, 71.25, H, 10.01, N, 7.83, /b). From the filtrate some more product (20 mg) plus starting material was recovered. 86.

2-Fluoropyridine-N-oxide.

2-Fluoropyridine-N-oxide was prepared from 2-fluoropyridine and trifluoroperacetic acid as it is described in the second part of this thesis.

Reaction of 2-fluoropyridine-N-oxide with glycine-cyclohexylamide.

Glycine cyclohexylamide (156 mg) and sodium bicarbonate (336 mg) in water (1.5 ml) was treated with 2-fluoropyridine- N-oxide (113 mg) and the mixture was stirred at room temperature for 20 hours. The reaction mixture was then extracted continuously with methylene dichloride and the crystalline solid obtained was recrystallised from acetone- ether, mp = 158°,1/ max (Nujol) 3400, 3230, 3060, 1680, 1630, 1570, 1540, cm-1.

(Found: C, 62.58, H, 7.53, N, 16.68, C10H12N203 requires C, 62.62, H, 7.68, N, 16.84, 5b).

Hydrolysis of N-(2-pyridyl-N-oxylycine cyclohexylamide.

The compound (1 millimole) was refluxed in 98/b formic acid (4 ml) for times varying from 1 to 3 hours. At the end of the reaction the solvent was evaporated in vacuo. The pyridyl-glycine-N-oxide obtained (quantitative yield) 87. was identified by TLC on silica gel (methyleydichloride- ether-n-butanol-formic acid, 3/3/2/2).

Reaction of 2-fluoro-pyridine-N-oxide with glycine.

A solution of glycine (401 mg) in aqueous sodium bicarbonate (1123 mg) (5 ml water) was treated with 2-fluoropyridine-N-oxide (604 mg) in water (2.3 ml) and the mixture was stirred for 18 hours at room temperature after which it was acidified carefully with formic acid. The separated solid (371 mg), quite soluble in water, was recrystallised from water, mp = 230° (dec.).

(Found: C, 50.02, H, 4.76, N, 16.53, C7H8N203 requires c, 50.00, H, 4.80, N, 16.66, 5b).

Chlorination of p-nitro-benzaldoxime25

Chlorine was passed into a suspension of p-nitrobenzaldoxime (5 g) in chloroform (40 ml) cooled to 0°, for 2 hours. The solvent was evaporated off at room temperature and the residue recrystallised from benzene as pale yellow prisms, mp = 125-126.5° (3.22 g).

Chlorination of benzaldoxime25

Benzaldoxime (18 g) in chloroform (80 ml) was treated with a current of chlorine for 6 hours. On working up as 88. above benzhydroximic chloride, mp = 48-50° (petrol) V max

(CHOI ) 3500, 3250, cm -, was obtained. 3

Reaction of p-nitro-hydroximic chloride with cyclohexylamine,

The hydroximic chloride (367 mg) in ether (20 ml) was mixed with cyclohexylamine (3 equivalents, 550 mg) in ether (10 ml) and the mixture was stirred overnight at room temperature. The thick pale yellow solid kOparated was extracted with dilute hydrochloric acid. The reeadue, a white solid (200 mg), crystallised fromiolaloroform-petrol, mp = 115-116° (Nujol) 3500, 3200, 2800, 1670, 1610, ,Vmax 1580, 1535, 1165, and 870, cm-1 The hydrochloric acid extract was made basic with aqueous sodium hydroxide and solid carbon dioxide was added in excess. The pale yellow solid separated (181 mg) was dissolved in the minimum quantity of 95/O ethanol. A brown precipitate which separated on cooling of the ethanolic solution, after two days, was filtered off and to the filtrate water was added to give pale-yellow crystalline solid, mp = 133-135°, V (Nujol) 3400, 3200 broad, 1635, 1600, 1530, cm max -1. (Found: C, 59.75, H, 6.52, C13H17N303 requires C, 59.32, H, 6.46, 5b). 89.

Reaction of p-nitrobenzhydroximic chloride with glycine cyclohexylamide.

The hydroximic chloride (186 mg) in ether (15 ml) was treated with glycine cyclohexylamide (291 mg) and the mixture was stirred at room temperature overnight. The pale yellow solid which separated was filtered (394 mg) and washed with water and crystallised from ethyl acetate, mp = 166-168°, V max (Nujol) 3300, 3100 weak, 1650, 1600, -1 1570, 1530, cm . (Found: C, 56.76, H, 5.42, N, 17.16, C15H20N04 requires C, 56.25, H, 6.25, N, 17.50, cb). A portion of this solid (48 mg) was dissolved in IN- sodium hydroxide and treated with solid carbon dioxide. The separated solid was filtered and recrystallised from ethyl acetate, mp = 185-187°, V max (Nujol) 3320, 3100, 1650, 1600, 1570, 1530, cm-1.

Reaction of benzhydroximic chloride with glycine cyclohexylamide.

Benzhydroximic chloride (118 mg) in ether (20 ml) was treated with two equivalents of glycine cyclohexylamide (236 mg) at room temperature and under stirring. After a few minutes a white solid separated. The mixture was 90. stirred overnight and then evaporated to dryness in vacuo. A portion of the residue (207 mg) was suspended in water, shaken well, and the insoluble solid (118 mg),' max (Nujol) w 3350, 1650, 1550, cm-1, was filtered off. From the aqueous filtrate after evaporation a white solid (165 mg) was obtained which was washed with methylene dichloride. The residue showed Vmax (Nujol) -,350, 2750, 2650, 1670, 1600, 1560, 1530, cm-1.

Reaction of benzonitrile-N-oxide with cyclohexylamine.

To a dry solution of benzonitrile-N-oxide (from 751 mg benzhydroximic chloride) in ether (60 ml), a solution of cyclohexylamine (485 mg) in ether (20 ml) was added and the mixture was left to stand overnight. On evaporation of the ether and washing the residue with petrol a crystalline white solid (238 mg) was obtained. %/ max (Nujol) 3350, 3200, 1630, 1570, cm-1.

Reaction of benzonitrile-N-oxide with glycine cyclohexylamide.

Benzhydroximic chloride (241 mg) was dissolved in ether (60 ml) and treated dropwise and under vigorous stirring with triethylamine (175 mg) in ether (15 ml). The separated triethylamine salt was filtered off and the filtrate was washed with water and dried. The ethereal solution, 91. of the N-oxide was treated with glycine cyclohexylamide and the mixture was stirred for 24 hours at room temperature after which the ether was evaporated and the residue (180 mg) crystallised by standing in the refrigerator. Recrystallised from ethyl acetate-petrol (60-80°) it had mp = 140-143°

max (Nujol) 3370, 3200 broad, 1640, 1540, cm-1. (Found: C, 65.36, H, 7.79, N, 15.21, C15H21N 02 requires C, 65.45, H, 7.63, N, 15.26, °M. (a) Acid hydrolysis. When this compound was left to stand for 6 days at room temperature with 98% formic acid or trifluoroacetic acid, it gave mainly starting material plus a pink pigment while reflux in 98°A formic acid resulted in a complex mixture of coloured products, as it appears on TLC on silica gel (chloroform-ethanol, 5:1). (b) Basic hydrolysis. (i) The above compound (84 mg) was dissolved in 0.5 N-sodium hydroxide in water-methanol (3:2) and left to stand for 3 days at room temperature. The alkaline solution was acidified with dilute hydrochloric acid and extracted with chloroform. The aqueous layer was evaporated to dryness and the residue extracted with chloroform f-om which on evaporation a semisolid residue was obtained showing)1 (Nujol) 2050 cm-1. max 92.

(ii) Treatment with sodium methoxide in methanol gave starting material plus a pink pigment, as shown on TLC on silica gel (chloroform).

Preparation of ethyl tosyl-hydrazimideo-carboxylate.27

Tosyl hydrazine (3.50 g) was mixed with ethyl orthoformate (3.50 g) and the mixture stirred at room temperature for 45 min. The white paste obtained was diluted with absolute ethanol and the separated solid collected and washed with ether, and recrystallised from chloroform- mp -1 112-118°, ),max (Nujol) 3250, 1660, 1600, cm (reported mp = 118°).

Reaction of ethyl-tosyl-hydrazimido-carboxylate with cyclohexylamine.

Ethyl hydrazimido carboxylate (199 mg) in ether (15 ml) was mixed with one equivalent of cyclohexylamine (83 mg) and the mixture was refluxed overnight. The insoluble solid in the reaction mixture was filtered and recrystallised twice from chloroform-petrol (40-600), mp = 149-151°,

V max (Nujol) 3400, 1660, 1600, cm-1. (Found: C, 56.49, H, 6.95, N, 14.32, 014H21N3S02 requires C, 56.90, H, 7.00, N, 14.20, %). 93.

Reaction of ethyl tosjl-hydrazimido-carboxylate with glycine cyclohexylamide.

The hydrazimido-compound (187 mg) in acetonitrile was treated with one equivalent of glycine cyclohexylamide (120 mg) and the mixture was stirred at room temperature for 18 hours. Evaporation of the solvent in vacuo gave a viscous oil which crystallised from chloroform-petrel ° = 3400, 1660, 1630, (40-60 ), mp 85-87°' V max (Nujol) 1560, cm-1.

(Found: C, 53.30, H, 7.23, N, 15.63, C16-24-- N 4S03 requires C, 54.54, H, 6.81, N, 16.00, %). This compound (132 mg) was dissolved in the minimum volume of 1 N-sodium hydroxide solution and the solution acidified with dilute hydrochloric acid. The separated crsytalline solid was recrystallised from methanol-petrol ° ° (60-80 ), mp = 180-181 , V(Nujol)max 3325, 2750, 1710, 1650, 1600, 1560, cm-l. (Found: C, 48.97, H, 6.46, N, 14.25, Cl, 8.22, C1025NOC103 requires, C, 49.48, H, 6.44, N, 14.40, Cl, 9.10, ,o). The previous compound (mp = 85-87°) (260 mg) was dissolved in redistilled liquid ammonia (300 ml) and treated with small pieces of metallic sodium Lntil the blue colour persisted longer than 15 mins. The excess sodium was 94. destroyed with addition of solid ammonium chloride and the ammonia evaporated. The residue was dissolved in 1 N- sodium hydroxide and extracted with chloroform. From the chloroform extract on working up a white solid was obtained, (107 mg), mp = 121-122° (from chloroform-petrol (40-60°)) 1/ max (Nujol) 3420, 3300, 1660, 1555, cm-1.

Preparation of p-nitro-phenyl-hydrazimido-carboxylate.

p-Nitro-phenyl-hydrazine (1.044 g) and ethyl orthoformate (1.100 g) was stirred at room temperature for 6 hours. The brown solid obtained (936 mg) was waaned with ether and recrystallised from chloroform-petrol, mp = 130-131°,

V max (Nujol) 3400, 1650, 1610, 1530, 1520, cm-1. (Found: C, 51.37, H, 5.50, N, 20.11, C9H11N303 requires C, 51.67, H, 5.26, N, 20.09, %). This solid (1.602 g) in chloroform was mixed with cyclohexylamine (1.550 g) and stirred overnight at room temperature. The red solid which separated (300 mg) was filtered off and recrystallised from lAnzene-petrol (40-60°), mp = 183-185°, ' max (Nujol) 3300, 1600, 1570, 1510, cm-1. The same product was obtained by treating the previous hydrazimido-carboxylate with glycine cyclohexylamide. 95.

Bromination of p-nitro-phenylhydrazone of benzaldehyde30

Bromine (1.660 g) in glacial acetic acid (50 ml) was added dropwise and under stirring to a solution of the hydrazone (2.410 g) in glacial acetic acid (250 ml) and at room temperature. A lemon-yellow solid separated (500 mg) which was filtered and recrystallised from chloroform- petrol, mp = 188-192° .

Reaction of the bromide with glycine cyclohexylamide.

The bromide prepared as in the previous preparation, (405 mg) in methylene dichloride (15 ml) was mixed with two equivalents of glycine cyclohexylamide (394 mg) and the mixture stirred overnight at room temperature. The separated crystalline material was filtered off and the filtrate evaporated to dryness to give a red oil which crystallised from chloroform ether (236 mg). On TLC on silica gel (chloroform-acetone, 10:1) shows four spots.

On a thick layer of silica gel developed as on TLC, the third band from the beginning was separated and recrystallised from chloroform-ether to give a red solid, mp = 137-138°

Vmax (Nujol) 3350, 1650, 1630, 1600, 1550, cm-1. This solid was dissolved in 0.5N-sodium hydroxide in water-methanol (1:1) and left to stand overnight at rocm 96. temperature. A pink solution is formed immediately from which a pale yellow solid separates. This solid was filtered, washed with water and dried, mp = 175-176°. On silica gel (chloroform) shows one spot. 97.

REFERENCES

(1) E.O.P. Thompson in "Advances in Organic Chemistry" Interscience Publishers, Inc., New York, 1960. (2) L. Goodman, and S. Winstein, J. Amer. Chem. Soc., 1957, 79, 4788. (3) H.K. Hall, J. Amer. Chem. Soc., 1956, 78, 2717. (4) C.H. Stammer, J. Org. Chem., 1961, 26, 2556. (5) S. Hoogewerff and W.A. van Dorp, Rec. tray. chim., 1892, 11, 84. (6) C.K. Sauers and R.J. Cotter, J. Org. Chem., 1961, 26, (7) D. Davidson and H. Skovronek, J. Amer. Chem. Soc., 1958, 80, 376. (8) T. Wieland and H. Bernhard, Annalen, 1951, 572, 190. (9) T. Sokolowska, G. Kupryszewski and E. Taschner, C. A., 1958, .51, 16236 g. (10) R. Adams and L.H. Ulich, J. Amer. Chem. Soc., 1920, 42, 599. (11) T. Figee, Rec. tray. chim., 1915, 21, 289. (12) R. Stolle and M. Luther, Chem. Ber., 1920, 53, 314. (13) M.A. Spielman in "The Chemistry of Penicillin", Princeton Univ. P2ess, N.J., 1949, p. 239. (14) G.S. Skinner and J.F. Perkins, J. Amer. Chem. Soc., 1950, 72, 5569. 98.

"(15) J.C. Sheehan and E.J. Corey, J. Allier. Chem. Soc., 1958, 74, 36o. (16) A.J. Speziale and I.R. Smith, J. Org. Chem., 1963, 28, 1805. (17) A.W. Ralston, E.W. Segebrecht and S.T. Bauer, J. Org. Chem., 1939, 4, 503. (18) F.W. Wenzel and E.E. Reid, J. Amer. Chem. Soc., 1937, .c1.2..„ 1089. (19) H. Seifert, J. prakt, Chem., 1885, [2] 31, 462. (20) R. Schwyzer, Hely. Chim. Acta, 1954, 2/, 647. (21) A.V. Oefele, Annalen, 1864, 132, 82. (22) R. Paul and A.S. Kende, J. Amer. Chem. Soc., 1964, 86, 4162. (23) W.J. Hickinbottom 'Reactions of Organic Compounds" Longmans, Green and Co., London, 1948, p. 113. (24) J.K. Sutherland and V. Tortorella, Unpublished results. (25) A. Werner, Chem. Ber., 1894, 27, 2847. (26) C. Grundmann and J. Dean, Angew. Chem., 1964, 76, 682. (27) R.N. McDonald and R.A. Krueger, Tetrahedron Letters, 1965, 851. (28) D. Pressman, J.H. Bryden and L. Pauling, J. Amer. Chem. 1948, /2, 1352. (29) P.L. de Benneville and L.J. Exner, C. A.,.1957, 5120. (30) F.L. Scott and J.B. Aylward, Tetrahedron Letters, 1965, 841. PART II 100.

Introduction

The use of "active esters" for the activation of the carboxyl of N-protected a-aminoacids and the aminolysis of these derivatives has been widely investigated since Schwyzer first prepared cyanomethyl esters'and Bodantzky, 2 p-nitro-phenyl esters of N-carbobenzoxy-aminoacids . The latest addition to the large group of active esters already investigated are the 0-acyl derivatives of hydroxylamines of the general formula (I) and the 0-acyl-oximes (II).

R1 R - OCOR C:=N - OCOR 2 2

RR = acyl group II or R1,R = alkyl group

(a) N,N-Diacyl-hydroxylamine esters.

Nefkens and Tesser- studied the reactions of esters of N-hydroxyphthalimide with some simple N-protected aminoacids. These esters are synthesised readily from N-carbobenzoxy- amino acids and N-hydroxyphthalimide4 in the presence of dicyclohexylcarbodiimide5 in good yields (40-80%). In the presence of an aminoacid ester, the active ester reacts quantitatively within seconds, at 0° C, to form a protected dipeptide according to Scheme 1. 101.

SCHEME 1

C 0 — OC OC FERNHZ + H NC HR C 0 OC C 2 1

ZNHCHRCONHCHR COOCH + I 1 NOH 1 3 - - c o

The by-product, i.e. N-hydroxyphthalimide, was readily removed with aqueous sodium bicarbonate. In this way four dipeptides and one tripeptide have been synthesised and showed ao trace of racemization, according to the Anderson 6 and Callahan test . A disadvantage of N-hydroxyphthalimide esters is the insolubility of the byproduct, N-hydroxyphthalimide. For this reason and because of the known solubility in water of N-hydroxysliccinimide7, Anderson et alb have prepared esters of this compound with a number of N-acylaminoacids which on aminolysis give excellent yields of easily purified products.

The new esters react with the salt of an aminoacid or peptide in aqueous solution, and are prepared in an analogous way to the N-hydroxyphthalimide esters, in yields of 50-90,0 after recrystallisation. They are stable crystalline solids and highly reactive towards the salts or esters of aminoacids or peptides, (Scheme 2). 102.

0 „fr,- --OCOCHR1NHZ H2NCHR2COONa > 0

ZNHCHR1CONHCHR2COONa 0

The activation of a peptide with N-hydroxy-succinimide has not been attempted and racemisation studies of such a synthesis are lacking. Recently Hofmann et al9 employed the N-hydroxysuocinimide NH0 ri4110011of Cbzo-Phe-Glu-Arg-Glu OH`rin a synthesis'. The p-nitrophenyl ester method in aqueous medium in this case gave poor results.

(b) 1-Acyloxy-2-pyridones.

Structurally related to the N,N-diacylhydroxy-lamine- 0-acyl esters are the 1-acyloxy-2(1H)-pyridones.

N—OCOR

Sutherland et al have shown that these compounds can 10 acylate amines . The 1-acyloxy-2-pyridones can be prepared from the N-hydroAypyridone, the carboxylic acid and dicyclohexylcarbodiimide as condensing agent. Recently, the preparation of pyridone ester of 103.

11 N-phthaloylphenylalanine has been reported . This ester was prepared by treatment of 2-ethoxypyridine-l-oxide with phthaloyl-L-phenylalanyl chloride, in quantitative yield. It condensed easily with

11 +.j• Phth.Phe.C1 It N OEt 0 OCOCHN _1 0 CO CH2Ph glycine ethyl ester to give the dipeptide in good yield (80%) and optically pure. The high reactivity and crystallinity of the esters make them particularly attractive.

Another approach to active acyl hydroxylamine derivatives has been reported12 and depends on the acylating power of N-alkyl monosubstituted and disubstituted hydroxylamine 0-acyl esters of the general formula:

R ---- OCOR R 2 where R1 = H, R2 = alkyl or R1, R2 = alkyl. N-Alkyl-0-acyl hydroxylamines rearrange to hydroxamic acids on standing at room temperature, by an 0 N acyl migration13. N,N-dialky1-0-acyl hydroxylamines being fully substituted, cannot undergo an 0 N acyl migration. 104.

Esters of N,N-diethylhydroxylamine with aminoacids have been prepared13 either by condensing the hydroxylamine hydrochloride with an N-carboxy-a-aminoacyl anhydride or with dicyclohexylcarbodiimide and the N-protected aminoacid. These esters have been coupled with aminoacid esters or salts to give peptides. However, the reaction proceeds slowly. Young et a114 prepared esters of 1-hydroxypiperidene and N-protected aminoacids, by the DCCI method or by reaction with the mixed carbonic anhydride of the aminoacid, or from the acid chloride and 1-hydroxypiperidine. The coupling reaction of these esters with aminoacid esters is unacceptably slow but the presence of acetic acid accelerates the reaction, and the use of aminoacid ester hydrochlorides has been shown to be advantageous. Presumably, N--protonation activates the piperidyl ester. The byproduct 1-hydroxypiperidThe presents no serious removal problems since it is soluble in acid and most of the common organic solvents. Optically active peptides retain their optical purity during peptide bond formation by this method. Further experience with these esters will show whether they rival the acyl-azide method a6 a means of peptide synthesis, as Young et al claim. 105.

(d) Hydroxamic acids.

Hoffmann and Faiferman15 regard hydroxamic acids of the N-acyl-a-aminoacids as activated derivatives suitable for peptide synthesis, and they prepared a number of them either by treatment of the N-protected aminoacid ester with hydroxylamine or from the mixed carbonic anhydride of the aminoacid and hydroxylamine. Anilides and benzylamides and some dipeptides of simple aminoacids were prepared in poor yields by refluxing the hydroxamic acid and the amine or aminoacid ester in dioxane, toluene, or tert.-butanol, according to the reaction:

OH R - CO.NHOH + HENR' ---> RCONHR' + NH2

Obviously, the reaction conditions are too sevRre for the more sensitive aminoacids and peptides.

(e) 0-(a-aminoacy1)-oximes.

The potential use of this class of compound in peptide 16 synthesis has been explored . The activated esters were prepared from the corresponding oxime and N-protected aminoacid either by the mixed anhydride method17 according to the reaction: 106.

H C1C02 CbzoNHCHRCO2 Et > CbzoNHCHRCOOCOOEt

R R 1 2C—I'NOH > CbzoNHCHRCO.ON:=1CR1R2

18 or, using diphenylketene which gave poor yields for steric reasons. Although 0-(a-aminoacy1)-oximes react more slowly than p-nitrophenyl esters, those of m-nitrobenzaldoxime, a-naphthaldoxime, and m-nitroacetophenoxime show rates comparable to those of p-nitrophenyl esters. Some simple dipeptides were prepared in good yield (60-855b). However, further work is needed with more sensitive aminoacids and large peptides to test the suitability of these esters for peptide synthesis. 107.

REFERENCES

(1) (a) R. Schwyzer, B. Iselin and M. Feurer, Hely. Chim. Acta, 1955, 38, 69. (b) R. Schwyzer, M. Feurer, B. Iselin and H. Mgi, Hely. Chim. Acta, 1955, 38, 80. (2) M. Bodanszky, Nature, 1959, 183, 1324. (3) (a) G.H.L. Nefkens, and G.I. Tesser, J. Amer. Chem. Soc., 1961, 83, 1263. (b) G.H.L. Nefkens, G.I. Tesser and R.J.F. Nivard, Rec. tray. chim., 1962, 81, 683. (4) (a) G.H.L. Nefkens, Nature, 1960, 185, 309. (b) G.H.L. Nefkens, G.I. Tesser and R.J.F. Nivard, Rec. tray. chim., 1960, 79, 688. (5) D.F. Elliot and D.W. Russell, Biochem. J., 1957, 66, 49P. (6) G.W. Anderson and F.M. Callahan, J. Amer. Chem. Soc., 1958, 80, 2902. (7) Beilstein, Vol. 21, p. 380. (8) (a) G.W. Anderson, J.E. Zimmerman and F.M. Callahan, J. Amer. Chem. Soc., 1963, 85, 3039. (b) G.W. Anderson, J.E. Zimmerman and F.M. Callahan, J. Amer. Chem. Soc., 1964, 86, 1839. (9) K. Hofmann, W. Haas, M.J. Smithers, R.D. Wells, Y. Wolman, N. Yanaihara and G. Zanetti, J. Amer.Chem. Soc., 1965, 87, 620. 108.

(10) J.K. Sutherland and D.A. Widdowson, J., 1964, 4651. (11) L.A. Paquette, J. Amer. Chem. Soc., 1965, 87, 1406. (12) S. Bittner, Y. Knobler and M. Frankel, Tetrahedron Letters, 1965, 95. (13) (a) W.P. Jencks, J. Amer. Chem. Soc., 1958, 80, 4581, 4585. (b) L.A. Carpino, C.A. Giza and B.A. Carpino, J. Amer. Chem. Soc., 1959, 81, 955. (14) S.M. Beaumont, B.O. Handford, J.H. Jones and G.T. Young, Chem. Commun., 1965, 95. (15) E. Hoffmann and I. Faiferman, J. Ord. Chem., 1964, 29, 748. (16) G. Losse, A. Barth and B. Heinke, Annalen, 1964, 677, 185. (17) (a) T. Wieland and B. Heinke, Annalen, 1958, 615, 184. (b) F. Weygand and W. Steglich, Chem. Ber., 1960, 93, 2983. (18) (a) G. Losse and E. Demuth, Chem. Ber., 1961, 94, 1762. (b) D.T. Elmore and J. Smyth, Proc. Chem. Soc. London, 1963, 18. 109.

Theoretical

One of the standard methods of peptide synthesis is the step-by-step one, that is, condensation of a N-protected and carboxyl activated aminoacid with the terminal aminogroup of a peptide or aminoacid, followed by removal of the protecting group. By repetition of this process long peptide chains can be built up.

ZNHCHR COX H NCHR C0NHCHR COOR ---> 1 2 2 3

---> ZNHCHR CONHCHRC0NHCHR COOR ---> 1 3

---> H NCHR CONHCHR CONHCHR COOH 2 1 2 3

where Z = amino-protective group X = activating species R = sodium cation or alkyl group. The synthesis of optically pure polypeptides is of great importance and even slight racemisation at each stage of the synthesis leads eventually to mixtures which are impossible to separate. Thus any new method of peptide synthesis must be tested for any possible racemisation in the bond forming step. The mechanism most commonly accepted to explain

110.

racemisation of carboxyl-activated acyl-aminoacids or peptides, postulates the intermediate formation of an oxazolone by intramolecular attack of oxygen at the activated carbonyl'.

NH____CHR\ 1 N CHR, H / 1 HY I /1 > RCONH-- C--R R — C...... :so a0--X R C 0 1 ''' I 0 COY _,-R, R N------q 1 HY I \ ' R --C. - `, H + > RCONH—C --H 0 COY N-Carbobenzoxy-aminoacids (R = PhCH2O-) do not form 2 oxazolones and give cyclic products (N-carboxyanhydrides) only under vigorous conditions so that in syntheses using this stepwise approach there is little or no danger Of

racemisation whatever the group X. However, when two peptides are being coupled or when a normal N-acylated (R = alkyl, aryl) aminoacid is being activated the oxazolone formation (and racemisation) can occur with great ease. Indeed only two methods, out of the multitude investigated have proved to be completely stereospecific, these are the azide and N-hydroxypiperidine methods of activation. It appeared possible that the absence of racemisation with N-hydroxypiperidine might extend to other hydroxylamine derivatives and that it might be worth while investigating methods for preparing these compounds. All of the published iia. methods for preparing these derivatives involve use of condensing agents which can themselves be used for peptide bond formation (and, of course, can cause racemisation). There- fore there is a need for methods for preparing these compounds which do not require such condensing reagents. A start was made using the reaction of a-chlorobenzaldoxime with carboxylate ions which led to the formation of

N,0-diacylhydroxylamines in yields varying from 10-7055 according to the solvent and the base used3. Cl PhC00 Ph C NOH > PhCONH.00OPh

Diacyl-hydroxylamines were shown to be satisfactory acylating agents, thus tetraethylammonium hippurate was treated with a-chlorobenzaldoxime in acetonitrile and after two hours cyclohexylamine was added. The cyclohexylamide of hippuric acid was formed in 26,'o yield. Obviously the yields are not high enough for peptide syntheses and in addition there is the possibility of Lossen rearrangement occurring as in the case of N,0-dibenzoyl-hydroxylamine which reacts only slowly with cyclohexylamine at room temperature in acetonitrile. On refluxing N-cyclohexyl-NI -phenylurea was isolated, being formed from the phenyl isocyanate generated by Lossen rearrangement. 112.

N-Substituted-N,O-diacylhydroxylamines (1) do not undergo the Lossen rearrangement since they cannot form the six- electron nitrogen necessary for the rearrangement, but they are good acylating agents.

VR2 R CON 1 \ OCOR (1) 3 Before any attempt was made to find new procedures of synthesis of (1) from the carboxylate ion, a study of the effect of structure on the rate of acylation seemed justified in order to determine which type of diacylhydroxylamine would be most satisfactory for peptide synthesis. N-Substituted-N,O-diacylhydroxylamines of the general formula (2) could give a clear picture of electronic and steric effects on the acylation rate of amines.

—00 _N \=71 OC OR (2) In the compound (2) the -I effect of the p-substituted atom decreases the electron density on the nitrogen and oxygen atoms of the hydroxylamine ester and consequently the breaking of the 0-COR bond is facilitated. !--- .. ,....„.:4, , .-%%111 0,„„...+ ,„...-.0 N--,i --C, <----> ,N--- C -....„ - 07' ---N - 0-COR 0.-7 N--0--COR // 1 CH CH 3 (2) 3 11:f.

Acid catalysis of acylation with N,N-dialky1-0-acyl- hydroxylamines due to protonation at the N-atom has been observed5 and is in line with this electronic effect. The p-methoxygroup in the compound (4) as in the case of anisic acid might be expected to increase the electron density on the nitrogen and oxygen atoms of the hydroxylamine and thus acylate more slowly.

CH3 N - 0 - COR CH (4) 3 In order to test these simple ideas in a somewhat more quantitative way, the preparation of the N,0-diacylhydroxylamines (5) (6, X = 0CH3) and (6, X = NO2) was carried out. The symmetrical N-methyl-N,0-dibenzoyl-hydroxylamine was prepared in one stage by treating N-methylhydroxylamine with two equivalents of benzoyl chloride6, while the two unsym- metrical, N-methyl-N-(anisoy1)-0-benzoyl-hydroxylamine (6, X = OCH ) and N-methyl-N(-p-nitrobenzoy1)-0-benzoyl- 3 hydroxylamine (6, x = NO2), were prepared from the hydroxamic acids (7, X = OCH3) and (7, X = NO2), and benzoic acid using dicyclohexylcarbodiimide as condensing agent7. The hydrox- ) were prepared from amic acids (7, X = OCH3) and (7, X = NO2 the N-methyl-hydroxylamine and one equivalent of the corresponding acid chloride in aqueous sodium bicarbonate solution. 114.

MeNH.OH.HC1

NaHCO 3

Me Me 7 PhCO.N'' OH OCOPh (5) PhC 02H DCCI.

\./

Me XI/2 OCOPh

(6) The half-lives of these diacylhydroxylamines in the presence of cyclohexylamine were determined as is described in the experimental and found to be as below:

Diacyl-hydroxylamine Half-life in hours (5) 7 (6, x OCH ) 20 3 (6, X = No2) 5

If the aminolysis of the above esters is considered to 115.

be a second order reaction8, then, for the half life of the

diacyl-hydroxylamines9,

t' - 1 or k' - 1 0.5 k'a' t' .al 0.5 where k' = rate constant a' = initial concentration and t' = half-life 0.5 From Hammett's equation we havel°

log 7-= P.O

where = rate constant of substituted compound o k = rate constant of the unsubstitut.ed compound

substituent constant

and P reaction constant (p-value) Introducing the t0,5, and the known initial concentrations we obtain: 0 a° log — - _Log = p.o a' t0! .5 The left term of the above equation was plotted against the corresponding c values for the methoxy- and nitro-group and the slope of the straight line through these points was measured and found to be positive (p = +0.7). 116.

tn Concen- to Diacyl- Sub- our 0.5 , compound stituent op, tration l # log —T hours a '0.5 a

7 0.058 m (6,x.ocil ) OCH3 20 3 - 0.27 0.044 m - 0.576 (6,X=NO2) NO2 + 0.78 5 0.055 m + 0.120

These results indicate that the aminolysis of the N,0- diacyl-hydroxylamines is aided by electron with drawing sub- stituents X (formula (6)) in an appreciably significant magnitude. In order to check the effect of group R in the structure (2) keeping all the other groups the same as before we tried to prepare a series of compounds of the general formula (8) CH / 3

OC OCH2NHC O2Et

(8) where X = H, OCH , or NO 3 2

The corresponding hydroxamic acid (7) was condensed with N-carbethoxyglycine using dicyclohexylcarbodiimide as condensing agent but although the resulting oily products had an infrared absorption consistent with the expected compounds, they could not be crystallised.

It was thought that the N-phenyl-N,0-diacyl-hydroxylamines might more readily give crystalline derivatives and this was found to be true. In the same way as before the diacyl-hydroxylamines (9) and (10) were prepared from the hydroxamic acid and the acid in the presence of dicyclohexylcarbodiimide. The hydroxamic

acid (11)__ was synthesised from freshly prepared phenyl- hydroxylamine and benzoyl chloride in ethanol in the presence of sodium acetate.

PhC0C1 V Ph PhCO H Ph PhNHOH > PhCON 2 > PhCON NaOAc NOH DCCI 'OCOPh (11) (9) Et02CNHCH2CO2H

DCCI Ph PhC ON OCOCH 2NHCO2Et (10)

Cyclohexylamine reacted with (9) and (10) too fast to measure the rate of the reaction, however glycine cyclohexylamide was found to react at a rate slow enough to be measurable. The later amine has one more advantage over cyclohexylamine in that it is a better model for an actual peptide unit. A convenient procedure for the synthesis of glycine cyclohexylamide was found to be: (a) Preparation of N-tosyl-glycyl chloride and

treatment with cyclohexylamine in chloroform to give N-tosyl-glycine cyclohexylamide in 90% yield and (b) Removal of the tosyl group by reduction with sodium in liquid ammoniall in 40% yield. The reaction conditions and procedure for the aminolysis, were followed as before, and the following half-lives were found:

Diacyl compound Half-lives

66 hours 7 min.

12 This result is not surprising since it is well known that the carbonyl of a benzoyl group is much less readily attacked by nucleophiles than corresponding aliphatic ones because of electron withdrawal by the phenyl ring. Qualitatively can be seen that the change of Y substituent in (2) from methyl to phenyl group, increased the rate of reaction with cyclohexylamine. It was interesting to see what would be the effect of nuclear substitution at the N-phenyl group of the compound (12). For this reason, the hydroxamic acid (13) was

119.

prepared, from one equivalent of benzoyl chloride and p- methoxyphenylhydroxylamine in methanol in the presence of sodium acetate. The hydroxamic acid obtained was condensed with benzoic acid in the presence of dicyclohexyloarbodiimide at room temperature. The procedure was successful for the unsubstituted compound (9) and it was hoped that the same would hold true for the compound (14). The crystalline compound obtained showed an infrared absorption at 1740 cm-1, which was suspiciously low for a hydroxylamine-0-acyl ester (14). x

- 000Ph (12) NHOH OCH 3 PhC OC 1 / PhC ON ., cr OH OCH 3 (13)

COOH PhCO2H tO2CNHCH2 DCCI // DCCI

OCH OCH 3 3 PhCON/ PhCON.„, -OCQCH2NHCO2Et 120,

The preparation of (15) was attempted in the same way and a crystalline compound was obtained with Amax = 1770 cm When this compound was treated with cyclohexylamine and the reaction product worked up, none of the hydroxamic acid (13) could be isolated. However, an alkali soluble isomeric compound was isolated which gape a pale green colour with ferric chloride. The structure of the hydroxamic acid (13) was confirmed by reducing it to the amide (16) with zinc in acetic acid and comparing the product with an authentic sample. Under the above conditions only the hydroxyl-nitrogen bond is hydrogenolysed and not a phenolic hydroxyl, which excludes a phenolic amide structure. The empirical formula, C14H13NO3, of the alkali soluble solid from (II), its infrared spectrum, 3420, 3100 broad, cm-1, and melting point, was consistent with the o-hydroxyamide (18)13. /1 '20CH 3 Zn PhC ON > PhCONH -OCH OH AcOH 3 (13) (16) Et0 2CNHCH2CO2H DCCI C H NH 6 11 2 > P1-10NH- --OCH C19H20N2 6 3 (17) HO (18)

121.

The compound (15), consequently, either rearranged to (17) during the cyclohexylamine treatment or (17) was formed during the synthesis. In order to test which one of these alternatives occurred, the alkali soluble compound (18) was condensed with N-carbethoxy-glycine in the presence of diayclohexyl- carbodiimide and the product obtained was found to be identical with (17), which means that (17) is not a N,0- diacylhydroxylamine, but an N,0-diacyl-(o-amino)-phenol. The possibility that the liberated hydroxamic acid (13) rearranged during hydrolysis, was excluded by a control experiment.

C-I NH PhCONH- p--OCH o 11 2 3 Et0 CNHCH COO 2 2 Et02CNHCH2CO2H DCCI (17)

The compound (19) was unaffected by treatment with cyclohexylamine but was hydrolysed by 2N-NaOH at room temperature to give (18) showing that a similar rearrangement had taken place in the formation of this compound. // 3 PhCO H PhCON N 2 -> PhCONH-- --OCH OH DCCI 3 PhC00 (12) (19)

122.

Compound (10) was found to be a true N,0-diacyl-hydroxylamine since treatment with cyclohexylamine gave the parent hydroxamic acid. This suggests that the p-methoxy-group is responsible for the rearrangement. However, when (11) was treated with tosyl chloride in pyridine the product obtained had rearranged to the diacyl-(o-amino)phenol (20). Compound (20) was synthesised from (21) and tosyl chloride in pyridine and found to be identical with that from the hydroxamic acid obtained. -.''''' 11 ,',, Tos Cl PhCON, > PhCONH < OH y. P C7H7S020'

(11) (2o)

PhCON OH Tos.Cl Py. (11)

Tos Cl PhCONH-N (20)

(21) 123.

This rearrangement was found to occur by treating the hydroxamic acid (12) with: (a) Benzoyl chloride in pyridine and (b) Ethyl chloroformate in pyridine. In both these cases the crystalline compounds obtained, showed infrared absorption at the NH region and analysed correctly for (22) and (g). Also treatment of (13) with phenyl isocyanate gave a crystalline product with an infra red spectrum and analysis consistent with (24). OCH 3 PhCON NOH --,,,.....,,,,, PhC0C1 /(12) \ Py PhNCO EtOCO.C1 Py

PhCONH PhCONH PhCONH ocb PhCOO --"'-')— OCH3 Et0C00---- c,,->------OCH3 PhNHC 00

(22) (?2 ) (24)

After the previous work had been completed we found that 14 Horner and Steppan had carried out similar work and they proposed the following mechanism for this rearrangement. 124.

COPh COPh

0 H li CH3 0 I I CY' Ph

_NHCOPh CH3O n OCOPh

The p-methoxy-group by pushing electrons towards the nitrogen atom facilitates the departure of the carboxyl grcup with concurrent attack of the oxygen atom at the o-positior of the benzene ring.

Subsequent tautomerisation results in the rearranged product observed. In the case of 0-tosyl-hydroxylamine ester the cleavage of the nitrogen-oxygen bond is facilitatrf by the strongly positive sulfur atom of the sulfonyl group. Since the acylating power of esters of N-hydroxypyridone (25) has been demonstrated3, a preparation of thee. esters under mild conditions would be highly desirable. 2-Chloropyridine-N-oxide does not react with the carboxylate ion3 however, 2-fluoropyridine-N-oxide prepared in this laboratory15, when treated with benzoic acid alone or in the presence of triethylamine gave the 0-benzoyl- hydroxypyridone (26) in 22-40o yield. 125

The rate of amminolysis of this ester with cyclohexylamine, benzylamine or glycine cyclohexylamide could not be followed owing to the rapidity of the reaction and would thus appear to be too powerful an acylating agent since thc stronger it is as an acylating agent the more likely it is to form oxazolones. In line with this it was found impossible to isolate the ethoxycarbonyl-glycine ester as a stable crystalline compound.

N 0 OH (25)

PhCO2 N F 0 I COPh 0 -

PhCO2H N 0 0 - COPh'

I I N N0 OH COPh 126,

From the relative reactivities of the diacyl compounds examined so far the following order of reactivity appears justified

1 Ph Me Ye / 'N-"v 0 >> PhCON >> pO2NC6H4CON > C H CON \ 6 5 \ \\ OC OR OCOR OCOR O0 0R

Me > Me0C6H4CONx OC OR

From the point of view of peptide synthesis, accepting that the reaction of glycine cyclohexylamide with ethoxy- carbonylglycine esters is a good model, it would appear that esters of N-phenylbenzhydroxamic acid are most suitable sine they react at a reasonable rate and are crystalline compounds. Crystalline esters of N-alkylhydroxamic esters might also be suitable. 127,

Experimental

Melting points were determined on a Ki5fler block and are uncorrected. Infrared spectra were determined on a Unicam S.P. 200 spectrometer. Nuclear magnetic resonance (n.m.r.) spectra were measured on a Varian A 60 instrument. Analyses were carried out by the staff of the micro- analytical laboratory (Imperial College). Organic solutions were dried using anhydrous magnesium or sodium sulfate. 128.

N-Substituted hydroxylamines

N-Methyl-hydroxylamine16, phenyl-hydroxylamine17 and p-methoxy-phenyl-hydroxylamine18, were prepared from the corresponding nitrocompounds by reduction with zinc dust and ammonium chloride in ethanol-water, as described in the literature.

6 N-Methyl-N,0-dibenzoyl-hydroxylamine (a)

N-Methyl-hydroxylamine hydrochloride (2.50 g) was dissolved in ethanol (50 ml) and potassium hydroxide was added (3.2 g) as a solution in ethanol (50 ml). To the suspension was then added benzoyl chloride (4.20 g) in ethat' (50 ml) and the mixture was stirred at room temperature for 2 hours before working up in the usual manner. Yield = 3.9 (875/o), bp 102-104,0.5 mm (reported bp = 103-105°/0.6 mm).

N-Methyl-N-anisoyl hydroxylamine6 (7, X = OCH ) 3 Anisoyl chloride (3.30 g) was dissolved in ether (10 ml) and added over a period of 15 min at room temperature to a stirred solution of N-methyl-hydroxylamine hydrochloride (1.80 g) in saturated sodium bicarbonate solution(80 ml) and ether (40 ml). The mixture was further stirred for 90 min. The aqueous layer was separated and extracted with ethyl 129.

acetate and the ethyl acetate extract was combined with the ether layer. On working up the combined extracts a white solid was obtained (1.50 g). Crystallised from chloroform- petrol (40-60°), mp = 108-110° (reported mp = 108°).

N-Methyl-N-(p-nitrobenzoyl)-hydroxylamine (7, X = NO2)

N-Methylhydroxylamine hydrochloride (1.67 g) and p-nitrobenzoyl chloride (3.71 g) were reacted as in the previous experiment. N-Methyl-N-(p-nitrobenzoy1)-hydroxylamine was (Nujol) 1630, 1600, obtained (2.25 g), mp = 182-184°, )max 1590, cm-1.

N-Methyl-N-(p-methoxybenzoy1)-0-benzoyl-hydroxTlamine (6, X OCH ) 3 Dicyclohexylcarbodiimide (200 mg) was added to a stirred solution of N-methyl-N-anisoyl-hydroxylamine (181 mg) and benzoic acid (122 mg) in methylene dichloride (3 ml) at room temperature, and the mixture was stirred further overnight. The separated dicyclohexylurea was filtered off and the filtrate evaporated to dryness. The sticky residue was crystallised from ether-petrol (40-60°) to give a white solid

(195 mg), mp = 91-92.5°,V max (_CH 2Cl2) 1760, 1660, 1610, 1580, cm-1. (Found: C, 67.23, H, 5.23, N, 4.99, C16H15NO1 requires C, 67.43, H, 5.23, N, 4.90, °A). 130.

N-Methyl-N-(p-nitrobenzoy1)-0-benzoyl-hydroxylamine (6, X = NO2 The same procedure as in the preceding experiment was followed. The product (75°,6 ) crystallised from ether-

petrol ()40-60°), mp = 79-80°,imax (CH2C12) 1760, 1670, 1600, 1540, crEl.

(Found: C, 59.57, H, 3.75, N, 9.82, C15H12N205 requires C, 59.94, H, 4.02, N, 9.32, Yo).

N-Tosyl-glycine cyclohexylamide 19 N-Tosylglycine was converted to N-tosylglycine acid 20 chloride and the later (1.2 g) was dissolved in chlorofol (15 ml) and added slowly to a stirred solution of cyclohexylamine in chloroform (15 ml). After standing for 2 hours the chloroform solution was washed with dilute hydrochloric acid, saturated sodium bicarbonate solution, dilute hydrochloric acid, and water, and on working up the chloroform solution, a white solid was obtained (1.24 g, 77%), Orystallised from chloroform-petrol (60-800), mp = 113-114°. (Found: C, 57.99, H, 7.00, N, 8.98, S, 10.86, C15H22N2S03 requires C, 58.06, H, 7.09, N, 9.03, S, 10.32, ,b).

Glycine cyclohexylamide

N-Tosylglycine cyclohexylamide (400 mg) was dissolved 131. in dry liquid ammonia (200 ml) and small pieces of clean sodium metal were added under stirring and until the blue colour of the dissolved sodium persisted longer than 15 min. The excess sodium was destroyed with ammonium chloride and the ammonia evaporated to dryness. The residue was dissolved in dilute aqueous sodium hydroxide and the aqueous layer extracted with chloroform. On working up the chloroform layer a white solid was obtained (190 mg, 40%), crystallised from chloroform-petrol (40-60°), needles, mp = 93-94°.

(Found: C, 61.77, H, 10.08, N, 17.71, C8H16N20 requires C, 61.53, H, 10.25, N, 17.94, 7O).

N-Phenyl-N,0-dibenzoyl-hydroxylamine 0)

N-Phenyl-N-benzoyl-hydroxylamine21 (288 mg), benzoic acid (165 mg) and dicyclohexyloarbodiimide (270 mg) were reacted as before. The product (132 mg) crystallised from methylene dichloride-petrol (40-60°), mp = 117-118° (reported.22 mp = 121° and 118-119°) `'max (CH2C12) 1760, 1675, 1600, cm*

N-Phenyl-N-benzoy1-0-(N-carbethoxyglycy1)-hydroxylamine (10)

Dicyclohexyloarboiiimide was added to an equimolecular mixture of N-phenvi-M_bonzuyl-hydroxylamine (213 mg) and N-carbethoxyglycine (175 mg) in methylene dichloride. The 132

separated dialkylurea was filtered off and the filtrate was evaporated to dryness. The sticky oil obtained (321 mg) was triturated with ether to give a white solid which was crysta:L- lised from chloroform-petrol (60-80°), mp = 97-98°, Ni rmax (Ku °1) 3350, 1800, 1700, 1660, 1590, 1540, cm-1. (Found: C, 63.49, H, 5.49, N, 7.99, C 18H18N205 requires C, 63.15, H, 5.30, N, 8.20, ,o).

Action of cyclohexylamine on N-phenyl-N,0-dibenzoyl-hydroxylamine

The diacylhydroxylamine (173 mg) in methylene dichloride

was treated with 2.5 equivalents of cyclohexylamine in ether, and the mixture was left to stand at room temperature for 2 days. At the end of this time there was no absorption at 1760, cm-1, and the solution was extracted with 2N-aqueous sodium hydroxide. The alkaline layer was separated and acidified with aqueous hydrochloric acid. A white solid separated (95 mg), mp = 110-114°, which gave positive ferric chloride test, and on TLC on silica gel (benzene-ether-formic acid, 14-4-1) moves as the authentic hydroxamic acid. The methylene dichloride layer was wahsed with dilute hydrochloric acid, water, dried and evaporated, to give a white solid (71 mg), mp = 148-150° (from ethyl acetate-petrol), Vmax (Nujol) 3250, 1640, 1620, 1560, cm-1. Mixed mp with authentic PhCONHC6H11, unchanged. 133,

Rate of reaction of N,0-diacyl-hydroxylamines with cyclohexylamine

A weighed amount of N,0-diacylhydroxylamine was dissolved in an accurately measured volume of methylene dichloride and to this solution two equivalents of cyclohexylamine in a measured volume of methylene dichloride were added, and the height of absorption at 1760, cm-1, in centimeters was recorded during suitable periods of time. The decrease of the height of the band in centimeters versus the time was obtained for each of the following diacyl-hydroxylamines. (i) N-Methyl-N,0-dibenzoyl-hydroxylamine (ii) N-Methyl-N-(anisoy1)-0-benzoyl-hydroxylamine (6, x = OcH,) 2 (iii) N-Methyl-N-(p-nitrobenzoy1)-0-benzoyl-hydroxylaminc. NO ) (6, x 2 Standard solutions of (6, x = OCH ), and (6 X =NO,) 3 in methylene dichloride were prepared and the band intensity in centimeters at 1760, cm-1, for a series of concentrations was measured so giving a calibration curve. In this way the half-life (t0.5) of the N,0-diacyl-hydroxylamines in the presence of cyclohexylamine was derived and the results are as in the following table. 134-

N-Methyl-N,0-dibenzoyl-hydroxylamine

Concentration 14 mg/ml methylene dichloride

Diacyl-hydroxylamine Height of absorption at 1760, cm-1 mg/ml cm. 15 14 12 12.5 9 11.3 6 8.5 3 5

Time in hours Height of absorption, in cm 0 13.5 5 12.0 17.5 7.1' 41.5 2.6 73.0 1.0

N-Methyl-N-(anisoy1)-0-benzoyl-hydroxylamine

Concentration 12.2 mg/ml methylene dichloride

Diacyl-hydroxylamine Height of absorption at 1760, cm-

mg/ml CM. 13 13 10 12 8 10 5 8 2 4.5

Time in hours Height of absorption, in cm. 0 13.0 15.5 10.3 39.5 6.5 71.0 3.5 95.o 2.3

N-Methyl-N-(p-nitrobenzoy1)-0-benzoyl-hydroxylamine

Concentration 16.6 mg/ml methylene dichloride

Diacyl-hydroxylamine Height of absorption at 1760, cm.- mg/ml cm 15 13 11 11.2 8 9.8 5 7 2 4 136.

Time in hours Height of absorption, in cm. 0 12.8 1 11.6 2 10.7 20 4.7 66 1.0

Rate of reaction of N,0-diacyl-hydroxylamines with glycine cyclohexylamide

The same procedure as before was followed. The following N,0-diacyl-hydroxylamines were treated with three equivalents of glycine cyclohexylamide (i) N-Phenyl-N-benzoy1-0-(N-carbethoxyglycy1)- hydroxylamine (ii) N-Phenyl-N,0-dibnnzoyl-hydroxylamine The results are as follow.

N-Phenyl-N-benzoy1-0-(N-earbethoxyglycy1)-hydroxylamine

Concentration 20.5 mg/ml methylene dichloride -1 Diacyl-hydroxylamine Height of absorption at 1790, cm mg/ml cm 20.50 10.2 16.4o 9.5 12.30 8.3 8.2o 6.4 4.10 4.o 137,

Tiwe, in minutes Height of absorption, in cm. 0 10.2 10 7.6 15 5.0 21 3.7 26 2.8 31 2.0 36 1.6

N-Phenyl-N,0-dibenzoyl-hydroxylamine

Concentration 11 mg/ml methylene dichloride

Diacyl-hydroxylamine Height of absorption at 1765, cm mg/ml cm. 10 10.3

8 9.1 6 8.0 4 6.2 2 4.0

Time, in hours Height of absorption, in cm. 0 10.5 20 9.0 44 8.0 140 4.2 188 3.o 132).

N-Cp-Methoxypheny1)-N-benzoyl-hydroxylamine (13)

A solution of p-methoxy-phenyl-hydroxylamine (1.61 g) and dry sodium acetate (0.95 g) in methanol (50 ml) was cooled in an ice-bath, and to the ice-cooled mixture, benzoyl chloride (1.60 g) was added dropwise under stirring. The solution was left at room temperature and stirred for a further 4 hours. Addition of an equal volume of water precipitated a small amount of yellow solid which was filtered off, and on further dilution with water, a pale brown solid separated (1.90 g). It was crystallised from chla20— form-petrol (40-60°), mp = 119-121°. Ferric chloride test positive. \1 (CHOI ) 3300 broad, 1620, 1575, 1525, cm max 3 (Found: C, 69.02, H, 5.36, N, 5.66, C14H13NO3 requires C, 69.13, H, 5.34, N, 5.76, jb).

Reflux of the N-(p-methoxypheny1)-N-benzoyl-hydroxylamine with zinc in acetic acid.

The hydroxamic acid (138 mg) and excess zinc dust in acetic acid, was refluxed for 2 hours. The excess acetic acid was evaporated in vacuo and the residue extracted with boiling chloroform, from which a solid was obtained containing zinc salts. .This solid was passed through silica gel column in chloroform, when a white solid was obtained 139

(98 mg) which was crystallised from ethyl acetate-petrol

(40-60°), mp = 158-159°, max (cHci3) 3450, 1670, 1600, 1580, 1535, cm-1, shown to be identical to N-benzoylanisidf

N, 0-Dibenzoyl-5-methoxy-2-amino-phenol (J)

N-(p-Methoxypheny1)-N-benzoyl-hydroxylamine (243 mg) benzoic acid (122 mg) and dicyclohexylcarbodiimide, were reacted as before. The sticky material obtained, on trituration with ether, crystallised to a white solid (240 mg) mp = 140-141° (from chloroform-petrol (40-60°), Vim (CHC1 ) 3450, 1740, 1675, 1620, 1600, 1530, 1490, cm ax 3 Chromatographed on a silica gel column (chloroform) had unchanged melting point. (Found: C, 71.71, H, 4.86, N, 4.55, $2Hr7N04 requires C, 72.60, H, 4.89, N, 4.03, %).

N-Benzoy1-0-(N-carbethoxyglycy1)-5-methoxy-2-amino-phenol

The hydroxamic acid (189 mg), N-carbethoxyglycine (11', and dicyclohexylcarbodiimide, were treated as before. Th() pale-brown solid obtained (151 mg) was recrystallised from methylene dichloride-petrol, mp = 131-132°,N)max (CH2 Cl2 ) 3480, 1770, 1720, 1680, 1620, 1600, 1590, 1535, cm-1. (Found: C, 61.27, H, 5.58, N, 7.64, C 1,H20N206 requires C, 61.28, H, 5.37, N, 7.52, (io). 140,

Action of 2N-sodium hydroxide on (la)

A suspension of the diacyl compound (241 mg) in 2N- sodium hydroxide (10 ml) and methanol (5 ml) was left to stand at room temperature for 3 hours. The alkaline solution was extracted with chloroform separated and acidified with dilute hydrochloric acid when

a white solid separated .(117 mg). Crystallised from ethyl acetate-petrol (40-60°), mp = 167-168°. With ferric chl (Nujol) 3420, solution it gives a pale green colour.\1 max 1640, 1605, 1580, 1540, cm-1. The above alkali soluble solid (124 mg) was dissolved in pyridine (5 ml) and treated carefully under cooling and stirring with benzoyl chloride (71 mg). The mixture was left to stand at room temperature overnight and the excess pyridine evaporated in vacuo to give an oily residue, which was taken up with methylene dichloride, washed with water dried and evaporated to dryness to give a solid from which starting material was isolated (large volume of methylene dichloride and petrol).

Action of cyclohexylamine on (I7)

The diacyl compound (109 mg) in methylene dichloride (10 ml) was mixed with two equivalents of cyclohexylamine 11r,

(59 mg) and the mixture was left to stand at room tempera" overnight. The methylene dichloride solution was extra:-.: with 2N-sodium hydroxide and the alkaline layer separated, and acidified with dilute hydrochloric'aCid. A pale grey solid separated (36 mg) and crystallised from ethyl acetat.

petrol (40-60°), mp = 167-168°, Vmax (Nujol) 5420, 1640, 1605, 1580, 1540, cm-1. With ferric chloride solution it gives pale green colour. (Found: C, 68.82, H, 5.01, N, 5.71, C141113NO3 requires C, 69.13, H, 5.36, N, 5.76, cA).

The above alkali soluble solid (137 mg) and N-carbetboXy- glycine (83 mg) in methylene dichloride (4 ml) was treated with dicyclohexylcarbodiimide (116 mg) as usual. On work.; up the reaction mixture a white solid was obtained (159 mg. shown to be identical to (17).

Reaction of N7(p-methoxypheny1)-N-benzoyl-hydroxylamine wit' (a) benzoyl chloride in pyridine and (b) ethyl chloroforma'- in pyridine.

(a) Benzoyl chloride (125 mg) was added carefully to a cooled solution of the hydroxamic acid (215 mg) in pyridi-:- (10 ml) and the reaction mixtrue was left to stand at room temperature overnight. At the end of this time a sherry red colour had developed and the mixture was poured into 142. ice-water and extracted with chloroform. The chloroform layer was separated, dried and evaporated to dryness to give an oil which crystallised from chloroform-petrol (4o-6oc:\ to a solid (151 mg) mp = 105-135°, 1,max C12) 3450, 1740, (CH 2 1675, 1620, 1600, 1530, 1490, cm-1. (b) Ethyl chloroformate (108 mg), and the hydroxamic acid (242 mg) in pyridine (10 ml) were reacted as before. The solid obtained (95 mg) crystallised from chloroform- ° petrol (40-60 ), mp = 118-119, \max (CH2C12) 3450, 1760, 1670, 1600 broad, 1520, 1480, cm-1. (Found: C, 64, 93, H, 5.33, N, 4.73, C17H17N05 requires C, 64. 69, H, 5.39, N, 4.44, /6).

Reaction of N-Thenyl-N-benzoyl-hydroxylamine with tosyl chloride in pyridine.

The hydroxamic acid (213 mg) and tosyl chloride (400 mg in pyridine (6 ml) was set aside overnight at room temperatl'r- The mixture was diluted with water, warmed on the steam bath. cooled and acidified. The solid obtained on scratching and cooling was filtered and extracted with boiling acetone. The acetone extract gave a solid on concentration which was extracted with petrol. The petrol extract was crystallised from acetone, mp = 111-112°, (Nujol) 3400, 1670, 1)max 1600, cm-1. 143.

Reaction of N-Cp-methoxypheny1)-N-benzoyl-hydroxylamine phenylisocyanate.

A mixture of the hydroxamic acid (334 mg) and phenylisocyanate (164 mg) in methylene dichloride (6 ml) was stirred for 3 hours at room temperature. A white compound which separated from the reaction mixture was collected and extracted with boiling ethanol-acetone (1:1). From the extract a white crystalline solid separated on cooling

(158 mg), mp = 163-168°,Vrmax (Nujol) 3350, 3320, 1715, 1650, 1600, 1540, 1510, cm-1. (Found: C, 69.47, H, 4.83, N, 7.76, C21Hi8N204 requires

C, 69.60, H, 4.90, N, 7.70, /b).

2-Fluoro-pyridine-N-oxide15

Trifluoroaaetic anhydride (7.8 ml) in one portion was added to an ice-cooled suspension of 850/o hydrogen peroxide (1.23 ml) in methylene dichloride (30 ml) and after 5 mine'-' the temperature of the external bath was raised slowly at room temperature. 2-Fluoro-pyridine (2.94 g) was added over a 30 minutes period and the mixture was refluxed for an hour and then left at room temperature overnight. The oil obtained by evaporation of the yellow solution was dissolved in water (10 ml) and solid sodium bicarbonate was 144 added until the solution was green. The. bicarbonate solution was extracted continuously with methylene dichloride for three days. The residue after evaporation of the methylene dichloride extract was recrystallised from methylene dichloride-anhydrous ether to give colourless crystalline 2-fluoropyridine-N-oxide (2.1 g).

Reaction of 2-fluoropyridine-N-oxide with benzoic acid.

(a) In the presence of triethylamine. 2-Fluoropyridine-N-oxide (0.791 g), benzoic acid (0.845 g) and triethylamine (0.96 ml) in acetonitrile was left to stand at room temperature for 110 hours. A dark blue solution had developed with a black precipitate which was filtered, extracted with saturated sodium bicarbonate solution, washed with water and evaporated to dryness. The blue residue was chromatographed on celite-water (1:1) and a crystalline product (0.300 g) was obtained. Melting point and infrared spectrum as reported23 for 1-benzoyloxy- 2(1H)-pyridone. From the alkaline solution, benzoic acid (0.38 g) was recovered. (b) Without triethylamine. 2-Fluoropyridine-N-oxide (0.30 g) and benzoic acid (0.324 g) was dissolved in methylene dichloride (3 ml) and 145

left to stand at room temperature for 40 hours. By extraction with sodium bicarbonate and evaporation of the organic layer 1-benzoyloxy-2(1H)-pyridone was obtained in 33/6 yield. 146

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