19F NMR Studies of the Interaction of ct-Chynotrypsin

with N-Trifluoroacetyl Arnino Acids.

A Thesis

presented for the degree of

Doctor of Philosophy

in

The University of Adelaide

by

Brenton Cunmings Nicholson, B. Sc. (Hons. )

Department of Organrc Chenistry

March, 7973.

(i)

Sununary.

Several aromatic cl-amino acicls and their N-trifluoroacetates, as

well as cinnarnic acids and 3-carbo4¡-7-fluorodihydroisocarbostyril were

synthesised. The interactions of these cornpounds as inhibitors with a-

chynotrypsin were studied ¡y 19f IJMR spectloscopy at 56,4 Wiz. These

studies allowed the dissociation constant of the enzyne-inhibitor complex,

KD, and the chemical shifts of the fluorine nuclei ín the enzyne-inhibitor

cornplex, A, to be calculated. These results were interpreted in terms

of the orientation of the inhibitor in the active site of the enzyme'

the nodel of the active site used being that determined previousLy by

X-ray diffraction stuclies of the crystal enzytne. A halogen aton on the

phenyl ring of N-trifluoroacetylphenylalanine greatly increased the

binding affinity, the degree depending on its nature and position. The

position of the halogen aton surprisingly influenced the position of the

N-trifluoroacetyl gïoup in the active site as evidenced by different

values of the chemical shift of these 19F ,rrr"l"i for different enzyme-

inhibitor complexes. The D and L isoners of a particular inhibitor

were also shown to bind in different ways with respect to the N-tri-

fluoroacetyl group.

KO and À were also calculated with the inclusion of an inactive

enzyme diner model in these systens, the enzyme dimer di.ssociation constant

being taken fron the literature. Such an inclusion had the effect of

greatly decreasing KO but hardly affecting A.

(ii)

Statenent.

This thesis contains no naterial which has been accepted

for the award of any other degree or diplorna in any University

and, to the best of ny knowledge and belief, no naterial

previously published or written by another person, except

where due reference is made in the text of the thesis.

B.C. NICHOLSON.

(iii)

Acknow le dsenents .

I would like to thank ny supervisor, Dr. T.M. Spotswood

for his encouragenent and guidance during the course of this

work, and his cornpilation and rrrtning of the computer

programnes.

I would also like to thank Mr. R.L. Paltridge for his

naintenance of the NMR spectroneter and construction of

the heteronuclear spin decoupler, and other nenbers of

the departnent for their help and advice.

Finally I would like to gratefully acknowledge the

support of a Connonwealth Postgraduate Award.

***

(iv)

Pub lication.

trNuclear Magnetic Resonance Studies of the Interaction

of Inhibitors with o-Chymotrypsin, N-Trifluoroacetyl

Derivatives of Phenylalanine.'r

B.C. Nicïrolson and T.M. Spo tswood, Aust.J.Chem.,

26, 13s (1975).

**lt

CONTENTS.

SI.IMMARY

STATEMENT

ACKNOI{ILEDGEMENTS

PUBLICATION

INTRODUCTION:

Applications of NMR Spectroscopy in the S'tudy of 1

Biological Problems.

(a) Historical ancl general. 1

(b) The spectra of amino acids, proteins, and nucleic 3

acids.

(c) Sma1l nolecule-rnacromolecule interactions. 5

'lo(d) '-F Nlr,ß. studies of biological systens. 11

The Chemistry of o-Chymotrypsin. t4

(a) General. L4

(b) Amino acid sequence and three-dimensional 15

structure of cl-chynotrypsin.

(c) the relevance of crystallographic data to the 19

mechanism of o,-chynotrypsin catalysed reacticns

in solution.

(d) 'Ihe activity and specificity of o-chyrnotrypsin 2L

towards organic molecules.

(e) The nature of the frmctional groups and residues 26

inportant in the catalytic process.

(f) A description of q-chymotr¿osin car.alysed 29

rcactions and proposed mechanisms.

Page

(i)

(ii)

(iii )

(iv)

1

2

CONTTNTS

RESULTS AND DISCUSSION:

Objectives. 33

Synthetic work. 35

(a) Preparation of inhibitors-acetates and 35

tri fluoroacetates .

(b) Preparation of substituted phenylalanines. - 38

(c) The preparation of fluorotlyptophans. 45

(d) Preparation of cinnamic acids. 54

(e) The synthesis of 3-carboxy-7-fluorodihydro- 56

isocarbostyril.

Quantitation of Chenical Shift Data. 59

The Interaction of Inhibitors wj.th o-Chynotrypsin - 64

NMR Studies.

(a) Bulk sr.¡sceptibility effects. 64

(b) The interaction of inhibitors with DFP - 68

o-cTrymotrypsin.

(c) Proton nagnetic resonance studies. 7L

(d) The interaction of N-trifluoroacetylphenylalanine 73

with o-chynotrypsin.

(e) The interaction of N-trifluoroacetyLtryptophan 78

with a-chymotrypsin.

(f) The interaction of N-trifluoroacetyl substituted 83

phenylalanines with o-chymotrypsin.

P. uæ-

1

2

3

(e)

(h)

CONTENTS

The interaction of the cinnanic acids with

cr,- chymotrypsin.

The effect of enzyne dinerisation and activity

on the binding Paraneters.

A nodel for cr-chymotrypsin inhibition.

The origin and cause of the down field shift on

binding of the 19F ,,r"l"i and the nature of the

Page

95

96

100

102

(i)

(i)

(a)

(b)

(c)

(d)

aromatic and acylanino binding sites.

(k) A nodel for enzyme-inhibitor complexes.

EXPERIMENTAL:

(e)

General

Acetylation of the anino acids.

Preparation of the N-trifluoroacetates.

Preparation of the anino acids and c-aceta¡nido-

cinnanic acids by the azlactone nethod.

Preparation of the anino acids by the nalonic

ester nethod.

5-FluorotryPtoPhan.

ó-FluorotryptoPhan:

(i) fron 4- amino-2-ni*'rotoluene .

(ii) from indoline.

Preparation of the cinnarnic acids.

(f)

(e)

108

111

LL4

115

1L9

L24

131

L33

L33

136

737(h)

(i)

CONTENTS

Preparation of 3-carboxy- 7- fluorodihydro-

isocarbostyril.

Miscellaneous.

REFERENCES.

APPENDIX.

Ptgq

I4L

L44

t47

(i)

INTRODUCTION

1

-1-

Applications of NMR SpectroscopY in the Study of Biological

Problens.

(a) Historical and general.

Although the fj.rst reports of nuclear magnetic resonance (NMR)

of hydrogen nuclei in solids were made in 1946 ,t'' ,, is only in the

last few years that NMR spectroscopy has really developed into a

powerful tool in the elucidation of problems of biological interest.

However NMR spectroscopy was applied to biological systens as early as

1950 by Shaw and coworkers ,3'4'5 M.t"tials such as proteins, carbo-

hydrates, Ðd vegetable tissues, were investigated, but all that was

recorded was a faitLy sharp water lesonance supe:'jmposed on a broad

envelope due to the rest of the protons. This allowed little more than

the water content to be estirnated. Odeblad et al.6'7 obr"rved the sane

phenonena in their studies of tissuer6 "rtd

hunan cervical *.,.,'.7 More

recently it has been shown in studies of tissues that the relaxation rates

of water protons in normal and nalignant tunours are different, and nay

serve as a diagnostic tool.8

Wertz and Jarde tzkyg-12 t""otded the 23N" *o^ spectra of sodiun

ions in solution and d::ew conclusions regardj-ng the extent of thej-r

binding with metabolites, ild state of ionisation; 23r" Ort* has sini-

IarLy been usecl ïecently to itrvestigate sodiun ion transport in

tissues. 13' 14 '

15

Although few meaningful results had been obtained, interest i.n

this new techn.ique was such, that, by the end of 1957, two reviews on

a-L-

the subject had appeared.L6't7 The reasons for this early lack of

success u/ith this technique, when applied to protons, are at once

apparent. The extrenely conplex naterials, studied in the solid state

in sorne cases, æd at low frequency would be expected to give broad

Teson¿mces with a large water resonance perhaps predoninating. Had fine

structure been observed, the assignment of resonances of particular nuclei

could not have been nade because the basic principles harl not been

established. This necessitated the stud)/ of simple molecules, the

building blocks of the complex biological molecules, and the elaboration

of such concepts as chemical shifts and coupling constants. It can be

said that, as these basic principles became appaicnt, then the study of

biological molecules started to yield rneaningful results. Progress in

this field has now begr.n to parallel the developnent of cornrnercial

spectrometers of higher frequency which ate capable of better resolution

and greater sensitivity. Now that 100 MHz machines are readily available,

the nunber of papers in the literature employing this technique in

biological research is enormous; JardetzkylS "stitates over 800 in his

review of LSTL With the ready availability of 220 and 300 MHz instru-

rnents, the rapid rise of pulsed Fourier transforrn (PFT) spectroscopi?'20'2I

especially t3c pFT spectroscopy ,22 ,h" nurnber should increase even more

rapidly.

The theory of NMR spectroscopy and its application to organic

chenistry is well documente &3-27 und theory will not be discussed

except where necessaïy. The basic theory is relevant to all facets of

-3-

chenistry, but the theory, pertinent to biological studies, has been

discussed separatelY. 28-34

(b) The spectla of amino acids, proteins, and nucleic acids.

The infornation necessary foÎ the interpretation of the NMR

spectra of proteins is, in part, enbodied in the spectra of their building

blocks, the anino acids. The first repolt of the spectra of anino acids

appeared in 195 7 ,35 f.ol\owed c1os."Iy ¡y the nore detailed studies of

the Jardetzkys36 and Bovey and Tiers.37 The following years saw many

nore papers appeal on amino acids; these have been extensively revier^red

by Roberts and Jardetzky3S *d Rowe et u1.39 Recently the 220 MHz spectra

of the anino acids have been reported,4o ", well as their 13C

'p""t tu'4L

Even with the spectra of arnino acids known, the interpretation of protein

spectra is sti1l complex. Because of its three-dinensional structure'

the protons of seemingly equivalent residues are usually not in nag-

netically equivalent environments and hence their resonances are not

coincidental. The large number of overlapping resonances thris gives tlre

spectrum of a protein the appearance of a broad envelope.

The first reported spectrum of a protein, the enzyne rjbonuclease,

appeared ín 1957.42 Very few assignnents could be made and subsequent

work by Kowalsky,4s Murrd"I,44 onð, others on proteins did not clarify the

situation. The later work of McDonald and Phittips4s at 220 MFIz was more

informative, but the arnount of information available from protein spect'l:a

was much less than that desired.. In some cases there are a smal1 number

of resonances to high or low fieid of the rnain resonance envelope which

-4-

Ìnay be very informative. Tht¡-s the study of the histidine residues of

ribonucle ^r" 146'47 of. whi ch the resonances of scme protons occur to

lowfield of the main resonance envelope, has resulted in their assign-

*"rrt46 and the proposal of a ribonuclease te"hurrisrn.4B Cytochrome c

has resonances to high and 1ow field of the main resonance envelope4s'49

a-s do other heme proteins.50-53 These can be rronitored in confornational

and other studies.

In some proteins, hydrogen bonding shifts the resonances of the

protons involved to low field of the main resorìaJìce envelope.54'55 The

1ow field resonance in &chyrnotrypsin has brrc'n attributed to the hydrogen

bonded proton between histidirre-57 and aspar.tic acid-102 inferred fron

the crystallographic data.55

With nucleic acids, the complex structures once again lead to

broad uni-nfornative spect"..56 '57 '58 In order to interpret these conpl-ex

spectra, most work has been directed towards their nitrogenous bases,

with evidence being fou'rd for base stacking and hydrogen bondinr.59-64

This work has been reviewed.SS'39'65

It seems that should instruments operating at frequencies higher

than 300 MHz becone available, the direct observation of a protein spec-

trum may not be much rnore informative. ltC N*O spectr-oscopy has been

appliecl recently to ribonu.l*ur"66 and Iysozynei'T the prelirninary

resurts indicate that this technique ma/ 1ris1¿ more information' The

applications of NMR spectroscopy in the study of proteins has been

extens ively reviewed. IB

'28-34 '38 '39 '68 '69 '70

-5-

In order to overcome the difficulties inherent in interpreting

NMR spectra of proteins, several nethods have been devised. Difference

spectroscopy has yielded more inforrnation on ribonuclease,Tl b,r, perhaps

the best method is the study of selectively deuterated proteins.

Deuterated proteins have been isolated fron algae glol.l¡n in DrO; the

residues containing hydrogen atons can be regulated by the addition of1n r*irro acids to the culture nediun. In this way phyco.y^nin7z and

staphylococcal rrrr"l""se73'74 h^u" been studied..

(c) Snrall rrrolecule-macromolecule interactions .

(i) Spectral changes inducecl by smal1 molecules.

In some cases the spectral changes in the macromolecule, induced

by a sma1l molecule (reporter rnolecule) exchanging between free solution

a¡rd a macromolecule-bound state, can be infornative. In this vray the

enzymes lysozyrne, ribonuclease, carbonic anhydrase, and staphylococcal

nuclease have been .studied by the spectral changes induced by inhibitors.

In lysozyme the interactions of an inhibitor h/ere investigated by

monitoring the change in the histidineTs "nd

aronaticTU'" ,.ronalìces.

Similarly with ribonuclease the histidine resonances were studieC in

detail ,78'79'80 ursigrred to specific residues46 *d a mechanisn for the

action of the enzyme ptopor"d.48 With staphylococcal nuclease, the

bi¡ding of inhibitors was studied by enploying the selectively deuterated

"nryr".81 The techniques emproyed tvith carbonic anhydt"r"82'83 ""t"

sinilar to tliose used with ribonuclease. 0nce again observatioll of the

-6-

macromolecule spectrum was feasib-1-e only rvhen there lvere resonances to

low field of the main resonance envelope or selectively deuterated

proteins could be ernployed.

(i i) Spectral chanses induced by ion probes a.:rd the use of ions

as reporter ions.

As rnentioned earlier, 23Nt ,ro spectroscopy has been used to

study sodiun ion transport in biological systetr.lS 't4'LS 35Ct l.lttlR

spectroscopy has also been used to cletermine the environment of chloride

ions in several biological systems.34-87 Those enzynes which contain

a paranagnetic ion have been extensively studied by observing the in-

fluence of the ion on the relaxation rates of protons in the enzyme or

substrate. This work has been recently reviewed by Mildvan and Cohn.88

Kayne and ReuberrS9 h"'t" used thalliun-2O5 as a probe in the study of the

pyruvate kinase reaction by nonitoring the thalliun-205 resonance.

(iii) Spectral cha¡rges of reporter molecules clue to their

association with macromolecrtles.

(o) Relaxation rate and line wrdth studies.

The nost fruitful application of NMR spectroscopy in the elucida-

tion of biological problens has been the study of the reporter molecule

exchanging between free solution and a macfomolecule-bound state. Such

applications have several advarrtages. The reporter molecule r¡sua11y has

a small number of r:esonances which can be uniquely assigned. In the

presence of a macromolecule they will be cliscerníble abo'/e the macromolecule

-7-

signal envelope if the exchange is rapid and the concentration of the

reporter molecule is sufficiently high. Such criteria have usually been

met in the systems so far stuclj-ed. Rosenberg et a1.90 olr"t.ane the

problern of protein resonances masking the reporter molecule resonances

by enploying a fully deuterated protein. In this way it was possible to

study the interaction of the surfactant, sodiun dodecyl sulpltate, with

deuterated phycocyanin.

Jardetzky and cowork"rs9l-94 rtrrdied antibiotic-protein interac-

tions by nonitoring changes in the line wídths of the antibiotic protons

on binding witir p-,'oteins. In this way penicillin-serum albumin inter-

actions9l rrrd others were shown to involve the arornatic ring of the anti-

biotic in hydrophobic binding. The nature of catecholamine-nucleotide

conplexes was also exarnined by NMR spectïoscopy.95 In these cases, the

interactions would produce different relaxation rates in different parts

of the molecules, depending on which parts were involved in the inter-

action. The increased relaxation rate of protons in the phenyl ring

was evidenced by the selective broaciening of their resonances. The

effect of nolecular interactions on relaxation rates and hence line

widths and the applications to pharmacology have been reviewed by

Jardetzky.96 '97

Also studied by relaxation rate processes, ffid to a srnall extent

chemical shift differences have been hapten-antibody interactions,9S'99

enzyme- coenzyme interactior,, , 100 '10l' birrding of sulphoacetanides ancl

sulphonamides to carbonic anhydrase ,t" ' 10t bi.rding of ribonuclease S

-B-

to ribonuclease S peptiderl04 birr,ling of succinate to aspartate tïaris-105

carbamylase, ^-- the interactions of reporter rnolecules with nucleic acid

he1ices,106 u.d substrate binding to G-chyrnotrypri.,,107-111 "u"bo*y-

peptidase A|12 and alcohol dehydrogenases -7t3'I74 The interaction of

nucleotides with ribonuclease has been studied using ptotorrll5 *d 51p

NMR spect"or.opy.116 Sykes and cowork"rr117-722 huu. employed relaxa-

tion rate studies along with chernj-cal shift differences to determine

exchange rates of inhibitors with lysozyrne ,'t'' 118' 119 aspartate trans-

720caroamyrase, o,-chynotrypsin,t27 ürd serum albu ín-122

(ß) Chenical shift differences.

In 1966, ThomasL2t t.potted that the chernica.l shift of the aceta-

nido resonance of N-acetyl-D-glucosanine was a fi.rrction of concentration

in the presence of lysozyne, and that this was due to binding between the

enzyme and sugar. Subsequent work investigated the binding process in

detail using nainly chenical shift differences, but some relaxation

effects .t24-L30 Apart from this, æd the 19F studies of rysozyrne and

other enzymes to be discussed later, there appears to be little work to

date in the biological field which is based primarily on chemical shift

differences due to association. Perhaps the only other quantitative

study has been the investigation cf the binding of serotonin and trypt-

anine to nuclei-. r.idr.131 0f course chemical shift ctifferences have

been used extensively in solute-solvent studies to determine binding

constants ,L32-137 ^nd the trea-tnent of dat.a is essentially the same.

-9-

(v) The infornation obtainable from line width, relaxation rate,

and chenical shift data.

From the preceding two sections it is clear that line width and

chemical shift studies have been carried out on a large nunber of sys-

tens. The parameters attainable are binding constants, cherníca1 shifts

of particular nuclei in the reporter molecule-macromolecule bound state

and, in some cases, exchange rates. The binding constant and shift on

binding for a particular systern are obtained in the follotving way:

For the equilibrium

a+b K

where a is the reporter molecule, b the nacronolecule, and c the complex,

then the dissociation constant for the complex

f'<-

k

c

1

Kn

Ko

t/K =a.b

c

("o-.) (bo-")c

(1)

where ao = initial concentration of a

bo = initial concentration of b

If the exchange is rapid, then the observed chernical shift, ô, will be

weighted aveïage of that in free solution, ôo, and that in the coinplex,

A. Thr:s

ô=pA * ôo

relative to ô as 0. If 6where P is the nole fraction bor.rrd an<l A iso

- 10-

is also neasurecl relative to ô then

ôc

oa

ô

Ã

0

A

o

oc

If this is sr-rbstituted in (1) with the assuntption that the tern in c

is nuch less than the other terns and can be ignored, then

2

^o= + oo¡) - (Ko*bo) Q)

Thus A, the shift on binding and KO, the dissociation constant of the

conplex, can be forrrd fron a knowledge of ao, bo, and ô. As D"t"rr1*urr138

points out, the accuracy of this nethod depends on which form of the

expression relating âo, bo, ô, A, an<l Ko fof which (2) is one form] is

treated graphically. Syk"r121 has overcone this by using a coïlputer

nethod, involving no approxirnations and based on the method of Groves

et al.159 fot the treatment of data obtained in solvent-solute interac-

tions studied by NMR spectroscopy. This will be discussed in more

detail later.

The use of line width and relaxation rate data to determine para-

meters for the system is nore conplex since it is often difficult to

distinguish between exchange broadening zurd that due to ¡nolecular

association. Jardetzky96 h", discussed in detail the use of line width

and relaxatj.on rate data to determine associatj.on parameters. His

assumption that the spin-lattice relaxation tine, T1, and the spin-spin

relaxation tilne, TZ, are equal leads to the sinrple relatiouship,

a

Av, = L/rT^ where Av'_ is the peak wiCth at halfireight. The nethod of4¿-'áSykes for the deternination of the exchange rates is based on spin-

lattice relaxation tines in the rotating frarne ,I40 '141 .orrpled with chenical

shift di.fferences.

- 11-

(d) 19F NMR Studies of Biolopical Systems.

rn 1967, spotswood et ^L.L42

first reported the use of 19r uuR

spectroscgpy in the biological fie1d. The interaction of fluorophen¡'l-

alanines with q-chymotrypsin was studied and the binding consiants and

shifts on binding of the fluorine nuclei in N-acetyl-rn- and p-fluoro-

phenylalanine reported. In the above case, the preferential binding of

the D isorner to the enzyme resulted in the fluorine resonance separating

into two resonances, corresponding to the D and L enantiomers of the DL

inhibitor used. A sinilar result was obtained by Zeffren and Reavi17743'!44

with N-trifluoroacetylphenylalanine in the presence of cr-chymotrypsin.

Syk"s12L'L45 investigated the systenì in more detail and reported exchange

rates. Trifluoroacetylated inhibitors and their 19F .h"n,íca1 shifts have

recently been used in studies of lyso ry^"r46 'L47 ' 148 and further studres

of o-clrynotrypsin .r49-L53 19r Nt* spectroscopy has also been u-sed in

the study of the interaction of fluoride ions with the active site of

carbo4ypepticlase o,1t4 the interaction of 8,8,8-trifluorooctylbenzene-g-

sulphonate ions with serum albuninr155 ah" fluorokinase ïeactiorrrl56

nicelle structure,157 "rd

the binding of trifluoroacetate ions to

aspartate transaminur". 158

-L2-

Raftery ancl colork""r148'159 h"rr" stu<lied conformational changes

in proteins using 19F. l'lt* spectroscopy. Selected residues were nodifiecl

to include fluorine atoms and th-e spectral changes of these nuclei as a

ftrnction of pH, and in the presence of inhibitors, measured. These were

then interpreted in terrns of conformational changes within the protein.

In a similar manner, Bittner and G"tig160 have modified o-chyrnotrypsin

by reaction with the three trifluoronethyl-substituted o-bromoacetanilides

(reaction. presumatrly occurs at rnethionine-I92). The resulting protei:rs

were inactive with the introduced groups nost likely near, or in, the

active site. Relaxation rate measurements then gave the order of

restricted rnolecular rotation of the trifluoromerhyl groups in the three

proteins.

Hturkapiller a¡rd Richardsl6l h"lr" followed the reaction between

pepsin and N-trifluoroacetyl anino acids and determined rate ccnstants

using ln, *ro specroscopy. \

The use of 19r' NMR spectroscopy has nany advantages. There are

no proton resonances of the macromolecules to interfere with the reso-

nances under investigation. Thus the problen of the broad macronolecule

spectrum envelope rnasking reporter molecule re-sonances is rernoved, The

snal1 size of the fluorine atom makes its incorporation into a protein

ideal as such incorporations shculd not, then, drastically affect the

rnolecular structure. This appears to be the case, as modified ribo-

nuclease S was shown to be ful1y active ,I4B '159 and modified hemoglobin

A had essentrally the sane properties as the tnmodified ptot"in.14B It

-t3-

is unfortunate that there are no naturally occurring fluorine containing

rnacrornolecules " Introduction of fluorine then produces a systen which

only approxinates a natural systen. However, the results so far,

indicate that this is an excellent approxirnation. 19F Ch"tical shifts

are also spread over a nuch wide" t"rrg"162'L63'L64 (150 p.p.rn. as against

15 p.p.n. for protons) and this reduces the problem of assignnent due to

overlapping resonances. However the nost important advantage is the

sensitivity of 19F .h"rical shifts to envitont"nt.25 small changes in

the environment of a fluori.ne nucleus can have a significant effect on

its chenical shift. Thus 19F .h"tical shifts serve as a .sensitive

probe in the orientation of an inhibitor in the active site of an enzyme.

a

-14-

The Chemistry of o-Chyrnotrypsin.

(a) General.

The enzyne chymotrypsin Ao occurs in rnammalian pancreatic juice

as its inactive precursor (zymogen) , chynotrypsinogen A. The zyrnogen is

enzymatícally activated in the duodenum by trypsin to give the various

forms of chymotrypsin A (o, ß, y, ô, and n forms).165'766'L67 For

sinplicity the o, ß, y, ô, and n forms of chynotrypsin A will be referred

to as o-chynotrypsin, S-chymotrypsin, and so on instead of chynotrYpsin Ao

chymotrypsin AU. Also occurring in the pancreas are precursors of a

nurnber of other enzymes; trypsinogen, chynotrypsinogen B, and proelastase,

which are also activated in the duodenun. Thosc enzynìes which have been

found to contain a reactive serine residue, inportant in the catalytic

process have been called the serine proteinasesr16B tlri, group includes

the chymcltrypsins, elastase, and thrombin. Other serine proteinases are

fotnd in other organisms and plants, and some, such as subtilisin BPN1,

are found in nicroorganísms.

In the main these are digestive enzymes; they hydrolyse peptide

bonds in proteins with remarkable activity and specificity. o-Chyrno-

trypsin hydrolyses the peptide bonds on tlìe carboxyl side of residues

with aronatic side chains, i.e. the carboxyl peptide bonds of phenyl-

alanine, tyrosine, ffid tryptophan residues. Trypsin is specific for the

carboxyl peptide bonds of basic amino acid residues, i.e., lysine and

arginine residues, while elastase is specific for smal1, rmcharged

amino acid residues. Thus these three enzymes are complementary in

- 15-

their specificities. Some serine proteinases have sinilar specifbities,

for example subtilisin BPN1 and o-chyrnotrypsin. Some have si-nilar amino

acid sequencesl'69'770 ancl sinilar three-dimensional structures, especially

in the region of the active site.LTt'L72'173 rt would seem then, that

the difference in specificities must be inherent in subtle differences

in the residues aror:nd the active si-te. Such sinilarities h¿rve also

invoked the idea of enzyne evolution .!74 '775 The homologies of amino

acid sequence and three-dinensional structure, as well as the subtle

structural differences between the zyrnogen'and free enzymerlT6 ,horrld

be of great imporr-ance in the elucidation of t-he catalytic processes.

In fact, a consideration of these processes is best carried out in terrns

of these honologies.

(b) Amino acid sequence and three-dirnensional structure of o-

chymotrypsin.

o-Chymotrypsin is a large nolecule; it has 247 amino acid resi-

dues and a molecular weight of 231000.t77 The prinary structure, the

amino acid sequence, was detennined by HartIeyLTS't79 from studies of

chymotrypsinogen, and apparently confirmed by Melor;n "t ul.180 However

a correction has been made in which residue 102, originally designated

asparagine. u/as found to be aspartic r.id.181 This was of great irnportance

as the catalytic activity of the enzyme could then be explained as due,

in part, to the increased nucleophilicity of serine-195 brought about by

the charge relay systern (Fig. 1) which was invoked; the residues involved

being in bonding distance (from the X-ray diffraction data).

ASP-10 ,¡

H IS-57

- 16-

Fig.1.

Charge relay systen, pHB.

As P -142

+---ù

HlS"57

SE R-19 5

181

o o

Ìa

HI

N

't

I

oI

H

N NER-t95.rù'tj o

canonical forms

o-Chynotrypsin is one of a number of enzymes of which the three-

dinensional structure has been deternined by X-ray diffraction' 182-185

these include elastase ,r7L '186 ' 187 trypsin, 1BB a"o subtilisi nt ,172 't73 '

L89-L92 and y-chymotrypsin. L93'794 'Ihe structure of chynotrypsinogen

has also been dete"tit"d.176 The three-dinensional structure of o-

chynotrypsin was deternined as its tosyl derivative by Blow and co-

. 19s- 199workers-"" -"- using X-ray diffraction techniques. In this derivative

the catalytically inportant residue, serine-195, had been tosylated.

The benzene ring of the tosyl gloup was formd to occupy a cleft in the

nolecule which has been called the'rtosyl hole'r. The'rtosyl holetrhas

also been shown to be the site of the indole ring of N-fornyl-L-

tryptophan in the X-ray diffraction studies of the N-fornyl-L-tryptophan:

-t7-

o-chynotrypsin comp1"*.200 These studies also showed the probable

existence of a hydrogen bond between the amido hydrogen of N-fornyl-L-

tryptophan and the carbonyl oxygen of serine -214 in the enz ne.199'200

The t'tosyl holeil was also occupied by the indole ring of indoleacryloy1-

u-chymotryprirr20l "rrd

the aromatic rings of other inhibitors in enzyme-

inhibitor "orpl"*"r.200 N-Formyl-L-tryptophan can be considered a virtual

substrate by analogy with N-acetyl-L-tryptophan which forlns an acyl-

enzyme as evidenced by 1BO

"*"hunge between the carbo4¡l group and the

202.203enzyne .'vL 'avr From the X-ray diffraction data it is difficult to say

whether: the N-r-orlyl-L-tryptophan: c-chynctrypsin couplex is an acyL-

enzyme; the coorclinates of the carboxyl group of the virtual sùbstrate

are not sufficiently well defined to deternine whether a bond exists

between it and serine-195. Indoleacryloyl-cr-chynotr)-psin is, however,

a stable acy|-enzyme which has been characterised.204'205 The crystalLine

acyl-enzyme, when dissolved, was shown to be the sane as the acyl-enzyne

which can be prepared in solution. Also deternined have been the struc-

tures of s-chymotrypsin in which histidine-57 had been neth tat"d206

and carbamyl-clt-chynotryp =in.207 Quite recently, Birktoft rttd Blow,20B

by refinenent of the X--ray diffraction data, have deternined the struc-

ture of o-chymotrypsin in nuch nore detail.

The homologies of anino acid sequence and crystal structure of

o-chynotrypsin, trypsin, and elastase make it possible to explain their

different specificities, by reference to the nature of the binding pocket

174.188in each.¿' r'rvv The hydrophobic binding pocket (r'tosyl holerr) of cr-

- 18-

chymotrypsin is large enough to accommodate an aromatic ring. In trypsin,

the carbo4¡l side chain of aspartic acid-189 is at the bottom of the

cleft. Under the conditions where trypsin is active, this will be nega-

tively charged and hence capable of bonding the ammonium ion of the

substrate side chain. In elastase, the resiáue at the mouth of the

pocket is valine-276. The steric size of its side chain will exclude all

but the smallest side chain, This is represented in Fig. 2 (after

shottonlT4¡ .

NH

>rCl{3

CH3-_,^

cH3'"

Tyrosinesubstrate

Lysinesubstrate

ASP18O

trypsinbinding pocket

Alaninesubstrate

oll_-¿C_ NH

OH

2

SER- I95

GLY-216

H 6¡y-226

-NlÉ-CH o{

CH 2

SE R.105

VAL-216

c THRE-?26

Rc

CH 2

H5

6CH,IxNHa

LY-z1

-2ER

G

t'H 26OH

cHlOHI

OH

OHI

c H2

sER-1Sl

chynotrypsinbinding pocket

+

Hc

o

oH

¿I

c

o-,

2

SE R-189

elastasebinding pocket

Fig. 2.

-19-

Ihe preceding structures involving o-chyrnotrypsin were all de-

termined at pll"v4. Vandlen and Tulinsky209 huu" reported X-ray diffrac-

tion studies of o;chyrnotrypsin at pH 3.9 and pH 6.7.

The enzyne-snal1 molecule corìplexes only approxinate the complexes

which must occur in the hydrolysis of large peptide substrates in vivo. In

these cases secondary binding sites probably become itpotta.t.210 To

this end, Huber et al .27I'2!2 h^u" rletermi.ned ttre stïucture of a naturally

occurring peptide, bovine pancreati-c trypsin i-nhibitor, which inhibits

both trypsin and cx,-chymotrypsin. From a knowledge of this structure and

that of cl-chynotrypsin, a model for the assccia.Cion between the inhibitor

and enzyne has been propor"d.213

(c) The relevance of crystallograph ic data to the mechanism of cr-

chynot sín catalys ed reactions in solution.

The large amount of inforrnation on the prinary, secondary, ffic

tertiary structure of a-chymotrypsin is of a comparatively ïecent nature.

Workers have been trying for over thirty years to elucidate the mechanisnt

of a-chynotrypsin catalysed reactions, æd determine the amino acid resi-

dues important in the catalytic process using physico-chemical means.

The information gained from crystallographic studies, especially t}:at

from the enzyme-virtual sr:bstrate , enzym?'inhibitor, Ðd acyl-enzyme

conplexes, is complenentary to the physico-chernical results if the enzyne

has the same structure in solution during reaction as it has in the solid

state. Kal1oszt4 t"¡to"ted low activity for reactions catalysed by solicl

cl-clrymotrypsin, but moïe recently Sluytermann and de Graaf ,2I5 und Rossi

-20-

and Bernhurd2'L6 have concluded that the solid enzyme has comparable

activity to that in solution. Comparable activity would most certainly

suggest comparable geonetry of the active site. The X-ray diffraction

studies also indicated only snall dj-fferences in the position of residues

arotrnd the active site between the native and inhibited enzy¡nes .200 '20I

There seems litt1e doubt then, that the structures in the solid state

and in solutj.on are similar, and that therttosyl holerris the arornatic

binding .siteHowever as rnentioned earlier, these results apply to the enzyme

at pHn4 whereas the catal.ytic activity is greatest arotnd pH 7.5.2t7

Vancllen and Tul-inrty209 have reported changes in crystal structure l¡etween

pH 3.9 and pH 6.7 comparable with the changes which occur on binding,

but neither details nor the magnitude of these changes lvere gi.len. Shiao

and Sturt evant2LS '2I9 have concluded fron their calorinetric studies that

binding of an inhibitor induces conformational changes in the enzyme.

This conclusion has been reached by other workers employing different

techniques.220-223 Since binding of inhibitors rnost certainly occurs

at the active site, these confornational changes must involve the active

site. This, however, is difficult to reconcile with the sna1l changes

which occur on binding, as evidenced fron the crystallographic data.

Evidence also exists for the occurrence of two conformations of

cr-chynotrypsin in solution with the inactive form predorninating at high

pH.222 '224-228 Th" x-ray diffraction results of vandlen and tulinsky209

support this change of conformation with pll.

-21-

A problern which is not solved by X-ray diffraction is that of

association of the enzyme in solution. There is much evidence for the

existence of diners ancl higher oligorners in solution .2IB '229-235

Different workers have reached different conclr¡sions regarding the

properties of these oligomers. Fa1ler and LaFon d233 '234 conclu<led that

only the monomeï was active and could bind an inhibitor. Martin and

Nierunrr256 concluded that the dirner was capable cf binding substrate,

but such complexes could not lead to product. The conclusion of Inagani

and Sturt "uunt237

was that the oligomers weïe partLy active whi.le Sarfare

et a|.238 "orr.luded

that oligonerisation did qot affect access to the

active site, i.e., the diner contains two active sites of normal activity,

the triner three active sites, md so on. Kézdy and Bendet23s "ot.1uded

that the dimer was inactive but readily activated in the presence of

substrate. These points will be further nentioned in the Ðiscussion.

(d) The activity and specificity of o-chymotrrpsin towards organic

molecules.

o-Chyrnotrypsin in solution hydrolyses, with narked activity and

specificity, the esters and amides of the acyl derivatives of the

naturally occurring aromatic amino acids. These can be considered

chernical substrates as distinct froin the nore conplex natural or physio-

logical substïates, the proteins. Atternpts to elucidate the nechanisn

of cr-chymotrypsin controlled catalysis ltave, for the most part, involved

the study of these chenical substrates. As such, ãny proposed mechanism

-22-

reaIly only applies to chenical substrates. Being smaller and sirnpler

molecules than the natnral substrates, they provide i.nformation only

about the catalytic site. I{ith the rnore cornplex peptide substrates,

secondary binding sites (subsites) probably becone irnportant in binding

the peptide to the enzyne surface, with the bond to be cleaved at the

active site.27o '239 '240 Morihara et al .24'J''242 h^u" shown the existence

of subsites in cx-chymotrypsin and srùtilisin BPN1. Subsites have also

been shown to be inportant in the cleavage of pepti.des by pupun,243

. 2I0 .244pepsln , ' carbo4¡peptidase o,t45 and erastase .246'247 Nevertheless

the seemingly ideal situation of using simple chenical substrates, instead

of the larger peptides or proteins, still leads to complex result.s.

The reactivity and specificity of chenical substrates towards

or-chynotrypsin has been extensively review"d,165 '168'248-255 brt th"

salient features will be discussed. As nentioned earlier, the enzyne

hydrolyses the esters and amides of the acyl derivatives of the naturall-y

occurring anino acids with high activity. The L isoners are rapidly

hydrolysed, while the D antipodes are virtually u:rreactive. The speci-

ficity is relative, not absolute; the D antipodes are hydrolysed at

rates of the order of 104 slower than their L counterparts. Also the

esters are hydrolysed nore rapidly than the amides. The aromatic ring

and the anido hydrogen are necessary for this high reactivity and

specificity. Replacenent of either of these groups l.eads to greatly

reduced reactivity with rather rnpredictable specificity. This has been

summarised in Tables I ancl II (due to Coh"n248¡.

-23-

TABLE I.

Hydrolysis of nethyl ß-sribstituted o-acetanidopropionates R CH, CHCO

NHCOCH

by o-chynotrypsin.

zc[s

3

ß-substituent, R_1 -1k".t/\ut (M 'sec *) Reference

CH t L

oHs

3N2

6.2 x L0

256

2574c L

TABLE II.

Hydrolysis of nethyl o,-substitutéd ß-phenylpropionates CUHTCHTCHCO2CHS

I

by a-chymotrYpsin R

o-sribstituent, R kcat

Reference-1 -1/KM (M ^sec ')

-NHCOCH 3'-H

-0c0cH

-cH2co

-cHg '-0H, L

-oH, D

L

3,L2R' ¡L

6.2 x L0

154

26b

gâ'c

sã'c

4

3b

257

258

259

258

258

259

259

260

q, lx lC

1lb--dó5-cl, D,L

a 10% ethanol.b 20% etli.anoL.c hydrolysis of L enantiomer of DL mixture.d 2C% methanol.

-24-

A similar table describing the effect of the ß-substituent on the rate

of reaction has been cotnpilecl by Krro*l"r.255 The const*a, k.ra. and K"

are defined and their neanitrg discussed in section II (f).

Of particular interest are the esters of 3-carbo4¡dihydroiso-

carbostyril (3-carbo4¡-1-keto- L,2,3,4-tetrahydroisoquinoline) (I) of

(r)

whiclr the D antipodes are hydrolysed at a :rate compa.rable with the esters

of N-acetyl-L-phenylalanine .261'262 This surprising re'¡ersal of speci-

ficity has 1ed to nuch work being done with this and similar compouilds

which can be clesignated substrates of restricted confornational freedon.

A conparison of the structure of (I) with that of N-acetylphenylal.anine

(II) shows the similarity of structure, with structure (I) having much

less confornational freedon and hence a restricted reactive conformation.

The similar reactivity, but different specificity, frãY then enable a study

of the behaviour of (I) to assist in the elucidation of the reactive

H

o

-25"

CO2H

CHr\-J

NH

(II)

conformation of (II). In order to define the conforlnation of (I) nore

precisely, Abdullaev et aI.263 have measuïed the isoquinoline ring

coupling constants in order to deternine the dihedral angles, ffid hence

confornation, in non-polar solvents (analogcus to an hydrophobic active

site). Cohen et al .248'258 and Silvet et aL.264 hur" exanined the

hydrolyses of other cyclic substrates in the presence of o-chynotrypsin

in order to elucidate the catalytic nechanisn. Cohen and cowork ers248 '258

have interpreted these results in terrns of an active site model with five

loci. Nienann and cowork.rt252'262'265 hru" explained the specificity

of o,-chymotrypsin in terns of microscopic binding sites. These loci and

rnicroscopic binding sites have yet to be identified rvith the fi-inctional

groups and residues important in the catalytic as determined by other

c'ilo

means.

The activity of a-chymotrypsin tolvards subsLrates is inhibited

-26-

by a large nunürer of compounds. A nunrber of these react with the enzyme

in a 1:1 ratio to give a stable, inactive protein ,'UU'267 '268 for example

the dialkyl fluorophosphate r,26g sulphonyl halides ,270 tulphonic acid

sulphones ,27L o-bromoacetanilides ,272 '273 .ltlotornethyl ketones ,274 und

carbarnyl chlorides.275 These are irreversible inhibitors. Apart from

these, the remainder of inhibitcrs fa1l into the category of competitive,

non-competitive, oï uncompetitive;276 by far the largest nurnber falling

into the category of conpetitive inhibitor. These bind at the active

site in competition with the substTate, i.e., they compete effectively

with the substrate for the active site. Since the enzyme-inhibitor

complex is non-productive; it does not lead to products, the reaction

between enzyme and substrate is slowed. The nost important are deriva-

tives of the N-acyl aromatic annino acids277-280 and a large number of287-284

aromatic compounds.

(e) The nature of the frnctional s and residues imPortant in the

cataTytic process.

Evidence so far has shown the frmctional groups of five residues

to be inportant in the catalytic pïocess '254 '285 this evidence also

correlates well with the stereochemistry and nature of residues arotrnd

the I'tosyl hole'r as determined by X-ray diffraction.

o-chynotrypsin reacts with dialkyl fluorophosphates in a 1:1

ratio. . The product of the reaction, the DFP-chynotrypsin, is an

enzymatically inactíve prot e'n.266-269 Degradation of this protein yielrls

a phosphorylated serine peptide.286 Cornparison of the sequence of this

-27-

peptide with the known sequence of the enzylrie leads to the lesult that

this is serine-l9S. Kirretic experirnents impiied the existence of a

catalytically important residue of which the side chain had a pKa n' 7.

This was ttrought to be the irnidazole ring of an histidine residue.

Reaction of the enzyme vrith the chloro- and brononethylketones derived

from N-tosyl-L-phenylalanine produced an inactive protein. Degradation

yielded a peptide in which one histidine had reacted with the y"tonZZ!'286

This was for¡nd to be hisiidine-S7. The fact that acetylation of the

free amino group, forlned in the activation step, results in the destruc-

tion of enzymaiic activity makes this residue important in the catalytic

process. This had also been inferred from kinetic studies - a gr.oup

with pKa,r,9 was found to be important and could only be a free amino

2B7.2BBgroup lhis is isoleucine-16. X-ray diffraction studies indicate

that the cl-arnmonium ion of the residue forms a salt bridge with the

carboxylate ion of aspartic acid-194, thus forming an hydrophobic.binding

pocket for aromatic side "h"irrr.?89 Deprotonation of this anino gïoup

above pH 9 would thus lead to the destruction of the salt bridge and

hence the hydrophobic binrling site. Thus the high pH, inactive torr?za

of the enzyne could be produc "d.225 Modification of a nethionine residue

(shown to be nethionine-192) was found to affect the enzyne-substrate

affinity but the enzyme was still appreciably t"tile.2B5 It appears,

then, that this residue may not be critically irnportant. The cïystal

structure of the enzyme indicates that nodification of this residue rnay

hincler access to the actir¡e site, as it is at the entrance. The importance

-28-

of a proton donor in o¿-chyrnotrypsin catalysis has been proposed.292'293

The recent arnendment to the amino acid sequence and a considerati.on of

the three-dinensional structure of the enzyne rnakes this proton donor most

likely aspartic acíd-702. The X-ray diffraction results show that

aspartic acid-1-02 is in the correct orientation to aid acylation of

serine-195 by increasing its nucleophilicity (the charge relay system181

Fig. 1).

Weiner and Koshl ürd294 have testeci the hypothesis that, in the

chenical nodification of the serine residue, loss of activity nay be

the result of the bulkier nodified group preventing access to the active

site (as is most certainly the case in the nodification of methionine-

L92). To this end, they have removed the elements of water fron the

serine residue and found the resulting enzyme to have appreciable binding

activity. This result has recently been reported by other *otk"tr.295

As access could not have been restricted relative to the native enzyme'

the hydro4¡nethyl group cannot be criticall.y irnportant in binding of

inhibitors.

From these results, it is clear that the enzymatíc activity results

fron folding of the three protein chains which brings the catalytically

irnportant residues, serine-195, histidine-57, md aspartic acíd-702 ínto

the correct orientation. Serine-195 is an important catalytic residue,

but nay not be critical for binding of inhibitors. The confornation of

the aronatic binding site and hence the active site, is controlled by

the isoleucine- 1-6-aspartic acid- 194 salt bri dge. Meth j.onine- 192 is at

-29-

the entrance to the active site.

(f) A descript ion of o-chyrnotrypsin catalysed reactions and proposed

rnechanisms.

A good Tepresentation of the reaction of substrates with cr-

chynotrypsin is the double displacernent reaction ,"t'296'297 '298 (scherne

I) where E represents enzyne, S the substrate, ESt the enzyme-sub.strate

E+S ESI

ESI EStt + Pl

E+Prf

Schene I.

complex, ESrr the acyl-enzyme intermediate, Pr the leaving grouP (an

alcohol or amine depending on whether the substrate was an ester or

amide) , æd Ptr the carbo4¡lic acid. Such a descríption is, however, not

a mechanism. A nechanisn, by definition, applies on an atomic leve1 and

is a description of all atornic parameters over the whole course of the

reaction. This description is irnportant, however, as thorough kinetic

evidence serves as the basis for reaction mechanisrns. In this case,

which is rare in rnechanistic studies, the physico-chenical evidence can

Ksk-><-

k

k->+k

2

5'+ESII

k--5

1

1

2

-50-

be directly compared with infornation on one of the reactants (the

enzyme) gained by X-ray diffraction studies '

In the case of competitive inhibition by N-acyl amino acids,

the enzyme-inhibitor cnmplex probably approxirnates an enzyme-sub'strate

conrplex or, in some cases an acyl-enzyme internediate, for example,

inhibition by virtual substrates .202'2'03 The major binding interactions

involve the aromatic ring and the anido group' a¡rd it seems logical that

D and L isomers of a particular inhibitor Ì:ind in a sinilar way át the

active site with regard to these substituents '

In o-chynotrypsin controlled catalysis of esters, deacylation

of the acyr-enzyrne intermediate is the rate-deternining step.299'300'30r

This may not be true for natural substrates; in fact in the hydrolysis

of anides, acyLation of the enzyme is the rate-deternining step'302'305

With esters, then, the acyl-enzyme is a very inportant internediate which

is reasonably stable - in some cases it can be isolated. Thus enzyne-

inhibitor studies, evelì those involr¡ing inhibitors of the unnatural (D)

configuration, should be of gïeat benefit in elucidating the first step

of the nechanisn.

The double displacernent reaction can be considered in a sinpler

forn (Schene II).

\'4 kcatl

E+S + ES.

->

E+P

Schene II.

In this case, the measurecl Michaelis-Menten constant is afì apparent

- 5l-

constant so that f, (ann) = KM ( k

k kz 2973*k3 )and kcat

2 3+ )

where k is the overall rate constant. This is the definition of thecat

parameters used in section II (d) to show the relationship between struc-

ture and reactivity of substrates. k""a/\, is measure of the efficiency

of the hydrolysis.

Recent v¡ork has shown that, in the hydrolysis of amides, there

exist other eintermediates before the acyl- enzyme internedi rtu.222'30L'

306-309 A similar situation has been shown in the hyclrolysis of N-

acetyl:N-methyltyrosine nethyl ester. 510

Fron the wealth of data available there seems lit'ule doubt that

the catalytic reaction involves acylation of serine-195. Early evidence

which purported to show acylation of irnidazol"311 '312 cart be repudiated.

The involvenent of the inidazole ring of histidi-ne-57 has been postulated

to occur in several ways. In conjurction with the inidazole ring of

the neighbouring histidine-56, a concerted acylation of histidine-57 was

ptopor"d312 (schene rrI).

R

2

N\.

llo

HN

^H

Scheme III.

X

-32-

Bender and t<6zdy3t3 proposed the involveaent of the two histidine resi-

due inidazole rings as charge transfer complexes. These two postulates

can be ruled out however. It has also been suggested that activation of

serine-l-95 involves L2-oxazoline fortttion.314 This, and other

mechanis¡ns involving heterocyclic active sitesr"t .* also be <lismissed,

The occurrence of oxazolinone internediates has, however, been postu-

1ated.316

As nentioned earlier, the X-ray diffraction studies explain the

high nucleophilicity of serine-195 (the charge relay systen). The use

of this charge relay system then overcomes a weakness of the pre-

transition state protonation theory in the mechanism of l¡Jarrg and

Parker.SLT'st\'319 Nevertheless, the nechanistic pïoposals are stil1

fairly complex, especially in the case of anides, ffid reference is ¡nade

to the individual papers .302 '306 '307 '3L3 '376 '320

RESULTS AND DISCUSSION.

-33-

0bj ectives .

Fron the preceding introduction it can be seen that, although

a vast amount 9f work has been carried out in atternpting to elucidate

the nechanism of or-chynotrypsin catalysis and much has been achieved,

there are still nany questions to be answered. NMR spectroscopy has

been shown to be a powerful tool in the elucidation of problems of

biological inte::est; especially in regard to sma1l nolecule-macro-

nolecule interactions of which enzyme-inhibitor interactions aÍe o]ìe

example. Quantitation of chenical shifts a¡rd line widths of inhibitcrs

binding to enzymes, then, can potentially give the environment of the

nuclei under investigatj-on and hence the orientatit¡n of the inhibitor

in the active site. Thus enzyrne inhibition can be studied by NMR

spectroscopy and, if the inhibition can be shown to resernble the first

stage in the catalytic mechanism of the hydrolysis of substrates, then

information on this nechanism can be gathered.

19, *to spectroscopy has also been shown to be of use in the

elucidation of these problerns if fluorine atoms can be introduced as

probes without serious disruptions. Moreover the use of 19p NMR spec-

troscopy removes the nuisance of the background I'envelope'r of the protein

ïesonances and, in some cases, the solvent resonances.

Thc ain, then, was to synthesise various fluorinated inliibitors

of cx,-chyrnotrypsin, for exanple N-trífluoroacetylated phenylalanine,

tryptophan and their clerivatives as well as several cinnamic acids, and

to study their interactiolt with the enzyne by nonitoring th" 19f' reso-

-34-

nances. From these studies it was hoped to be able to fornulate a nodel

for the enzyrne-inhibitor conplex fornation'

-35-

1 Synthetic l\rork.

(a) Preparation of inhibitors - acetates ancl trifluoroacetates.

Phenylalanine was most conveniently acetylated with acetic

anhydride in dilute acetic acid according to the nethod of To*n.322

The reaction carríed out over 30 minutes at 40o, gave cornparable yields

of products to those reported in the acetylation reactions r.mder basic

conditiont ^t 0o.323 However wi.th tryptophan, this reaction was trnsatis-

factory as the product was often coloured and of a low purity. Acetyla-

tion of tryptophan under weakly alkaline conditions at 0o according to

du Vigneaud and Sealock324 g u" excellent yields of the N-acetyltryptophans.

The results are surunarised in Table (III).

TAtsLE III.

Conpound Method ofPreparation

Yield (%)

N- acetylpheny 1 a1 anine

N-acetyl-D- rr

N-acetyl-L- rt

N- acetyltrypt ophan

N- acetyl -D- rr

N-acetyl-L- rr

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

322

322

322

322

324

324

74

72

68

81

73

70

Trifluoroacetylation of phenylalarrine and its derivatives was

best achieved using trifluoroacetic anhydride in dry benzene .325 '326 '327

The reactions were rapid; the yields in nost cases were 70-B0eo and

-36-

purity was excellent as evidenced by the fact that further recrystallisa-

tion clid not raise the nelting points more than two degress, if at aII.

ülitli tryptophan, the acid instability of the indole ring was again

encountered and this method was unsatisfactory. The nethod of Weygand

z.t oand Geiger"' employing trifluoroacetic anhydride in trifluoroacetic

a.cid/ether, at 0o, was also rursatisfactory. However the nethod of

Schallenberg and Calvin ,t29 "rrrployi-ng

ethyl thioltrifluoroacetate as

tine acyTating agent gave excellent yields of the desired compounds when

the reaction tirne was lengthened from 24 to 72 hours. The results are

sumrnarised. in Table IV. With tyrosine, it was found tlnat a twofold

excess of trifluoroacetic anhydride was necessary to obtain good yields

when the reaction was carried out in benzene. Using one equivalent of

trifluoroacetic anhydride, or trifluoroacetic anhydride in trifluoro-

acetic acid/ether according to the literature ,t" -"n" yields were lorv.

With the two equivalents of acylating agent, no evidence was found for

O-acylation. Recently weyand and Rdps"h530'33L 1'u o reported the

conversion of most amino acids to their N-trífluoroacetates in good

yields using phenyl trifluoroacetate.

An attenpt was nade to prepare N-trj.fluoroacetyl-D-g-rnethoxy-

phenylalanine by the silver oxide catalysed methylation of l{-trifluoro-

acetyl-D-tyrosine with methyl iodide in acetone. Under the reacti-on

conclitions, the trifluoroacetate noiety was retained, but the product was

the nethyl ester. This is not surprising when compare<l to the work of

Mehta,332 *ho has enployed nethyl i.odide in the preparation of rnethyl

esters of a number of carboxylic acids.

N-trifluoroacetateof

-37 -

TABLE IV.

Method ofpreparation

Yield (%) n.p

phenylalanine

phenyl al anin

D-phenylalanine

L-phenylalanine

n-nethyl phenyl ai anine

ffnethylpþenyIa1anine

o- fluorophenyl a'!. anine

n- fluorophenyl a1 anine

g- fluoropheny 1 al anine

n-bromophenylalanine

p-bronophenyl al anine

2, 4 - di fltoropheny I al an j-ne

¡n- t ri f 1 uo rornethy lphenY 1 -alanine

p-t ri f1 uorornethy lPhenY I -alanine

phenylglycine

trypto;ohan

D-tryptophan

L-trptophan

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

Ref.

327

329

327

327

327

327

327

327

327

327

327

327

327

327

327

329

329

329

74

76

68

72

7L

76

6B

73

73

B8

85

67

82

76

67

75

73

66

L23-LzS

L26-L2B

L6s-t64

L6s-L64

o

o

L20-Lzro

119 - 1200

Lr6-tfio

L34.5-L360

132-L330

119- 1200

L44.5-145.50rss-1370

760-L620

tsL-t330

L43-L450

t42-L440

159 - 1600

155-1570

o

o

(b)

- 38-

Preparation of substituted phenylalar'.ines.

i) Tlie azlactone nethod.(

The fa.ct that benzaldehyde condenses with hippuric acid in the

presence of sodiun acetate and acetic anhydride to give 4-benzylidene-2-

phenyl-5-oxazolone (I) was reported by P1öch1535 'n 1883. Et1"r,*"y"t534

subsequently discovered that the oxazolone (azlactone) could be reduced

to give phenylalanine (Scheme I).

CH^CO^H

l'¿RCHO+

R = C-H--65

P lHr

->

R-CH=C-CCOCH( )zo 4.o3

NHCOR cHrc0rNa

R-CH^CHCO^Htl ¿

NHz

/\*\rroI

R

(rr r)

Schene I.

The reaction also occurs between benzaldehyde, aceturic acid (acetyl-

glycine), sodiurn a,cetate, and acetic anhydride to give 4-benzylidene-

2-nethyl-5-oxazolor'r",535'336 *iní.h is best converted to the anino acid

in 3 steps; nanely hydrolysis to the o-acetamidocinna¡nic acid with

aqueous acetotìe, hydrogenation to give the N-acetyl amino acid, and

finally acid hydrolysis (Scheme II).

-59-

cH.co^H|"

,L(c[|co)

20R-CHO +

(il)

R = aryl

(CH

4,oNHCOCH

3

R-CH=C-C0

(VI)

R-CI{=C -Ccrlrc0rNa I*\ ,T

clc Hg

(v)

COìzPd/c

R-CH2 CHCO

NHCOCH

(vrr)

211 2HHzo

CHNHCOJ

+H

R cHzCHCO

I

NHz

2H

(vr r r)

Schene II.

This procedure was adopted initiall-y as the aldehydes were available,

or easily prepared, and some o-acetamidocinnamic acids and acetyl anino

acids were required. As the stability of the 2-nethyl-5-oxazolones is

considered less than that of the 2-phenyl analogues ,tt7 '338 thi, probably

accoturts for the lower yields obtained in the first step of the reaction

(of the order of 50%). Atternpts to inprove the yields of azLactones

v¡ere unsuccessful and purification was difficult as they had to be freed

of water and trnreacted aldehyde. The IR spectra also indicated that

hydrolysis to the cx,-acetalnidocinnamic acid had occurred to some extent.

-40-

As this was folrned in the next step of the reaction sequence, its

removal was not necessary. The next step proceeded readily with the

crude azlactone and the high neltíng point. of the product formed nade

purification by recrystallisation sinple. Xhis, and subsequent steps,

proceeded in excellent yields. The nethylphenyl and n-trifluorornethyl-

phenylalanines, as well as o-acetanidocinnamic and a-acetamido-$- (g-

fluorophenyl)-acrylic acid were prepared by this nethod. The results

are surnmarised in Table V.

TABLE V.

Yields (%) of compounds formed in azlactone synthesis(Scheme II)

Aldehyde

II

Azlactone

III

Acetanido-cinnarnic acid

IV

Acetyl-phenylalanine

V

Phenyl-ala¡ine

VI

Benzaldehyde

n-tolualdehyde

p-tolualdehyde

n-tri fluoronethyl -- benzaldehyde

52

50

s4

67

82

85

77

76

81

77

81

91

98

5386

g- fluorob enzal-dehyde

56

(ii) The nalonic ester rnethod.

In 1945 it was simultaneously reported by Albertson and At.h"",339

and Snyder et a1 .340 th^t benzyl chloride reactecl r^/ith diethyl acetaniclo-

-41-

malonate in the, presence of sodiun ethoxide and that the product of the

reaction unden¡ent acid hydrolysis to give phenl'1alanine. As both

starting materials were readily available and the yield in each step rvas

excellent, this constituted probably the sinrplest and best method for

preparíng phenylalanine. It has been shown by Bennett and Ni"t*ns41

tbat this method is superi.or to t.he azlactone method fo:: the synthe"sis

of fluorophenylalanines. These workers have also shown that in the

preparation of substituted phenylalanines by the azlactone nethod,

transacylation occurs in the step leading to the oxazo \on..342 This

lowers the yield of the desired oxazolone still further and nakes the

nalonic ester synthesis rnore attractive. The najority of anrj-no acids

used in this work weTe prepared by this nethod which is outlined in

Schene III.

Of these, only g-'bronophenylalanine had been prepared previously

by this nethod but no details were given.343 p-Trifluorornethylphenyl-

alanine harJ previously been prepared by the azlactone meth od,344 but

this present nethod produced better yields in a snaller nuinber of steps.

The synthesis of g-trifluoronethylphenylalanine is represented in

Scheme IV. Conversion of (XI) to (XII) and (XIII) to (XIV) both involve

strongly acidic conditions. Llnder these conditions, no hydrolysis of

the trifluoromethyl group to a carboxyl group was detected. This agrees

with the results of Filter and Novar344 ,ho prepared rn-trifluoïomethyl-

benzyl bromide from m-trifluorobenzyl alcohol rmder acidic conditions.

However several workers have reported hydrolysis of this group with

CH 29r

-42-

R

c H2

+cH (c OzczïÐz c2H5O-

NHCOCH3(vr r r)

CH

R

efHcorn. NHcoCH3

R

(ur) 11* ./r' \ou-, H*

\

CH

(IX) (x)

Scheme III.

concentrated sulphuric acid345 '346 '347 ürd concentrated hydrobronic

acid,34B'349 but it seens rnore forcing conditions are required than

those used here.

The results of Schene III are summarised in Table VI.

Phenylalanines have also been widely synthesised using cyano-

acetic ester instead of diethyl acetanidomalonat"350 "rrd

other l.ess

general rnethods rvhich have been recorded by Greenstein and lvinitz.551

More recently Kidwai and D"lrasia352 h^r" rcported the synthesis of

phenyl al anine us ing 2 r 4- ðisubstítut ed- 2 - ímidazolin- S-ones, the nitro gen

equivalent of the oxazolone, but it is doubtful whether there are any

-43-

coct

cFg

HBr---ù

H2SO4

H+

--+

C¿H5 OH

c H28r

(xrr)

cFg

(xrv)

02c2H5

c,f3

LiAtH4

ol-l

o -4

F3

(xr)

cFr

zltco2czH izCH(C02C2H5)2NHCOCHoI

NHCOCH3__>c&H5o-

CF¡

(xrr I)

fHcorHNH2

Scheme IV.

R

(WI)

-44-

TABLE VI.

Yield (%) of the Yj-eld (eo) of thediethyl o,- acetamido- N- acetylphenyl-¡¡-benzylnalonate alanine(vrrr) (x)

Yield (2"¡ of thephenylalanine

(IX)

o- fluoro-

2 r4-difluoro-

rn-bromo-

g-brono-

¿-tri fluoroneth¡'1-

74

74 65

5B

76

79 64

75 100

60

353 -

advantages over other syntheses. Yanada et al.""" have reported a-

anination of carbo4¡lic acids via the ct-lithio derivat.ive but this, too,

does not seem to have any advantages in the synthesis of phenylalanines.

(c) The preparation of fluorotryptophans .

(i) General and nethods of preparation of tryptophan.

The synthesis of tryptophan derivatives, in which a fluorine

atom was substituted in the aronatic ring (XV) presented an interesting

challenge. The approaches involve introduction of fluorine int'o

tryptophan itself or a precursor which can be readily converted into a

fluorotryptophan. Tryptophan can be synthesised fron indole-S-aldehyde

by an azlactone t"thod354'355 (sinilar to Schene I for the preparation

of phenylalarrines). Diethyl acetarnidomalonate reacts with gramine

64

-45-

cHzc HCO 2H

H

(XV)

nethiodide (xvr¡356 (or ethioaiae55T¡ in the presence of sodium ethoxide

to give a product (XVII) which can be readily cotlvertecl to tryp+"ophan

(xvIII) (Schene v). Gramine methiodide is readily prepared fron

gramine, ild both indole-$-aldehyde and grarnine are readily prepared

frorn indole. Thus the synthesis of fluorotryptophans could also involve

introduction of fluorine into granine, j.ndole-3-alcle,hyde, or indole

on synthesis using a fluoroindole.

A method for the preparation of tryptophan, which does not' 358,359

require the preformed indole ring, is that reported by Moe and Warner'

The phenylhydrazone of the condensation product between acrolein and

diethyl acetarnidomalonate also gives (xvlI) (scherne VI).

Thus the g-fluorophenylhydrazone of (XIX) should give 5-fluoro-

tryptophan, the m-fluorophenylhydrazone should give a rnixture of the

4- and 6-isomers, whil-e the o-fluorophenylhydt:azone should give 7-fluoro-

tryptophan. Such an approa.ch hacl been reported by Rinderknecht and

FI

NHz

-46-

+H2N ( cH3)2 I

+

cH2f ßo2c2H5)2

cH(CO2c2H{2

CH3

cH cO 2H

NHz

czHso

..-*3.oH-

4oHCN

H

(XV)

1. oH-2. H+

NHCOCH3

H

(xvrr)

(xvr r )

eHz

NH

Schene V.

-47-

cH2=cHcHo + cH:co2c2ql2 t44,.NHcOcH3

H2cH2cH O

ßo2c2\ l2NHcoCH3

(xIX)

H 2CH2C H= N NH

iC'a2.C2H512

cI

C

I

N HNH2

(xrx)

cI

cI

+ _--+

N Hc OCH3

cH21 ßt)2c2H'5)2H2 so4__+Hzo NHcocH3

NH

(xvr r)

Schene VI.

-48-

Niernur,.,360 for the synthesis of 5-fluorotryptophan and was utilised in

this work. As a mixture of isomers most probabl¡' would be forned in the

cyclisation of the n-phenylhydtazone of (XIX), this method was deemed

ursatisfactory for the preparation of 4- and 6-fluorotryptophan, ffid

was not investigated as a method for the preparation of these compounds.

The approaches which were investigated were those involving ì.ntroduction

of fluorine into tryptophan, indole-3-aldehycie, gramine or indole, and

the synthesis of fluoroindoles.

(ii) Approaches involving introduciion of fluorine into

tryptophan indole-3-aldehyde, or granine.

The gsual rnethod for synthesis of ar aromatic fluoro cornpound

is nitration, reduction of the nitro group to an amino group, convetsion

of this group to a diazonium fluoborate, followed by thermal deconposi-

tion (Schene VII).

Ar--.-Þ

Ar NO2 .._---Þ Ar NH2

Rr N2*eFi +-----Ð

Schene VII.

ATF

-49-

Conversion of (XXIII) to (XXIV) is the Schiemann reaction.36L'362'363

This, and ot-her methods for the introductj on of fluorine into orgailic

compounds, have been rer¡ierved.364'365 Methods other than the Schiemann

reaction are not feasible in any of the approaches towards the synthesis

of fluorotryptophans.

h" nittation of tryptophan has been reported, the nitro group

entering the 6-positiorr.366 The reported yield was, hovrever, low.

Reduction of this nitro group to an anino group should proceed in good

yield; reduction of the 2-3 bond of the indole ring would probably not

be a conpeting reaction as the reduction of thisr bond in indole itself

requires fairly fo'lcing condition t.367 llowcver conversion of the amino

group to the diazoniun fluoborate would necessitate protection of the

o-anino group. It has also been shown that diazonium fluoborates con-

taining carboxyl groups are formed in lorv yield and their subsequent

decompositions proceed in poor yi.td.362 The yield of both steps could

possibly be increased by esterification of the carbo>q¡1 group. Overal1,

the preclicted lol yields of some steps if they occurred at all, coupled

with the protection and deprotection reactions, nade this approach

unattractive for the synthesis of 6-fluorotryptophan and it was not

carried out experimentallY.

Nitration of indoles, substituted in the 2- and 5'positions

proceeds reaclily in good yie1d,368 '369 '370 while substitution in the

3-position only leads to a mixture of isomers ,tuu'369-373 the yielcl

of each depending on the nitrating agent. Indole-3-aldehyde can be

-50-

372,373nitrated to give the 5- or 6-isorner, depending on reaction conditions.

5-Nitroindole-3-aldehyde would lead to S-fluorotryptophan which can be

rnoTe conveniently synthesised by a different route. 6-Nitroindole-3-

aldehyde (which would lead to 6-fluorotlyptophan) is urrfoltunately

formed in lol yþf,d.373 Similar results are obtainecl for gramine. Thus

syntheses of fluorotryptophans involving grarnine or indole-3-aldehyde

as starting naterials rvere also unattractive and not carrieC out

experimental ly.

(iii) App roaches involving cyclisation of phenyl¡y¿Tazones

the synthesis of 5- fluorotryptophan .

The synthesis of 5-fluorotry¡rtopham according to Schene VI was

the nethod enployed in this work. g-Fluorophenyl-hydrazine was readily

prepared in high yield from g-fluoroaniline by sodiu¡n sulphite reduc-

tion of the diazo¡i,-6 rult.374 The use of stannous chloride as reported

by Suschit"ky375 was curbelsome. No product was obtained, however,

when an attempt was made to reduce the diazoniun salt of o-fluoroaniline

(a logical precursor of 7-fluorotryptopharr) with sodíum sulphite. This

agrees with the result of Suschí trky375 who found sodium bisulphite

ineffecti-ve, and Bullock and Handr376 ,ho found that different reducing

agents had different reactivities. Suverov et a1 .377 h^u. reported

inproved yields in the cyclisation of phenylhydrazones of the t¡pe (XXII)

by enploying sulphosalicylic acid instead of dilute sulphuric acid but

this nodification was not atternpted.

Cyclisation of phenylhydrazones is a method of great versatility

- 51-

in the synthesis of indoler.378'379'380 îhese nethocls require the

phenylhydTazone to have a structure such tliat an indole, -substituted

in the 2- o'r 3-position, is formed. Without this requireinent, the

reaction fails. Ethyl pyruvate phenylhydrazones can undergo this

Fischer indole-type cyclisation to give ethyl indole-2-carboxylate, which

can be saponi.fied and decarboxyiated to give indole. Indole is ::eadily

converted to granine methiodide fron which.tryptophan is best synthe-

sised (Schene V). 0nce again, the ¿-substituted phenylhydTazone lvould

lead to a S-substituted indole, an o-substituted phenylhydTazone would

lead to an indole substituted in the 7-position while a mixture of the

4- and 6-isomerswould be formed from the meta-substituted phenylhydra-

zone, This nethod would thus be satisfacLory for the synthesis of 5-

and 7-fluorotryptophan, but these two compounds would most certainly

be rnore conveniently prepared as already discussed (Schene VI).

(iv) Other syn theses of fluoroindoles.

As nentioned earlier, indole is readily converted to tr)'ptophan

via grarnine. One nethod of synthesis of indole, the cyclisation of ethyl

pyruvate phenylhydrazorle and subsequent reactions, has a1.so been dis-

cussed. Although this is not the nethod of choice in the synthesis of

5- and 7-fluorotryptophan, it has been used for the synthesis of '5- an<l'

7-fluoroindol".3B1 6-Fluoroindole and 6-fluorotryptophan had been

prepared by a reaction sequence involving oxidation of 4-fluoro-2-nitro-

toluene to 4-fluoro-2-nitrobenzaldehyde. SB2 '383 The yield of this step

was reported to be 1ow by either of the methods employed for tl-re oxidation.

-52-

6-Fluoroinclole had also been prepared fron indoline.3B4 Nitration of

indoline gave the 6-nitro derivative38s "hi.h could be smoothly converted

to 6-fluoroindoline essentially as described in Scheme VII. Dehydrogena-

tion was reported to give 6-fluoroirtdole in good yi"td.3B5 Indole

itself could not be used, as the products of nitration were reported

to be tars and polyrneric naterials when the reaction was carried out

r¡rder acidic conditiont.56T Nitration using benzoyl niirate was reported

to give 3-nitroindo1"386 and not nitration in the benzene ring. Reaction

of potassamide with halogenoindoles was reported to give the amino

indoles in good yiclds,3B7 a mixture of isomers indicated a¡r aTyne-

type mechanism. It rvould be interesting to see whether these amino

indoles would undergo the Schiemann reaction. An atternpt rvas nade to

synthesise 6-fluoroindole from indoline but the near explosive decomposi-

tion of the diazoniun fluoborate 1ed to its abandonment.

Attention hras then directed towards the Reissert synth"rir,SBB

an approach which had been used previously for the synthesis of 6-fluoro-

indo1"381 (Scherne VIII). Conversion of (XXV) to (XXVI) by thermal

decarboxylation proceeded in a yiel d of. 52"'". A later method enploying

copper chromite in quinolirr"389 as the decarboxylating agent failed in

this case. The failure of this reaction has, however, been reported

to be due to irnpurities in the ryrt"rn.390 Nevertheless, the yield of

indole from the thernal decarbo4¡lation was sufficiently high to nake

this procedure worthwhile. Others steps proceeded srnoothly. Another

procedure reported for the preparation of 6-fluoroindole involved 1,4-

-53-

o K'I

CH 3

F

+

Noex* õc2 H5--4

cH=c CO2C2H5

Noz

co2 H

(xxv)

+ (CO 2C2H{2

+

H zczH's F

OH

F

NHoF

H

A

NH

F

(xxvr)

Scheme VIII.

-54-

difluoro-2-nitrobenzene as starting nìaterial, but as the reported yields

' 391were very lowr""^ it was not investigated. the preparation of -fluoro-

indole from 6-fluoro-2-nitrobenzaldehyde had been reported,t9' b.ra *u,

not investigated.

(d) Preparation of cj,nnamic acids.

For the purpose of this wor:k, it was desirable to synthesise

fluorinated cinnanic acids. o-Fluorocinnamic acid had been prepared as

early as 1885 by the reaction of diazotised o-aninocinnamic acj-d with

hydrofluoric a"id.39B rhis reaction was utiliscd by Kindler394't9u fot

the preparation of g- and g-fluorocinnamj-c acids but no yietrds were

reported. g-Fluorocinnamic acid had also been prepared by the Perkin

. 396 397reaction""" a¡rd from a Schienann reaction of ethyl p-aminocinnamate..--'

o-Fluorocinnamic acid had been prepared by the Knoevenagel t"..tio.t398

while the method enployed for m-trifluorornethylcinnamic acid had been

the Doebner modification of the ltaoevenagel reaction^399 The nnethod

employed in the literature for the preparation of 2, -difluorocinn¿¡-míc

acid was the Perkin reaction.400 The long reaction ti.nes and low yields

in some cases nade these approaches rursuitable for the synthesis of a

series of cinnarnic acids. It h/as reported that the Wittig reaction

of benzaldehyde with carbomethoxyrnethylidenetriphenylphosphor¿Ìne gave a.

quantitative yield cif ethyl "inr,r*ut".401 As saponification of the ester

should give the corresponding acid in good yäd, and each step is easily

carried out, this method was chosen (Scheme IX) .

o

-55-

+ Ph3P=C HCO2C2Hb

cH=c HCOTCcHxLLI

(xxvrr)

(xxvr r r)

__>

R

cH=c H c ozH

Scheme IX.

The results are surnmarised in Table V.

TABLE V

Yield (%)of (XXVII)

R Yield (%)

of (XXVIII).

g-methyl

o-fluoro

n-fLuoro

g-fluoro

2,4-difluoro

m-trifluorornethyl

73

85

80

82

81

74

87

86

B4

83

82

B6

-56-

The preparation of o-fluorocinnanic acid was also investigated. This

acid had been prepared in 2% yield by the Perkin reaction402 ^nd

in fair

yield by the reaction of ethyl fluoroacetate a¡rd benzaldehyde in the

presence of sodium hydride.403 The ethyl ester of this acid had also

been prepared in poor yield by the Refornatsky reaction of benzaldehyde

and ethyl brornofluoroacetate ,404 *d in good yield by the Claisen reac-

tion of benzaldehyde, ethyl oxalate, æd ethyl fluoroacetut".'405 This

latter rnethod was.chosen. The ester l4/as prepared in 65% yield and the

acid in 75e" yíe7.d

g-F'luorophenylpropionic acid was prepared fron p-fluorocinnamic

acid by catalytic hydrogenation. This apPears to be superior to the

-406reported nethod+"" involving a variation of the Willergodt reaction.

(e) The synthes is of 3-carboxy - 7- fluorodihyCrois ocarbostyril .

ozH

F

o

(xxrx)

As a¡r example of a fluori.ne labelled subst:rate of restricted conforna-

tional geometry ,'Ut'262'264 3-carboxy-7-fluorodihydroi-socarbostyril (XIX)

-57-

(3-carbo4¡-7-f.luoro-1-keto-712r3r4-tetrahydloisoquinoline) was synthe-

sised. The conditions followed those for the parent compo.*d26' u.ty

closely. The synthesis can be represented by Schene X.

Nitration of o-toh.¡nitrile with nitric acid/sulphuric urid407

gave 2-cyano-4-nitrotoluene (XXX) in 77% yield. This was catalytically

reduced using palladium on charcoal as catalyst to give 4-amino-2-cyano-

toluene (XXXI) in 84% yield. Conversion of this proceeded srnoothly to

give the diazoniun fluoborate (XXXII), thermal deconposition of which

gave 2-cyano-4-fluorotoluene (XXXIII) in 52% yield. This is quite a

good yield for fluoro compounds prepared by the Schiema¡rn reaction. Also

there appear to be no reports in the literature of the Schienarrn reac-

tion being carried out with a compound containing a cyano group. Free-

radical bromination of 2-cyano- -fluorotoluene gave 2-cyano-4-fluoro-

benzyl bromide (XXXIV) in 65% yield. This compound was found to be a

powerful lachrymator and skin irritant. Condensation of this, with

diethy 1 acet ami domalonat e, produced di ethyl c- acet ani do - o,- (2 - cy ano - 4 -

fluorobenzyl)-nalonate (XXXV) ín 76% yield. The next step of the

sequence, saponification and cyclisation of (XXXV) to give (XXXVI)

proceeded with a better yield (45%) than that reported in the lit-erature

for the parent conpound (35%). 5-Carboxy-7-fluorodihydroisocarbostyril

was readily esterified, using thionyl chloride in nethanol (92u" yield).

H NQg_+H2SOa

--Þ

ilo

-5 B-

CN

CN

H3

NHz

(xxxr)

CH 29t

-_+

H33

NN t. HNo2

2. No BF4

CH 3

+

ON

N2BFa

(xxxrr)

(xxxrv) +

Noz

(xxx)

CH 3

(xxxrrr)

QzH

CNA

F F

(xxxrv)

cH (c o2c 2Hl2

NHcocH3

H 2C(Co2czïùz Or-NHCOCH3 €

CN

(xxxv)

+F

---}-.F

F

(xxxvr)

Schene X.

H

c 02cH3

-59-

2 Qua:rtitation of Chenical Shifts.

For an inhibitor, I, exchanging between solution and the active

site of an enzyme, E,

E+I ES

if a 1:1 conplex is forned, an<l the dissociation constant for the enzyne-

inhibitor complex is related to the enzyme and inhibitor concentrations

by the expression

KD= +If the exchange is rapid, then the observed chenical shift, ô, vri1l be

the weighted average of the shift in the conplexed form, At, and that .

in free solution, ôfr""

1k-+<-

k1

Thus, ô =EIT_

ofree (1)

If the cheni.cal shift of the conplexed forn is to be deternined relative

ao ôf""" = O, then Ä = Ar - ôf""" a¡d (1) sinplifies to

^r +

I -EIoT-

o

ô

ô

fln.oo

free(2)

(3)

If ô is also rneasured relative to 6 = 0 thenfree

$= EI

^o

E.I_ET

- Er) (r

I

K¡

(Eo

EIo

- EI)(4)

From (2)

-60-

EI= Io

ô

Ã

Kn[Eo (ô/A)ro] lro - (ô/A)ro]

(o/N ro

Ko

luo (ô/A) rol [1-ô/^ ]

ô/A

If ô<<A , then I << other terrns

Hence (E +I )

I (^Eo)-(Eo+Ko) (s)

Thus a plot of Io vs will be a straight line of slope AEo and inter-

cept - (fO + Eo) . Hence A ancl KO can be founct by graphical nethods. The

approxinatíon by Spotswood et al .,'O' that, in the equilibrium expression..

EI << I^ and ca¡r be ignored leads to the expressiono

However this approximation is not particularly valid as it v¡as found

that KO r Eo. Hence using this approxination can lead to large errors

in KO.

Ao

ô

Ã

E -E -r +r Iooooa

o

A

ô

1.Fô

1

Ò

Eooo

o

^Eo(L

6-IO ) Kn

Equation (5) can also be derived frorn (4) and (3) by making the

-6 1-

assumption that in (4), terms in EI

be ignored.

2 are sufficiently small that they nay

The calculation of KO and A requires a knowledge of 6fr"u, the

chemical shift of the resonance in free solution. As this is often

difficult to deternine, use was nade of the nethod of SykesltT 'L21 fo"

the calculation of KO and Â. This rnethod has the advantage that no

approxirnations are involved. The method is as follows:

(4) can be expanded to give

E I -E EI-I EI * EI2oo o o

EI

Solving for EI in (6) gives

KD=

.,

(6)

(7)(E +I + Ko + lcEonIo*KO) 4ETEI= o o oo

2

KO is assumed and EI calculated according to (7) with the condition

o < ËI << 1 for each value of Io. The value of EI is used to calculateo

the least squares error in (2) for the experimental values of ô and Io.

The value of KO then, which best fits the values of ô and EI/I. to a

straight line in (2) is chosen as the true value. This also allows A

and 6 ^ to be calculated.treeThis was rnost convenj-ently carried out by computer. KO was

assurned and EI calculated. The RMS error corresponding to this value

of KO and hence EI for the data points was calculated according to (2).

KO was then incremented (or decrenented) r¡ntil the RlvlS errot reached a

-62-

minimum. This corresponds to the best value of KO and hence A. KO and

A rvere also calculated assuming inactive enzyme diner fornation was also

taking place. The calculation was essentially the same, excePt that

the added restriction of the enzyme diner equilibriurn had to be maintained.

The value of the enzyme diner dissociation constant was taken fron the

literature, In this systern, the added equation is

->E+E <- EE Konø (B)

Klttr,l

where EE is enzyme dirner

E =E+EI+2.EEo

E.EEE

(Eo-Er-2EE) 2

EËKort,r

Solving for EE in (9) gives

(e)

(10)EE=

(E'-EI * Koil¡) tT

(Eo-EI * KUlt,l)-T-

(E . EI)o

2

Similarly (5) with Eo = E + Er + 2EE becornes

(Eo-Er-2EE) (Io-Er-2EE)

EIKo

Solving for EI in (10) gives

-o5-

(E -2EE + I L

o oEI=2

EI a¡rd EE are calculated by the following set of operations. KO is set'

Calculate EI fron (11) with EE = 0

Calculate EE frorn (10) with EI value frorn step 1.

Calculate EI using previous calculated EE value.

Continue until there is a convergence to 0.0001 in EI'

KO is iterated and A calculated frorn (2) as described earlier., The

value of KO for which there is the mininum least sguares deviation in

(2) is the true value.

(E.-2EE + Io + KO) ! . 4T TE -2EE). o' o (11)Ko)

1

2

3

4

-64-

The Interaction of Inhibit ors with cr-Chynotryps in - NMR Studies.3

(a) Bulk susceptibilitY effects.

It has been shown in the previous section that a computer treat-

rnent of the data requires only an accurate deterr.rination of the chemical

shift relative to a fixed reference. For a graphical treatment of data

it is necessaïy to know ôfr"". Most of the inhibitors studied here were

used as DL racemates. In nost cases, in the presence of q-chynotrypsin,

th" 19F NMR spectrum consisted of tv¡o resonances. Whereas the position

of the resonance to 1ow field was a fr:nction of inhibitor concentration,

it was for.¡nd that the position of the resorìârrcê'to high fiel<l was vir-

tually rmmoved by ch.anging inhibitol concentration. This phenomenon vies

also reported by Zeffren and Reavi LIl43 and by Spotswood "t uI '742 As

the chenical shift of this high field resonance was considerably differ-

ent between solutions with and without enzyme, and subsequent shifts with

changing inhibitor concentrations weïe vely sma1l, it rvas thought that

this initial large shift on the addition of enzyme to an inhibitor solu-

tion may have been a bulk susceptibiLity effect. The chenica-l shift of

the 19F resonance of t¡e inhibitor N-trifluoroacetyl-g-bronophenylalanine

(XXXVII) was nonitored in the presence of various en.zymes (Table VI) '

In the presence of o-chyrnotrypsin there wele two 1esonances ' By analogy

with the resuLts obtained with N-trifluoroacetylphenylalanine to be

discussed 1ater, this was due to pr:eferential binding of the D enantiomer

of the racenate used.

-65 -

TABLE VI.

Chenical shifts of the 190 r"rorrances of N-trifluoroacetyl-g-brornophenyl-

ala¡rine (XXXVII) in the pïesence of enzyme (0.lM citrate buffer' pH 5.3)

at 56 .4 l4flz

EnzyneConcen-tration(ñ)

Conc. of(xxxvrr)

(rnNr)

Cheqr[cal sh.iftof Ii leSO-nance (Hz)a

Change inshift (Hz)

pepsr-n

lysozyme

trypsin

DIIP-s-chymo-trypsin

ol-chymo-trypsin

2.3

2.4

2.s

2.6

2.6

50

30

30

9

30

9

30

9

186. 3

188.6

188. 5

188.0

187.8

189 .6

190. 1

188. 3

188.2

188.4 , r93 .7

188.7 , 203.4

2.2 ))

2.3 )

1.s ))

t.7 )

1.e ))

2,0 )

2.L, 14.1 ))

2.4, 7.4 )

3.8 ))

3.3 )

9

30

I

a Relative to trifluoroacetic acid lock.

It can be seen that addition of an enzyme to an inhibitor solution causes

a shift to low field of the 19, t"ronance of the inhibitor. For a

particular' enzyme concentration, this shift is virtually independent of

the irrhibitor concentration, but depends on the nature of the enzyme.

-66-

If the enzyme is o-chymotrypsin, this result applies only to the high

field resonance, the 1ow field resonance position depends markedly on

inhibitor concentlation. Thus it appears that the L antipode of the

racernic inhibitor is only weakly binding to ü-chymotrypsin, or that the

chemical shift of the enzyne-inhibitor conplex is not significantly

different from that of the inhibitor in free soiution. In this case it

appears valid to use the chenical shj.ft of the L enantiolnerr as ôfr"",

the initial shift of 2.1 Hz being a bulk susceptibilitl'effect. This

is arnplj_fied by the results obtained with the other enzymes.

With the other enzytnes, it is urrlikel.y that the observed shift

could be due to N-trifluoroacetyl-g-brornophenylalanine functioning as

an inhibitor. Lysozynre is an enzyme which hydrolyses molecules containing

sugar noieties.130 It also binds sugars; in fact these lysozyrne-sugall

comprexes have been extensively studied by NMR spectrosc orr.t23-r30 ' t46 '

t47 rL4B The likelihood of its binding an acyl amino acid is er-trenely

remote. The change in shift measured, then, ca:r only be explained in

tenns of a bulk susceptibility change. The fact that the change is not

concentïation dependent suppolts this conclusion; the formation of an

enzyme-sna11 nolecule complex would be evidenced by a concentration

dependent shift.

In the case of pepsin and trypsin, the conclusj-ons are not as

clear cut. Pepsin is a peptidase which hydrolyses peptide bonds on the

amino side of residues with aronatic side chains ,'lO ..f., o-chymotrypsin'

The fact that the initial observed shift is not then concentration deperL-

-67 -

dent nakes it most likely due to the nature of the enzymers specificity,

but apparently is not occurring to an extent whereby it can be detected

by NMR techniques. With trypsin binding is again a possibility. X-ray

diffraction results shorr¡ the bin<iing site to be somewhat larger than

that of cr,-chymotryprinrlBB Naturally occurring bovíne pancreatic

trypsin inhibitor also inhibj-ts cl-chymotrypsin;2L3 as such it seens

possible that inhj-bitors of o-chynotrypsin nay inhibit trypsin. Also

trypsin is inhibited by aromatic "otpo.urdr.408 These compounds are

amines, however, which fulfil the requirement of the basic sícle chain

necess ary for binding of substrates. In thj,s case there is probably

hydrogen bonding between the annonium ion gloup of the inhibitor, ffid

the carboxylate anion of aspartic acid-189 at the bottom of the binding

pocket. The concentration independent shift once again suggests only a

bulk susceptibility effect.

Thus any attempt to measure a chernical shift of an inhibitor

resonance in the presence of an enzyme nust make allowances for a chenical

shift due to solution properties different for solutions with, anC

without, enzyme.

(b) The int eraction of inhibitors with DFP-cx,-chymotrypsin.

The results obtained with DFP-o-.:trymotrypsin, in which the

catalytically inportant residue of cl-chymotrypsin, serine-195, has been

esterified with diisopropyl fluorophosphate, were sinilar to those

obtained with the enzyrnes pepsin, lysozyme, and trypsi-n. That is, there

was a significant change in the chenical shift of the 19, t"rorrance on

-68-

addition of the enzyne to the irrhibitor sol.ution. Subsequent changes

in inhibitor concentration, lvith the same concentratiotr of DFP-g-

chymotrypsin, failed to affect the chemical shift further. Once again,

only a bulk susceptibility effect v¡as opeïating; there hras no evidence

of binding.

The interaction betweelr an inhibitor and DFP-cl-chynotrypsin was

investigated at pH 7.7 ín 0.1M Tris buffer, usíng N-trifluoroacetyl-g-

fluorophenylalanine as the irrhio-itor. With this inhibitor, under the

same conditions but with or-chymotrypsin, the NMR spectrurn consisted of

two resona:lces. For a 20nlr{ solution of the inhibitor, the peak sepai:a-

tion was 5.9 Hz, due to the preferential bindin4 of the D enantiomer.

In the presence of DFP-o-chynotrypsin there Ï/as no separation, indicating

no binding or, more correctly, no preferential binding of either isomer.

As it was possible that both enantiomers could bind equally weli to

DFP-a-chymotrypsin, and this binding could be evideneed by alnnost

identical shifts on binding, the interaction of N-trifluoroacetyl-D-

tryptophan was studied.

Results with this inhibitor and o-chynotrypsin at pH 7.6 showed

a substantial concentration dependent chenical shift ('ì' sHz over the

concentration range studied). However in the presence of DFP-o-

chyrnotrypsin, the chemical shift l^¡as concentration independent. There

was, however, a bulk susceptibility shift of 2.1 Hz on addition of the

enzyne to a 50mM inhibitor solution; decreasing this concentration to

10nù{ effected only a further 0.1- Hz change. SykesT2L rruu t"pott"d 19F

-69-.

NMR studies of the binding of N-tri'fluoroacetlrl-D-phenylalanine to DFP-

o,-chyrnotrypsin at pH 7.8. The results reported depended on the lneasure-

ment of very snal1 chemical shift changes (< 0.5 Hz). With the large

errors thus invoJ.ved, the report appears dubi.ous; such smal1 changes

in chenical shift could easily arise from a snall concentration effect

due ro the inhibitor: slightly affecting bulk susceptibitity.

The wealth of information on DFP-tt-chyrnotrypsin leaves rro doubt

that this enzyme is totally inactiu".269 Whether or not it is capable

of binding an inhibitor or substrate is another question. Modification

of serine-195 wou.Ld preclude its acylation by a substtate (or inhibi-tor,

if the inhibitor is a virtual substrate). Horvever the aromatic binding

site, the "tosyl hole't, is sti1l intact in tosyl-cx-.chy¡ne1¡yps1n195-199

and rnost probably would be so in other serine-195 nodified enzymes. Bind-

ing could be precluded by the diisopropyl phosphorylo group either being

in, or preventing access to the aromatic bincling site. A consideration

of the three-dimensional structure of N-formyl-L-tr)?tophan:o-ch1z¡¡e¿rypsin

shows the aromatic ring of the virtual substrate to occupy tire !'tosyl

hole?'. the anido hydrogen forms a hydrogen bond with the carbonyl

olygerì of serine -21,4 and the carboxyl group is near serine-195.200

Steric interactions between the carboxyl and diisopropyt phospho'rylo

groups are then inevita-ble. Thus binding at the active site becones

unlikely. Confornational changes of a degree necessary to relieve these

interactions would most certainly disrupt the aromatic bincling site. 0n

these bases, binding to DFP-o-chynrotrypsin by N-acy1 aromatic alnino acicl

inhibitors is ur:lil<e1y.

-70-

Proflavine has been shor\rn to bind weakly to DFP-o-chymotrypsin

and chymotryps j-nogen by ecluilibriun dialysis ttudíer"294 However the

stuclies using visible absorption spectroscopy 'fenonstratecl no bi"ding'409

With the dye thionine, an inhibitor, binding both to cl-chymotrypsin and

chyno'trypsinogen was reported, a:rd postulated to occur at sites other than

the catalytíc site.410 The catalytic site was sirown to be the only site

of binding for the dye, Biebrich scarle'c41l ho*"",r"t. Thus it seems there

are other binding sites in cr-chyniotrypsin lvhj-ch may or xray not be usecl

if the catalytic site is unavailable, for exarnple in chymotrypsinogen.

Howe\¡er, as our 19f'rutm. stuclies failed to delronstrate any bindi-ng at all

to DFP-a-chymotrypsin, these secondary sites coul-d not be further eluci-

dated.

Anhydro o-chynotrypsin, in which the elenents of water had been294,295

removed from serine-195, was fotuid to be capable of binding inhibitors.

It is clear, then, that phosphorylation of scrine-l-95 has the effect

of preventing access to the arornatic binding site. Serine-195 cannot

be inportant in binding by forming an a cyl-enzyne as nany strong inhi-

bitors such as índole and profLavine lack a carbo4¡l group.

Thus binding to the arornatic binding site must be of rnajor inportance.

To test this further, the binding of N-trifluoroacetyl-g-

bromophenylalanine, a stïongly bound inhibitor, to DFP-o-chymotrypsin

was studied at pH 5.3 where the binding of both inhíbitort279 '4r2 '4r3

and subst r^ates277 '4L4 is knourn to be stronger than at alkaline pH's .

No separation of th" 191' signal into two resonances due to preferential.

binding of one isomer could be detected. Apart from a sma1l initial

-7L-.

bulk susceptibilit¡, change, there was no concentration dependent slrift

which indicates no detectable binding by this nethod'

(c) Proton naqnetic resonance studies.

The interaction of several inhibitors with o,-chymotrypsin was

studied by proton nagnetic reson,ance spectloscopy. The resonances of

the protons in the acetyl groups of N-acetYl-D,L and Dl-phenylalanine,

N:acet/1-D,L and DL-tr¡,ptophan, and q,-acetanidocinnamic acid, and th.e

acetyl and nethyl groups of o¿-acetamido-ß- (m-methylpheny! ecrylic acid

were observed. Addition of cl-chynotrypsin to 50nM solutions of these

inhibitors in 0.1M citrate buffer in DrO, PD 5.4, such that the concen-

tration of the enzyne was 2.6N, caused shifts of 0.5-1.0 Hz in these

resonances. The changes in chenical shifts on decreasing the inhibitor

concentration to 10rM, in the plesence of the same concentration of

enzyne, varied. Over this range, the acetyl proton resonances of N-

acetyl-D and L-phenylalanine, and o-acetamidocj-nnanic acid noved to 1ow

field. However this shift ltlas 10'5H2, which was withi-n the limits of

e1.ro1. of there being no change in shift. 1'lie same I'esult u¡as obtained

with the acetyl and methyl proton resonances of o-acetanido-ß- (rn-rnethyl-

phenyl)-acrylic acid. With N-acetyl-Dl-phenylalanine and N-.acetyl-Dl-

tryptophan, no separation of the acetyl proton resonances due to prefer-

ential binding of one isomer was observed. The initial low field shift

of the Tesonances on the addition of enzyrne is, once again' nost probably

due to bulk susceptibil ity changes. Such shifts were also observed in

-72-

th" 19p studies and are describecl in sectiot 3a.

A chemical shift cha:rge of a particular resonance in the presence

of onzyne (after bulk susceptibility effects have been allorved for) is

a sufficient, but not a necessaly criterion for bindi.ng. N-acetyl-D-

phenylalanine has been shottn to ben an inhibitor of o-chyrnotrypsin by

these techniques.L42 The lcw value of A obtained irnplies that very snall

chemical shift changes must have been measur:ed, and hence rnust be subject

to large eïrors due to the possibility of snral1 concentration dependent

shifts arising from bulk susc.eptibility cha:rges. As a change in the

observed chenical shift of a nucl.eus will occr:r only if the bindi.ng is

strong and there is a reasonable different betl4/een the free solution and

enzyme bornd shifts, these criteria nust be satisfied if accurate results

are to be obtained.

N-Acetyl.-D and L-tryptophan have been shown to be inhibitors of

o-chyrnotrypsin,'77'278'27L'4L2'416 although N-acetyl-L-tryptophan has

been terned a virtual subst rate by virtue of its 180 "*"h*ge

properties

which have been interpreted in terrns of acyl-enzyme formation.203 The

changes in chenical shifts of the acetyl proton resonances were snall

oveï the concentration range studied (10-50mM). These shifts were too

small (1.0 Hz to 1ow field and 0.7 Hz to high field for the D and L

isomers respectively) to allow a quantitative determination of KO and A.

Recently, Gerig and Rimermanlll have reported studies of this system at

100 MIlz and pD 6.6. The reported values of A were -0.13 and 0.17 pprn

for the D and L enantiorneïs. This corresponds to shifts on binding of

-73-

7.8 Hz to low field and 10.2 Hz to high field at 60 MIlz. Once again

the measured changes of chenical shift rnust have been small. These

workers have also investigated the binding of N-acetyl-D and L-tryptophan

to tosyl-cx,-chyrnotrypsin. As discussed in section 3b, any binding which

presurnably occurs with nodified enzymes, does so at sites other than the

active site when access to the aromatic binding pocket is precluded. In

tosyl-cr-chynotrypsin, this pocket is tu-rdoubtedly occupied by the phenyi

ring of the tosyl group. Their reported values of A are so srnall that

any measured changes in chenical shift rnust have been so sma1l that they

could easily be due to the bulk susceptibilj.ty changes which occur on

varying the inhibitor concentration. The reported values for the tosyl-

cr-chyrnotrypsin systen must, then, be accepted with reservation.

Overall, the rather small changes on binding of the proton

ïesonances which could be readily observed with these inhibitors (< 1 Hz)

made ít inpossible to study quantitatively the binding process at 60 MIJz"

Aloo any data reported to have been obtained by these techniques must

be treated cautiously.

(d) The interaction of N-trifluoroacetvlÞhenylalanine with cr-chyno-

trypsin.

The interaction of N-tri.fluoroacetylphenylalanine with cl-chymo-

trypsin had been studied by SykesLzL'\45 and Zerfren and Reavil L.t43'744

The 94. L MHz studies of Zeffren and Reavi11145 ""t" carried otrt at pH 6

with the racemic inhibitor. They repo::ted that the l9r rOnra spectrum of

-7 4-

N-trifluoroacetylphenylalanine, in the presence of u-chymotrypsin,

consisted of trtro resonances, the positions of which l^Iere concentration

dependent. Theír interpretation cf the data neglected bulk susceptibility

effects; the shifts weïe neasured relative to the chenical shift in

solution in the absence of enzyme. In this system, the change in chenical

shift due to bulk susceptibility chariges is a major contribution to the

observed shift. Hence the quantitative interpretations are incorrect.

The work of Sykesl2!'145 at 94.1Ìr{Hz and pH 7.7 involrtred very

sna1l chenical shift differences; this was carried out using the D

enantiomer. An attempt was nade to repeat this work but failed; within

the linits of error' (t 0.2 Hz), there was no trend of the 19F t"rorru.r."

to move to high or low field as a fi:nction of inhibitor concentration,

except for the usual bulk susceptibility phenomenon.

This system was then jnvestigated at pH 5.5 in 0.1M citrate

buffe::. The changes in chenical shift of the N-trif1uotoa."ty1 19F

ïesonance were srnall, but could be determj-ned nore precisely by averaging

a number of results. The results obtained are shown in Table VII. From

these data, KO and À were calculated according to the nethod described

in section 2. 'Jhe values were 8 t l rrM and -9 t 1Hz* respectively.

The shift on binding, a, was to low field which is in the opposite

direction to that found by Sykes]-zl'L4s at pH 7.8. However, the value

reported is considerably different in his two papers purporting to

describe the sane system under the same conditions (41 vs 24 flz).

* Negative values indicate shifts on binding to 1ow fielcl.

-75-

TABLE VII.

Chemical shifta of 19f' ïesonance of N-trifluoroacetyl-D-phenylalanine in

the presence of o,-chynotrypsin, Eo = 2.6 nM; pH 5.3 0.1-M citrate, at

56.4 MHz.

19Concentration of N-trifluoro-acetyl -D-phenylalanine, Io (rM)

Chernical shift of F trí-fluoroacetyl resonance (Hr)a

25

20

15

10

5

a relative to trifluoroacetic acid lock

18B. 1

188. 3

1BB. 5

188. 7

789.2

The activity of o-chymotrypsin has been shown to be a ntaximum

at pH 7.s-8 .0.2t7 '4I3'4I4'475 As this activity is probably associ.ated

with the conformational changes (arid also with the state of ionisation

of histidine-S7) it is possible that the trifluoroacetyl gloup has a

different environnent between pH 5.3 and pH 7.8. As the shifts

rneasured by sykes727'r45 brere very small and his work could not be

repeated, coupled rvith the fact that the shifts measured at pH 5.3 were

also smal1, detailed interpretation of these results can only be nade

with reservation.

Ashton and Capon149 h..r" reportecl studies of this systent also.

-76-

They found a concentration dependent shift at pH 6.34 but no evidence of

binding at plJ 7.96. their values of KO (45 ÍM from the NMR s'tuclies and

30 nM from kinetic studies) are some fivefold larger than those de'Ler-

mined here. It is difficult to know whether bulk susceptibility effects

were included in the consideration of NMR data by these workers.

The interaction of N-trifluoroacetyl-Dl-phenylalanine with c¿-

chynotrypsin was also studied at pH 5.3. In the plesence of enzyme, twc

separate resonances u/ere observed. The chenical shift of the resonaltce

to high field was independent of the inhibj.tor concentration, while the

separation of the resonances, and the chemical shift of the low field

reson¿mce were concentration dependent. Addition of N-trifluoloacetyl-

D or L-phenylalanine to a solution of this inhíbitor in the presence of

the enzyme showed that the resonance at lower field was due to the D

enantiomer while that at higher field was due to the L isoner. As the

position of the ïesonance of the L isoner was invariant, its position

hras a convenient reference and the posi-tion of the D isomer resonance

was measured relative to it, i.e., the separation was measured" The

position of the L isomer resonance was thus assumed to be equivalent to

ô.--. for the D antipode for graphical treatnent of the data. It wastree

also assumed 1,hat the L 'isorner was binding nuch more weakly and its effect

could be ignored. CIearLy this assumption is only a first approxination,

but it is strongly supported by the fact that the separation of the D

a¡rd L isomer resonances at corrstant D isomer and enzyme concentration was

independent of the concentration of L isomer, withiu experimental error.

-77 -

The results obtaíned are shown in Table VIII.

TABLE VIII.

Interaction of N-tri fluoroacetyl - Dl-phenyl aI anine with cr- chynotryps in,

Eo = 2.6 rM; pH 5.5 in 0.1-M citrate buffer at 56.4 lvftlz.

Concentration of D isomer ofN-tri fluoroacetyl- DL-PhenYl -alanine, fo (rM)

Separation of resonances

(Hz)

t0

7.5

6.25

5.0

4.0

2.5

L.25 ! 0;L

1.55

1.6s

L.75

2.00

2.25

The graph of this dat.a (ro E 1/ô) is shown in the appendix (Fig. 1).

From this graph KD = 5.4 N, A = -9-0 Hz.

KO and A were also calculated usi.ng the method utilised for the

D isoner alone. If the L isomer were binding weakly, a change in shift

may not be apparent but its measured shift nay stiIl be slightly differ-

ent fron the free solution shíft. The position of the L enantioner

Tesonance was thus taken as a convenient reference, md Kp, A, ..d ôfr""

were calculatecl by the iterative proceclure clescribed in secti on 2. ôrr""

-7 8-

was found to be within t 0.5 Hz of the L j,somer resonance position in

this, and all the other systems involving racemic derivatives of phenyl-

alanine. The values of KO and A obt.ained were 8.5 t 0.5 nM and -I7 ! 2 Hz

respectively. The good agreement of these results, with those obtainerl

by graphical nethocls and the iterative computer procedure, on the D

isomer alone confirm that the assurnptions are valid and the approxima-

tions excellent.

It is difficult to interpret these results in absolute terms;

the effect of the environment on 19f' .h"rical shifts is not suffic.iently

well docunented to a1low these results to interpl'eted in terns of specific

interactions. These results are best discussed in terms of relative

effects by cc'nparison with results obtained with other inhibitors.

(e) The interaction of N- tri fluoroacetyltryptophan with cx,- chynotryps in.

The interaction of N-trifluoroacetyl-D-tryptophan with o,-chymo-

trypsin gave rise to phenomena sinilar to those discussed earlier for

N-trifluoroacetyl-D-phenylalanine. In this case, at pFl 5.3, the observed

shift to lol field of the 19F ""rorrance

over the concentration range

studied was much larger (t4.3 Hz vs_ I.t Hz for N-trifluoroacetyl-D-

phenylalanine) which allowerl the systein to be studied in greater detail.

The results are shown in Table IX. Treatment of these data according

to the nethod described earlier Bave KO < 0.1 nM and A = -30.4 t 0.2 Hz.

Under essentially the sane conditions, Ashton and Capon ,L49 hru" subse-

quently reported KD = 6.5 mM and A = -7I2 Ílz (at 94.1MHz) respect:'.ve1y.

Thís corresponds to a shift of. 67 Hz to low field at 56.4 NL\Iz.

-79-

TABLE IX.

Interaction of N-trifluoroacetyl-Ð-tryptophan with o-chyrnotrypsin, Eo =

2.6 nù{; pH 5.3 in 0.1M citrate buffer at 56.4 Hz,

Concentration of N-trifluoro- Chenical shifta of 19P

trifluoroacetyl reson¿mce

(Hz) .

acetyl-D-tryptophan, Io (InM)

50.0

40 .0

30.0

25.0

20.0

15.0

10.0

5.0

184.0 ! 0.2

784.7

185.0

185.4

T86.4

187. 5

L90.2

198. 3

a relative to trifluoroacetic acid lock.

Under the sane conditions, the change in chemical shift of the

19F t"ror,a¡ce of the L isomer (1.7 Hz) was ntuch snaller over the

concentration range studied. The results are shown ín Table X. The

calculated values of KO and  by the iterative procedure are 5.6 t 1.6

mM and -7.8 ! 0.6 ÍIz respectj,vely. As the rneasured shifts were smal1,

the calculated constants are subject to the same turcertainties as those

en count ere d with N- tri f 1 uo roacetyl - D-phenyl a1 anine .

-80-

TABLE X

Interaction of N-trifluoroacetyl-L-t::yptophan with cr-chymotrypsin, Eo

2.6 nù{; pH 5.3 in 0.l.M citrate buffer at 56.4 \'4H2.

Concentration of N-trifluoro-acetyl-L-tryptophan, Io (ûM)

Chenical shift of N-tri-fluoroacetyl resonance

(Hz) "

50

40

30

25

20

10

L82.9 ! 0.2

185.0

L83.2

183. 5

5

L83.4

1,83.7

L84.3

a relative to trifluoroaceti.c acid lock

This system was also investigated a+- pH 7.6, At thís pFI, no

binding of the L isoner was detected; i.e., the chemical shift was

concentration independent. For the D isomer, the concentration depen-

dent change in chenical shift was srnaller than that observed at pH 5.3

(6.9 vs. L4.3 Hz to low field). This is sti1l sufficiently large for

reliable and accurate data for the systen tc be calculated. The results

are shown in Table XII. KO and A were calculated fron these data to

be 4.3 t 1.6 mM anð, -34 ! ! Hz respectively. Ashton and Capon149 t"pott

values of 28.6 mM and L48 I1z respectively (at 94. L lvltlz and pH 7.72).

- 81-

the shift on binding with pH, will be discussed in a subsequent section.

The interaction of N-tri fluoroacetyl-Dl-tryptophan with cx,-

chymotrypsin was studied at pH 5.5. The separation of the 19F t"rorl*""

into two, in the plesence of enzyme, showed the preferentíal binding of

one isomer or the possible differentiat.ion of the environment of the

fluorine nuclei betrveen the enzyine-D-inhibitor and enzyne-L-inhibitor

complexes. The separation, over the concentration range 5-50 rnM of the

racemic inhibitor, varied between 1.8 and 19.0 Hz. The resonance to

lower field was shown to be that of the D enantiorner by the fact that

its intensity was increased by the addition of this enantiorner. The

chenical shi.ft of the L enanti-omer ïesonance also rnoved to low field on

decreasing the inhibitor concentration. This shift v¡as snall (L.l Hz

over the concentration range studied). The concentration dependent

chenical shift, although smal1, indicated that the L isorner was binding

to sorne extent; this was also proved by the fact that peak separation

was also dependent on the concentration of the L enantiomer present at

constant D isoner and enzyne concerltration. Thus, in this case' the L

enantiomer must be cornpeting effectivelv with the D enantioner for the

enzyme, although there is only a small shift on binding. A graph of

the separation of resonances as a fiurction of the concentration of the

D enantioner was rnade according to equation (5) (Appendix, Fig. 2)

although the assunption that the L isorner is not bindi.ng is not good.

A Was found to be -22 Hz for: the D isoner by this rnethod, while the

clissociation constant calculatecl was less thal zero. As this is inrpossible,

-82-

it also indicates that the assunption is invalid md/or the approximation

of ignoring ter:ns in EI2 is poor. As the work shotnred with the D enan-

tioner a1one, KO to be very sna11, the approxination involving the

rernoval of the tern in EI2 will be poor at 1ow concentrations of Io as

EI2 q, E EI.o

Treatment of the data, according to the rnethod described for N-

trifluoroacetyl-Dl-phenylalanine, involves the assunption of the L

isomer not binding, but no mathematical approxinations. KD and A were

fowrd to be < 0.1 nM and -22 ! 1 Hz respectively. The agreement between

these results, md those obtained for the D enantiomer alone, is still

not good, and shows that the assumption of the L isoner not binding is

not valid.

(f) The j,nteraction of N-trifluoroacetyl substituted phenylal anines

with o-chynotrypsin.

The interactions of a nunber of N-trifluoroacetyl substituted

phenylalanines v¡ith cr,-chyrnotrypsin were studied. These inhibitors were

used as their racenic mixtures. îhe 19F t"rorrances separatecl irrto two

resonaflces in the plesence of q-chynptrypsin. The 1ow field resonance,

the position of which hras concentration dependent, uras assumed to be lhat

of the D enarrtiorner by virtue of its preferred bindi-ng. There is 1itt1e

doubt of this being so; it has been reported previously in observations

of the ring fluorine nuclei on Uindingl42 ^rrd

has been shown to be the

case in this rvork wi.th the N-trifluoroacetyl¡-.,henylalanine and N-tr:ifluoro-

acetyltryptophan. The L enantiomers of these substituted phenylalanines

- 85-

were assumed not to bind. Their resonance positions (the high field

Tesonance) did not change rvith changing inhibitor concentration and

it nay reasonably be assumed that the L isoner is ineffective as a

cornpetitive inhibitor.

The N-trifluoroacetyl fluorinated phenyi alanines were rnainly

stu,Jie<l, as these inhibitors had at least tlo fluorine labe1s. th" 19¡

Tesonances of the ring fluorine nuclei were the expected nultiplets.

In the presence of enzyne, extremely complex spectra were produced due

to the overlapping multiplets of the D ar¡d L isomer resonances. The

complex spectra were an indication that there wa: preferential binding

of the D isoner. However, the complexity of the sPectla did not a1low

the separation of the resonances to be measured with any certainty.

With the aid of anhderonuclea.r spin decoupler, operating at the 1H

frequency of 60 MHz, the rnultiplets r^rere collapsed into two singlets,

the separations of which were measured as a fr¡rction of inhibitor concen-

tration for the para isorner. Instability in the heteronuclear spin

decoupler, as well as the tine needed to accumulate sufficierlt spectra

in the CAT, nade this a veay tedious and frustrating procedure which,

because of this, ü/as not applied to other systems. The separation of

the ring fluorine resonances of N-trifluoroacetyl-DL-g*fluorophenylalanine

varied from 4.8 - 70.4 Hz over the concentration range 20-50 mM for the

racernic rnixture at pH 5.3. Fron these data, KO and A were calculated to

be 1.5 t 1 mM and -55 I 2 Hz respectively. KD and A for the corresponding

N-acetyl compound lvere ïeported as 6 ! 2 rnM and -83 t .5 Hz respectively

-84-

L42at pD 6. The linited data available fron kinetic studies indicate

enzyne-inhibitor dissociation constants for acetates and trifluoro-

acetates of a particular inhibitor to be simi1a.r,278'279 '425 '420 which

a comparison of these results also shows.

The trifluoroacetyt 19p NMR spectrun of the above compound was

also monitored in the presence of o-chymotrypsin at pH 5.5. The separa-

tion of the resonances varied fron 5.1 to 13.6 Hz over the concentration

range 5-50 mM of the racemic inhibitor. KD and  were calculated to be

1.5 t 0.2 nM md -26 ! 7 Hz respectively. The values of the dissociation

constarit calculated fron the two kinds of fluorine nuclei are in good

agreement, Ðd of an order expected, while the ring fluorine trndergoes

a substantially greater shift on binding to the enzy¡ne (Table XIV).

Garnmon et a1.150 ""port

values of 2.19 nM, -65 flz, md -115 I 15 Hz for*

KD, ATFA, ttd ARTNG respectively for this systen at PH 5.5 and 94.L lvlllz,

These values were obtained from calculations which included a nodel for

dinerisation. Such an incl-usion in a calculation of the binding para-

neters has the effect of markedly altering KD but hardly altering A.

This will be further discussion in section (h). This corresponds to

shifts on binding of -39 and -69 Hz at 56.4 MHz. When this systen was

studied in DrO instead of water, values of 0.7 t 1.5 mM and -26 ! 4 Hz

were formd for KO and A respectively at pD 5.4. As these parameters

are the same, ürithin experimental error, for the two systems, there does

ArrO ancl ARf¡,¡C refer to the shifts on binding for the trifluoro-

acetyl and ring fluorine nuclei lespectively.

*

- 85-

not appear to be any change bethleen the state of ionisation of any resi-

due inportant in bi-nding, or any conformatjonal differences of the enzyrne

in the tlo systems. A similar concl.usion was reachecl by Bender "t uL2t7

fron kinetic studies.

At pH 7.7, the separation of the 19U atifluoroacetyl resonances

was rnuch less (3.5 - 9.4 Hz) over the concentration range studied (10-30

nM for the racenic inhibitor). The calculated values were 2.5 t 0"5 rnM

and -34 ! 3 Hz for KO and A respectively. Once again the weaker binding

at alkaline pll was observed.

With N-trifluoroacetyl-m-fluorophenylalanine in the presence of

c-chynotrypsin, the complex spectra of the overlanping n',ultiplets of the

ring fluorine nuclei could not be satisfactorily resolved into those of

the D and L isomers. At pH 5.3 the separation of the N-trifluoroacetyl19_¡ Tesonances varled fror¡ 1.7 - 4.3 Hz over the concentrati.on range 5-30

nM of the racemic inhibitor in the presence of 2.6 TrI4 cr-cliynotrypsin.

KO and A were calculated to be 2.7 ! 0.2 rnM and -11 t 0.4 Hz respectively.

The values from the graph (Appendix, Fig. 4) were found to be 3.4 mM

and - L4 Hz respectively indicating the usefulness of the graphical rnechod

when KO is not too sna11.

Fron these results it can be seen that the ring fluorine atom

influences both the enzyne-inhibitor cornplex dissociation constant (Kn)

and the chemical shift of N- trifluoroacetyl fluorine nuclei in the enzyme-

inhibitor coinplex (A). The decreased values of KO relative to that found

rvith N-trifluoroacetylphenylalanine show tira-t the presence of the ring

- 86-

fluorine atom aids binding to the enzyme. Presumably formation of the

enzyne-inhibjtor conplex is favoured by added interaction of the ring

fluorine atom with the hydroxyl hydrogen of serine-189 which the

crystallographi c data shows to be at the botton of the aromatic binding

pocket, the 'rtosyl holerr. This hydrogen bonding could also orient the

inhibitor in the active site. Fiydrogen bonding of the meta and Bara

ring fluorine atoms would, by necessity, place the trifluorornethyl group

of the respective inhibitors j.n different environments which the diff-

erent values of A show. Such a conclusion would also explain the stronger

binding of N-acetyf-p=arninophenylalanine rnethyl ester relative to N-

acetylphenylalanine methyl ester at pH 8.42L At this pH, the free amino

group with a pq t 4.6422 would most certainly be ,nprotonated.

With N-trifluoroacetyl-o-fluorophenylalanine, no separation of

the 19p trifluoroacetyl resonances could be detected by NMR nethods. The

19a atiftuoroacetyl resonance remained a singlet over the concen'uration

range 5-50 rnM of the racemic inhibitor. It has been reportecl that the

ring fluorine resonances of N-acetyl-o-fluorophenylalanine separate into

those due to the D and L isomers in the presence of cr-chymotrypsin, but

quantitative results cou1c1 not be obt"irl"d.423 îhus one isoner is

preferentially binding to the enzyme. It appears then, that bj'nding is

occurring, but that the environnent of the trifluoromethyl group in the

enzyme-inhibitor complexes must be very close to its free soluti.on

environments for both enantiomers.

In order to overcome the problem of overlappi-ng multiplets in

-87 -

the spectra of the ring fluorine nuclei of inhibitors in the presence

of a-chymotrypsin, the interactions of N-trj-fluoroacetyl-g- and g-

trifluoromethylphenylalanine with this enzyme were investigated. lVith

these probable inhibitors, the aronatic ring is norv lalrel1ed with

fluorine nuclei of which th" 19t, Nx{R resonances are singlets. Thus in

the present of cx-chymotrypsin, preferential binding of one enantiolner

should result ir, 19p NMR spectra consisting of two resonances. Hottever,

over the concentr:ation range studied (5-2A nM of racemic inhi-bitor at pH

5.3, enzyme concent.ration = 2.6 nM) tl-,e 19p ïesonance remained a single

peak both for the aromatic ring ar'd trifl-uoroacetyl trifluoromethyl

groups of both cornpottnds. Once again, eithe:: wep-k binditlg or binding

of each enarrtiomer equally well such that the environment of the nuclei

in the enzyme-inhibj.tor complex approximates that in free solution is

possible. However the arontatic ring, which is of najor inportance in

binding, must occupy tTre I'tosy1 hol.e". By analogy with the results ob-

tained with N-trifluoroacetyl-p-fluorophenylalanine, the shift in the

enzyme-inhibitor complex could not be sinilar to that in free solution.

Thus the only c.onclusion that can be leached is that these two compou-nds

do not function as inhibitors. This conclusion is strengthenecl bv recent

work on the o:-chymotrypsin catalysed hydrolysìs of tyrosine derivatives.

It was found that replacement of the phenolic hydrogen atom by a bulky

alkyl or alkyloxy gïoup 1ed to the abolition of t.aca,ity.424 Hence

steric requirements of a furctional group on the aromatic ring of a

substrate severely influence its binding abil ity.

- BB-

In the case of substitution by an a1kyl group, however, these

results are in contradiction to those of Pcterson et ^t.425

who reported

high reactivity.

However the trifluoromethyl group j.s not much larger than a

rnethyl group or bronine atom, the incorporation of either of which onto

the aromatic ring of N-trifluoroacetyl phenylalanine actually increases

the binding affinity. It seerns unlikely that the slightly larger size

of the trifluoromethyl group relative to the rnethyl group would preclude

its binding altogether. The trifluoronethyl grcup has more hyclrophobic

clraracter tha¡r the rnethyl group or bronin. ^tu^,426

Its insertion into

the hydrophobic binding pocket should be favoured. The possibility of

both ísomers preferentially binding at a site other than the active site

such that the environment is essentially the same ¿rs that in aqueous

solution seems fairly rernote. Thus the lack of binding carl only be

attributed to the unfavourable steric size of the aromatic ring with

a trifluoronethyl group.

N-Trifluoroacetyl-m- a:rd g-rnethylphenylalanine were fot¡nd to

bind strongly to ol-chymotrypsin. The W2 isomer showed a separation

of 1. B - 5 .9 Hz of the 19, ariftuoroacetyl resonances in the concentrati.on

range 10-50 nM of racemic inhibitor. KD and A were calculated to be 1.5 1

0.6 ÍM and -16 t 1.5 Hz respectively. The values fron the graph (Appen-

dix, Fig. 5) were 1.0 nM and -I9 Hz, respectively. With the rneta conpound

the separation varied fron 1.8 - 4.5 ÍIz ovel the sane concentration range.

KO and  rvere calculated as 4.5 t 0.2 nlil and -'20.1- 1 0.2 Hz respecti.vej.y.

- 89-

The values from the graph (Appenclix, Fig. 6) were 2.4.nM and -1'8 LIz

respectively. 0nce again {gta sribstitution leads to wealter binding.

In this case, horvever, the substituent does not orientate the inhíbitor

in the active site; the shifts on t-¡inding are virtrrally the same for

both inhibitors. Tne presence of the rnethyl group does strengthen the

binding relative to the unsubstituted inhibitor (Tab1e XIV). Presunab1y

interaction betr{een the residues constituting the "tosyl holeil ancl- the

methytpherryl group of the j.nhibitor, are enhanced relative to the ur-

substituted inhibitor, by virtue of the formerrs increased hydrophobicitl'.

The hydrophobicity of inhibitor side chain with an attached substituent

decreases in the order cF3 , Br > Cl , CHs > diF , p.426 This is not

quite the order found for binding constants indicating the hydrophobic

interactions are not the only factors influencing binding. No indication

of the trifluorornethyl substituted inhibitors binding to the enzyme

could be demonstrated. The bj-nding of fluoro substituted inhibitolls lvas

stronger than expected, with the ring fluorine atom also orienting the

trifluoroacetyl gïoup indicating interaction of the ring fluorine with

some resiC.ue of the enzyne. Thus the bind'ing of inhibitors with

essentially non-pol a-¡ substituents paral leled their hydr:ophobic charac-

ter indicating the importance of this interactíon.

N-Trifluoroacetyl-2,4-díf1-uorophenylalanine was shot^¡n to be an

inhibitor of o-chymotrypsin, using this NMR technique. The separation

of the l9p atiftuoroacetyl resonances varied fron 1.9 - 4,5 llz over the

collcentïation range 10-40 lrr\{ of racemic inhibitor. KD and A r¡ere

-90 -

calculated as 4.5 i 1.5 rnM and - n ! 7 Hz respectively. The graphed

values (Appendix, Fig. 9) were 3.9 nM and -21- Hz respectivel¡,. These

results show that, with N-trifluoroacetyl-o-fluorophenylalanine, a

failure to bind cannot be due to the steric intelference of the ortho

fluorine aton with a residue side chain of s-chynotrypsin. In fact N-

trifluoroacetyl- 2,4-dífluorophenylalanj-ne has a very sinilar shift or

binding to that of N-trifluoroacetyl-g-fluorophenylalanine, and the

binding is stronger than that of N-trifluoroacetylphenylalanine itself

(Table XIV). It appears, then, that ín N-tríflucroacetyl-o-fluorophenyl-

ala¡i.ne, the ortho ring fluorine has an urfavourable intelaction with the

enzynìe which results in very weak binding. A further fluorine in the

para position compensates this effect, by j-nteracting strongly with,

perhaps, the hydro4¡1 hydrogen of serine-189. The resulting complex is

orientated by this para ring fluorine-serine-189 hydrogen bond. Thus

the enzyne-inhibitor conplexes involving the N-trifluoroacetates of p-

and 2,4-difluorophenylalanine would have sini-lar geonetry. This is

evidenced by the similar shifts on binding, A (Table XIV).

Both N-trifluoroacetyl-rn- and g-bromophenyl alanine bor.nd strongly

to o-chynotrypsin. At pH 5.5, over the concentration range 9-30 nM of

racernic inhibitor, the separatíon of the 19, t"ro.rances of the neta

isomer varied between 4.4 and 9.5 Hz. KD and A were calculated as 1.8

t 0.6 nM and -26 ! 3 Hz respectively. The graphed values (Appendix,

Fig. B) were 2.2'rIM and -32 Hz respectively. For the para conpound at

tlris pH, the measured separation was 3.8 - 1,4.2 llz over the concentration

-9 1-

range l)-40 rd\4 of the racemic inhibitor. KD and A rvere calculated as < l-

mM and -26 t L Hz respectively. The graphed value (Appendix, Fig. 7) of

 was -33 Hz. KD was determined as < 0 fron the graph, indicating the

approximation involved to be only fair. Relative to N-trifluoroacetyl-

phenylalanine, the addition of a bromine atom to the aromatic ring

gteatLy enhances the binding of the inhibitor. The para compound binds

rnuch rnore strongly than the neta isoner; in fact the para compc'und

probably binds as strongly as tryptophan. lVith these bromophenylalanines,

hydrogen bonding to the bronine atom cannot be of najor inportance in

binding as the trifluoromethyl group of each inhibitor occupies a very

sirnilar environment, c.f., binding of N-trifluoroacetyl-n- and g-fluoro-

phenylalanine where the environment of the trifluoromethyl groups was

quite different.

At pH 7.6 the binding of both N-trifluoroacetyl-m- and g-bromo-

phenylalanine rva.s much weaker. The resonarrce separations varied from

4.1 - 8.L Hz and 5.i - I0.7 Hz for the rneta and pa-ra isorners respectively

over the concentra-tion range 9-30 mli{ of racemic inhibitors. KO and A

were calculated as 4.6 I 0.5 rnM and -33 ! 7.5 Hz, and 3.2 ! 0.4 nM and

-37 ! 2 Hz for the neta and para isomers respectively. At pFI 7.6, then,

the environments of the trifluoronethyL groups in the two conplexes are

still very similar. There are slight increases in the shifts on binding

for both compounds, indicating perhaps that the trifluoronethyl groups

are nore buried at the higher pH.

The interaction of N-t.rifluoroacet)'l-p-bromophenylala¡ine with s-

chyrnotrypsin v¡as also studied at a number of other pHts; in all cases

-92-

the concentration of o-chymotrypsin was 2.6 nil'{.

in Table XIII.

The results are shown

TABLE XIII.

Binding pararneters of N-trifluoroacetyl-D-p-bronophenylalanine as a

function of pH

prl KD (n'rM)

^ (Hz at 56.4 }4Jz)

5.0

5.5

6.0

6.ó

7.6

8.2

<1

<1

<-1

<1

3.2 ! 0,4

9.0 t 1.5

-30

:26

-3L

-28

-5t

!2

r1!2

!3

!2

-25 ! L.5

At all pHrs < 7, the enzyne-inhibitor dissociation constant was

snall (< 1 nM). At pH 7.6 and B.2o KO was quite large, indicating that

the binding was nuch weaker at these alkaline pH's. At pH 8.2, the shift

on binding, A, was substantially smaller than at the other pHts. As has

been mentioned, considerable arnounts of the inactive enzyme are believed

to exist at high pH.222'224-228 rhe different value of A at pÍr 8.2

rnay be an art.ifact of the ignorance of this phenomenon in a consideration

of the data obtained.

The values of KO as a function of plì are plotted in Fig. 3.

The graph of KO vs pH for N-trifluoroacetyl--D-g-bromophenylalaaine -shotvs

-93-

I

K¡(mM )

6

1

2

5 I6 7

p l-l

Fig. 3, Variation of KO with pH for N-trifluoroacetyl-D-p-bronophenyl-

alanine.

a break point at pH tu 7.5 consistent with the ionisation of a group in

the enzyme with a pKa of this value. This is presumably the inidazole

ring of histidine-S7.

The interaction of N-trifluoroacetyl-D-tyrosine with cr-chymotryp-

sin was studied at pH 5.5. Except for the initial shift due to bulk

susceptibility effects, the chemical shift of the 19, atiftuoroacetyl

resonance rvas concentration independent indicating very weak binding, or

the shift of the fluorine nuclei in the enzyme-inhibitor complex to be

sirnilar to the free solution shift. As nentioned earlier, the acetate

and trifluoroacetate of a particular amino acid have similar enzyme-

-94-

inhibitor disscciation constants ,278'279 '420 '427 0n this basis, and with

a considerat.ion of the trencls in KO for amino acids and their *id"rr27B279 -2BOLtr'aov

KO for N-trifluoroacetyl-D-tyrosine would be expected to be t 50

mM at pH 7.9. At pl-l 5.3 this could be expected to be ru 1.0 mM. If the

shift on binding were also srnal1, then there would be no apParent change

in shift. The pKa of the phenolic hydroxyl would NLO.42B Th* it is

the phenolic, and not the anionic phenoxide forn which is bínding at pHrs

< 8. The weaker interaction of this inhibitor with cl-chynotrypsin mr:st

be clue to tntfavourable interactions of the hydroxyl group with the aro-

natic binding site.

Another compound with whj-ch no interaction with o-chynotrypsin

could be Cetected, was N-trifluoroacetylphenylglycile. There ivas neithe::

change in the position of the resonance over the concentration range

studied, nor did the resonance separate into two. The ß-methylene group

in the derivatives of phenylalanines is therefore irnportant in binding.

Without this ß-nethylene group, binding of the aromatic ring, if it

occurred, would place the anido hydrogen tclo far from serine-Zl4 fot

hydrogen boncling to further stabilise the conplex. The size, rather

than the nature of the p-nethylene group has been shown to be inportant

by the fact that its replacement by sulphur or an amino group does not

decrease a substraters binding ability; in fact it is slightly

"nh*."d.429 Hov¡ever the rate of hydrolysis of the esters of the L

enantiomers is greatly d".r"^"d.429 A similar lack of reactivity when

the p-hydrogens are replaced by methyl groups has been obr"tu"d.430

-95 -

The values of KO and A obtained for the series of compotmds

studied are sunmarised in Table XIV.

TABLE XIV.

Binding parameters of substituted N-trifluoroacetyl-D-phenylalanines at

pll 5.3 and 56,4 MHz, 0.1M in citrate buffer (deterrnined fron DL racenates)

Substituent h (nM) A (Hz) of N-trifluoro-acetyl 19F nuclei.

H

p-F

n-F

2,4-díF

P-81

m-Br

Bt1

L.3 ! 0.2

2.7 ! A.2

4.5 I 1.5

<L

1.8 r 0.6

1.5 t 0.5

4.5 ! 0,2

no binding detected

no binding detected

no binding detected

-9t1

-26!L

-11 t 0.4

-20!l

-26!L

-26!3

-16 t 1.5

-20 ! 0.2

P-cHs

n-CIl3

P-CFE

n-CF3

o-F

(e) Interaction of the cinnanic acids rvith ct-chynotrypsin

The previous reports of the inhibition of cr-chymotrypsin by

cinnami. u.i,1108'282 nrð. dihydrocinnarnic acid,2l9 '43L'432 sug1ested that

interaction of their fluoro derivatives with cx,-chymotrypsin could be

-96 -

1ôJ.Jstuclied using "F NMR spectroscopy. These inhibitors vrere not suffi-

ciently soluble in a buffer of plì 5.3. Since binding should be stronger

at this pFI, the solubility in a 30% methanol citrate buffer of pti 5.3

was investigated, as o,:-chymotrypsi.n was known to be fu1ly active in

this solvent syster".433 A sufficiently high concentration (50 nM) of

the inhibitor could be obtained, but addition of o-chyrnotrypsín to this

solution such that the concentration of enzyme was 2.6 nil\'I, gave a floccu-

lent solution, rnsuitable for quantitative studies. No furthe:: investj--

gations were carried out.

(h) The effect of enzyne dinerisation and activity on the binding

parameters.

As nentioned in the introduction, the properties of the dimers

and oligorners of o-chymotrypsin is sti11 an open question. Evidence has

been presented in favour of inactive o1igom"rr,233'234 partially active

oligomers ,"',434 otigorners with the number of binding sj-tes equar to

the nr.¡rnbei: of associa.ted nonoïnersr"t drr"rs binding substrate but unable

to deconpose to give produ"ts,236 and oiigoners dissociating jn the

presence of substrut.,235 The nethods used for deternining the activity,

oï concentration of active sites, of the enzyme, depend on the measure-

ment of the co:rcentration of a leaving group forned in the initial hurst

of the catalysed hydrolysis of a substrate ,435 '436 '437 under the

conditions, this may be consiciered as irreversible fornntiorr of an acyl-

etÌzyme. If conversion of oli.gonels to mononters is reasonably rapicl,

-97-

then all the potentially available enzyne rvi.ll be present as the acyl-

enzyme. Rao and Kegel .r'3I h",0" .o.,.1-uded fron their studies of o1i-

gomerisation of o-chymotrypsin that the equilibriun is truly reversible.

Thus the nethods reported for the measurement of activity actua1-J.y

determine the concentration of potential nonomer, i.e., the anount of

molecules present as mono¡ner, dimer, and trímer. Irreversibly forned

oligomers are, of course, implicit inthe determined activity.

A consideration of reverslbLe enzyne inhibition is thus complex,

as either of the models for oligomerisation can be chosen wit-h equal

justification. By far the simplest is that pertai.ning to iitactive

oligomers. The data obtained were treated by a conputer method, the

details of which are clescribed in section 2. The treatment of the data

using other nodels is nuch more complex and not worthrvhile until the

real moclel is knonn. The values of the diner dissociation constant used

were based on,effective I'dimerisationil constants reported by Garunon et

"1.150 i.e., the constant is weighted average of all oligorner disso-

ciation const.ants. They were 0.05, 0.1, and 0.4 rnM at pHrs 5.0 - 5.3,

6.0 - 6.6, and 7.6 - 8.2 respectively.

A comparison of the results obtai.ned, with and without the inac-

tive diner rnodel at pH 5.3, are shown in Table XV. It can be seen that

inclusion of such a no<iel in the calculations reduces KO by about a

factor of ten but has litt1e effect on A. A similar result is obtained

with the parameters of N-trifluoroacetyl-D-g-bromophenylalanine at

various pFIrs (Tab1e XVi) and N-trifluoroacetyl-D-tryptopl-ran (Tab1e XVII)

-9 B-

TABLE XV.

A comparison of the binding parameters of N-trifluoroacetyl substituted

D-phenylalanines with a:rd without an inactive enzyme diner nodel i at

pH 5.3 and 56.4 \fr12, trifluoroacetyl 19F ,r,lclei, dimer dissociation

constant = 0.05 nM.

Substituen.t Dimer not includedA (Hz) KD (mM)

Dirner includedA (Hz) Ko (rM)

H

rn- F

p-F

2,4-diF

-9 11-10.6 ! L.4

-1L ! 0.4

-26!L

-20!L

-20 ! 0.2

-16 1 1.5

-26!3

-26!L

-5 .9 t 0.2

-6. L t 0.5

-9.7 ! 0.4

-24 ! L.2

a

-16 t 0.5

-13,6 t 0.1

-15 10.8

-It!2

-27 ! I.7

0.32 ! 0.a3

0.45 r 0.054

0.25c

0. 15c

o " 4b,c

0. 5 t 0.03

0. 3c

0.2c

0.18 r 0.05

< 0.1

n-CH-J

P.-cHs

n-Br

P-BT

8+" 8.0

2.7

L.3

1.5

4.5

4.5

1.5

L.8

0. ga

0.2

0.2

2b

1.5

4.2

0.5

0.6

1

t

I

+

+

t+

+

+

1

(a)

(b)

(c)

Fron pure D isoner.

Fron ring fluorine studies.

Error < step range (0.1 nM)

-99-

TABLE XVI.

Binding paranìeters of N-trifluoroacetyl-D-P-"bronophenylalanine as a

firrction of pH wíth and rvithout an inactlve enzyme dimer nodel; ^t

56.4 nIIz, trifluoïoacetyl 19F nrcl"i, dimer dissociation constants of

0.05 mM (pH 5.0 and 5.3), 0.1nM (pH 6.0 and 6.6), and 0.4 mM (pH 7.6

and 8.2).

pHDiner not included

A (Hz) KD (IIIM)

Dirner included

A (Hz) KD (rM)

5.0

5.5

6.0

6.6

7.6

8.2

-30 !2

-26!L

-3I!2

-28!3

-37!2

-25 I 1.5

<1

<1

<1

<1

3.2 ! 0.4

9.0 I 1.5

-28

-27

-29

-27

-32

-19

L,4

1,.7

1.5

))

1.5

1.1

+

+

I

+

+

+

< 0;1

< 0.1

0. L t 0.02

< 0.1

0.8 10.1

1.7 ! O.t

TABLE XVII.

Binding parameters of N-trifluoroacetyl-D-tryptophan as a firnction of pH

with and without an inactive enzyme climer modeli at 56.4 Mlz trifluoro-'

acetyl 19F ,,.r.l"i, diner dissociation constants of 0.05 nM (pH 5.3) and

0.4 nM (pH 7 .6) .

pHDimer not included

A (llz) Ko (rM)

Diner included

A (Hz) Ko (InM)

5.3

7.6

-30.4 ! 0.2 < 0.1

-34!L 4.3t7.6

-30 r 0.8

-28!I

< 0.1

0.92 r 0.06

- 100-

Ko and  for N-trifluoroacetyl-D-n-bromophenylalanine at pH 7.6

were 1.02 ! 0.10 rnM and -27 ! 7.3 Ítz respectively. For N-trifluoro-

acetyl-L-tryptophan at pH 5.3 the values were 0.4 t 0.1rnN'l and -5.5 !

0.7 Hz respectively. The inclusion of the enzyne-dimer inodel in the

calculations does not, for the most part, affect the relative values of

KO and A. As such, conclusions reached on binding modes and affinities

from results calculated in the presence, or absence, of the diner model

are the same.

(i) A model for o¿-chymotrypsin inhibition.

By far the greatest factor influencing binding of inhibitors

(and substrates) to q,-chyrnotrypsin is the hydrophobic interaction of the

inhibitor or substrate side chain with the enzyme.248'255'438-443 The

energy changes on binding have been found to be sinilar to those which

occuï on tTansferring the substrate from htater to certain orgalic

solvents, e.g. n-octanol.440'444'445'446 This, and other partition

^od.Lr447-450 hruu pronpted suggestions that the active site polarity

is conparable to that of these organic solvents. The hydrophobic

interactions of the inhj-bitor (or substrate) enzyme result fr:om the

more favourabl-e interaction of the inhibitor (or substrate) side chain

with a non-polar environment rather than with the polar h/ater rnolecule.;

of the solvent ,4sL'452

The amido group is probably only important in orienting the

1abile group of the subst,rate in the correct position for hydrolysis to

occur. The catalytically inportant Tesidues are serine-195 and

'¿\

- 101-.1.

'a\'.

histidjne-S7. Modification of either residue side chain may or may not

affect binding of ínhibitors and substrates. Greatl.)' decreased binding

appeaïs to be due to the nodification of the residue side chain pre-

venting access to the hydrophobic binding site. Of course the binding

site foT the hydrophobic side chain wi11, itself be hydrophobrc. Its

hydrophobicity will be evidenced by lack of reactive polar furctional

glcoups. As such, it is virtually inpossible to identify residues in

the hydrophobic pocket by chemical rneans" Fortunately X-ray diffraction

data have identifíed the hydrophobic pocket as the I'tosyl holerr and alloled

the residues comprising it to be deternined. Yet the dotr'n field sltift

of the ring fluorine nuclei on binding, suggests a more polar environ-

ment. This, and other spectrophotornetric evidence purporting to be the

result of a polar,300,453 o" non-po1ar active site,220,223'409 '454'455'456

really only applies to that part of the site which is being J.nvestigated.

It seems logical that, as the stereospecificity of o¿-chymotrypsin demands

a three point interaction of the substrate with the enzyme, these three

points could be associated with loci of great1-y different nature. the

negligible difference in A of the inhibitors studj-ed between pH 5.0.ard

7.6 infers that there is negligible change in the envir:onnent over this

pH range. No change in envirorrment suggests possibly no change in

conformation of the active si.te. It seems that el.ectrostatic interac-

tions pLay a major part in the binding of inhibitors to o.-chyrnotrypsin.

Whereas the binding of neutral inhibitors (arnides) is virtually pH

independent, that of charged inhi.bitors acyl amino acids at pl{ts above

-102-

5 (where the anion will be the predoninant fott428¡ is narkedly so. As

the bincling clecreases rnarkedly above pft 7, this has been interpïeted as

due to a change of ionisation of a residue sicle chain of the enzyme

with this pKa. Presumably this Ís deprotonation of the inidazole ring

of histidine-57. As this phenonenon also occurs wíth neutral substrates

2L7 '414'457 '458'459 and inhibit.rs,419 brr, to a lesser extent, the

decreased binding of charged inhibitors at alakline pHts cannot be due

to purely electrostati.c interactions .

changes in confonnation of the active site with pH220'224'222'

29r.4t4---,'-'or on binding of inhibitorr2lB'2ln h^u. been the interpretations

of rnany workers from results obtained by other nethods. TIle sinilar À

values at these two pHrs, then, must be due to the trifluoronethyl gloup

of the inhibitor occupying a similar environment, but the renaining

part of the nolecule could be experiencing a different environment.

The work of Gamnon et al.150 ,ho*s that the ring fluorine nuclei of

N-trifluoroacety 1 -D-g- fluorophenyl al anine have a s imi 1 ar environrnent

over the pH range 5.0 - 8.0. Also, the possibility exists that the

conformational changes of the active site do not involve the residues

inportant in binding an inhibitor or substrate, but do involve the

orientation of the groups inrportant for the catalytic activity.

The ori and cause of the down field shift on bincling of theU)

Whereas factors influencing proton chemical shifts are well

19 F nuclei and the nature of the aronatic and acvlanino binding

sites.

-103-

docunented, such is not the case with flucrirre chemical shifts.L62'763'L64

The large amount of infbrnation rvhich has been gatheled in studies of

proton ItrMR spectroscopy has allowed fairly precise explanations of fac-

tors influencing chemical shifts in terms of srxceptibilitles, hydrogen

bonding, ring currents, and so forth. Holever, the arìotÍit of informa-

tion available or, 19F chemical shifts is nuch less, and the ínfluence

of external factors such as those cited above has not been elaborated.

Also a sinple relationship between chemical shift and st.ructure c¿urnot

be established by references to the trends in proton chemical shifts as

a function of structure. 19t. Chenical shifts as a function of structure

bear no relationship to those of protons in a conparison of what type of

nuclei resonate at high or low fíeld. Whereas protons on aromatic rings

resonate at low fields relative to those in rnethyl groups, the reverse

is the case for fluorine nuclei in sinilaÌ structures.

The down field shift on binding of the ring fluorine nuclei of

the inhibitors has tentatively been attributed to hydrogen bonding of

the fluorine aton to the hydroxyl hydrogen of serine-189. This was

discussed in section (e). However, hydrogen bonding effects have been

the explanation of shifts both to 1ow and high field ir, 19p NMR studies

of fluorine containing ions .460 '46L rt is also impossible to decide

what influence the hydrophobic interaction of the aromatic ring of the

inhibitor with the aromatic binding site would have on the ring fluorine

chenical shift. Presumably this interaction would not influence the

chemical shift of the trifluoroacetyl fluorine nuclej- as they are removed

-104-

fron the arornatic binding site. However the fact that both kinds of

fluorine nucleii nove to low field on binding suggests that the sane

factors are operating on these nuclei, but to a diffeTent extent.

Thus it is inpossible from this, and previous work, to decide

. _19the origin of -"F chenical shifts induced by binding. The magnitudes

of these shífts can be usecl to discuss relative effects, i.e. the

greater the induced shift, the more removed fron the aqueous en''¡iron-

rnent are the nuclei in question.

zeffrenT44 fo*rd that the trifluoroacetyl 19, t"ro,,ance of N-

trifluoroacetylphenylalanine is upfield in an hydrophobíc solvent,

relative to its position in aqueous buffer. However in the presence of

an electrolyte, the shift was to low field. His inlerpretation of thjs

phenonenon was that the binding site was hydrophobic but a high charge

density was conferred on it. However, as these shifts were rneasured

relative to an external reference with no susceptibi-1ity cortections,

the rneasuïements are suspect and cannot be used as evidence. The work

of Arrington et ut.462 shows that, for the ring fluorine of fluorobenzene'

the chemical shift is upfield in aqueous solvents relative to that in

non-po1ar solvents. Sinila.r phenomena have- been found in studies of

fluorofor^1463,464 ^nd

other fluoro .onrpo*dr.465 This suggests that

the factors causing the low field shift of the ring fluorine nuclei carr

be attributed to non-polar (hydrophobic) interactions. Such explanations

however, nust be accepted v¡ith reservation.

As mentioned earlier, evidence has been presented both for a

-105-

polar and non-polar active site. It seens logical though, that in the

binding of substrates oï inhibitors, the aromatic ring is bourd in an

hydrophobic pocket such that the carbox,vl (or carboalkoxy) and anido

groups are in the correct orientation at, or near the surface of the

enzyme, and in the correct orientation to rmdergo hydrogen bonding with

particular polar residue side chains in the enzyne. Martinek et a1.432'

444 have conctuded that the polar noieties of the substrate do not

interact with hydrophobic regions of the enzyme. These workers have

shown that, if the polar gloup is on a hydrocarbon chain, however, Í-t

will readily enter the hydrophobic binding ,it".466 This conclusion is

well supported by the crystal structure of the N-forrnyl-L-tryptophan :

cr-chyrnotrypsin complex in which the amido hydrogen of the inhíbitor and

the carbonyl o4ygen of serine-2!4 in the enzyme are ín hydrogen bonding

distance near the surface, and the aromatic ring of the inhibitor

occupies a narrow cleft which extends towards the centre of the enzyme

rnolecu1e.199'20Q'20L Previous work which purported to shol the active

site to be polar or non-polar really applies only to that part of the

site which is interacting with the inhibitor, or other molecule of

which the change in properties due to the inte::action is being moni-

tored. Similar clisadvantages apply to the work of Martinek, Berezin,

and coworkers which demonstrates that the active site is as polar as

n-octanol. Such neasurernents really give only the average of the inter-

actions.

The specificity of o-chymotrypsin for bindi-ng sttbstrates is due,

in part, to hydrophobic as well as a charge-transfer interaction of the

-106-

phenyl ring of the aronatic side chain of the suhstrate with the enzyme.

Substrates with alkyl side chains of the form Ctlj(CH2)nCHCO2CIlg bindI

with the er.zylne, especially when n = 4,467 but not ,t .Ti;?iy as the

arornatic substrates. Delivatives of tryptophan and polycyclic hyclro-

carbons are very strongly bound suggesting that charge-transfer inter-

actions may also be of irnportance, 'Ihis shows that the alkyl group must

be sufficiently long enough for binding to be at a maximum, but that an

optinum length is required for the amido and carboethox¡r group to

hydrogen bond to the enzyme. That is when n < 4, tlne antido and carboxyl

groups may bond to the enzyme, but the hydrophobic binding rtill be weak.

If n > 4, the complex is destabi.lj.sed because th:: hydrophobic pocket is

linited in size, c¿rrr accommodate only a side chaj-n the size of the CH,

(CH2)O- tnit, md no favourable interactions of the anido and carboxyl

groups can occur.

Also the strength of binding of atiphatic alcohols to o¿-

chynotrypsin is proportional to their chain length, i.e., their h)'dro-

phobicity.446,468 If the acyl group is l.arge in comparison with the

side chain (i.e., more hydrophobic), kinetic data suggests that the acyl

group nay bind at the aromatic binding site.469 Such a mode of b-i-ncling

has been called rrwrong way binding'r as such a conplex is in the vtrong

orientation for hydrolysis to occur. This probably orrly occurs with

aryl derivatives of aliphatic amino acids, e.9", hycirolysis of N- 256

picolinylalanine methyl ester where some inversion of specificity occurs

ancl is certainll'neglígib1e in the interaction of acyl derivatives of

-107-

aronatic amino acids. As such it can be neglected in the-se studies .

It also seems feasible that the walis of thebinding pocket are

hydrophobic, while the bottom is hydrophilic. This woulci explain the

pre ferent i al ly b incling o f the N- tri f luoro ace'cy1 fl uoropheny I al anines

(relative to N-trifluoroacetylphenylalanine) . Such an explanation has

precedent in the specificity of trypsin. Since the binding depends

on a basic substrate side chain of a part-Lcular length, e.g.

NFr2 (cH2),",cH-cor-I

NH-

where n = 4 for optimum binding (the sub-strat.e becomes a derivative of

the naturally occu:,:^ing basic amino acid, lysine, thete appears to be

hydrophobic interactions betrveen the hydrocarbon part of the si<le chaj-n

and the enzyme, and also hydrogen bonding between the ammonium ion of

the substrate side chain (at the optirnum pFI) and the ellzylne. In fact

the binding pockets of trypsin and o-chymotrypsin are of simila't síze

and comprised of similar residues except at the botton where the aspartic

acid-189 of trypsin becomes the serine-l-89 of o-chymctrypsin. In the

presence of anmonium ions, trypsin behaves rernarkably like cr-chyno-

tryps in.470 Presunably aspartic acid-189 at the bottom of the ar:omatic

bindi.ng pocket j-s neutralised, thus leaving the renaining part of the

site like that of o-chymotrypsin" Trypsin also binds aromatic amines

particularly wel1, again showing the homologies of the side chairr binding

pocket as regards size and length !".t. Introduction 2(a)1.

rf, in thc inter:action of ac)'i anino acicl substrates with a-

-L0B-

chyïnotrypsin, it is postulated that hyclrophobic binding is the prirnary

binding force, followed by hydrogen bonding of the anido hydrogen to

the carbonyl o4¡gen of serine-2L4, the activity would ari-se from the

ester or arnide bond being in the correct orientation for catalytic

scission of the 1abile bond to occur with the aid of the chaige relay

system. l\lith the D antipode of a substrate, the hydrophobic and anido

bonds would lead to the labile bond being in the wrong orientation,

i.e., away fron, instead of towards, the nucleophilic serine-19S of

the charge relay system, for hydrolysis to occur. ltlith acyl amino aciûs,

an acyl-enzyne could thus forn wj.th the L, but not the D antipocle.

However, the NMR results of this research show that this cannot

be strictly correct. The different values of A of the 19p,g-atifluoro-

acetyl fluorine nuclei with different inhibitors show that the aryl

side chain orients the trifluoroacetyl group. The vastly different

values for the D and L enantioners show that the environments of the

trifluoroacetyl group is vastly different between these forns. For the

L isomer, the snall value for the shift on binding indicates an environ-

rnent litt1e different fron that in free solution. However the tri-

fluoroacetyl group o.f the D isoner is possible in a substantially more

polar but at the sane time hydrophobic environrnent.

(k) A model for enzyrne-inhibitor conplexes.

The nature of the residues forming the active site has been

determined with some ceïtainty for the crystalline enzyme. lhis appears

to be a good nodel for the systern in solution. The approximate size and

-109-

shape of the aromatic binding site had also been determined previously

from the optinun, and liniting dimensions of molecules whj-ch can act

as stùstr ut"t r47r or inhibitors.2Bs '472 Hi.stidine-S7, like serine-L95,

has been shown to be important in catalysis, but its modification does

not affect binding of substrates and ínhibitors.473'474 Hence evidence

gathered by chernical means has not demonstrated the inportance of any

polar residue in the enzyme critically inportant in binding of inhibitors

or substrates, the nain interaction is that of the hydrophobic substrate

or inhibitor side chain with the trydrophobic pocket of the enzvme. Part

of the inhibitor which is polar (the acyl or carboxyl groups) nay inter-

act near serine-195 as bulky rnodificatíon of this residue prevents

binding.

The X-ray diffraction results show that the amido hydrogen of the

inhibitor probably forms a hydrogen bond with the carbonyl oxygen of

serine-214. The carboryl group of the inhibitor is then near histidine-

57 and serine-19S. Such an arrangement fulfi.ls the requirement of

stereospecificity and places the labile group of an ester in the co.rlect

situation for hydrolysis. For a D inhibitor, the hydrophobic interac-

tions nust stay the sane; there is only one hydrophobic cleft. The

interactions of the amido and carbo4¡I groups cannot- be reversed, i. e. ..

the amido group cannot interact with serine-195 and the carboxyl group

with serine-2l4, as the polarities are reversed. At all the pFIrs

studied, the inhibitor will be present as the carboxylate anion. This

anion probably interacts rvith a neutral active site bel-ow pFI 7 artd a

-110-

negative acti.ve site above pH 7 when histidine-S7 is deprotonated. The

carbo4¡l oxygen of the L inhibitor i.s probabty hydrogen bonded to the

anido hydrogens of glycine-193 and serine-l9S. Such a nodel places the

N-trifluoroacetyl gloup of an L anino acid in essentially an aqueous

environment, i.e., it protrudes into the aqueous solution fron the active

site. Theoretical calculations have also shown this nodel to be nost

stable .475'476 This is consistent with these NMR results which shorv

the environment ,of the 19p ariftuoroacetyl nuclei to be in a¡r environ-

nent vely similar to a free solution environment.

With a¡r inhibitor of the D configuration it is unlikely that the

carbo4yl group stil1 interacts with serine-195. If its orientation we.re

the sane as that for the L enantiomer, then the estcr of D antipodes

should be appreciably active. Presumably the position of thc carboxyl

group is nearer to the inidazole ring of histidine-S7. This then puts

the acyl group neaï to glycine-19S. If a hydrogen bond between the

amido hydrogen of the inhibitor and the backbone carbonyl of glycine-193

were formed, the trifluoroacetyl group would be flanked on one sicle by

part of the enzyne molecule. The N-trifluoroacetyt 19f nuclei wou.1d

thus be partly protected fron the solvent, ild such interactions could

explain the down field shifts on binding of the D isomers.

EXPERIIvIENTAI,.

- 111-

(a) General.

19, ,r,* spectïa h¡eïe recorded on a varian DA-60 (1.L.) N.M.R.

spectrometer operating at 56.4 MHz in the frequency sweep node' Probe

temperature was 29o. Spectra at low concentrations of inhibi'[or were

obtained with the aíd of a Varian C-L024 conputer of average transients

(cAT) which was used to drive a Hewlett-Packard 3310-A low frequency

fturction gener:ator. The spectra were obtained by sweeping a frequency

ïange of 20-50 Hz at a rate of one scan per 25 seconds. Peak positions

ancl peak separations weI.e detennined by electronic cotnting with a

RACAL SA35 r.¡nir.ersal counter-tirner and are accurate to ! 0'2 FIz in nost

cases.

The heteronuclear spin decoupler was a unit constructed by

Mr. R.L. Paltridge of this departnent.

rn order to observe the trifluoroacetyl 19r ,ignuls of the

inhibitors, a capillary of trifluoroacetic acj-d was used as the refer-

ence lock signal. A capillary of benzotrifluoride ldas used as the

reference lock signal in the recording of the aromatic triflu'oromethyl

resonances. In order to o¡serve the aromalic l'9F t"ro.tances, a capillary

of 11 1.12r2-tetrachlorotetrafluorocyclobutane in carbon tetrachloride was

used. The sane capillary rvas used for each determination in a part-

icular experiment; peak positions and peak separations were conparable

on1.y when the sane capillary was used'1q

With some ne.!g-fluoro compouncls, the '"F 1esonances were coinci-

dental with the lock. In these cases, a capiJlary of tlifluoroacetj.c

-ttz-

acid was usecl as the lock and the rnanual oscillator frequency was supplied

by a lt{uirhead D-8904 deca<ie oscillator.

Solutions weïe prepared by dissolving a weighed anor¡rt of the

enzyme in a known volume of a mixture of the inhibitor in buffer sol.u-

tion, and a solution of the buffer such that the total volume of the

solution was 0.5 nl.

o-chymotrypsin (three times crystallised), DFP-chymotrypsin and

the other enzymes used rve:ie purchased fron l\lorthington Bioche¡nical

Corpolation. The activity of cl-chymotrypsin was assumed to be 100e";

no correc1ion rras nacle for inactive inpurities. Atthough the activíty

is'ì,BO%, the values of KO and A deternined are inportant in relative,

rather than absolute terms so are unaffected by such an assumption. It

was also assumed that the activity remained constant throughout differ-

ent batches.

n-FluorophenylaIanine and g-fluorophenylalanine were purchased

fron Sigrna Chenical. Co., Missourí.

The buffers used rnrere prepared accordíng to Wolff arid Vander.^t4'17

or t'Biochemistsr Handbook"4TB and the pH checked with a Pye universal

pl{ meter. For solutions prepared in DrO, pD = meteÌ' reading +0.4

according to Glascoe *d Lorrg.479

Optical rotations were determined on a Hilger M4I2 polarimeter.

petroleun ether fractions, b.p. 40-600 and 60-80o "t" X-4 and

X-60 respectively. All melting points were deternined on a Kofler hot

stage melting point appa::atus and are uncorrected.

-tts-

Analyses were perforned by the Australia¡r Microanalytical

Service, It'felbourne.

In the naming of racemic inhibitors, the prefix DL was not

always used.

D-Phenylalanine gave, by the above nethod, N-acetyl-D-phenyl-

alanine , 72%, fl.p. r70-r7ro., t"]3t = -48.50, c = 2% in absolute

ethanol (lit'323 "'o,

[n]3u = -tto in absolute ethanoll'

-7L4-

(b) ACETYLATION OF THE AMINO ACIDS

N- Acety lpheny 1 a1 an ine .

322Phenylalanine was acetylated according to Town to give N-

acetylphenytalanine, Z4%, m.p. r4s-147o (lit.322'480 L46o, 150-1510).

N-Acetyl - D-phenyl al anine .

N-Acetyl -L- phen-¿1 a1 anine.

N-Acet 1t an

322Tryptophan was aeetylated by the nethod of Town to give N-

acetyltryptophan, B1%, m.p. 204-2060 (1it.324 zos-206o).

N- Acety 1 - D- t rypt oph an .

L-Phenyialanine gave, by the above nethod, N-acetyl-L-phenyl-

alanine , 6g%, m.p. I7I-I7,2}, to]35= 47.Io, c = I.gS% in absolute ethanol

(lit.323 ,rro, lg]rru = -5lo in absolute ethanol for the D isoner).

324D-Tryptophan hras acetylated according to du VJ-gneaud and Sealock

to give N-acetyl-D-tryptophan, 70e", rn.p. t7g-1,82o, [cl]lu = -"o, c = Leo

in water with l equivalent of soclium hydroxide (1it.324'277 ßg-1-900;

to]31 = 29o, c = I% in water with L equivalent of sodiun hyclroxide for

the L isomer : 180-181-0; to]35 = 50 ! 10' c = 1'23% ín water with 1

equivalent of soCium h¡'c1t.t"td" for the L i-sorner').

178- 181

-115-

N-Acety 1 - L- tryotoph an .

L-Tryptophan sinilarly gave N-acetyl-L-tryptoplian, 73'o, m.p.

o, tol35 = 2Lo c = L% in water with 1 equivalent of sodiun

hydroxide (1it. 32 as above).

(c)PREPARATION OF T}IE N-TRIFLUORCACETATES.

N- Tri fluoroacetylpheny lalanine .

327

The nethod employed was essentially that of Weygand and Leising.

Phenylalanine (0.825 g, 0.005 mole) rÀras suspended in dry benzene (20 nl)

and, with vigorous stirring, trifluoroacetic anhydride (0.8 nl, 0.005

nole) added. The solution rvas warmed to 70o , t1,r: solvent, trifluoroacetic

acid, and unreacted trifluoroacetic anhydride removed urder reduced

pressure and the residue crystallised from benzene. N-Trifluoroacetyl-

pherrylalanine, 0.96 g (74%) was obtained; rn.p. L23-725o (lit.4BL'329

L2L-I23o , !25.6-126.Ao). This compound was also prepared by the nethod

of Schallenberg and Calvin.329 Phenylalanine (0.825 g, 0.005 nole) tvas

dissolved j-lt 1N sodiun hydroxide (5 nl, 0.005 mole) watel (20 ml), 'and

ethyl thioltrifluoroacetate (1.0 nl, 0.008 rnole) added. The mixture

was shaken for 72 hours, made acid with concentrated hydrochloric acid,

cooled in ice, ffid the solid filtered off. Recrystallisation fron

benzene gave N-trifluoroacetylphenylalanine, 0.98 g (76v") n.p. t26-728o

(1ir. 4Bt'329 r2L-tzso, Lzs.6-126. ao).

Also prepared by the forrner nethod wcre:-

4,277

-LL6.

N- Tri fluoroacety l-m-nethylphenylalanine , 7L%, m. p. tt6-Lfio. Calc.

for CrrHrZNO¡FS : C, 52.37; H, 4.36; N, 5.09; F, 20.7. Found: C

52.33ì H,4.21; N,5.20; F, 20.Beo.

N-Tri fI uoloacetyl -p-methylphenylalaline, 769o, m. p. t34.5-1360. Cal c.

N-Trifluoroacetyl-o- fluorophenylalanine, 689o, m. p. 1,32-I33o. Calc. for

for CTTHTZNOSF¡ : C 52.37 ; H, 4. 36; N, 5.09 ; F , 20 .7 .

52.65; H,4.39; N, 5.20; F, 20.Aeo.

CttHgNOSF4 t C, 47.3L; H, 3.23; N, 5.02; F, 27.2

H, 3. t6; N, 4 . 78 ; F, 26 .9%.

N-Trifluoroacetyl-n- fluorophenylalanine, 7 39o, m. p.

by solidification and finally nelting at 119- 7200.

Found: C,

Found: C, 47 .481,

Lt2-7.730, folloured

Calc. for CrrFInlJO

C, 47.52; H, 3.2'/; N, 4.87;

op. 744 .s- 14s . s Calc.

Found : C, 47.511'

Calc. for

Fourid:

E3'4

C, 47.31; H, 3.23; N, 5.02; F,27.2. Found:

F, 27,2eo.

N-Trifluo:roacetyl- p-fluorophenylalanine, 73eo, m.

for CrrHnNO3F4 : C,47.3t; H,3.23i N, 5.02; F,27.2"

H, 3. 58; N , 4.96; F , 27 .teo.

N-Trifluo roacety!-m-bromophenylalanine, BB%, n.p. I35-L37o .

CrrHnNOaFrBr i C, 38,83; H, 2.65; N, 4.12; F, 16.7;8r,23.5.

C, 38.84;11,2.70; N, 4.23; F, L6.4;Fr,23.7%.

-tt7-

N- Trifluoroacetyl- p-brornophenylalanine , B5%, m.p. 147't4B

final-ly rnelting at t6O-I62o. Calc. for C

23,5, Found: C

o and then

,HrNOrFrBr:

,38.98; H,

l_resol idiffing,

C, 38.83; H, 2

2.47; N, 4.25;

, 4.t2; F, 16.7; Br,

.3; Bt , 23.2'o.

65;

F, L6

N

N-Trifluoroacet L-2 4-difluo ronhenv I al anine 67e; , m.p.

F, 32.5.for CTTHUNOSFS : C, 44,44:' H, 2.69; N, 4.71;

H,2.70; N, 4,73; F, 32.5%.

N-Tri fluoroacety 1 -n- trifluorometh),lpheny1al an ine., B2'o, m.p.

Calc. for CrrHntOaFr., z C,43.77; H,2.74; N,4.26; F, 34.7

C, 44. 00; H, 2 . 85; N, 4.27 ; F , 34.8%.

N-Tri fluoroacetyl-p- tri fluoromethylpheny 1 a! anine, 769o , m,p.

F, 34,7.

LsL-Ls3o. Calc.

For¡rd: C , 44 ,35 ;

1.43-L45

Formd:

L42-144

Fourd:

o

o

Calc. for CrrHnN0SF6

C, 43.84; H, 2.631, N,

C, 43,77 i H, 2.74; N, 4.26;

4. 15 ; F, 34 .9%.

N-Trifluoroacet I t 1 c].ne

C, 48.59; H, 3,24; N, 5.67; F

F , 22.9%.

Formdr C, 48.70; H, 3.1-B;

N-Trifluoroacetyl-D-phenylalanine, 68%, m p.L2o-!2f, t"l3t

s7%,

23.1.

n.p. 159-160o. Ca1c. for CrO HrNOrFr:

N, 5.87;

-29o , c=

I% in water with I equivalent of sodiurn hydroxide (Iit.329 Iß.4-L2O.60

to]35 = 36.40, c = 0.0187 g in 5.0 ml of glacial acetic acid for the L

isomer) .

- 11B-

N-Trifluoroacetyl-L-phenylalanine, 72%, m.P. rts-tzoo, [o]3u 32o

,v

329L% in wateï with 1 equivalent of sodiurn hydroxide (lit. as above).

N- Tri fluo ro acety 1 - D- t ryptophan .

D-Tryptopharr was reacted with ethyl thioltrifluoroacetate

according to Schallenberg and Calvirr,329 but with a reaction tirne of

72 hou-rs. N-Trifluoroacetyl-D-:tryptophan was obtained in a yield of

73eo, m.p. 163-1640, [o]'rt = -tro¡ c = 1% in water with l equivalent of

soclium hydroxide (lit.329 t6z-163o for the L isomer).

N-Tri fluoroacetyl - L- tryptophan.

In the same manner, N-trifluoroacetyL-L-tryptophan was obtained

in 66% yield, n.p. t63-I64o, [a]'rt =r1or c = LYo ín hrater v¡ith 1 equiva-

lent of sodiun hydroxicle (1it.329 tøz-163o).

N-Tri fluoroacetylt rYPtophan .

This conpound was likewise prepared in 75eo yield, m.P. 155-1570

(Lft.482 1s2-1s4o)

N- Tri fluoroa cetyl - L- tYros ine .

L-Tlrosine (1.81 g, 0.01 rnole) was suspended in dry benzene (20

nrl) and trifluoroacetic anhydride (3.2 nL, 0.02 mole) adrled with stirring.

The solution was war:med to 70o, the solvent' excess anhydride, and tri-

fluoroacetic acid rernoved in vacuo, and the residue crystallised fron1E

benzene to give N-trifluoroacetyl-L-tyrosine, 79'o, m.p. 195-1960, [o]í'=

430 ,

-119-

c = !% in water with 1 equivalent of sodiun hydroxide (lit. 329

44 go, c = 0.4eo in water with 1 equivalent of sodiumts2-rs30, t"l3o

hydroxicle.

N- Tri fl uoroacetyl - D- tyrosine .

D-Tyrosine gave in a¡r analogous manner, N-trifluoroacetyl-D-

tyrosine , 78%, m.P. 193-1950, t"]3t = -52o, c = 1% in water with 1

equivalent of sodiun hydroxide (Lft.329 tg2-7g3o, [o]'ro = oo.to , c =

0.47% in water with 1 ecluivalent of sodium hydroxide for the L isorner).

N-Tri fluoroacetyl - p-nethoxyphenylalanine nethyl ester.

N-Trifluoroacetyl-D-tyrosine (0.227 g, 0.00L noIe)' methyl

iodide (2.5 nl) , and silver oxide (1 g) in dry acetone (10 rnl) were

stirred overnight. The silver oxide was removed by filtration and thc

residue, after rernoval of the solvent, hras sublimed at 14Oo/L5 run. hl-

Trifluoroacetyl-g-methoxyphenylalanine methyl ester, 0.210 g (69e"),

rn.p. 76-78(J after a further sublination was obtained; mass spectrum,

M+, m/e = 305 (n.wt. = 305); Calc. for CrrHr+NO+Fg I C, 51.l-5i H, 4.59;

N,4.59; F, 78.7. For¡rd: C,51.05; H, 4.64; Nr 4.37; F, t9.Seo.

(d) PREPARATION OF TTIE A},IINO ACIDS .¡U\D A-ACETAT'IIDOCINNA}{IC ACIDS

VIA TIIE AZLACTONE IVIETTIOD.

g-ACETAMIDOCINNAMIC ACID.

The nethod employed was that of Herbst and Shemj-n.550

Benz al -

-t20-

dehyde (2t.2 g, 0.2 nole), aceturic acid (15.6 9, 0.134 nole), sodium

acetate (8.2 g, 0.L nole), and acetic anhyclride (36 g, 0.34 nole) were

heated on the water-bath until solution becarne coÍp1ete (20-30 minutes).

The resulting s<llution was heated under reflux for t hour and then

cooled in the refrigerator overnight. Water (40 ml) was added to the

solid mass which was broken rrp z:nd filterecl off. Recrystallisation

fron ethyl acetate /X-60 gave 4-benzylidene-2-nethyl-5-oxazolone, 1.3.0 g

(52%), m.p. I4g-152o. The azlactone (10 g) was heated turder reflux for

4 hours with acetone (35 nl) and water (90 ml) ' acetone added until

solution was complete, ffid the solution boíled for a further few rninutes

in the presence of charcoal. The solution was fj l-tered and o,-acetanido-

cinnanic crystallised on cooling. The yield was 9.5 g (36%) rn.p. 1960

(lir.336 rgtrs2o).

o-ACETAMIDO-ß- (p - FLUOROPI]ENYL) -ACRYLIC ACID.

2-MethvL-4- p-Fluorobenzylidene-5-oxazolone was similarly prepared fron

g-fluorobenzaldehyde. The yield was 56e¿, fl.p . t52-153o. Ca1c. for

C'1HSNO2F ! C, 64.39! H, 3.90; N, 6.83; F' 9'3' Found: C' 64'49;

H, 3.95; N, 6.91; F , 9.0%. Hydrolysis gave cr-acetamido-ß-(g-fluoro-

phenyl)-acrylic acid, 8L%, m.p. 216-2200. Calc' for CrtHi.oNOsF : C'

Sg.2O; Í1, 4.48; N , 6.28; F, 8.5. Found: C, 59'07; H, 4'73; N, 5'99;

F, B.L%.

n- Ii,IETI{YLPTIENYLAI,AN I NE .

n-Tolualdehyde.

^1r^L,,Ja l- ^ o"-ooo r1,7 -^ (1-'* 483 oz-o/4olrz --ì

-L21,-

prepaïed in 42% yield from n-bromotoluene by the nethod of Smith and

Bayliss. 484

2-Methy 1-4-m-nethylbenzyl idene-5 - oxazo I one ¡vas prepared in 50% yield

from m-tolualdehyde according to Herbst anrl Shemin.336 The m.p. hlas

105-1080. Further purification was difficufi: and was not perseverecl

with.

o-Acet amicio- ß - methylphenyl) - acryLic acid.

'Ihe crude azlactone (10 g) in water (35 nl) and acetone (100 nl)

was heated gnder reflux for 4 hours. The solvent wa-s removcd in vacuo,

and the residue c::,'stallised fron acetone/water to give the acrylic

acid, 85eo, m.p. 186-187o as yellow plates. Recrystallisation from

chloroform/acetone or water gave white needles, m.P. 186-187o. Calc.

for CrrHrrlJO, : C, 65.76i H, 5.98; N, 6.39. Found: C, 65.82; Iì, 5.90;

N,6.27%.

N-Acetyl - rn- methy lpheny 1 a 1 anine .

The acrylic acid (6.8 g) vras suspended in 95% ethanol (150 nl)

and hydrogenated in the presence of 5% palladiun on carbon (1 g) at 4

atmospheres for 4 irours. The catalyst was removed by filtration through

Celite and, after removal of the solvent in vacuo, the residue was

crystallised from water to give N-acetyl-n-methylphenylalanine , 5.3 ;

(77%), m.p. 149-1s1o (1it.485 14s-149o).

-t22-

rn-Methylphenyl al a:rine .

The acetylated anino acid (5 g) was heated under reflux for LB

hours with LN hydrochloric acid (100 rnl). The acid and water were

rernoved in vacuo, water (50 nl) added, and that removed in vacuo. The

resiclue was dissolved in a little hot water', the pH brought to 6 with conc.

amnonia, and the arnino acid filtered off after cooling. The yield was

9!%, n.p. 2Lo-21go d (lir.486 z+z-2460 d).

p-MET}IYLPHENYLALAN]NE .

2-Methyl-4-p-methylben zylídene-S-oxazalone, 54%, m.p. 150-1350 was sini-

larIy prepared. Further recrystallisation raised the neltj-ng point to

L36-137o. Ca1c. for CtZHttNOZ ! C, 7t.65i H,5.47; N,6.98. Formd:

C, 71. 95 ! H, 5 . 47 ; N, 7 .05eo.

cr-Acetamido-ß- (p -methylphenyl)-acrylic acid was obtained in a yield of

77%, n.p. 225-23Oo. Recrystallisation from chloroform/acetone raised

the rneLting point to 234-2350, Ca1c. for CrrHrrNo, I C, 65.76; H, 5.98;

N,6.59. For¡rd: Cr 66.04; II,5.83; N,6.26Yo.

N-Acetyl-p-methylphenylalanile., n.p. L66. 5-1680 (1it.485 to¡.5-164. 50

was obtained in a yield of B1%.

p-Methylphenylalanine, n.

)

in 98% yield

p. zL4-zLao d (tir.486 276-279o d ) was obtained

-123-

rû- TRI F LUOROME TIIY LPIIENY LALANI NE .

n- B ronobenzotri fl uori de .

487Benzotrifluoride was brolninated to give the above comPormd

in 55eo yield, b.p. L54-L57o (lit.487 sz%, b.p. 151-1520).

m-Tri fluoronethylbenz aI dehyde

m-Bromobenzotrifluoride was converted to its Grígnard reagent

by the method of Smith and Baylirr.4B4 The Grignard reagent was reacted

with N-methylfonnanilide according to Gilrnan et 41.4U8 ,o girre m-

trifluorornethylben zaìè,ehyde, SO%, b.p. 72-730/ 17 nn (1it.488 5 L-sg%, b.p.

64-660/ 1o nm).

2-Methy 1 -4-m-trifluoronethylbenzyl idene- 5 -oxaz alone .

m-Trífluorornethylbenzaldehyde, aceturic acíd, acetic a:rhydride,

and soditun acetate were reacted according to Fi1ler an<l Novar344 to giu"

the azractone, 67%, m.p. 123-L250 (lit.344 84%, ^-p. 150-1310).

c¡¿-Acetanido-ß- (n-trifluoromethylphenyl) -acrylic acid.

Hydrolysis of the azlactone according to Fi1ler and Novar344

gave the acryl1c acid, 76eo, m.p. 2rg-220o (1it.344 etz, m.p. 225-2260).

N-Acety I -m- tri fluoromethylphenvl al anine .

o-Acetanido-ß- (rytriftuoromethylphenyl) -acrylic acid was hydro-

genated according t.o Fil-1e:: anrl No't ut344 to give N-acetyl-n-trifluoro-

nethylphenylalanine, 86%, m.p. t2g-730o (lit.344 eO%, m.p . 1.sz_igso).

-124-

n-Trif1uoro¡nethyl.pheny1al .

This anino acid was prepared fron the N-acetyl compound b1r 1¡s

nethod described for the preparation of n-methylphenylalanine. The yield

was 53%. Recrystallisation from v\¡ater gave the pure amino acid, rn.p.

lgl-202o d (lir.344 2ß-222c d). calc. for croHroNozFg i c, 51.50;

H, 4.29; N, 6.01; F, 24.5%. Fou¡rd! C, 51.85i H, 4.4I:' N, 5.7t; F,

25.0%.

(e) PREPARATION OF TTIE AMINO ACIDS BY THE I"{ALONIC ESTER METI]OD.

o - F LUOROPHENYLALAN INE .

o-Fl uorob enzy I bromide .

The nethod used was essentially that of Olah et "t.489 I

Fluorotoluene (1L.0 g, 0.1 nole) was dissolved in dry benzene (15 tnl).

The solution was heated under reflux, irradiated nith a 200W incandes-

cent light, and dry bromine (4.5 nl, 0.09 nole) added dropwise over 1

hour. After a further hour, heated utder reflux, the solution was

fractionated rnder reduced pressuïe to give o-fluorobenzyL brornide,

72.s g (74%), b.p.96-Lolo/25 mm (tit.489 tg5-202o).

Diethyl o-acetanúdo-q- (o- fluorob enzyl) -ma1 onate .

sodiun (0.69 g, 0.03 mole) was dissolved in dry ethanol (50 m1),

diethyl acetanidornalonate (6.51 g, 0.03 rnole), md o-fluorobenzyl

bromicle (4.9 g, 0.026 rnole) were added. The mixture was heated urder

reflux for 4 hours, filtered hot, and r{ater (100 m1) adcled to the filtrate.

-tzs-

The diethyl a-acetamid.o-o- (o-fluorobenzyl) -malonate was filtered off

after cooling, anct was crystallised from aqueous ethanol. The yield of

material, m.p. 1.L7-11.2o (Iit.34t toz-108o) was 60%.

o-Ir luorophenyl a1 anine .

Diethyl o-acetanido-cr,- (o-fluorobenzyl)-malonate (3.25 g, 0.01

rnole) and 48s¿ hydrobrornid acid (50 ml) were heated under reflux for 6

hours, th-e solution brought to pH 6 with concentrated amrnonia, and

concentrated in vacuo until crystallisation began. The amino acid was

filtered off ard washed well with water to remove ammonium bromide. The

yield of acicl, n.p. 2rz-214o d (lit.490 2s8.5-259o d) was 1.1 g (5s%).

2 4 - DI F LUOROPIJENY LALAN INE .

4-Amino- 2 - fluorotoLuene.

2-Amino-4-nitrotoluene, m.p. i.05-1080 (rit.49t toro was prepared

by nitration of o-toluidine according to Ullmann "t ul.491 2-Amino-4-

nitrotoluene (t52 g, 1rnole) u/a.s suspended in a rnixture of concentrated

hydrochloric acid (210 rnl) and water (300 rnl). The mixture v¡as stirred

vigorously, cooled to OoC with a dry íce/acetone bath, and tl-iis te-npera-

ture rnaintained while a solution of sodiun nitrite (87 g) in wat'er

(220 nI) was added dropwise. The filtered diazonium solution was stirred

at OoC while an ice-cold soluti.on of sodium fluoborate (138 g, 1.25 rnole)

in rvater (S00 ml) was adclecl over 10 minutes. After stirling f-or 1¿ hout,

the diazonj-tun fluoborate was filtered off , washed successively rvith a

-726-

little cold water, rnethanol, ether, and dlied. The yie1d was 160 g

(85%). The diazonium salt was decomposed in dry chlorobenzene according

to Brown et al .492 ^nd

the resul tlng 2-l.Iuoro-4-nitrotoluene reducecl

in situ with iron ancl aceti. ^"id492

to give 4-amhno-2-fluo::otoluene,

78%, b.p. g4-g7o/ 16 nm (1i1.492 zoz-204o).

2 ,4-Dífluorotoluene.

4-Amino-2-fluorotoluene was conr.rerted to tl¡.e diazonium fluoborate

in the manner described above for 2-anino-4-nitrotoluene. The yield was

79%. Thermal decomposition of the dry cliazonium salt in the usual *uy493

gâve, 2 ,4-difluorotoluen e, 64%, b .p. 113- 115o (Ift..492 tl3-7úo) .

2, -Difluorobenzyl bromide.

Free radical bromination of 2 14-difluorotoluene by the nethod of

01ah et "1.489

gave 2, -difluorobenzyl bronide, 70%, b.P. 74-76o/15 nm.

A sample, b.p. 75o/13 nm was submitted for analysis. Ca1c. for CrHrFrBr

C,40.57;11,2.42; F, 18.4. Found: C, 40.47; H,2.47; F, LB.4%.

Diethyl cx-acetarnido-cr- (2,4-difluorobenzyl) -mal onate .

The reaction of 2,4-dífltorobenzyl bronide (5.42 8, 0.026 nole),

diethyl acetamidomalonate (6.51 g, 0.03 nole), and sodium (0.69 gr 0.03

nole) in dry ethanol (50 ml) under the condi-uions described in the

preparation of o-fluoropl-renylalanine gave diethyl q-acetamido-cr¿- (2 '4-

difluorobenzyl)-nalonal-.e, 6.6 g (74e"), n.p. 133-t34o. Ca1c. for

-L27 -

C'OH'gNOSFZ : C, 55,98; H, 5.54; N, 4.08; F, 11.1. Found! C, 56.09; H,

5.65; N, 4.01; F, 1,7.3%.

2 ,4-Dífluorophenyl alanine .

'Ihe nalonic ester v¡as converted to the anino acLd by the nrethod

described in the synthesis of o-fluorophenylalanine. The yield of anino

acid, m.p. 2t5-220o d, was 76%. Recrystallisation from water raised the

melting point to 230-2340 d, Calc. for CnHnNorF, i C, 53-73; H, 4.48;

N, 6.97; F, 18.9. Fou:rd: C, 53.73; H, 4.35; N, 7.lLi F, 78.9e".

N-Acetyl - 2 .4- di fluorophenyl al anine .

Diethyl o-acetamido-a- (2,4-dîfluorobenzyl) -nalonate (L.72 g,

0.005 nole) was heated under reflux fot 4 hours with 2.5N sodium

hydroxide (25 nl), the pH adjusted to 2-3 with concentrated hydr-ochloric

acid, a:rd reflux continued for a further hour. On cooling the product

crystallised. 'Ihe yield was 0 .78 g (6s%), il.p. 148-1510. Calc. for

CtlHt'NOEFZ, C, 54.33; H, 4.53; N, 5.76; F, 15.6. Found: C, 53'96;

H,4.59; N, 5.70; F, 75.4%.

n- BROMOPHENYLALAN INE .

m-Bromobenzyl bronide.

Tris cornpor.¡nd was obtained ín 75eo yield by free-radical bromina-

tion of m-bromotoluene. It had b.p. 722-L26o/ 12 rnm, m.p. 35-5Bo Gjt.494

+oo) .

-tzg-

Diethy I cr- acetaniclo-cr- (m-bronob enzyl) - ma1 onat e .

m-Bromobenzyl bronide (6.75 g, A.027 mole), dietl'ry1 acetamido-

rnalonate (6.51 g, 0.03 mole), and sodiun (0.69 g, 0.03 mole) in dry

ethanol (50 nl) wele reacted as described in the preparation of o-

fluorophenylalanine to give diethyl o-acetamido-'r- (m-bronob enzyL) -

rnal.onate , 7.6 g QgÐ, m.P. 90-960. Further recïystallisation from

aqueous ethanol raised the nelting point to 100-1010. Ca1c. for

C'.HZONOSBT :' C, 49.73; H, 5.18; N , 3.(>3; Br, 20.8%' Foundl C, 50'06;

H, 5.40; N , 3,52i Br, 20.6%.

m-Bromophen)¡l al aninc .

The nalonic ester (5.8 g, 0.0L5 rnole) and 48% hydrobrornic acíd

(S0 nl) were heated r.rnder reflux for 6 hours" The acid was rernoved r¡nder

aeduced pressure, water (50 nl) added, and that removed under reduced

pTessure. The residue was dissolved in hot water (30 nl) and the pH

brought to 6 with concentrated arnmonia. The anino acid was filtered off

after cooling and crystallised from water, ß.P . 21'5-2200 d'. The yield

was 64%. Calc. for C9H1ONOTBT i C, 44.26; 11, 4' 10; N, 5' 74; Br, 32'B'

For.rrd! C, 44.30i H, 4.11; N, 5.42! Br, 32.9vo'

p- BROMOPIIENYLALANINE.

p- Bromobenzyl bromide.

Free-radical bromination of g-bromotoluene by the nethod of

olah et "t.489 gave g-brornobenzyl bronicle , g6eo, b.p. l-40 -1460f22 mm,

n.p. ss-6oo (1it.o" oto).

-129-

Diethyl u,- acetamido- o- (p-brornob en zy 1 )'-rna1 onat e.

This cornpound was prepared fron g-bronobenzyl bronide by the

method described in the preparation of o-fluorophenylalanine. The yield

was 7S%, n.p. 125-7320. Recrystalli.satj-on from aqueous ethanol raised

the melting point to I33-134o, Calc. fo'r CTUHTONOTBT : C, 49,73; H, 5'18;

N, 3,63i 8r,20.8. Fornd: C, 50.09; H, 5.41; N, 3.88; Br, 2Q'5v"

P: Bronophenyl alanine .

Diethyl o¿-acetamido-q,- (p-bronob enzyL) -malonate (19 . 3 8, 0.05 rnole)

was heated r:nder reflux for 6 hours with 48% hydrobronic acid (75 m1).

The pH was adjusted to 6 with concentrated ammonia, the acid ttta*t"U ¿nU

off, and washed with water; yield L2.2 g (quant.) m.p. 2L5-21-9o d (lit.

2s8o d).

p- TRI F LUOROIVÍE THYLPHENY LALAN INE .

Ethyl p- tri fluoromgthylben zo?tg.

p-Trifluorornethylb enzcyl chloride (10 g) was added to dry pyli-

dine (10 g) and anhydrous etha¡ol (25 m1) cautiously added. The rnixture

was heated under reflux for 3 hours, poured into dilute hydrochloric

acid and the ester extracted with ether. After drying the extracts with

nagnesiurn sulphate, the ether was removed under reduced pressure a¡rd the

residue dj.stilled in vacuo to give ethyl g-trifluoromethylbenzoate,

9.1 g (s4%), b.p . ro2-704o/20 rnm (1it.497 so-80.sols.5 mn).

- 130-

p -Tri fluoronethvlbenzyl alcohol .

Theester(9.1g)indryethel(50nl)wasaddedtoastirred

suspension of liihiun aluminium hyrlride (1.7L Ð in dry ether (50 ml) '

After the addition, stirring was continued for 15 ninutes and wet ether,

wateÍ, and dilute sulphuric acid added successively. The ether l-ayet

u¡as separatecl, the aqueous layer extracted with ether, and the extracts

dried. Removal of the ether in vacuo follorved by distil.lation in vacuo

gave E-trifluoronethylbenzyl alcohol , 6'7 g (S5%), b'p' Llz-tlso/24 mn

(1it.497 78. s-eoo/+ mn) .

p-Trifluoromethvlbenzyl bronide .

Thealcohol(6.7g,0.038rnole),4Sgohydrobrornicacid(20g'

0.12 rnole), and concentrated sulphuric acid (3 n1) were heated under

reflux for 5 hours. The nixture was poured into water and the benzyl

bromide extracted with ether. The ether extracts were dried, the ether

removed, and the resi.due distilled to give g-trifluorornethylbenzyl

bromide , 8.4 g (90%), b.p . 96-970/22 mm (lit.497 as-OOo7s mrnl.

Diethy1 cr,-acetamido-o,- (p - tri fluoromet Lbenz 1 -malonate

g-Tri-fluoromethylb enzyl bronicle, diethyl acetanidomalonate, ffid

sodium ethoxide gave, in the usual way, the malonic ester, 64vo, m.p'

L2g-Lsro

point to

F, 75.2.

Recrystallisation from aqueous ethanol raised the melting

132-I33o. Ca1c. fot CrrHrON0sFs I C, 54 '40; H, 5' 33; N, 3'73;

Fonnd: C, 54.10; H, 5.60; N, 4'00; F, 15't%'

-73L-

p- Tri fluoronethylphenyl al ani ne .

p-Trifluoromethylphenylalaaine was prepared froin the above ester

by the same rnethod employed in the preparation of m-bromophenylalanine.

The yield of recrystallised amino acid, n"p. 203-2OBo d, was 60%.

Calc. for CTOHTONOZFS I C, 51.50i H, 4.29; N, 6.01; F, 24.5. For¡rd:

C. 51. 30 ; H, 4 .34; N, 5 . 82 ; F, 24.2%.

(f) 5-FLUOROTRYPTOPHAN.

p- Fluorophenylhydr azine .

g-Fluoroaniline (22 g, 0.2 rnole) h/as converted to the diazonium

salt and reduced with sodium sulphite according to Schiemann and Winkel-

rntllters74 to give 19.3 g (77%) of p-fluorophenylhydrazine, b.p . !25-

L28o/77 mn (1it.374 Isso/25 nn) which solidified on cooling.

o-Fluorophenylhydrazone of y-acetarnido-y,y-dicarbetho4¡butyraldehyde.

The method of this and subsequent steps is essentially that of

Rinderknecht and Ni"o,rr,r, " 560 Diethyl acetamiclornalonate (21 .7 E, 0 . 1

mole) v¡as suspended in benzene (35 m1) c.ontaining a catalytic anount of

sodium nethoxide fsodium (20 ng) in dry nethanol (1 n1)]. The mixture

was cooled to 10o and freshly distilled acrolein (6.9 mI, 5.6 g, 0.1

mole) in benzene (10 ml) added droplvi.se with stirring. The temperature

Tose to 40o and stirring was cont.inuecl for l- hour. The resulting solution

was filtered and to the filtrate acetic acid (3 ml) and g-fluorophenyl-

hydtazine (72.6 g,0.1mo1e) were added, the solution warmecl to 50o ancl

-L32-

then cooled in the refrigerator for 2 days. contrary to the literature,

no precipj-tate of the g-fluorophenylhydrazone was obtained. Renoval of

the solvent in vacuo gave a ye1low gum'

Ethvl o-acetamido-ß - l5-fluoro - 3- indole) -propionate .

The yellow gum fron the preceding step vras suspended ín water

(200 n1) and concentrated sulphuric acid (10 ml), and the rnixture heated

under reflux, with stirring, for 3 hours. The resulting mixture tuas

extracted with ether, the ether extracts dried, and the ether removed by

distillation. A red gum was obtained which, after tritu::ation v¡ith

ethanol, partly crystallised. The solid was filtered off and recrys-

tallised frorn aqueous ethanol to give the above cornpound, 7 't g (20%

based on g-fluorophenylhydrazine), D.p . tsg-L40o çtia.360 137-1380).

N-Acety 1 - 5 - f 1 uorotrYPtoPh an .

Ethyl o-acetanido-ß-(5-fluoro-5-indole)-p::opionate (5.9 g) and

10% sodium hydroxide solution (45 nl) were heated under reflux for 4'

hours, the solltion cooled and acidified. The cl-acetamido-o-carbo4¡-$-

(5-fluoro-3-indole)-propionic acicl which crystallised was collected and

heated rncler reflux with water (60 nl) for L hour. 0n cooling N-acetyl-

5-fluororryptophan crysta11ised, 2.84 g (66%) of naterial, n.p. 193-1960

(1it.360 tag- Lg2o) was obtained.

-L33-

(e) 6 - FLUOROTRYPTOPHAN

(i) from 4- anino-2 -nitrotoluene .

4- Flucro- 2 -nitrotoluene .

4-funino-2-nitrotoluene was converted to the above compound in an

overall yield of 4Seo according to Steck and Fletch"t'Ont The yield of

the diazoniun fluoborate was 80%, thermal decornpc'sitíon of lvhich gave

4-ftuoro-2-nirrotoluene (s6%), b.p, Lo2-Lo30 /20 nn (1it.498 toa-109o1'

23 run) .

Potassiunt salt of ethv1 4 - f luoro- 2 -nit ropheny lpy ruvate .

Potassium (19.5 g, 0.5 nole) was cautiously dissolved in dry

ethanol (125 ml) under nitrogen. To this solution of potassium e'thoxide'

clry ether (600 rnl) rt¡as added, followed by redistilled ethyl oxalate

(65.7 B, 0.45 nole) and 4-fluoro-2-nitrotoluene (77'5 9, 0'5 nole)'

The red potassium salt of ethyl 4-fluoro-2-nitrophenylpyruvate, 13'0 g

(66%) was filtered off after 2 days.

6-Fluoroindole-2- carboxylic acid.

The nethod enployed was essentially that of Allen et tl.381

The potassium salt from the preceding section (93 g) was suspended in

0.880 arnmonia (630 ml) and water (770 nl), and a hot solution of ferrous

sulphate (660 g) in water (750 m1) aclded. The nixtuÏe l{as heated under

reflux for 5 hours and filtered. The filtrate was acidified, cooled, and

the 6-fluoroindole-Z-carboxylic acid filtered off. The solid nat'eria1

-t34-

fr6m the ferrous sulphate-anmon.ia reduction was dried, powCered, placed

in a so>rhlet and extracted rvith ether for 24 }rrouts. Remova.l of the

ether by distillation gave crude ethyl 6-fluoroindole-2-carboxylate

which was heated under reflux with 2N sodiurn hydroxíde (300 rnl) attd ethanol

(200 ml) for t hour. The result.ing solution was cooled, acidified, and

the 6-fluoroindole-2-carboxylic acid filtered off. The conrbined aci<l

preparations were recrystallised from aqueous ethanol. 6-Fluoroindole-

2-ca-rboxylic aci<l, 29.4 g, (s2eo), m.p. 2ss-240o d (rit.38r z+s-246o d).

6-Fluoroindole.

(a) 6-Fluroindole-2-carboxylic acid (11.0 g) was heated strongly in

a 250 nl flask fitted with a reflux condenser. Heating was continued

until no more gaseous products were evolved and sublination ceased.

Steam distillation of the residue and the material rvhich had subiin''ed

gave 6-fluoroindole (4.3 e, 52%), r.p. 74.5-760 (1it.ttt tto).

(b) 6-FluoroindoLe-2-carboxylic acid (26.9 8, 0.15 nole), copper

chromite (18.1 B, 0.075 rnole), and freshly distilled quinoline (100 mi)

were heated under reflux for B hours, ild the hot solutj.on poured onto

a rnixture of ice and hydrochloric acid. The nixture l^/as filtered, the

filtrate extractecl with ethe::, the extracts dried, atld the ether removed

in vacuo. Steam distillation of the residue gave 6-fluoroindole, 1.35 g

(7%), n.p. 74-7so (lit."t tto).

- l_35-

6-Fluorosramine,

The rnethod employed in this and subsequent steps is essentially

that of Bergrnann ancl Floffmur-,r.383 lVith cooling ancl stirriug, 6-fluoro-

indole (4 g) in dioxan (30 ¡nl) was adrled to a mixture of dioxan (30 nf) '

acetic acid (30 ml) , formalin solution (2.5 m1) , and aqueous dimethyl-

anine solution (55%, 3.5 nl) over 30 mi¡rutes. After standing for 12

hours, the 6-fluorogranine, 5.0 g (87%),n.p. 732-I37o (lit.383 tslol

was filtere<i off.

Ethyl o,- acet amido-ß- [6 - fluoro- 3- indol e) - propicnate .

A nixture c:E 6-fluorogramine (5.0 g), diethyl acetanridornalonate

(5.6 g), sodiutn hydroxide (0.4 e), ffid toluene (50 nl) were heated under

reflux, in an atmosphere of nitrogen, for 4 hours. 0n cooling ethyl

o-acetanido-ß-(6-fluoro-5-indole)-propionate crystallised. The crystals

were collected and recrystallised from isopropanol to gi-ve this compound,

o 3834.3 e (45e"), m.p.201-205 (lit. 20 to) .

N- Acetyl - 6 - f luorotryptophan .

The above ester (4.0 g) and I0% sodiurn hydrc.xide solution (35 nl)

were heated under reflux fot 4 hours. The resulting solution was

filtered, cooled, and acidified. o-Carboxy-ß- (6-ffuoro-3-indole)-

propionic acid, 2.36 g (BOrø1 was obtained. The acid was decarboxylated

by heating turder reflux with water (60 rnl). The solution, on cooling,

gave N-acetyl-6-fluorotryuroplìan , 7"74 g (86rø), m.p , 7g2,-IB4o (1it.383

L7B-L79o¡

-136-

(ii) from indoline.

6-Nitroindoline.

The nethod enployed was essentialLy tli'at <iescribed in the litera-

ture.5B4'385 rndoline (40 g) was dissolved, with stirring, in con-

centrated sulphuric acid (530 rnl), the tenrperature being kept below 50

with an ice salt bath. At the sane temperature, a ntixture of concen-

trated nitric (15 rnl) and concentrated sulphuric acid (330 nI) was added

dropwise. After stirring for a further hour, the nixture was poureC

onto ice (1.5 kg) and the solution obtained made alkaline rvith sodiun

hydroxide solution, the tenperature being kept below 40o. Contra'ry Lo

the report of Ikan et al.385 the procìuct was p:i'+cipitatecl before the

solution reached pH 6, and not in the range pH 6-8. The 6-nitroindoline

obtained hras ïecïystallised from X-60/etheri 43.9 g (80%) of materjal

n.p. 68-690 (1it.385 66 .s-67.5o¡ was obtained.

1-Acetyl -6-nitroindol ine .

6-Nitroindoline (24.6 B, 0.15 nole) was cautiously addecl to

refluxing acetic anhydride (L00 nl). The mixture was heated under

reflux for a further hour, cooled, ærd the solid which crystallised,

filtered off. This was washed with water and dried to give 1-acetyl-6-

nitroincloline, 25.0 g (BIr'), m.p. 159-160o (1it.585 154.5-1550).

1-Acety I - 6- arninoindol ine .

L-Acetyl-6-nitroindoline (30.9 B, 0.15 mole) was suspended in

absolute ethanol (1S0 n1) , 5% paLladium on carbon (a g) added, a¡rd the

-L37-

nixture hydrogenated for 2 hours while irradiated wi.th an IR lamp. '[tre

reaction mixture was cliluted to 400 ¡nl with ethanol, heated to boiling,

and filterecl quickly through a hot lluchner funnel. 1-Acetyl-6-a¡nino-

indoline , 21.6 g (84e"), n.p. tLz-t}4o (lit.385 tato¡ was obtained.

1- Acety l, - 6 - f luoroindol ine .

1_-Acetyl-6-aminoindole (I7.6 g, 0.1 nole) was converted to the493

diazonium fluoborate, 23.9 g (Alu,¡ in the usual way. The diazoniuin

fluoborate (23.9 g) was nixed with sand (50 g) and decomposed ther-

^"11y.493 Deconposition was extremely vigorous and the reaction was

not f.urther j-nvestigated.

(h) PREPARATION OI.' FI,UORINATED CINNAMIC ACIDS.

Efþyl p-nethyl cinnanate .

The nethod enployed was essentially that of Kucherov et .1.401

A solution of g-tolualdeh¡,de (2.40 g, 0.02 nole) and carboethoxynethyli-

clenetriphenylphosphorære (16.06 g, 0.046 nole) in benzene (200 ml) was

heated under reflux, in an atmosphere of nitrogen for 6 horrrs. The

benzene was ïemoved under reduced pïessure, the residue stirred well

with X-4 and filtered. The solid triphenylphosphine oxide which was

filtered off was washed well rvith X-4. Removal of the solvent fron the

conbinecl filtrates gave the crude ester. Distillation r¡nder reduced

pressure gave ethyl E-methylcinnamate, 3.32 g (B7ed), b.p. 766-tego/Zq nwt

(lit.499 1sB-1s9o/17 nun).

- 138-

p-Methylcinnamic acid.

(a) The ester (2.0 e) and L0% sodium hydroxide (30 ml) were heated

wrder reflux until solution becarne conplete. The solution was cooled,

made acid, cooled in ice, ild the acid filtered off. Recrystallisation

fron acetic acicl/water gave g-methylcinnamic acid, L.25 g (73e,), m.P.

196-i.9zo qrit.soo tszol.

þ)598 g-Tolualclehyde (2.40 g, 0.02 mole), rnalonic acid (2.18 g, 0.02

nole), pyridine (0.4 nl), md ethanol (10 m1) were heated under reflux

for 3 days. Ir/ater (50 m1) was added, the solution cooled, the acid

filtered off and recrystallised frorn acetl-c acid/water. g-Methylcinnanic

acid, 1.48 g (45%), n.p. 197-1990 (lit.500 197o7 was obtained.

o-Fluorocinnanic aci d.

Reaction of o-fluorobenzaldehyde (2.4s g, 0.02 nole) with the

phosphorane as described in the preparation of ethyl g-nethylcinnamate

gave ethyl o-fluorocinnamate, 3.37 g (86%), b.p. tsO-LS2o /25 rnn (lit.394

L40-141.o/ 11 mrn). Saponification of the ester (2.0 Ð gave the acid, 1'"43 g

(83%), m.p. 179-1830 (lir.394 ttso¡.

m-Fluorocinnamic acid.

n-Fluorobenzaldehyde (2.48 g, 0.02 mole) was teacted with carbo-

nethoxymethylidenetriphenylphosphorane (16.06 g, 0.046 nole) as des-

cribecl in the preparation of ethyl p_-methylcinnanate. Ethyl m_-fluoro-

cinnamate , (84v") was obtainecl, b.p. L54-ß6o /26 nn (lit.395 ß7o/ 11 rnm).

-139-

Saponification of the ester (2.0 Ð gave the acid, 7.34'g (80%), m.P.

160- i.66o (1ir . 395

' 501 ts+- 1sso, 166. 5o) .

p-Fluorocinnamic acid.

g-Fluorobenzaldehyde (2.48 g,0.02 nole) sinilarly gave ethyl

g-fluorocinnamate, z.sL g (Bs%), b.p . rs6-r3|o/i.5 nn (l.it.594 tss-L4}o/

11 rnm). Saponification of the ester (2.0 g) gave ¿-fluorocinnamirc acici,

1.40 g (Bze"), m.p. 204-206o 1tit.394'397 2o|o, 2o2o)

2 4-Di fluorocinnamic acid.

2, -DifTuorobenzaldehyde (5.10 g, 0.218 r'ro1e) was reacted as

described previously to give ethyl 2, -difluorocinnarnate, 3.79 g (82%),

b.p. t4i-Ls2o/24 nnrvhich solidified on cooling, rn.p. 35-40o. Recrys-

tallisation from X-4 raised the melting point to 40-41o. Calc. for

CttHtOOZFZ ; C, 62.27 ; H, 4.72; F , L'7 .9 . Foundl C, 62.25; H, 4.61;

F, 17.g%. Saponification of the ester (2 Ð gave 2,4-difluorocinnamic

acid, 1.40 g (81%), m.p. 205-2060 (lit.400 zooo¡.

n-Trifluoromethylcinnamic acid.

m-Trifl.uoromethylbenzaldehyde (3.48 8, 0.02 rnole) sinilarly gave

ethyl m-trifluoromethylcinnantate , 4.!7 g (86%), b.p. Lsl-t-;óo /22 mm

which solidified on cooling, m.p. 3B-40o. Recrystallisation from X-4

raised the rnelting point to 40-410. calc. for crrH, t}zFs I c, 59.02;

11,4.51; F,23,4. Fo'.mcl: C,58.78; H,4.60; F,23.3%. Saponífication

-t40-

of the ester (2.0 Ð gave the acid, 1.31 g (74%), m'P'

135. s- 136.5o) .

1ss- 1350 (1 ir . 399

p-Fluorophenylpropionic acid.

g-Fluorocinnanic acid (1 g) in ethanol (50 nl) was hydrogenated

at 4 atmospheres for l- hour in the presence of 5% pa]_]-adium on carbon

(100 rng). The reaction mixture was filtered through celite, and the

residue, after removal of the etha:rol in vacuo, crystallised from X-4'

g-Fluorophenyl-propionic acid, 0.86 g (86%), m.p. B9-90o (1it.oou nto)

was obtained.

cr,-Fluorocinnamic aci d.

The method employed was essential:-y that of Bergnann and

Shahak.405 To a suspension of sodium hydride (50% dispersion in oi1,

2,4 g,0.05 note) in dry xylene (50 ml) at 40-500, a few drops of dry

ethanol were added, followed by ethyl fluoroacetate (8.0 g, 0.055

rnole) r,rith stirring. At the sane tenperature and with stirring, a

solution of ethyl fluoroacetate (5.5 g, 0.05 nole) was added dropwise'

After ! hour, the ethanol fornecl was distilled off; the reaction mixture

being heated to the boiling point of 4¡1ene. A smal1 quantity ("t' 5 nl)

of xylene was distilled off and after 14hou[=, benzaldehyde (5.3 g, 0.05

mole) in dry xylene (15 mf) was added. The mixture was hea+-ed under

reflux for 15 ninutes and poured into watel (250 m1). The xylene layex

was sepaTated, washed successively with dilute sodíun bicarbonate

-'L4L-

solution and water: *d clried. The lylene hras removed in vacuo and the

residue clistil1ed. Ethyl cy,-fluorocinnanate, 6.7 g (65%), b.P. 158-1500/

32 mnt (1ir.405,403 L2o-.L22o/20 rnn, Ls2-ß4o/ 13 nrn). The ester (5.8 g,

0.03 nole) was dissolved in ethanol (25 nl) a¡rd Ïrot 25% potassiuin

hydroxide solution (15 rnl) added. After standing for 1¿ hout, the ethanol

was removed in vacuo and concentrated hydrotJrloric aci.d (15 ml) added

to the residue. the acicl was filtered off and rec.rystallised from

acetic acid/vrater; 4. 5 g (7sv") of acid, m. p . 1-57- 1600 (lit . 405

15 70)

was obtained.

(i) PREPARATION OF 3- CARBOXY- 7 ^. FLUOROD II-IY DRO I SOCARtsO STYRI L .

2-Cyano- 4-nitroto luene .

Nitration of o-tolunitrile (L77 g, 1 rnole) using concentrated

nitric acid (S.G. L.42,64 nl) and concentrated sulphuric acid (500 m1)

according to Ruggli and Meyer4O' f^u" 2-cyana'4-nitrotoluene' L26 g

(72%), m.p. 103-1070 (1it.407 toso¡.

4-Anino - 2 - cyanot o1 uene .

2-Cyano-4-nitrotoluene (40.5 8, 0.25 rnole) was dissolved in ethyl

acetate (550 n1) , 5% palladium on carbon (a g) added, and the rnixture

hydrogenated at 4-5 atnospheres fo'r L\ hours while heated with an IR lamp.

The catalyst l{as rernoved by filtrati.on through Celite, t.he solvent

removed in vacuo, ffid the residue crystallised from benzene/hexane. 4-

o I )2Amino-2-cyanotoluene, 28.I g (A4u,1 was obtained, rn'p' 89-91402.(1ír. o

-t42-

2-cv ano- 4- fluorotoluene .

4-A:nino-2-cyanotoluene (79.2 g, 0.6 rnole) was suspellded in a

mixture of concentrated hydrochloric acid (125 n1) and water (1-50 rn1),

and diazotised at 0o with a solution of sodium nitrite (52,2 g) in water

(150 rnl). To the filtered díazoniun solution at 0o, a solution of sodirun

fluoborate (BB g) in water (180 rnl) was added with stirring. After:

stirring for a further 1.., lnout, the diazoniun fluoborate was filtered off ,

washecl successively with water, ethanol and ether, ffid dried. The yiel d

was Ll-6 g GaÐ. Thermal decomposition in the usual *^y493 gave 2-

cyano-4-fluorotolur:ne (52%), b.p . g2-g4o /20 mm, which solidified on

cooling. A sanrple was purified by recrystallisation fron X-4, m'p' 44-

45o. Calc. for COIlNF : C, 7L,t2i H,4.45; N, 10.37; F, t4'L' Found:

C, 77.30; H, 4.74; ii, 10.61; F, 75.}eo.

2 - Cy arto - 4- f luorobenzy 1 b ronide .

2-Cyano-4-fluorotoluene (28.g 8, 0.21-4 mole) was heated to 1400

and irradiated lvith a 200W globe. With stirring, br:omine (35 g, 11'3 rn1,

O.2Ig mole) was added over 2 hours. After the addition, the nixture lttas

fractionated under reduced pressure to give 2-cyano'4-fluorobenzyl

bronicle, 29.8 g (65%), b.p. LsZ-1S6o/I2 nn. A sanple b.p. Isso¡Lz nm

r^¡as subnitted for analysis. Ca1c. for CTHTNBTF z C, 44. 84; H, 2.34; N'

6.54; Br, 37.4. Found: C, 45.24i H, 2.56; N, 6'40; Br, 36'9%'

-743-

Diethyl o-acetami <1o-cr- (2-cyano-4 - f luoroben zY1 ) -malonate .

To a solution of sodiun (3.45 8, 0.15 mole) in dry ethanol (L20

m1), diethyJ. acetamidomalonate (32.6 9, 0.15 mole) was added ¿Dd the

mixture maj.ntained at 50o for L hour. 2-Cyano-4-fluorobenzyl bronide

(27,8 g, 0.1-3 mole) in the rnininum amount of absolute ethanol wa-s added

dropwise and the solution naintained at 50o for 5 hours. The solution

was then pour.ed into water (450 nrl). The product which crystallised on

cooling was collected and ïecrystallised from chloroforrn/hexane; 34.6 g

(7 6%) of diethyl o,-acetamido-q- (2 - cyano-4- fluorob enzyl) - malonate, n' P'

725-130() was obtaiuecl. Further recrystallisation fron aqueous ethanol

raised the melting point to 150-1310. Calc. for CrrHrgNZ0SFi C,

58.28iH,5.43;N,8.00;F,5.4.Found!C,58'01'iH,5'45;N,7'89;

F , 5.I9o.

3-Carboxy - 7- fluorodihydrois ocarbos tyri 1 .

The prec.eding compound (30 g) in 6N sodiun hydroxide (50 n1)

was heated under reflux for 50 hours. The pH was adjusted to 2 and

reflux continued f.o-r a further 2 hours. The solution was then evaporated

to dryness, 48% hyclrobrornic acid (70 rnl) addedr and the resulting solu-

tion heated to boíling. The hot solution l{as pouled onto ice, the

resulting solid collected and recrystallised from water with decolourj'sing

clr.arcoal. 3-Carb oxy-7-fluorodihydroisocarbostyril, 8.0 s Ø5%), m.P.

22g-23Lo d rr,as obtained. Calc. for CrnFI*NoaF : C, 57 '43; lI, 3' 83; N,

6,70; F, 9.1. Foundi C, 57.32;11,3"87; N, 6'81; F, 8'9%'

-744-

3-CarbonethoxY- 7- fl uorodihydro j.socarbostYri I .

The preceding acid (5 g) rvas added to a solution, prepa::ed by

the careful dropwise addition of thionyl chloride (2.2 nI) to ice cold

absolute nethanol (25 rnl) at 0o. 'Ihe resulting solution was then nrain-

tained at 40o for t hour, the excess rnethanol removed in vacuo and

the resídue dissolved in chloroform. The chloroform solution rvas washed

successively with t^/ater, sodiun bicarbonate solution, water, Ðd dried'

Renoval of the chloroform in vacuo gave a crystalline residue which was

recrystallised fron chloroforn/irexaue to give 4.9 g (92%) of 3-carbo-

netho4¡-7-fluorodihycli'oisocarbostyril, n.p. 1-06-1080. Further IecÏys-

tallisation frorn cJlloroforn/hexane raised the melting point to 107-1080'

Calc. for CrrHr0N0:,.]-r : C, 59.20; H, 4.48; N, 6'28; F, B'5' Found: C'

59.56; I1, 4.57; Ì'i, 6,'iJ-; F, B.SYo'

(i ) MISCELLANEOUS.

Ethyl thioltri fluoroacet at e .

Ethyl rhiolt:rifluoroacetate, b.p. 90-910 Git?47 90.50) was

prepared frorn ethane thiol and trifluoroacetic anhydride by the method

of Hauprschein .t u!.347 The yielcl was 59% (lit.347 t+21.

Diethyl acet anidomalonate .

The nethod ernployed was that of shaw and Nol*.505 Diethyl

acetanidomalonate, m.p. 94-960 (1it.503 gO-gZo¡ *ro, obtained in a¡r

overall yield of. 65%,

- 14s-

Aceturic acid.

Aceturic acid, n.p. 207-2ogo (1it.504 207-2080) rvas prepared

504 504by the method of Flerbst and Shemin.

89-92,").

Phenylglycine.

Phenylglycine was prepaled in 30vo yield (1it

the method of Steig"rl0s

'Ihe yield was 90% (lit.

50533-37e") by

C arb oneth o xyme thy I i den e t ri ph eny 1 p¡o t pho ran e .

This compouncl, rn.p. L25-r2Bo (1it.506 tzs- 127.5o) was prepared

in 90% overall yield from ethyl bromoacetate and triphenylphosphine

according to Denny *d Rorr.506

2, A-Dífluorob enz al dehyde .

The nethod employed was essentially that of Liebermann and

Connot.507 2,4-DífLttorotoluene (9.2 g, 0.072 mo|e), glacial acetic

acid (114 ml), and acetic anhydride (113 nl) were cooled in an ice-

salt bath. Concentrated sulphuric acict (17rn1) was added dropwise, with

stirring, followed by chrom-ium trioxide (20 g), the temperature being

naintained below 0o. Stining was continued for l-0 minutes and the

reaction mixture poured onto crushed ice (800 g). The volune of the solu-

tion was nade up to 1200 nl, the solid filtered off, and washed with v¡ater

until the r'¡ashings were c.olourless. After drying, 2,4-dif).uorobenzaldi-

-L46-

acetate, 7.t g (40%), was obtained. The crurle diacetate (6.1 g,0.025

rnole), vrater (10 rn1), ethanol (10 nl), æd concentrated sulphuric acid

(1 ml) were heated rurder reflux for 30 ninutes and the nixture then

steam distilled. The distillate was extracted with ether, the ether

extracts dried, a¡rd the ether rernoved under reduced pressule. Dis-

tillation of the residue in vacuo under nitrogen gave 214-difluoro-

benzalctehyde, s.10 g (s7%), b.p . 6B-7f /26 nm (Iit.400 oz-osoltt t*).

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451.

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45s.

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462.

463.

469.

470.

471.

472.

473.

47 4.

476.

477 .

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idem, J.Amer.Chem.S_99., P, 250 (1967).

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W.B. Snith and A.M. Ihrig, ibid, 75,497 (1971).

and E. H.

464. D"F. Evans, J.Chen.Soc., 877 (1960).

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466.

467.

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J.R. Rapp, C. Niemann,

T. Vadja and T. Szabo,

28s (7s72).

6, 520

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Berezin,

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S.P. Da-tta and A.K. Grzybowski. in "Biochemj.stsI Handbookrr,

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478.

479.

480.

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490.

49L.

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487.

4BB.

492. J.H. Brown, C.W. Suckling, and l{. B. l{rhal1ey,

489

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J. Chen. Soc 95S

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494.

49s.

496.

497.

49 B.

499.

500.

501"

502.

50 3.

504.

505.

506.

507.

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A.I. Voge1, rrA Text-Book of Practical Organic Chenistry",

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APPENDIX.

Graphs of Io vs. 1/ô for the racentic inhibitors studied. Io

is the concentration of the D enantiorner (nM) and ô is the separation

of the resonances (in Hz at 56.4 lvlHz). The slope is EoA and the

intercept - ¡fO + Eo) . Hence A = slope /Eo and Kn = intercept -Eo

(section 2). These data refer to the trifluoroacetyl 19F ,r.r"l"i- except

wheré otherwise stated.

Figure Inhibitor, N-trifluoroacetate of KD(mM) A(Hz)

*

7

2

3

4

5

6

7

B

9

*

ú

L0

11

L2

13

phenylal-anine, pFI 5. 3

tryptophan, pH 5.3

g-fluorophenyl.alanine, pH 5. 3

n-fluorophenylalanine, pH 5. 3

g-nethylphenylalanine, pH 5. 3

n-methylphenylalanine, pH 5.3

g-bromoptrenylalanine, pH 5. 3

rn-bromophenyl alanine, pH 5. 5

2,4-difluorophenylalanine, pH 5, 3

g-bromophenylalanine, pH 8. 2

g-fluorophenylalanine, pH 5. 5,

ring fluorine nuclei

g-blomophenylalanine, pH 5.0

g-bromophenylalanine, pll 6 .0

4.4

t.2

5.4

0.6

2.4

*

2.2

3.9

6.4

tr

-8.4

-21

-JJ

-74

-19

-18

-JJ

-32

-2L

-23

-51

-3L

-31

Figure Inhibitor, N-trifluoroacetate of KD(rM) A(Hz)

74

15

15

L7

18

g-bronophenylalanine, pH 6. 6

g-bronophenylalanine, pH 7.6

p-fluorophenylalanine, pH 7 .7

rn-bromophenyl alanine , pH 7. 6

g-fluorophenylalani.ne, pD 5 .4

(in Dro)

*

3.0

2.5

4.4

0.7

-3!

-4r

-28

-36

-37

* KO rJetermined as < 0, indicating weakness in

approximations.

10

6

lu(r'rM )

20

15

lo(mM )

10

I

t,

2

/,0 50 60 70 80

5 10 15 20

ll5ixlo2)

Så- 1

?6

1/ 6 (x102 )

!js.-z,

30 35 40

o

o

5

25

20

to(mM ) 15

10

20

lo(m M) ,U

1O

5

5 10 15 ?0'll 6 (x10

lig-é.

lr0

1/5 (x10

25 30 35

50 60

)2

2

5

)

10 20 30

Fig.4.

lo( mM)

t0 20 30 4ü

1t s 1x1g?-1

pie. l-.

30 A0

1¡g 1xto2)Fiå-f'

o

50 60

25

20

15

10

5

20

15

10

5

lo(nrM)

20 50 60

20

15lo(nr ¡q¡

10

c

20

15

lo(m¡4¡

10

o

15 20

1/6(x10

o

5 10 '15 20 25 30

1/ 6txlo2 )

Fig. 7.

o

o

5 10

)

R

li-9,- q

225 30

20

15

10

5

o

o

lo (m M)

20 30 /'0

1t 6$rc2)

Fig.9.

50 60

25 o

o20

15mM)lo(

þ

o

20 30 L0 50 60

t/ 6 (x102)

Ss.,--1q'

10

5

1

lo(m M )

25

20

tÉ,l.)

10

5 10 15 20

116 (xß2)

5

Ii€.J-l

25 30

20

l5

l0( m M)

10

5

20

15

to(mM)10

510

o

o

15 20

1l G (xi O2)

Fig. 12r.

20

o2

25

o

o

1/6(x1

5

5 10 15

)

li€.'Ji

25

2a

15

lo(mM )

10

5

15

to(mM )

10

5 .i0 15 20

I ¡ ô (x ro2 )

Fie. .4.

15 20

1t6 ({02)FiE_._- 1s"

2

25

5

510 25

20

15

lo (m¡4 )

10

0

o

5

20

t5lo(lnM)

10

5 10 15 20 25

1/ 6 (x102)

Fig=_-ry".

15 20

1/6 (x102)

Ii-&.-17.

30

o

o

5

10 25

l+0

,lq

30

o

35 t+0 45

2s

lo(mM )

20

15

10

E.

o

5 10 15 20 ZÊr 30

1/ 6 (x102)

Fig. I 8"

Nicholson, B. C. & Spotswood, T. M. (1973). Nuclear magnetic resonance

studies of the interaction of inhibitors with chymotrypsin. N-trifluoroacetyl

derivatives of phenylalanine. Australian Journal of Chemistry 26(1), 135-145.

NOTE:

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