..n;.: '~~;iiv:. Of:"~~i1:'~,i'Cr~f~~":is, ~~viJY 'de~d~~t"', :. ~ 'GUI;i;e'dHe~icrofiche '~pe~d:9'~~~~i

CANADIANTHESES ONMICROFICHE

I.S.B.N.

.THESESCANADIENNESSURMICROFICHE ,J

-.

'Nablal'~~~"

Q!llfdiDns Diwtlolll'N"lIB.f'\Ch

. "~.,:. ; . ' . ~ ':" '"~d~E'' ' : "

..n;.: '~~;iiv:. Of:"~~i1:'~,i'Cr~f~~":is, ~~viJY 'de~d~~t"', :. ~ 'GUI;i;e'dHe~icrofiche '~pe~d:9'~~~~i

^{de' .. '}

upon,tm.'"qulIlit y.c t~tlfigin81theSissubmiltedlor IIIqul llt4deII't hb!sou,mise,au,rnlcrofllm aqe;NC)lJs.

',microtilming..'Every.dfort'M been m.llde'loelJsu re I\lonstouttailpour.fSsurer-"UlleqUllit' supirieu..

',~th.hlllh~quIIljtyof repr-oduction pouible.'. · .de rep.-Oducti Dll.: .

.. :n,.,;_"..,',f....t. .

"' .'~~.~ng..IXII'I~

_....V'''''' ^tl-.

un~r~ly·whic:h

^'S;I.'

m~~-:

^des

~:

^veulllez'

comri,u~iquer

^.,'

avecI'unive"itequilton fer4legrade.

.

~ ~ges

^'ma;

~ve' ind~nC1 ~int ~lIy ;"

^...

~" qualiti' d'im~~ :~

^certaines

~

^{peut' ,}

';~~~o~~i~~:e":~ =i~i~=t=it!r ·".laiswr 'desirer,SUrtoulsites~ origi~les^anteli '

' ,/ ' . .'.. . '. _. _ . '\ ~I:tv,::~~= ~~r ~: =~U:ie~de'i~~ .,

':/\ "r~-' .• ': q~81\~ .

.Previ Ously.copYrightedmiteria l.Oournaltrticles.;' P.'IJbli.h edJe5Is.e tc.larenotnlriled.: ·

.. :.A'e~rodui::tionJ~;full^0;:

iniiar-t

~f.thit^filmi.r1o~: ^{" La}reproduetio n,"rimepartiejte,

de oe

miaofilm

.... Inedby·the CllnildianCopyright Act,R.S.C,'9~O;::::,-estsoumlsB'IiLolcanadiennewrled roit,d'8\Jleur,".

c,.C:30,JPlease·reed.the authorl1;llianf~~ v.:h~ch." :.SRC.1970 ,e,{;:30.Veuillerprel;ldrecof\f\aiss/lnudes

; ..'"a7p.a~.,..t'~ilitQ.e.sjs., ' , . :~.formul~d'~ulor'SlItlon- qui accomp~gf\e~temet~!iB.

•'.,',"HAS.BEEN MICROFILMEO

;~'SQ;SSERTATiiiN ,

^,

MICRLOAFITLHMiE~EETAE'L ELTE~

^QU,E'

::,:EXACTlY',A S'RECEIVEO', .NOUSL"AVQ NSRE,CUE

~,:

" ) \t. ,.:, ;;~:,\' :X,;,f'±:wT-."~\:":"':;'",:,;":",<:':" ~'''T-:7~~:~'-'''' ' : ": 'i' - :

STRUCTUAAl STUOIES·ONTHE"

LlPOPOlYSACQlAR IOES ISOLATEDFROM. BACTERIOPHAGESElECll:D"STRAUSOf~ ~

::::

-

..

:~

..

"

.. '

..'

;

.. ..

Arnest ssu~m1t~d fn.p~~~f·~lfulff"11.IIlf.nt ."

-',o~"there~ufr~ntsforpie.~egree'·~f

·Doc~Or"ofP~'10S~~Y

-

. ~.,

... ..

^:'

0"~p~r"~~t"~roCt!e"l1 st.ry

o. : ~":.

. ..'.~ (.

.'- < .".:

~. hYdrO~hil~' ~ep~~.~e~ts 'a' ,~r~\: ~f ·b~ct~.~,f~, -pa-t~~~enf.C':t~,:'·· ·

^.

fish•.'R~c~·ntlY.the samebacteria'havebe.~nlncrea~lng ly'1I11plf~ate,d

·..

1~; h~~~n ~'ni~~t1o~~

^•.

pa~t1CIJ1~rlY

^.

~~. a s~~'~nit~~', 1'nvad~~:~;i~~:"~re:'~OW.:

.b eing retog·nl~~d:'~s .~\~r10Us pat~dgen·· ~n'd~r·:·tI;~s~:,'~ircu~~~ances'.:·" ~s"

·

·.\l~~~e.·,

1

s";,i:~o~, ~f.:th~-'b;'6ch~~;~~'i\baS~'S'.~~'."~~t~'~9'~~1c~~·

.

^of:~~

ti- :

· ~."~H'~~:s;~':Yn~re~~.:"l

^n'.

~.~:" .~~:~~,c:tu,~e".'~nd,'l~~~O:l'~'~~'~~1 ",'~r~~~:r~I~.s'.~f~.\ . ' : .

~~ 'C~l' '~u'rfa~e; ~y'~accharfd~S " Oft~

^"d1

ff~~ent ' cheRlotYP~S ~f"

^{,t h'f}

~ :::

':

~~~:m~jri~g'~t1:~" ba~~~/i'~'~ ,h~~~:ii~c:r;~a~:~;d:"~

^:..

~.~:,',:'-'

^{. -,:..,:,:';':.}

. T he'three

bact~'rl~l co' oril es:'~~edfor.ih1s'fnvestIg~ti,o~'.were

'\~le'c't~~'

^.

~~O'('~'.'bfOl:h~f~~l'i/'pure ~·uit~~.e"'~f ~. ·~Yd'rOphf;'i"

^...

o~e

~~pr~~ent~'~~~ ~~~r~i'i'::s'{~afn.~here~~th~'~~her'~', ~utan~~'.-::w~'~~'

f'so~'ated' as::'.~rp~o~ ^{091Cal . l; '} dl.;~fe~ent ~~~~.e~~.:~.Th~ Orf!l~

^na',,'

St'~~:fn.::.

':'aOO . ,

o~, fh~ clon~s ·w;-~:.s~·n;ft1~e 'to Ph~~' ' wh 1le 'ih~ ot ' her ~~(I~~" .

'::,',

'~a's: ~~'Si, s~~t

^..

~

^..

th,e:"::s~.~ ' -~h~·ge.

l1P~'p~1¥saC~ha'd de~"we~e.eJ(t~acte4^from.~~ 1^,.t,h;e,e:varla.n.:~';~'and·';"

·.~th'e.\lasj~:.~t~u~t~re_S::~f:.the·foiysacch.a~lde::Por.~f(ln.:fr~.,:~'~'~h',O,~''t,he ':

t~ree~tr~in s' eJ(h1b'it~d' dfsti~ct 'st'ru~tural ,dlffere~ces-.,· · ~

^... .... '..' .'., ">" :~" " "..--:.'._",.': "....:." .'.. ':

C.~erI.lca~aria'y~,is.l me~hylat1(1n..p~.rfoda.~,oxf ~~tf oni,~d

.\

,...

. . \\

^"

~~"::--.>

...

...

~

:.': '..':~:.

'~,.:;.' .. .:-. .',''." .: r. -: ^..,..

,

,"

. .':a ,

STR,UCTU~STUDI~~.'Dlt.ni·E:·SEMI;~ROIIG~·,.:OLlG'OSAcCitA~ID~

P.ORTIQN'JJlD~N".t:HEL1NlAGEBE~E.ElC",rHEo-SPECIF~C·'.

..

~~Y~~~HARI~EAND

^{:'TliE ¢iRE}'REGIONof

~) HTDR~PH'ILA

^-',

STRAIN $01-55

Int r odUC

.

,tton•.,,'•••.•.'•••• • ,•••^, ,.,•••• • ••• 119 ,Sugar l4J"!posf tt onof thl!SR:'011gosaccharf de. .• •~.'.•.• 1'19

4.'STRUCTURAl"INVESTiGATIONS ON THECOREOLIGOSACCHARIDE OF

~.:"~DROPHIlASTAAINSJ~47

.,.":4;1

'_int~odu~tfon

^•'.".:•

: ,~

^{:., ' .•:: . ',' .• .}

j' .. ~'-: - ;'" ,

^124'-

., ~~.2 "Isol~tfon ~f:,SJ~47

^Core

Oi1gosaCCharl de~

^{•.•;. ..••"••}

~24

4.3. theml

ca

^1,Ana1Y5'1'5 of. theCore'Dl1gosac'charf de •••••• ••,.124

.:40' Met""i ,,~; Aj'1,~ji ^{'h O ';'" 0} 1l'''"i' h''''O'' , " ',' 127

~:5.'.Conelust cn."•'. .•.~'•'.:._.:. •:..• . • •.••.-.• •129

•,'.•:. ••~:..• ••..'',' .• '-131.'

-xi-

Tab"feYII: Sugar.analysis ofSJ-55~SelDiRough"(SR)

:.TableI: '.,~~hOdS.fOr

re

ext ract f on of.l~PopolY,SacCha~f ~es..•

.< "

8·

"Table"l l: Corestructu~.that,have,bee'part1al_1Yevet uete e..';15

Page LISTOfTABLES

.Analyt1ca~assay s.QUSJ- 48 1p5 , cor e and,li pi dA.",.". 69 Sugar analysis'ofSJ~48core olfg'osaccharldeand

derived product s .! ' 70

Table III : PARTJ

Me~~~tlonanalys hof SJ-48coreoligosaccharide an~ derl ~ed ,products. ••.". .• . •..."•.' ... 74 AI1ll..1Ytfcafdata,onthe fract ionsof~. ~Ydrophf1a ,

SJ- 55l ps.".',••·•..\•..•• • ••• •• ••••• 95 Tabl e V:' Sugar I1na1Y51s of SJ-55core oligosaccharideand

derived~rOducts.. . . . .• .,",• . •. . •.:-.~. 97 Tabl e VI: M;thYh t fon analys i s of.SJ-55core oligosaccharide

andderived products ••• • •. . . •"',' 101

011gosac~h:ar1 de'.'.••• ...• ..•: .•.. . ..120 TableVIII:HethYlati'on analys is of5.1- 55(SR} ol1gosaccharide •• .·120 -

TableIX: An.alyt1cal'-assaysonSJ-47 ,lps coreandlipi d A·. ....125 TableX: .Sugar analY51 sofSJ-47core oligos accharfdeand

derivedfra9lllent s..~•.•~•••..•.• •. ..•. .126 TableXI: MethylationanaJy.sfsofSJ-47cereol1goSacch.~rfde.•.12&

!)

•PART II' Table'I:

Tabl e II:

-xtt - LISTOF FIGURES

Page PART I

F~~ure1. Structure'~fthebactedalcell'membrane::••

'F1g~re

^{2. SChelna-;1·ci'.}

'~t~'~t'ure' Of" ~~l,~riei-l a : ·"f'P~p~,y.sa_cc·harides,'

^',

~

^,S'

.'Ffgure3 .:

stru~ture -of t~ nPOPol~sa"c~harf.de

^of'

;alll~!Iel la

typhimuriLlII_..,. • ..•••• .'.•.•_•....'----'10 Figure 4. Str'Uct ures,o f enterobacter ialcoretypes:.Ra-R4alld

1:.-12..•. ... ..• •.•• •.• • ••.... • .14 FIgure5. The structurecore defective (chemotypeofO-Challl

..

derect tveR.b-Re)lipopolysacchari des(chemotypeRajandof

Salmonel1a"'R-lIUtants .. .;.... . :•..• .•...;.~7 Figure6. Struct ureoflipi dAfro.mSalilonell a.• ·.••.','. .•19 PARTII

Figure 1. IH_n.lI. r.spectruaof the originalcore frolll Strain'SJ-48 .•-.... .. . • •... ....•..•;-72,.

Figur.e 2. 13C_n.m.r.spectrumof,the origin alcerefrom 'Str~1n SJ~48••'. .•... . :'., ;.'..•;••••'.

!Z

Figure3. M,i'ss spect rUillof 1,5-di-O-acetyl-6- 0 or 7_0_(3,4,6_trl_o.,.methYl_2_( N-meth~lai:etamido)._

.D-:gl~copYl'anosyl]- tetra~~-m.ethYl_L-glycero-D-. manno-heptttol.

I""

^~-.--

_ -_ . .

~.

I

^{-l 111.}

""

Ft~~4. 13c_n•••r,~Ktr.oftntorlgtlWl' ld"" ttl!

_2.3,4_t r1_Q_methylg1ucftol•. : • ,,'•••, 104.

Ftfllr'l8. Stpblciea 6-50t",....to9rl~Y_of 11,eeerte&Cf4 • hydrolysisfrl~ntsoftilel1popOlyucc:h.r1dt

Figure12. toreol1gosa~ch4r1 destru~ture's f.~m ~,hyd.roph(la Stra1nSJ-S5.SJ-4:andSJ -48...•~,.r; .. '

' I '"

0/

.- , , ;,

oU goSi cc nar1de·troll.St rai nSJ-55...~.•:',~•.••.99

if,,,,

10:13C" , .,;.•

,,,t,,,';;, "" ';" ''''

^{eer..'' 1,}

ol lgosIcc llar1de'1"01IStrl l~.SJ-55•. •"."..•.• •• 99 frOll~.I!¥droph1h5.1- 55•••"••• •,.•:-•• • • • • ?4 Fi gurego;'llf..n,••r:·spectr. ofttl!ori ginalcore

F1f1lre6•."'U~^{SJlKt rw}

o f the

perati.yh ted'S.lttl

01190SllC~bl'tck~f5tralll.~-4a^{• • •••• : ••}~^{•• • 83.} FtrJllre 7. llc- n.• •r-,$pK t r. ofthel)rl gtna l(.)&ftCltM ...

pirthl1y Itydrol,sed Ib) coreoltllOuccblrftltof Sir.11lISJ-48•• • • •• ••• •••••••••1... 81

Figure11:Mu sspectMII.ofthe 1,5.,d1-.Q.-acetyl-6.-.Q- (4,6- dl- D-.cetyl- 2.3I7-trl- O-methyl-heptopyranosylJ.

. . -

,"'

.-

v.,

NOTATIONSAND'ABBREYlATl 08S

SJ-~~.' ~:47', :~_~ss ,r~~r.ese,~t.:~

^•.

· hidr~ph 1;:a

^..

~tr~1n.,A6.~"., ,~6a,

^'f.nd

..A6

~os: ori9;n :f~ 'dii'c~s's~d i~

^.the

:ex~er1!,"!'ntal . par~ _I S~ctf(\n'

^1,.

P~" ~7 1 ' ,.:,~.- :

._t-" ""',',:,:;','.,;:".": .:':'.,..:'..:''" :':,-." .''.-.'".;: ' .:~ ,.,·R-.end.SR;- standfor,~r.oLl9h·j4[Hl':selllf-~~9h·01f90saccharf d~s•.^,'J

·~pS',.,,~o~

^..

1\~~~,O'Y~,aC~,ha~1:d~

^..

~,·;

,.,:'.':: .-:,'" ,' ','..

:: : ,.:,;~ '"

,..'

:_~ '~';LD~He~~ , ~~~ndS

^'for,

:i.~~~'Y~~ro~~~m~nn6~h'ePt~~~~an~se,

^.;

. '~~DO:He,pi·~~~~~~·~~r ~~ilYCe.ro~~:~~m~~no~h~PtO·pr;~'~·~~·e"

^{",: '}

eKOo'

o~·d~~lA ~t"a'nds 'fO~ 3-de,~Xj'~.D~manno:..2-~~;tulosonf~,

^.actd..-

;,Gl c!M

' s hnds

f~~ i~amf~~-,Z-deO~~D~91uc?pyr~'ncis~

•

Ma~.2.N

^'-;ta'ndS"

f,Q~ z,:am!n.c-Z-deoXy-~~ma~_n~~Yr~,no.~e

.·.G1C.2.,~t{n'ds,for'gl ucQPyranos'~

•Gall!.stands'for'galactepyranosl!'

•Rha.2.· 'si~ndS for'rh~IDllQPyrariose ."M,a~'st~n-~~-f~~,:ma~~:nO~yran~'se,~,^.

•',A~a'stands'for,ar~bfnose ^{.' "}

·.;' ",. , '.. ',''.. ··.. ,:;.i:'

Thefoll llW1 n9 abbre~1at1Qns";'111.a150beused:

:-.. '

!

J'

.-

..

,:' '.~.

~~RfAt. Ll;O'O!-l,~ACCH~lDES AMD·

^-.

STR.UCTUU.LIJlAlYSlsOFPOll~~ioES

, .

,:~;.

.. .

..~.

".".-;

' -'< '

., ' : '

",~.

, ./ .<_.

.

:-:-:,

I ,

'.!

',=. <.

'I.··· ·:· ':.' ' , " ..

'

'

1'

.1' ,(C,l'~eceter.«rnibr~ne,contai ni ng'a lipoprotein,pho.sphol1pidand the

l1popolysacchar1delayer·IFfg.1).

\ . .. ':'. , '. ' " .~0

,Th.~.:c.a.~bohydr~te cha,i.~~,~f:.~~e,l p~e~ten,~outw~~~.-:~,th~.,outer i

·.rlerilbrane,1ayer U7ramfarerespons, bl e>,for

the

antigenicspec-H1.c1ty_of

"'.':':

th~:'O;g~"j~m: (18)~'; -: i~' ;~di t"~o'ri'; 'ina~:"':ba~t~~f~ -'5'~C:h ~~,: P~'~~OC~~U~'i':'

"' ~1 ~b."~::·~:; :S:;::~7":::':;~:::':: ·.:h:·::::· ~.~,;"~ L:,;,~~; ^{.. .,"}

co~tains',carbohyd,r at e'li'n'tig~~s:'whf~hindu'c~,:tfIe''format t on,of"~~tib~dies;~

'~ii~ :~~~h','r~'a~~,~~'r~l';~'f~'111'~:;':~ ~~; t~~~~; 'a~'~;'~~d·f~'s'1'n:bo'~h"~;~::'~a~·~.

^'_'

\nt~~l,::.~:~~):·: :'·;·;h.~:.~~l';~·~~v,e'~'o~' .aJs~ ,~·O~~.~i~~' ~~c~~'~~'~~,' ;or~~c't~~1~­

Pha!les~~h~~'~e,'U;~,bacteri al'surfa~e'~orit~1,~s':cbmpl?n~~ts,^'wh'1ch'pl

it'

an

"illp~r~ant.;';Ole·1,"',the:1~u,~:'reC09~f.t.~ciner ecess."Th~',besf"stud~ edat'.

" t~es~' C'~mp~n'ents:

^.;

·th~'lYS~C'cha·~·fde~. ~fcti

fom ihe,'cap'sules"and

~~e

,

kn.o~

^{es clIpsul}

~~' ~nt1 g~;n~

^or

K,;,~'~t~

^gens'and'

~he l ,fPO~O~y~a'C~harl

^des

k~~n

,asc o<'or

'·R-a:nt19~~s

•.

1~-:-h::~'~Jl~y· ~~OU9h K-~ " 0-

and

R'~ant'lgens

.~

,'t ha't

.i;~ct~~1~.·~xp'r~ss :the~·r:.frmi~nol~'!ii,dil

'.

,t'ndiV~'dua11 iy'.- (i;~':, 16'i~"

,"

, " ',''.'

."

.'" ,. " ,' , "" " ,--

' .

..~

.

lipGPoiYS3'CCharide I" Cll@tJ:$;cL,;.,

':t'jp'~p';~\~;n'"

^PhGSpholipid

PeP:lja~g'lycan ,Layer"

..,:': '\.~:,') "....:

.

C~I~~~a$~iCMem~rane'"

C~tDplas~

.,.;

TheO- s l dec.llal~is the serologi call ydO!llfnant.pa r t ofthe

•moleculeresponsibleforantigenicspecifi city . It15 composed of repeatin goligosacc~ar1 deunits l~fte<contai n l ngrare sugars)"'f~which

.the seroTogfcaldetennl nant·resides.,It ts',absent frontheR-fornl

1 1.POP01Y:S_~.c~~arfde~- fs~lai~~ - ~~oii·-ioU'9:h:·s~..ra'1~S. b.ut~

^-IS-

us~ailY p~Oduced

'~ -a~,.a_hap-~n b¥~''ml/ia~t~_":f~'~hfCh~:g~n.~tlc·i:r,~c~~,ie,~d.^to.'~~ b~OS!n~.~eSIS~ '^.

Of'"an'fn~:Omplete:coreun.' ,... ".' ,. -, .

,

~_

"

,T~e:o,~a:'vg~~,~:rtl o~

'. ? f

_'.~e'~ps,~~_le~.u~,~:.:_,,~·pOt~,n't'faH!;

. .

_·~:~fqr,e:

.:;

for'each~a~terl~l,~p~c.{~<and^{as 56'ell}lmpar~s serylog·f.cal'.;:5·pe~1 ffCl~Y'^,'A

'-dnd'identityto'IIpa~tlci.lTarorganism.

The

o-an'tl~en;~ l1nk~d

^{tothe core}

01i90saCC~i1rfde_wh1~h ';'S:: ~f~~~

s~ecles

^specific

b~~

"can'vary bothwithin andbet ween

bacterhl· s.P!ci~~:" ' P~tlcUl~':(VwtJ"en

non:Ent.,rOtlact eriacea are

.co"~·~f~~rei.,. 'J~.~" :,~~l~~

J"j 0l,.j90SaCCharlde. comP,osedofdfff ererlt'slIgarUrlf.~s.>ma;._be ..'

~ . 1Y1~ed,

^;nto.'an

·ollterrr~gion"

to'whfch_"fhe

o.,.anti~en, '~s a~t~.c~~d

^•.

and.an "1nner reglon"llnked.tcl~pidA. lhh,o11 90~accharide h,._

~ene'~al1y^{linkedto'thelipid}comp~nent,~f^Ip's~11~1d^'A"j'thro~g·ti^a trl

saccha'~i

^de

o~,,2"ke.~O-J~~eoxy~g-~ oct~1~Sq.ni~' ~Cid

^..

(kDO_,::~O,~lAI

;t :

though~1;~Ji'nl:age"ay"not

be

uriive rsal" Lip'idA 1s'~pparent1y responSible.,fortileendotox1C,act~\{1·il.~ o~_t,~)fPO~~lYSaC~ha:rf~.^and appear-sto.be.very si mi,la rffnottdent.tcal in'manybacter fa] species (19) . : ', ' . .,." .. '.. •,., .. :. "' -., .

The":'1.fP~POTYS~CChar1 de Of::S'~lmon~ll:a" 'ha$been

^{Wide'; usedas a-}

..::~,..

:.,.:.~:1.3.2

"i { { Z o :' ^{it"; ;,.;;.;; ;,;} _" "Ch;;~'::-. ::;~, :/,:: oi :\i:t;:>:'D:>'"

..~'.- :

:(3{il;~-:::The~;;~n):t'~~~5uaJly-·con{atn· :dff-ferent.-:~Ugai-: co'n~tf~ent~.-a~_»

the " ''. .. '..'

."('~.'

__. ~f~.a_fr.: :~s ^... the.~~.~.o~,:·~.'~._te.~~~~~s"~.c~~~~r~.:· "'~~;~ ,st.~-.~~'~~_~.·:~~~_ev.~~~ ~: i~"'~~~" :.~:.~~;.:~k~::

....".i ~ ·o-ch~1n·~',be:-~_:.h~poly~_"ccharide_:c~n$h~1ng"ofun1~s·of an:.Ql~ 9~r;._:;:_•.:.';:';".J

< i ~~;~~) % f~b1'~1~~f~~;~,~

{

.,-. -As

1ndtcat"e~ .

^{'LfptdA:core.andl)-Ch'at: are}Interli nkeil-

b~ · .~:

^{--. ...}

.., . .'... . . ,- ' : .jr":''t '

t) .

covale nt l1n,kage_{5. ,~he}c- chatnccneetnsrepea,t1ng_un~ts;ofa ..~-> •••• • ~ ~ , : . penta 5accharHlewithlI\IInno5yl, rhamnosyl'at1d galattosy lresidues1n the ~.". '.,:

; ; j:~c::::"; '~"d'Z;t~ce:1i~t~d :~~ ~u~sii f'd/;);;;:i~) ;'~;~;dU;s Ac; :,\ ,; :f);:: :~:

;.," " -.

..:,"<,~,:':

. . . ..

;::.:: ~.,

. .. '".

-"::.:' ;:.',.'<'

>.';.:

.~':~',

- .-.:'.'.;

"~'

; ,." ... '.

.... .::'.~~ ••-' . ,..- ..': • .'.,'!;: . : '...• : ..

. ' -, ;' '-".: ' {;, :: . ' /' . . :\:~; :-:':,~"}~~<~':'\:;; .. ~:,?}~.>;·2L

. , '~ ::;: .' ^{••.. .•; ;!) : <"' .;;;;'; ;; ':' ,} · ; ;tg;:;,(i

:·.i.·

._ ~

.'

.~~

I r ; . .... \ -~--

~

..

11

Theoligosacc~arldeJwhi ch 1sfOrnle~and polymerizedduring the bt~synthesfsrepresents"t he bi ologicalrepeating unit'. Due to the acidlabilityofthe rhamnosyl'an~abeqaesyllinkages, mildacid hydrt'lTysfsofthelipopolysacc har ide yieldsthe "ehemlcal"repe~t1 ng

LtTlft2withadifferentsequenceIll.12). 'Tile conceptoftwo"types of

. , r--J

' re~ea:l.n~·unft

^isquite fllportant"as

ch~1ca'

^analYSf,S

~U, on~y _rewea~

^-", "

theCherp1 C~' re pea tt ~gunitsandonlybyluck. wm.t hey,beident i calto .the.bi,ological ones,OZI•.

I

/ ,,,

^Ab,I Rtla .. ^"GleGa⁺l

'Gle

,.,

+ ^(Abe)Man⁺ .. Rha •

1.3.2.2 . TheHeterogeneit yofa-Chains

. .

T!l econs t ituent softhe trueb1~log1cal repeating uni tAbe.Man, -, Rha and GalofFigure 3 are ccnsrene. Incontras tboththe ace-tylgroup of the eceeucse,:si-due and thesidecmm glucoseunft, whichare.added inthe latter stages of thebiosynthesis, vary~ndareoftennot p.resent

....

in equilllOlarratio(31).,Fur t hennor e,theirprese nceorabsencemaybe subject tovariation for ot.herreasons(e.g:antigen fc conversion by phages ) . This tnceeotetesubst i tu tionof the basic c-cnat ncause s the wellk.nown microheterogeM1tyfoundinlfpopolysaccharfdes flll •.

. ' .

..

12

1.3. 2.3. St r uctureof the Core

The corestructur eof1.typhtmur11J111(fi g.3)

1s

COOIlIO."toall Salilonell a speci esand also occursfnother enterobacterialltpopoly- s'acch~r1 desIll,12, 201. TheSal 1l1Onel 1acoreccn t et nsa1fpfd; a hexoseoligosaccharid e(out er cor e) consisting ofg-:-glu\=osanilne.

g-"

.gi ue,osea,nd.~-ga'llcto_se;andaninnercore.regioncon~i5tf ngof an ol1go~accha rf deof the corespec~ftc ;ugar,£';'~~~~heptOse ILO':'Hep)and3-dedxY-D-lianno~f..octulo$onlcacid(KOOordoclAl ,'ea~h. . 'r: :1ilg a

bran~red tr7saCCharf ~e' (1~.'j2·1 :

^this

r~g1on f·sc_1fn~ed

^directly

tothelfpfdwhlch, hycommonagreement,Isal ways refer red toas l.1pfdA. The exactlinkages tn the KDOtrfsecchar-tdehave not,ye tbeen. def fn.ltf,velyesta blished (331.

The reducing end.of the KDDtr1sacch~ddelinks thepolysaccharide tothe lipi d Ain arel at ively acid labile(ketos id i cllinkage. The0- chains are attached

:a

^{thetermi nal.gl ucos:unit ofthe coreoligosac-}

chari de.

A'chara ct er is ti cfeature ofthecoreisits substitutionby pola r groups such as phosphateandeth~noh.llline. Tilecarboxyl group of .KDDcontritiu~es^{to the}~~.idic charac terof the llpopolys acchar ideand

thisnegathe charge. appear s.tobephYSiO~ical1yimport ant(12)•.rhe .l1popolysacchoir1 des 1IIClY,bei~terl1nked^{byioniclink'agesto·ot her}

~ompone~tsoft~ecell wailthroughd1valentcationsand p·olyallllnes •.If· · this-isthecase-suchanarrangellll.'nt wouldprovi deintegri tya~d stabil ityto thecell wall .

I I

13

Thecoreregionoflipopol y sacchar i deseKh1bH sahfgher degreeof het e rogeneity. Substl tuents of the cor-e,fm:ludl ng P,EtN,andG1cNAc, are notnece ssarily presentin molaramounts . F"urthermore,aH:alf labtl~subs tttuents suchas glycine haveoccasionall y been dete ctedon

~ .thecore(32,34l.

In recentyec:arsnumerouscoretypesh~vebeentdentif ledin Ent erobacterhcealll,!2, ZO,35). Figure 4_ilhstratesa selectio n of thesecereo1f ~osa:ccharfdes^(Ra-R4a~d^r-iz).'

They all showlIaj ordiffer ences fnthehexose,r eglon route-orcere!

but

a~e quite '~fmfhr

ⁱⁿ

'the H~p-kOO

^re9'1on

(~5l .

^{iable'!1lists}

.df f fe rent

'strlfn~

^havfng

i:ltffe\rent'core ' ~l1g0SaCCha~ides struct~res,

^,

someofthemare R.lIIJunts withfncompl ete?tosynthe s fs.

:ompos itio nal anal ys es of thecoreofltpopclysacchar tdes isolated from variousbacte rial species;have revealed the presenceof many unus ualconstituent s'suchas~.~...,g.~~-~ePtose,(441, uron1c acfd(45) , or amino ac1d(4Z,I,and1n thecaseofHbrlocholerae

st~afns~ fru~~se ^(16,461Orsedoheptulo~^may.~^{present. These}

possi b1.rreplace KDQ In llnklngthe core~^{the lipfd A.} Lfpopo~Jsaccharldes,contai nlnga coredevoid ofheptose orK OO have also been identlf fe d(W,"21,47-491 .'·

<:

I;'.Z ,' u:"",,,,,';r",, c f '-,,"h" ; (

R-m iJ t~ n tsare defectheInli popol y saccha ri de biosynthesis.

Depe~dln9on the defect,these 'mUtants_'syntlles 1secompleteorf~comp l ete

Ra

RI '

14

. ,

.; "

L~

^{'.: . :-}

:::'rl:~l,~~:'.

':':'".

z.. .

o.lJJ. lilrJ1.; Gli-.u...,Jt-.,.,,-!..1...;...+llCDOI_ ~

. . . . ^{Ri *~: ~¥~ f .}

fl~ .

^4.

Strllctur~

^ofenterobacteria l

-co~e' types

Ra -Kll.(12l. .. ,". . .

.. .t:

15

Table11. Cor e strcctares thathn e been parti ally eV/lluated (121.

• •' "0"

._ .,oI. ·- 'M '

""1;' )" "

", '.:".

.

_._~_ I /.

.; .\

..

^{- . , ( -"}

...·c,.

a. . _._

_.

.-

_•

_ .--.. ..

^01.(00)

~11'."".

.... ^,

.e>. . ..,

.'_ ,...,..,....;.c.

c '> .

_to,...;.:",. . - . ,__ .~__c...":"".'_ ,"

.... . L... ~ ..

. .,;;,." ...u ..:.... ....

-t"~.

~

', .. ,

·'....,;-u_ ...,;.;-

. ..

'

.

...._~-_.-"

',.." "--. .',.''','

.1.3.2.5~·struc~ure:~flipid calf(39 ),Shigella(50)an,~p;.otei.is.(511o

core structures stl11 1hkedtollpjd..'~.•butall de~oldofthec-cnatn.

Ff9U~⁵shows a sertesofR-forml1popolysaccharidesderfve~^(rem Sallllonelta ootantseerecuve tndifferentstepsof thebfosynthetfc

- - -

^.

.

^{. .\ "- ."}

pathw.ll.Y,'Simflar,ser i e sofR llIut antshaveal~obeen:1sDlatedfrom 16

~'a mutari~:are.'~~f;ec,~·i ~e-

^'In

.O~~ h·~l~. s:yni·'he~si~.:

ami

.~ro~i1~'e l'f~ciP01'YSaC~~~rl~e'~;'

^wi

t~" 'o;~~let~' ~6~e'/-; A ll _'~/,~h.~'

-

~~t~n'is

^.

from,.,R~

^';:".:'

, . .. . . • ···;:c:4;::Z:1: ne:~~'::::;::::.;;:e:y;::~~t1::C;::::::l;'::, • ..• .•

conia1~'

'o ' nlY

~DO'an~

'L 'f'"p 1d : : ,i.;

':·'The;df ffe~~·t~R.~~·tants:·~;'d:ih'eir.re~~~~".,,·::··"

·tfv(R.-1'1

popol~s:~dc~artd'~~

^-

~aX,':~e ,~f,~e'r~~t1,~t!d:'~nd ':jd,~li~t~f.;~.d. ~Y:~.an~'

of'lK.ttns(5'21.end~nt1b6d1~s:(53)'~~thete~ln~l,sugar.sequen~~s (whfch'are'

d1ff~;ent·~" ,are.', ~~~~nf·Ze~.':bY':S~eh· , ~~~,~~~.dS.

^":

~he. ~tant~,

^..

~,~

a-l~o'·be . fdent1~t~

^by.'

ChenitC~l

^.

analY~f~ '

^of,

t~eir' 11p~p~lisaccha·rf.de~ , a'n~'

^'"

.bythei r:

'ph~ge ·-pa~te,.n

^'(54).

~c:ur~e~~e"O f'the" R-fO~S--:'S ~~~~ ,:1.~·'

.

nature,~'t~o'U~h':

^they;

ian'

^be.'..CUl

~I~at~d 'Jl~der '~~b~'rat~~ :c~ndf~f.oni

^'a.nd...:

ar~ge:ner~'ny,n~·n.vfrul.e'rit.,'P~~9ocYt1i;dby:~cniPha9:~~'a~d a:r~ .sen~l:':·

~i1'~e ·.~o.' ;io~1C' ~g~n~s':'U.2\;

IB

, ',, "

·acYl'a~U;eami no,.gro upsofthe,dh,;c'ehar1de,.,Theremat~fn9^{ttvemol es}

offa i t y

'~~iJl~:are 'e'~t~'; ;t.nk~ ·but

^,not,

necessa~ny

^{d1;ec't lyto;t h:·}

hy~r~XY1;

^g;.o,ups

;~f the '91u~OSalnlne

^(55).

"

. .

In s1l1li1ar'.annertoO·t/:lal osand core',lipfd Aalso exhfbi ts

struc·tli;~lheter~gene·1ty.

^In

approl(;~~tely',50'

^0'-

th~

^U'p1d'JlIIO l eCules-

~f:sal~~nena.

^the'

P-hos~~a~

^..

~rOU~ ·l1n~~. w" .iuco~'a~!~l

^1s'sub,stf-

tu~ed

by

a,.~ho~phoryl:etha,no}am1nere~idue^{with a'fr e e.}amIno group .

~1..6. Bothg~~cosami n eunitsare substit ute dby aph~S pl'la tegroup; one be\"g bound in esterlinkagetoC-4 of thenonreduct ng glucos411in e (gll.icosamlneII), aodtheo~hertoC- l,ofthe reduci nggloc osdmfne (gluc.oH.lneIl. .thusoccupyi ng-th,eglycosf dlC hydro xylgr oupand nmderf ngthe'molecu le.nlin-re~'uclng . 'inthe"l'lpopolysacc har fde";the

·

tel'lltnal{~oo -un~~ .oftheco~ 1nttac~~'"toC-3.: ~r : glucosamf~

^II

(1~.

;i6.,.19',20)~ Thtp.Gl~N'{;'CN':p""lIOte·tY ·ofu,e-'1fp1·d·,^Al's.~:"ed"lip id'

" . .'.

'.".

^'

' .

^{- '},".. -.",

. .

^{" -}

A,backbone"'IF19•.61•

.".

i~~";~ptd:l

b ackbone

'~6~~~'1i1~ ~~'t~,:'~s;~,;'~~d''a~~d~-'11n~~d

:.tattY'

,~':;

>~~fd~'-:~ nd

^.ts

·.~'~tr~y ~ro~hobt~, ., f~~fC'- ~,~~-,~. :'t~e:-:'~t:e~~~ce\i ,~:, l~~g''''

~ha.in · ~at·~i:ac1.6s:

^and'

ne~~tf,ve'lY' ,~h;r~ed,' py~op,~~~p~~'te '·g~~ups.

^,

l Sllllru)nel l aHp1dAcon~1nsUlefollo~f ngseve nmoles of long ellain

fatt;~ adds:

^4,moles.of

g.;hydrOXYtet~adeeanIl1c

^<,3:"00-14:0or'a-

.:hydroXymY~i~tiC',aci d),~,andapproximat elyimole.cj-~'e ach of.'.dodecanol ~

acid!l2:O.latlrka~i df,·tetr adecano1'cacid'114:0.'nrt ristfc'acid),'and'

·hexadecanoi c'~c1d^{'(16:'0 .}palmH1~^acid), (12'). T....omol es ofJ-OH-14:0

i-, ...

;-

---

19

"\

J

. . " . 8'6 . 1

®-® ., .

~.~_ArQN__ ®--:,~G1C~,.~-Q-GlcN- p.. ~- EtN

¥#~.1r l~: ^... ~ ^.

. .It11G

1~lla "

, ,

-.c-c .~.O

.l~tllO·^{.:1l? t1"}

. CHI CHJ

-

' - . ..

, Fig.6. -StruCtureof lipId AfnllllSalmnella. DottedIink~gl'5indicate

incompl ete5ubS'~I,tlltton( 11)~" -.- - .

. \

......: ..r, .7:' "

20

InaBout30-:-601 ofthelfp1,dAmoleccl esthephosphatebound to. glucosMl1nerr.ts subst i t utedby4~ am1 no-4 - deollY-;,- arab1nose(4 - AraN)i theltnkagebe ingthr ough e--l. Boththe amino gr oup'andthehydroxyl

!I1'l)~PSof4-Ar aN appear"tobeunsubstit uted(56).

1.3. 3.'8iosy nthesis oflfpo polys accharf des'

,, ", .

1.3.i.l pr.1.nCf'iies.o,f

, we

.Bf~sint~esis.~f,t~~,o-Chafo·s·.

The\f_~~ynt~·~S1~^.:

'? f

'_O-~_h.a~ns fs'·"cata.'Yse,~·.bY^--a."11I1t1-,eriZy~:..s~st_~m Uat ut flhes,'

I'

~~i>l"~ne-bou·~d.-po1~1;o'pr~nOld,~OOipo,u,~d,'a~.:,·(;.'rrf~r_-f~,~

.:"~^91YCOSYl··.'r.eSfdue~;~:· .. .: .•. . :.

'In

'salm;n~l'fa: grOUps'·A.D~:·ani t.

^(57).

t~~ Syn~he9of S

^begi-n.s

~1th '

tilereYerslble-tra~sferof galactosel-,~ho~pha-te frOllU~P;..g'a1iJc 1;OSl!to thl!phosphoryl ated carrier li pid.

S'equent fa l tran s fer of,J-e'~dd1t1Qnalsugar sto the-Hpi d"-lint ed lnte nnedlate'lead:tothefonnat"t on of

the O":c~a1n 'rep_~a~inf

^unitlinked

throughapyr~pMsphllt'l!'bri dgetotill!carrfer-~'ecule'-" ' :'Therepeati ng uni ts arethen:pol yrtlerlsed prIor"totheaddit 10no~'othe-r_;SUbst1 tul!~ts (e.g . eeetetel

1l,2) '- ''''POlym~"rf5at.10n \cur~

^by

,con~~a~t~ l·trAnsf.e~~fng

_thegrowi ngchain~^a'newly act fv at edrepeatingui'iidg~owtli^atuti·

reduci ng endl., This,'resurtsfna~long cha lnpOTymerl~'1nterled hie"that

. .

.

^{, .}

ts st i ll Ifnlced. attheredlt'~,ln9 en~of the.carr fe r ltptd, Asi!l:ffn~."· step.tileO:-chalns' are transfer re d (rOIlthelIp1 d-carr~e.rtothe:tude-

"

pendent lysynt hesis ed core1l pldAtofonntheCbmpletemol ecule(56).

11

1.3 .3.Z. Biosynthesis ofthe Core

In aeanne rsimfla rtoO-cbainbiosynth esis elipfd~ca.rrlerts i,nvolved,Inthi s case . lipi d A, wh1c;H.NlIIa1nslinked,'tothe cor.eas partor thelfpOpolysaCCharfde·~lecU·le. ^ThebfOSynthetf~^pathway.oL_ cor e-lip idA';sy'nt heslsprOCeedsf'r~

the

,Re,'io'Ra'rm,;tants~

:

the su gar'

~~~st~~u~_~t,s:~be,1~.9_ s.~·qU~~t'l~.~·_~~_.~~~_n~:f'~rred, ;:by 'te~~~,I'~·?s',::~.~,ialY'~,ed::~ : ', ~ ~.;::.'~": .

'...;:",ttje'

, resp_~~,~lve' ~~ga~ tran~f~.ra·se5· (1~. ·16:

^..

2Q{ , O.,.i:~a1n ~~~~e~~,s ; :!

^;

F,:oRlpu1.se/~hase expe~1~e!,~5,,,t~ebiosrnthes~sof'li pi d,A appearsto occurv1~aprecursor:moleculeIIhlc hcontaf ris the',dtphosphorylated~-

. 1.6~J,1 n~~d -df9~·u~~~~mt~e:b·ac~ ~o~. ~~i;~'tit~i_~

^·bY.,..

~o~r 3~:oH~1~.:O reS1d~~s,,,:O .'11J

^{eefde linkage:and}

'~ll ;n

^ester

1~nkage (58J ~ .~;he p"i-e~~r~~r -d~~~·. ~~t,,'to_~t~',i~·,~~'·-~~d; , ; ac~s

^-

~~~. 'sat~r~i~d ' fattia~i

^ds.

. i

>.

',""!

;-",'

".~

.:. '

1.4.:if~1091c&1 an~ [nd~todc.-Pro;.ert1·~s_of^l1popolysa t tllir1d eS-':

, .-

" . '

':-: ,. .' ~ .~'

..

^~ " " "

.;·sra. fI!gat h ebatte~~.a~~sep~leshOc'k"t~N.~

art'-":

direct·'

r~sultOJf.the",,~act_I_O.to·.Ncterhl.~d0t4dns·I60I,. Early.~tOms'of ...". gr a. .neg~tiftse.PSh,1nclllCiefever ,dYSpnfal·ollgurla .'.IlllhyRote~s_'Oll.;:'~^{-, '}

r',

" ,"" ,/:::.' ;'.;'.: . . ::;:.::: .:7.~i.; ^{. ...:;: .} : .··.:~ .·.: .t.;.( .'.t:} .:~ ^..• ~ .f.: . ;~~ ^{. . •} ~.~:r .~: ^{. . . ; . •} ~ .~~~;·.r .;.r ."+ ^{. .} ; . : ,:~.:/%i:r'; ·, , ;.;~

.-.. ',:~> :.:..: .'.

^thegastro1 ntest1na'tract>'andgeileralhedthi-ciilbl.tn

hpln a'rl~s

^{,'-i·:'-,· · -:.;.. :.}

; : , . > :':~',:' . ;: 160 1; ['bO"~'~ :;f"d;::' f"'j"de LLs:,;L::".;:i" '~f ;d'om.~ · ^{;' ':. .'}

" 'i\'. ....

n~_~~ '~~.'c·~pl~n~. ·de;t~1.e~c; ~~·. 't~' ~~ve;'d' C~'oitl~ ·fact;·r~

^•.

:;eu~~ ,

cytoshand·dK:!'e.llsrd plateletcOlln.is'.: ·> . .' ." . .~:~.•.',' TheSyndromeb7wh1ch-local

US:su~ ~.age otcllr~a~·.a

^{result of"J:. : '}

in~~else~ ' Vlstllllli llf~a'b(ll;Y iog~U;~!". ·~~ · th~ fonn.atl~/Of _fi i:!,1~:

.

;

. .. .. .. :."

.;":

•tton{OI'::!.: i6ll

• . ' . " . , _ '."..

_-~""

^..

~" :

. . •Severalaspectsof thls.pathOlogyunbe-'.lc:ke d,inlIn f.ll1Odfls·

.;,

b~

^the

_f.nj-;c.t1~ ~f

^..

1P~.' ~ S~lI~ ~~ml: g~'r~" lleg~ii~ ~acie~'I:~.:~:

^..

'~T~~~~ :,"~ ^.

'. "genenll~edSCllwlIrtP;nn,nacHon,In''t1i~rabbtt ,\'11Ch:1~el Icited,.by;:·,

."'> ~wO PI'Gp'~rl)'. SP~~ed.1ntr~~e~~ .1·~~~tf·~ns;~f.::;·P's, re~ul~~ ,l~ : b~t:~'i'~~at ^".

· ',rerial",cor t fc¹11necrosli,.d~~:-td<:f1brlnd'eposltt~n IS.~.

result.

_O(~lffU~·:..-:

·

·\~\~aV.I.sc~~_lr ~; ~t~;ng.(6·2·j'.::· Th~'~e'~~'f~~ts' 1~ . acc-~~~·~~le~ :bi.:~ ';~~~;i~···:·~ :

"

"-

14

pol yclo nalactiv atio nof bonemar row der iv edly~phOCytes(68), lIet1Yatlonof the class icalcOlIIplementpat hway(69).and inductionof

~acrophagecytolyticcapacity nOlo

Inadditio ntopo)ysacchar ide def icientpreparation's,nat urlilYar1- .a tf~ns

in

theO-chaincoilponent of smoothlipopolysaccharf des providea

'~ld~

^spectrumof

1llD',eCLl'~s

^whlch

' ~a.n .~

useete Investigatethe • ^j

cont r f butfon,.of'thepartie.uh f'carbohydrate-groups-to'Ia)b1o~ogfcal 'at.Uv!ty.in1t'ated

~t

^,the

polysll,c~ha.ride ltse~ f'-a~~

^_

(~l _

^the

_.~dUl a~10n

oflipidAactiv ity.·,'Thefract1on~ttonof

i.'

cal fseratypesOlll:~and K235 (71,721", ntoniolecu la r'specie scontaf ~~grea te rorlesser. amounts

0 . '

r~pea tlngO-'poly,saCchar~ de1IIl1! provepa rt~cul~r'lyvaluable. Morr 1sonand.Lei ve (71)"demenstrat ed thattpsfrac;tion J~romf·.£2l!

se.rotypes 01\1:64'(long"'o--tha1nl1~ con~1d~rab'lYless tox'k'than

f~actlon

ⁿ

l$hO;~ ~~hiiin).

^Onthefother hand,

fra~t1on I '~a's

^{fOLi nd}

to

beamore

po~ent

^{actiYat()1'of}

' ~er~ c;ompl~erft

^dueto' act fvat fonoithe .. alter-natl'

~e

.

'path;ay

;, "

~~

^tile

. ,p~l;sa~charf~~-c~onen.t. ^,

^{. ..'}

'.

^{" .. '.}

1.552. 1"Acfd'Hydrolys1 ~

.:$". .r" , .; .~

.

^{0 . • :•.-,•}~ ^{, ". ""}

Mild acid-hydrolysis(n ,~c;et,lcaci d;IOO' C)creeves the KDO-lfpi d A'linkage lIber a-ting'

·fre~.\1P.1~ A~)l~, .12 ~·

^16:2Ql••'prior.to tile

. .. . . '

^- .'"

.. ...

^{:., -.'.':" , . ' "} :.- .

.

a_~afl~~~l.ftyofpolysa~tharfd~ ~d.l ~ f~,ntnutants,.1nvesUg~t:10~SLls1ng

-'sLiCh<1s~hted"lJpfd^,A,pre~aratf'ons^'prov1ded:" e beste't'fd~nce'that^many

b1ologf~'aooendot olf c'propert fes''IIereassocf'ate d, w{th thelfpfd_ .

:"

^'

-

^"." ~,

'. . . ' - "

• 25

component . Itshoul dbe noted howeverthatR~!,ee J.1~~

,, -

;l,sn ot ne~e ss a rfl 'yidenticaltothe~~_boundlipi dA" of the-originalraclecule,

1.5.2.2 . Alkali neHydr? ly"sfs

Shor t'periods ofre1a~hely^{mil daHal}j:ne hydrolysis-

(O.03W,

HaQH.

J7'C)causeasubst ant ial reduct tcnfn',th'~e5~erbound [at tyad d c~ponMtofIps(]Jr•.However,

trn;

b101 og 1 c~1 ac~iv~t.1:eswhic h,ar e ,as~odatedwithlipi d,'"and'whichmight be''exP&<;~'dtodepend'ueen the

.

.

hydr.olysfsresulttnthelossof biologic prapertfes associatedw1~lLthe lipid Acomponen~ofIps, includin gmftogene~fs(7J).

1.5.2.3 . PolymyxinB

.,. .

'~'th"

'PO''':''

.Wht~'·^,esP""'~

"t".," ""rp,.",

^tho'''~!"'-. tlan ofstruc t ur e-funhfo nrelat1onS:h fpsf nvolves theu~eof the.cycl ic'

pePt~de

antibi ot i c

PtO;'~YXin

^B.

~~l~Xfn'

^a

ha~

^been

~epo~t~d

^to

,~

1nh1.b1 t'var 1cius1n,'vivoeffeqts o(,l ps(74,751. Ithils rio....~ee~·est eb- l'1shed

th~t tfi1~-:~ot1~;

." .. ,

.

~

btnds ;~fth

."

'lfPid' ' . ~ fnterferin~' '

^" ' ^with

~e'le;al . .

of the bfolog 1cal-proper'tl e ~of the lps IOOlecule(761.

o. • 0 ~ , : •

2. STRUCTURAL,AHA.LTSESOF!POlYS~CCk"'RIDES'

Thi1ll11iu nolog1c~l ,serolo~fcal.and phage"re'c4lptorp'~oper,t1esOf bact eri al_{• . '}

P01~sa~chaddes

_j ^{are usuall y}expr esse d

thrO~gh

^•.

reg1~~S ~f

^the

< ' . .

polysacchal'lfdes-:knownasthe.,antf genll; determfnants,(12.16 ; ZD). The .basis_•'of_'.

~e

_..'

ari~1g,n-an~1bod~ ;nrnUnOl~g1C4,~

_{• ;,I•.1} •'.' " I'~

'p~'en',OlI1enon

. - ^is, ."^the

. ....

/ '

.

26

recognitionof partialstructuresof thepolysaccllar1 de antI gens by antibodymolecu les orflllllu necell recepto rs. Fora quanti.t athe consl~erat1o nof tile differen tspecificiti esof theantfgenicdete r- rstnant s,the elucfda t fon ofthe struc t ure ofthe polysaccharlde antfgen is necessary02.16,20).

Inorderto fullyCharacterf~ea.polysacchari de,consi st ing of .'"repeati ngunitsor"anol1 gosacc~adde .a st ructural study·should

det ermine:

a) thenat ureandnumber ofs,ugar s resteues andthe irrela t iv e proport ions;

b) tileposI t i onofthe lfnkages of tile sugarsresid ues;

c} tilesequenceofthecOOIponent'sugars;

el) the anomertccon ft!iur~tionsoftile sugarspresent:

el theconformatf onof-thepolysacchar ide.

Thelitel"ll. t ure descr1b1ng the techniquesusedto ecntevethese requfreeentscannot be reviewedadequatelyin a dt scusstenof this

n~tu r~.

There fore,onlythetechntquesusedInthe courseof this work will be~1scussed"witha~attempt tore fer to thelatest'crf ttcat .revt ees;andtoupdate th~applicat i ons ofthose'yechn lques forwhtch

significa nt improvements haverecent lybeenmad~~

Whi le some ofthe follo-':lngdi scus sio nwill deal wfth repeat i ng units,ofpolysa cchar id es ,IllOst'of the met hodol ogy descrlbed'canbe

'"

equally ..ell applfedto theinvestigatio n ofoli gosa ccharide structures.

2.1. Total Hydrolysis

The hydr ol y sis of"poIYS8c~hartde.andthe subsequentaI'l.lyshof the hydrol ys isproducts is'of te nperfomedlJ,Ial lta tt'ely . However ,the total

~YdrO'ysh

^or

POly~aCC·h·arfd~S.

^{'particularlythose}

~ith r!Sfst~nt glYCOSfd~C l1 ~.kages. ,h ~- 'mo~~

^{d1ff1CUltoper ation1111.}

HydrOChl~~fC.

s~l furfc

^and

t~1f'uor~acet1 C'

^{.ect es}

ar~ ~~only ,used\~ ~drol)'sls " a n d

are sUfficfen t toc~pl~telYhydr ol;se aneut ral polysa cc hari de intoit s monl)flerl c sugarunits:'.

HostOf th'econstituent~n~saccha rf des"re1easeddur ingthe addle hydrolysishavetr"d1t1on~11ybeenfdent f f f led~Ypaper chrolliltogr aphy"

and e. f ,e,'(78 ) .but'tlleq,l.lt tathe andq,l.nt1 t.theanalys is is·no.

lIOn!

us~allyperfOlWd

^by9.1.C.,and

g.1.e.~II.s .

^of

~ut-table de~hathes

(79. BOI. The sugarsart.Us~~l1Ytransforale-d"intoae.ety1ated-"l df ~o1s or ildonftrfl es. ster eet seee rs ghe si _narIllaSSspectraand 'Whenmts .1nfo~tton^{15eoupl edwiththe';;lathe}retentt~n.^{UIll@Son g.1.e .}

, '

~de~ttf1eat.lonofthetndiv idu al sugarispo~st ~le,"-w{ ththeexe~ptton of the absol ut e'eonf1'gurat1on(79-831.

2.2. Methylatf on Analysis

Methylat10nanaly~~ s.developedbyHawort handeo-worker s, 1~st tll.

the"most fmportant sfngl e IIlethod 1nst~ue tu"ral"c:~rbohydratechemtstrY". It1nvolv esmeth ylattonof all fr e-ehydroxylgroups inthepoly saecha- rreeand

tlYd"O~Y~1's,

^of"the

h!thy1a~d POl~~a~Chi'r~de

^{to .iafxture of}

,.

28

partially IIIl'thy latedsonus eccnert des• The freehydroxyl-groups In these

'"""

malt thep.osltfol'lsatwhichthe correspondingsugarresi dues were

substftuteQ1n the polysaccharide. Qualf ta t fve and quant f ta t fv e analysisof thismixture therefore gives1nfannationon,how the d~.fferentsugars.are jtnked..It doesnot.~however,giv e fnfonnat1C)1'lon the sequence oron the anomericcOr:lflguratfons (81-83). Duetothe , tnsolubil itY'Of hf 9h

lllOleCll·l.ar: ~~9ht O~thal'n' poly~r-

^In

~r-9a~'ic

SOl ~en~~,

^the

~ihOd. Of'-~lIwort~ ^{(in," ,}

Purdfe lBS)"-'nd

K~'hn

^(86')

reti~ired'

several>teps

~" a~h;eY'~ complet~ meth~1at1ori. -~he:reas

^now

th~ H~k-om~~l

ilethylatlon(a7l achievescomp.l.~_t;methyletten_1n'j ~stonestep.>The latter method consists oftr eat ing the po1-ysaccha,rfdefl'!,0

dhllethyl sulfox lde'WIththe anfonofsod11J1J1 methylsul flnylmethan1de and subsequentl ywit~methyliod1de(87). TheHa1<omorf methylation, unl1kethe.K,uhnand Pllrdieprocedures, .cannotbeusedon'a polysaCCharidecont afning alkalflabile subst ftuentswhichshouldbe preserved.,In the latte rcase,methylationwith,met hyl.' .tr f f1uorolllethanesulfona'teintr lmethYl phosphate,',usf"g',2;6-di..' t:

.butyl Hyrfdi"neas"aproton scavenger

't~

^an'

~legant ,aHe~n'att:ve

^(881-.

The methyla tionanalYstsisbasedonth~^ability'~fractionatE!'and char-acter-lzettlepar 'ffall ymet tlyl at ed monosaccharidesgeneratedby hydrolYSiSof tile.fUllYmethYlatE!d'polysac,ch~ride. ^{Thts is"!Ost} con~enfentlYaccomplishedby .:omb fnedgas.ltQllfdchroma tograp';Y/~ 5S spectrollletry of the methylet~erald1tdJ~cetatesderivatives(81-83, 89).

TheinterpretationofttJemas5sp,~ctr.aIs1l5~anyasimpletask.

Pr flllary fragments~and~are'-,fonoedb/f,iss1onofth~carbon',cha1nof"

"

component1conta in in gvtct netmethoxylate dcarbons , therfssf cn between ,.

these carbonatoms is pref err ed; eith er fragment may carrythe.positive charge. Whl'!n a methoxyla t edcarbonhas anacetoxy1ate d neig hbour such asstructure~ . teneencytcfis si on'betweenthemethoKylated and acetox ylatedca r~oil.a1tho~gh^not·as,\g.rea~^as.be~we.envfcf r(i cal".methoxy¥

lat edcarbon,~ives'sf'gnfficantp~1R1aryions and,t herraginent'wh.ht~e

lIl~th-oxy'

^Aect.

'~arbon ~

^always

carr~es

^{_,t he-}

~sf' tl~echlii'ge

^.IscneeeO.

',1~

^'general

~ _ 1 ~

^II

~e:,s.ldue ~o~t~.1~.5,··a··,~th~)(:y. gr{)~P.' f~a;.ne~~at~o~

-

^{. - . -.'}

consecuti ve el_c flllliat1 0ns of me_.t hanol,"hVz_. 321'. aceticaci d(m/z 60) and. ket ene-(.Rl/z42), Elfmfnat lon sofaethancl,keteneIlnd'~e~lCacidfrolll twop~f mar.rfragmentsof1'1/1.161(coirmonin massspect ra.Dfmethyl at ed sugar s)'are,illustrate dIn-Scheme2•

.A,nalys1s andst r uctu r al·char acter f zat fonof par t fally~thY-1.ated al~ftol,.

aceta te s ofami nosugar s-hesbeen.tne'~ubj ectofsever al investig at io ns (BO,9O-~3)•.For aldftolacetates d~rh,edfrOlll2"acetamfdo-2.d~xy ~ sug'arsg.,t he'pri ma?"fragll~~t^m/zissand'th'~t~'sec~ndary fra~nts m/zU6:andrn/z. 9lI pr edOlll f na.t e. Other fr a!pents er e ,IIowever,'strong .enough to,i dent ifythe met hylationpat t.em (Scheme 3) .

Othermod1fi cati ?ns ofthe methylati onprocedure such asthe

J

·i nt r oduct fon of deut~riUIII^{at CoOl 'fnorder to·obta in}'unsY!IJ!et ri cal deri vativ es, did'euteratfonOfC-6, trl deute~fomethYlat1on.

.'. .

. •..

" " .

",:

2a:..·

;"":':-s

. .

HC=OMe

'C=I O

Ct!;)

I

'm,~

&7

·HC=

~

OM.

H C- cile •

R

I

Z..

>...:

, ~: ^.

HC=OMe

,": '.-8 ,

.H~'::'O~C RZ"

!:..

~.

fH Z

f?Me,. HC=OMe.

e m'.IOI

"

... .

^:

. ~ . :.

. '.. HC-OMe ":"-e-:)..._~

'. ' "

^{\ ." " .}

. ~k:OAC ' "

z .1

HC"OeM.

tH _{s .} . . ~ .

HCOMe ''''' . 1.01

.-

^";.

.;.~,._,:.'r••

j

-

JO

\11 I . r

^l

H~-OM. ~ ^HC-Ot-le

~

^.

^.

^HC-Cl'l.

^{. . .}

Hr-,- Ot-le

^I

RI

Z R

Z

~~

,L

!l

t-toeutertcecetyteercn, or ethylat10ngive'deriv ati ves,the fragmen- tation patterns,'of which are quiteanalogoustothosedisc ussedearl.ter

182-.:94, 1021.

-'The reductive'cl e a vageat' thepennethYlat~dpolysaccharideby boron

• I . .

,atr1'fluor1:4e.trfethyhf1a~eand trffluoroacetf cacfd ,toa mixture of

.

~anhydr~~idft01S '"

^can

be ·us~d.

^todf stf ngui:h

be~we:' 4-11~'ked

"al dohexo;

PYr~:~o'~~

^:a'nd

,~~

^linked'al

dOhexofur~'~~se

^.r'e;s'f

duest~ -P01Ysil~charf

^des.

-- .·::~~rm·~t~Yl ~te.d·.PO!y~~ci.ha~fde$·'conta't,;'t~~:,th_e~se" ~std~e·s'. W 1 '1'"9f:v~·:onl,;,.·.

:--O~·':''part'fl' 1.v~.~et~l at~d'-su9ar;'213.~6~·tr1-.Q.~~·~l hex~:seafter -r;omal.

i~:fd: hY'd~DIy.s.fs.

^-

:-Thi ~ ~t~~¥

^c'an'

al~o be.~p~11e-d t~ 4-

^And

5~;fnked

aldopentosere"sfduesandtoS-and6-lfnl:.lffi'ketoheJ<.oseresldues(95);' Asthe methylatIo nanalysishas become sc.s tepte, Itis also used In connection'~f thd,lffe~ent,specHf-cdegr ad.lIt 1ons of.polysaCChar1deS perf?nned'f n order..tcdetenni.~etbe.secuenees~fthe sugar resi due s(96-98 ).

seque nce'Analy~i s

Mci.'nyd1fferentde g..radatfontechnfques areusec-rcr-sequence

.ailal~~i,\^of·pO!YsacC~~'r~de~'.^"There~s^nos.tandard.meth~d^{s.and each}

polysaccharideprese'nts',its0Wll probleins_ Among't he'ol d,estmethods are th.epartialac1dhydrolysis and analyslS"ofth~reSU1ting011g05ai::- chari.. .des:.the SlIlft b" " . ,'de gr adati on and the'" .

dealiin~t~on

" ^{of the aminosligar}

residues . 'These' diffe rent methods hav'ebeens~....arfzed1 nthe l1te ra-

.Ca pon'(100 ) has cceprehenstvelyrevfewed:the"firstorder.j-ate.

.32

1llOnosacchar ldes i and severalgeneralizatfonslll/lybelllade:

a) - furanosldesaremorelabile than pyranosfdes b) - /feOltysugars eremore easily hydrolysedthannexeses c) •uron1c acids:~re^very~eststant

to

^hydrolysts

.due"to"the'triduct j,ve'eff ect.'of't heprot onated aminogroup'

"eo)'-

pe~~~Py!,ano~'f1_e~' ar'~' -~r~:.1ab1,i~

^than

'hex~~;ranOS1de:s

.fI,':II-g1yc.o:$i(i~c~~~nds'

' a . re

~~ually_~ore"'~blle

,than

"Il:~fyc'OS~ d1'C -bonds

': ',. ' ,

.

It'5thereforelogicaltIlat'"given a.hete:ropolysaccharld'e.•there w111 be

s~ g'YCos.~iI1C

^bO;;dS.•

th~t ,~~

^relat'hely"resls tanttoacid hydrolysfsandoth'lOrsthatar~:compari t 1vely susceptible.Therefore, -.polys'aCChar1de~c?ntatn1'nglink.agesofef thertypewf11-tend,t o produce"

deff_.nedoli gomeric 5ubun1;~5'whtChcan'be"fsol'ated'and1dentfrted. Co~

, '. ' " " .- : ', ' ,' , .

.51~erablymorestr,uc tural,~nf.ol'lMtfon'canbe cbtefnediftlle:llethyl a"t ed.

o11gosa cchar,fdes,.der h,ed"{ram,aCfdic'tiydro"lysis'ofpermethyl'~ted^{'pol y•.}

sac~harf~: ~:~: ~e,du~ed'

^'and

re~i~;hted u~{Ag t;td~u"terf ~thY~

^or

, e~hYl

.fod'Ide."'.

ThiJ~,·th~·'. 1'~oht~O~:: o~

^,the

,a:l~y.~a~d ~fsa'~Cha~ide ald.ftol.\~' ·

^"

d~monstrates:

^{·'a )}

"':h~t th~ ~l~sac't~t1de. ~~~,tafris ,th~ d;~aCCh~'~t'de

^unft 1i,b)that".tile:~on-redu~t^ng,galactosy1gro~p'~,as^{11nk.ed'to,the 3-}

.

^{.. ' " . "} .": ^{' ,'}

_:posft1on''fn ·the,Polys·accharide;.clthatthe.reduc1ng r es1due 'oIasfurano-, stdfc; dla~',thiJtbothsuga,:s'we~'chaf~restdues'a~d~ot bra~chlng

33

Scheme 4',

I

r

I

1

34

residues(JOl)(Scheme4) . Thislatter techniqueha sbeen further devel opedby Albershelmand his cc-eercers{lOZI.

mtalternathetopartialacidhydrolysisis acetoly sis(97,103) arid the relativeratesby whichthe diffe rentglycosid iclinkagesare cleaved differ for:thfs and.the' !:'revlous.IIIl!thod. Pyranos1dfc(h- 6 )- li nkages arecleav'ed-more'readily' thanoth er types ofpyr~nOsfd1C

onthereactfo~tempe.ra t,ureused.ISthem~

st.

35

~POlyalde~Ydi:\!Ihich'~'wti~n',;'~du'cid',wi~ii.'~o~iUII,·~·o~hyd,r,f;de;··,-Y'~J.~s:^{'a..' .'}

~p~1'YOH~,'Thls'.'def h ed"p~lYO':,'~~n-proY1'de'bans"!der~bl'ti "i'~f~nI~t1olr-(in"

formed.

by perfodate \!Ii t h't he formationofcarb?nylc9111~ollndsasf11ustrat ed in Scheme.6i the. simplestfra911~nt.sb~1ng'fonnal dehyde(R,~nd~l^{•H): In}

.the.specia lcase of three contiguous hydroxy.groups.fOMlfc,acid is elsc

'"0' ,""

.'::~:';...'.,.'':: c : ', ~' ':

···~···':~-t; \T···;:

.. : .. " .:- ·<·:;::~·:~:,~t

stnlCtl.l:~l

^elnent,ll.

^9'.;';S· the,,2~·~~;~-·gllaC~PY~anOS1'1~!-eryth~'.tol · ~~"~:':/

^",F

.

I'~

,

!l·Yia·~'~lya;c~oj,g'(1C)~ )~~Ch~-:7). ^{- -} -r-,~'. : '.'. :~..

' ,;~sg~f; i:~~ ::~~ ~~ i:, ~ t;,;{,/;i :

. . ' '.'(.:...,::':".)•.;. ,'; ..'~^...1"., ';;:~.\^...~^','.-

.:'. '2~3:3: ',: D~amfna't1 o~~' :

^{,,: ;..}

' .;::~':,:;>::,:, " : ',,' :' :.'J

. . ,. s;;ve';'~';";~h"i;e'c"ti,n";no<;";;;;;.t,ich· i" :;;;"'l\~N~ ; ; ; ...: :. ) .:

aC~~1a~d, 't~~~ ':'~~I ~U~S

^{ofle r-a'}

~tar~fng

^po'fntc

~'r ~~~ff1~ de~ra~a~,.. ·

...",:.

'~5~~~f~ffJ (~~"~1des'and- gfYCoconjllgates'~;i 2~anriIl,O::2.:~CJXy,:,~glucos@' ~~~~~ and ·2~';1no- ~f',,'I ~ .~.~;: .

^-:'

·.~~~·~QXi~~~~)~{~~~;~.:;~~~,~~~:~~·i.i~. ~f ~~.~ '~~~~f.n~~~d~~~~~~i~.e~~.~~;·' · ·;

^'::

<'.j-, :->

.~hel r-_, lleltopyr~ nos1des_{" . '.} ...1':1'ltlich the,alIi,no group_{'.', •,~.. ' ' ,-"} isequa tDrh lly·'or1ente d_{•. ,' '" - ,c "}.. :, "

:""n't~'.o·stst~blefoj,D,,Y1 elds·2.5-a~llydro::res1ciues~,',1h1's'reaction'hils

.'

..

^{' " ,} ', ,' ' . - _.

""

^{'- '} '.' -.

,'.

been"a,~~quiteljrevf!wed.•tnthe'1 1tfra~joe: 197~.98~11~~:113 ),,"::',The·':,:·,-.":.' ,.::r'

.'.'•.:'.'rea~~an'geai~nt,:·,r~s~l,ts·:fr~,·an·^{att aCk,:}oiI,"~he^"1;nu"nn! dt,a,ted~a~orii~:f,on^{,:::. '..,;,'.}

(. ,;.~~~

^.";::":'.'i·'',\•.,.::-':

i>

^by

~~ rt ~~~'~xYg";~ 'a~~' (~;Ch

^-1$i'n"a'

tra~s a~d' antt~~~'~;lel'

^{t) ,. " :., "}

"

dfsPtisft;o~l .'

^ThW$.deamfna tfonofa

2-amlno-'2-t1eOx~-Q-gIU~Opyranostde

^."

~ '. ' :;'.,t_~.;'.:'

~ ~~;~,~,"·, ;~ ·:;~Y;~: :I~:; - :,); i~\+

.:'.

...

~.'

",:~

' ,.

38

...

\~

I . HQ

HO~ ~, H~ -

' " ~ ' ,0" '

^{, CH,oH0 0 .;,.0 .}

" - 0 "

~ ~\. ,

HO ' . .

OH.~

. ',' .OH . . OH

11 [ :

. CH.oH

H~ O OhOH

^{• •}

H~ '

^.'

o-at

.." OM '

&.oH ·

. n

- . ,'1:.:-

,'j

,

\ '

^"

:

Scheme7 :'.' :

,"",

"

.r-";

39 withrel ease ofthe aglycon(Scheme 8).

Similarly,2:.amlno-2-deoxy-g-g alactos fdeyie lds2,5-a nhydr o-g- tetcse , St udi eshave revealedthatpartof thedeaflfnatf on reecrtcnof 2- aml no-2-deoxy -D-g lucos ide 14 Ilayalso= - ,ta ke anot her cour se with, fomationofa2-deOXY-2-f.- fOrtlYl ~g-rl b.O-heXO.5:id~This,u~.expected rearrangelllent.-_resultsfromtheattacl:ofcarbOn~hichisa1sotrans

.

'~nd' ~nt1 parallel'to

^{the d.1azonfum1ion(97.9B,}112, -113). · - -.

AlIlinosugar-i ncor por atedtn a polysacc haridechai nalsc reac ts in thesam; manner. rne.u-speettrcpolysac charidefro.. Shtgel.lafle~ne rf. pol ysa cc hari de variant Y,is ccepusedofthe.tet rasacchar iderepeati ng unit !J!(114). The N;.deacety latedpolys acch ar-i de easdeamfn'a-tedby treatm ent withnitrousaci d yieldingtetrasaccharideIIasthema i n productJscneee91. · Met hyla ti onanalysfsofth~reducedtet~ a s ac cllarl d e showedthatoneof.{ he~-sl.ibstitu tedo""k- rhamn,opyr anosylresidues inthe origfnalpol y s.accha r1 de hadteceeethetermlnal'non-reduci~9o-k - rham~OPYra nosy\^{group in.t hetetrasacc haride,proviog.t,hatthe2-"}

ac~tam1 dO-~,.deo~-g- 91Ucos·ewasl1n-ke~ .to 0-2of thiskrhamnose·. On", treatnient oftetrasaccharideIIwjth base,thetr'is~cCharidelinked to 0-3of the 2.5-anhyd ro-mannose(produced'f romthe deilllf na t t on of the amino sugar res1 due)wasrej eesed; M~thYl a tj onanaly sls

of

trfsaccha

. .

.

ridealdito l.@provi ded the.seqce nce ofthe,threerhaillnosyl resi due fn~.,

neentn ett cn reactio ns ,havealso been appliedtopolysacc har fde .contai ni ng, ot herami noS,ugar s"such, as the polysacch,arfdeJrom Vibr i o c.hol~raecont aini nga polymercomposedof(1+2)linked4-amfl)o-4 ,6~

\

c••

1 ..

_v

. ..

;

., ';

40

Schelae 8

.2)~-""Rh~.P:(~.2~-~L::h"-(11·')-"'.P:·~~l~ )-.~l>-Cl" N~ (l"

. '. M02

. . H.flH

",-L-.kMrCi+2)-o-L-R

.

. .hap;-U+3)__L-RhaZ- . ..

",!.!

a-L-RhtlJ!.-( l+Z )-ct""L-Rha,e- (l+) l -:QPL-Rhnnltol

I .

2~

Schelle9

...

" :

'i