Note: Descriptions are shown in the official language in which they were submitted.
-~ WO g4/1~388 21 ~ 8 ~ 9 ~ PCI/AU93/005q6
~ .
~ .
Te~hn .1l Fiekl ~ .
Thi- in~entioll rei;lle.~ to ~ .~olid sur~ce for the synthesis of pep~des
~hereon. ;Ind in paru~ u!;lr rel;~ ro(1 or pin h~vin~ a pluraliitv of surface~ on
which peptides may be synthesi~e~ ;lnà ;~ssayed efficiently and econon~ically.
1(~ _
Geysen. EP I ~XX55. disclosed ~ method ~r the simul~neous syn~hesis
of a lar~e number of different peptides. B~SjL~11Y. ~hiS metbod involves the synthesis
of pepddes on ~ solid po~ymeric ~surf~ce~ such as polye~ylene. which may be molded
nto the sh;lpe o~ a rod or pin. ln ;l pre~oIred embod~nent of the method. these rods
5 or pins Dre positiQned in a holder so ~ha~ they fonn a 12 b~ X ma~ix. with the rods
or pins bein~ po~i~ioned so that the ~pacin~ ~orresponds to that of ~he wells of micro-
iter pl~tes ~which are wldely used for ELISA (enzyrne-linked immunosorbent assay)
~ tests.
: ~ The method discloxed in thls prior specif1cation was based on the real-
~: ~ 2(~tion th~t~for the~solid-ph;l!ie synthes~s~of ~y pep~ide, almost all of ~the~s~eps of the
1~ ;ynthesi~ ~re e~ctly the s;lmie for any piepiide ~nd a: independen~ of the se~uence
¦ ot' the pep~ide bein~ x~nthesized. Thus. wi~h the rods or pins arranBed in ~he pre-
ferred forrn~t ,so that ~ ;~ in ~ holder. ~11 sups in the synthesis such~ as washing
step`c. neu~llz~ion sleps and deprote~oon steps ~n be ca~ied out sim~leaneously
5~ in the synthe!;is ot' ~h (~ feren~ peptide~i. .The only steps which must be earried out
; s~p;~tely t`or e;l~h ~dl~t'feren~ peptide~ ;Ire the eoupling of the appropri~te ~nirlo~ dcld
residues. E;l~:h of the~ t~p.~ is~onveniently ~;lrned out by dispensing ~ppropr~e
W094/113B8 2148~9 ~ P~/AU93~00546
i
- 2 -
aL:tivated amino acid solution into the correspondin~ wells o~` ~ mi~ronter pl~te or the
like. Thus. ~he appropri~te amino acid is coupled to the pep~de for e~ch of the rod~.
The ~uantitv o~ peptide ~:ov;llently bonded to the solid polymer surf~ce
by this method is suffi~ierlt to allow re;lction of the peptide with speci~lc bindiny
5 enuties ~u~:h ;IS ;mtibodie.~ to be re~dily dele~ted. Althou h the ~uantity of peptide
~ynthe~ized on elch rod i~ relativelv sm~ll (typi~:~lly less than I umole~ the abilitv
. tO reuse the syn~hesized peplide atter ;I test ~ompensates tor the sm~ uantity ot
peptide on the rod. However~ In some applit:~tions the ~uantity of peptide needs to
be ~reater. E~c~mples of su~:h appli~:a~ons include the removal of the peptide from
It] the rod and recoverv ol` speLific bindiny en~tie~ ~or further te.~ing. Modificauons tO
the process of synthesis ~nd. testin~ of the peptides were diselosed in PCT
wo~l/()4~
lt is ;m oblecl of the present invention to provide means by which the
~mount of pepnde~ or ~or that mat~er ;lny other polymeric eompound such as nucleic
15 ~cid which can be synthesized on a solid suppor~ c~n be in~reased while retaining
. the adv~nt~e of beinv ~ble to ~ynthesize many pep~dçs with different sequences
simultaneously.
In the e;!rlier method polyacrylic a~:id was ~ted ~o the surf~ce of a
~olid polyethylene ~upport usiny g~a-irradia~oll. In th~t earlier system. ~he region
'~0 on the rod on which pep~itle was ~r~ted wa~ ~ornpletely defined by the volume of
re;l~ent used for the eouplin~ of speeifi~: amino ;l~:ids to the ~rowing pepide. or more
;l~eu~tely~ the depth into whieh the rod dipped into the amino ~ei~d solu~ons. As this
depth inevitably v~ned ~ htly ~rom ~,yele to eyele in the ~ynthe~is. the re~ult wa~
sm;~ll re~ion on the rod where the peptide synthesized m~y h~ve h;ld ~ppreci~ble~5 ;lmounts of deleuon peptides (th;lt i~i. pep~de~ who~ie :;e~uen~e vaned from that
intended by h~vill~ one vr more residue~ ~bsent~ present because sli~htly less ot` one
: ~ ()f the ;lc~iv~ted ;lmlno ;leid solutiorls w~s dispensed in one or more cycles.
A~further disadv;m~e of the e;lrlier ~ystem i~ th~t the polymer l~yer
ed onto the rods durin~ the r;~di;ltion proee~s is re~dily solv~ed by m~ny sol-
_~v~
L~!
.. .
~ WO 94/11388 Z 14 8 ~ 9 :1 PCI/AU93/00546
ven~ nd ;~ conse~uen~,e~ .~olven~i ~ill migr~te upwards through thi~ l~yer by ~,;lp-
i.l~ry ~ction. This re~ults in ~eple~ion ot` the reservoir of re~ent and ~:onse4uently~
~s described ~bove~ rt~er zone oi uncer~n synthesis quality is created. Ln addi-tion. unless extreme ~re is t~ken in extensively washing these rods. the polymerl;lyer ~ts ;lS .1 re.ser~oir ot the .~oiven~ u.~ed in synthesis leadin~ to the contan~ination
of subseL~uent ~olution~.
Disclo~sure of the ~nv Qon
One aspec~ ot` the Invenuon is an improved synthesis support. having
1() ~ plur;~lity of indepenaent synehe~ urf~ces~ which pern~its the simul~neous~ syn-
theses of pep~des (or other nlodul~r polyrners~ havin~ d~ferent N- or C-~roups, hav-
in~ different link~ges to ~he support ~ .. pemlanent links. eleavable ~s. ~tc.), or
otherwi~e differin~ in ~e;l~ures other than se~3uence. Addi~on~lly, one can employ the
new supports to m~kè mulùple copies of ~ eollec~on of polymcrs.
~Another ~speet of ~he invcnion is a cleavable linlc which enables one
! tO ele~ve modul;lr polvmers from the syn~esis suppores of the irlven~ion, leaving the
modul~r polymer with ~n amide .le the cle;lved end~
Summ~r~s~L~1~i~' '
:
Figurc I depicts pl~n arld cross-section views of an ac~ive sur~ace com-
20 ponent of the inventian.
Figure 1 depi~ pl~n vlew.s of support rod of the inveMinn.
Fi~ure ~ depi~t~; ELISA resul~s ob~ined using mul~ple epitope lib-
raries prep~red by the method of the inven~ion.
Fl~ure :~ d~pi~; ELISA binding inhibi~on results obt~ined using mul-
'75 ~ tiple epitope librlries prep;lred by the method of the invention.
: ` : : :
:: ~
::
: ~ ~
: ~ ___
::
WO 94/11388 2 1 4 8 ~ 9 1 p~ U93/~546 ~1 ~
Mode~s of C~rrvinY Out The InventionA. Definition.~ .
The term modul;~r polymer" re~ers to ~ polymer ~omposed of non-
identic~l subunits selected ~rom ~ Vroup of rnonomers. Modul~r polymers ~re ~en-er;llly .~nthesized one monomer ;It ;l nme.
The term monomer" ax u~ed hereirl refers ~o ~ molecule whieh may
be ~oupled or conden.~ied ~o ~nrm ~n oli~omer. To provide diversity. monomer.~ re
selected from sets whi~:h cont~in ;lt leas~ ~our dis~nct members. Sui~ble monomer
~ets include ~:onvention~ nd ~ mino acids. nucleic ;lcids/nu~leotides. c~rbo-
I() hydrate.~ non~onven~ion~ nc~ L-;lminb ~cids (~ yclohexylal~nine. ~enzhyctryl-
~Iycine~ ~:hloroal~nine. ;md the like)~ ~nd "peptoids" as described in WO~ 735
(ineorporated herein by re~eren-:e).
The term "~onvennonal anuno acid" refers ~o the ~r~no acids alanine
~A). cysteine (C), ~sp~ic acid (D)~ ~lu~mic ~cid (E), phenylalanine (F)~ glycine ~
IS histidine (H), isoleucine (I)~ Iysine (K)~ leucine (L), methionine (M), ~sp~gine (N),
proline (P), glutamine ~Q)~ ;~inine (R~. serine ~S)I ~hreonine (T), valine (V), trYPtQ-
phan ~W), and tyrosine (Y).
The term "nonconvennonal ~mino acid" ~e~ers to amino acids other
than ~onverl~on~ no acids. Presently p~f~edl nonconven~aonal anr~ino acids are
'~() Me = l~-norleu~ine: Aabu = a-an~inobu~Tic ~cid:
Hphe = L-homophenyl;ll~nine; NYa = L-norv~line;
Cl~bu = ~-an~inobutvri~ ;leid: D~la ~ - U~alanine;
Dey~ = L)-oysteine: r)asp = D-aspar~is: ~uid:
I Dglu = v-~lu~ id: ` Dphe~= L)-phenyl~lanille~
'~5 Dhi~ = u-his~dine; Dile = [)-isoleu~ine:
Dlys ~ lysine; ~ Dleu = v-leuc~ne:
Dmet = v-me~hiot~ e: D~sn = v-~sp~ine;
Dpro - L)-proline: D~ln - v-~lu~mine:
Darv - v~ inine: ~ Dser- D-xerine;
~() Vthr = v-threonine: Dval -i U-valine;
Dtrp = v-tryploph~m: Dtyr = L)-tyrosine;
Dorn = L)-ornithine: Aib = ;lmin~i~obutyrie ;leid:
Etj~ = L-~thyl~iye-ne; ~ Tbu~ = L-~-bu~yI~ly~:ine:
~ T
--~ W~ 9~/1138~ 9 1 P~/~U93/~0546
Pen = penieill~mine: Anap = a-naphthylalanine;
Chex~ y~:lohexylalanine: Cpen = eyclopentylalanine:
Cpro = ;lminoeycloprop~ne e~rbo~vi~te: Norb = ~minonorbornylcarboxylate;
M.lla = L-a-methyl~ nine: Mcys = L-a-methyleysteLne;
S Masp = L-a-methyl~sp~rtie ;leid: M~lu = L-a-methyl~lu~sni~ aeid;
Mphe - L-a-methylphenyl~l~nine: Mhis = L-a^methylhistidine:
Mile = I.-a-methyli~oleuLine: Mly.~ = L-a-methyllysine:
Mleu = L-a-methylleu~ine: Mmet = L-a-methylme~hion~ne;
Ma~n = L-a-me~hyl~xp;~ ine: ~ Mpro = L-a-methy}proline;
ln = L-a-methyll~lut~mine: M.lr ~ L-a-me~hy~ inine:
~vlser = L-a-methyl~;enne: .~thr~ L-a-methylthreonine;
Mv~l = L-a-methylv~line: Mtrp = L-a-methyl~yptophan:
Mtvr = L-a-methyltyrosine: Morn = L-oc-met~yloTTIithine:
IMnle = L-a-methylnorleu~ e: Maabu - ~-amino a-methylbutyn~ acid;
Mnva = L-a-methvlnorvaline: Mhphe = L-a-methylhomophenylalar~ine;
Met~ = L-a-methylethyl~lycine: Mgabu = a-metbyl-~-aminobu~ic acid;
Maib = a-methylaminoisobutyric ~cid: Mtbug -- L-a-m~thyl-t-blltylglycine;
Mpen = a-methylper~icill~r~ine: ~ MaIIap = a-methyl-a-naphthylalanine;
Mchexa = a-methylcyclohe~ylal;mine; Mcpen - a-methylcyclopentylalanine;~l) Dmala = t)-a-methylal;mine: : ~ Dmorn = [~^a-methylornithine;
DmL:ys = L~-a-methylcys~eine: D~asp = D-a-meehylaspamc acid,
Dm~lu - ~ -methyl~lutamic acid: Dmphe - D-a-methylphenylala~ine;
Dmhis = v-a-methylhi~dine: Dtnile = E3-a-methylisoleucine;
Dmlys = D-a-methyllysine: I:)~eu = D-a-methylleucine;
~5 I)~et = ~-a-methylmethionine: Dmasn = l)-a-methylaspara~ine;
Dmpro = D-a-methylpraline: ~ : Dm~ln c D-a~methylglu e;
Dm~rg = D-a-methyl~ir~ine: Dmser- ~-a-methylse~ine;
I:~mthr - v-a-methykhreonine; . Dmval = D-a-methylvaline;
Dmtrp - ~-a-methyltrypEo~han: Dm~yr - D-a-methyltyros~e;
3~J Nm~la - L-N-methyl~l~nine: Nmcys:= L-N-me~hylcyste~ne;
~: Nm~sp = L-N-methyl~p;lru~: ;u:id:: Nmglu = L~ methylglut~nic ~eid;
Nmphe - L-N-methylphenvl~l;mine: Nmhis = L-N-me~hylhis~idine;
Nmile = L-N-methylisoleue,ine: ~ ~ Nmlys = L-N-methyll:ysu~e~
Nmleu = L-l~l-me~hylleueine: Nmmet = L-N-m~uhylme~hionine;
Nmasn - L-N-mel:hylasp~ine: Nrnchex~ = ~-m~thylcyclohexylalanine;
Nm_ln = L-N-methyl~lut~n~ine: ~:Nmar~ = L-N-medhylar~e;
Nmser ~ L-N-methyl~erine: ~: : Nmthr - L-N-methylthxeorline:
Nm~ral - L-l~l-methvlvllline: ~ Nmtrp: = L-N-me~hyltryptophan;
Nm~r = L-N-methvltYro~ine: ~morn = L~ me~hylomith~ne;
() Nmnle - L-N-methylnorl~u~ine: : Nma~bu-N-amin~a-me~hylbu~yricacid;
Nmnva = L-~l-methylnorvaline: Nmhphe-L N-methylhomophenyl~lanirle:
Nmet~ = L-l~l-tnethylethylYly~ine: ~ ~ Nm~ u=l~l-methyl-~-an~inobutyric~cîd;
Nmepen = N-methyl~vclopen~yl;~ nme: Nm~bug = L-N~me~hyl-~-butylglycine;
~m~T~ T
: -
WO 94/11388 2 1 4 ~3 ~11 9 1 PCI/AU93/00546
^ f, -
.
.~impen = N-methylpenieillamlne: Nmanap = N-methyl-a-nsphthyl~lanine:
?Imaib = N-mei:hyl,lrninoi~obutvrie ;lcid:
Dnmala = v-N-methylalanine: Dnmorn - v-N-methylornithine:
Dnmey.~; = L)-N-meth~ teine: Dnma~p = L)-N-methylasp~rtic a~:id:
Dnm~lu = v-N-methyl~lu~m~ : Dnmphe = D-~l-methylphenylal~nine:
Dnmhi~ - V-l~i-methvlhistidine: Dnmile = v-N-methyli~;oleucine:
Dnmly.~i = D-N-meth~llv.~ine: Dnmleu = D-N-methylieucine:
Dnmmet = v-N-methvlmethionine: Dnmasn = ~-N-methylasp~ra~ine:
Dnmpro = V~ methylproline: Dnmgln = D-N-inethyl~lutamine:
. I() Dnmarg = v-l~l-melh~ rg~inine: Dnm~er - D-l~l-methvlserine:
Dnm~hr = v N-methylthreonine: Dnmv~l = D-l~-methylvaline;
Dmn~p = L)-N-methyltryptophan: Dnmtyr = u-N-methyltyrosine: ..
I`iala = N-methyl~lycine ~s;lrcosine~: Nasp = N-(c~rboxymethyl)~lycine:
~IY1U - N-~2-c~boxyethvl)glycine: ~ Nphe = N-benzylgly~:ine:
~hhis = N-(imidazolvlethvl~lvcine: Nile = N-~l-methylpropyl)~lycine:
Nlys = N-~4-~rr~nobutyl)~lycine; ~ Meu = N-(2-methylpropyl),~lycine;
~1met = N-(2-methylthioethyl)gly~:~ne: Nhser- N-(hydroxyethyl)glycine;
Nasn - N-(c2rbamylmethyl)~1ycine: Ngln = N-(2-carb?mylethyl)glycine: :
Nv~l = N~ methylethyl)glycine: Nar~ = N-(3-gual~idinopropyl)glycine:
; 7() Nhtrp - N-(3^indolylethyl)glycine: Nhtyr-:N-~p-hydroxyphenethyl)glycine;
: Nth~- N-(l-hydroxyethyl)&lycine; Ncys = N-(thiomethyl)glycine: ~nd
: ` Norn - N-(3-aminopropyl)~lycine: Ncpro - N-cyclopropyl~lycine; ~:
Ncbut = lY-~:yelobuty~lycine: Nchex = N-cyclohexyl~lycine; ~.
:Nchep = N-cyclohept~lglycine: ~coct- N-cyclooc~ lycine; j-
Ncdec = N-cyclodecylglyeine: Ncunc~ = N-cyclouJIdecylglycine:
Nedod - N-cy~lododecylg~lycine: : Nbhm = N-(2.2-diphellyle~hyl)glycine;Nbhe = N-(3,3-diphenylpropyl)gly~;ine:
~ ` Nnbhm = N-(N-(2,2-diphenylethyl)carbaI3lylmethyl)glyeine: ;
: Nnbhe = N-(N~(3~3:~iphenylpropyl)carbamylmethyl)glycine;
3() Nbmc = I-c;lrboxy-l-(~.~-diphenyle~hylan~ino)cy~lopropane: and
N;le~ = N-(2-~oethyl)~lycine.
The ~enn ";Icuve ~;urf;lce" me;m~ urf~ce which i~ deriv~zed or other- :
wi~e rendered ~ui~ble t`or: ~iynthe.~ of modul;lr polymers. It is ";~dapted" for u~e in
the synthesis of modul~r polymer~ ff modul~r polymers ean be effî~iently syn~hesiæd
35 thoreon. The ~omponent whil:h e;~ssie~; the aenve surfa~e may be homogeneous or
:~ ~ : hetero~eneou.~ in: compo~ on. For e~cample. the a~ti~e su~aee m~y be bonded or
r;lfted to ~supportin~ ue~ure nr.~ur~ee. In pnneiple. the ;le~ive ~urf~oe m;ly be
:r~dl~tlon ~r;lfted~to~nv ~uppomn~ tru~lure ~whieh m~y be~ for ex~rnple. ;m "inert
urt~ee" ~s defined below~.
; ~
~ ~ : SUEI~iTlllJT: Sl ItET
:~ :
.-~ ) WO94/113~ 2 1 4 8 ~ 9 1 PCI/~Vg3/0054
The te:rm iner~ surr;lee re~ers to ~ surfa~:e whieh is s~able to the mod-
ul~r polymer synthesis l ondition.~ nd does not reae~ Suitable inert surf~ces inelude
withoul limit~lion polve~hvlene~. polyolefins~ ~ellulose acet~te~ wooL L~otton. chiun.
~nd the like.
3 :~
B. Gener~l Metho(!
In a first~embo(iimell~ T sl~e Dresent invention~ there is provided ~ plur-
;-
;llity ot` supports for use in the svnlhe~ils of peptides or other polymenc compounds
thereon~ whiL~h support~ eompnse ~n Inert ~urfaee and a set of active surfaoes eauh
10eomprisin~ an 2l~;tive re_ion on whien s~id synthesis may take place. '~
e ine~ s~e~ls typi~:ally provided in the forrn of a rod or pin of
g~n~rally cylindri~l sh~pe~ :h;lving space for a plurality of acdve~ surface componen~s.
Pr~ferably. the inert SUppOlt is c;lpable of re~ining 2 :to 20 ac~ve surfac~ compon-
I ents. moD preferably ibout 5 :~cuve ~ e componen~ e suppor~ may have a
IS; : ~Tos~;-section th~t~is circul~r. ree~n~ular~ or any other sh~pe: cir~ular and squarc are
prefeJTed~ SUppO2~ m;ly turther be provided with proiectio~s or protrusions to:
SSiSt in placin~, loc~n~ ;md re~ining the ac~ve sur~ace componeflts. ~ Fur~her, ~tlie
suppo~ m~y~be~provld~d wlth mden~nons and/or incisions to irnpart:sufficient flex-
ibility to the supportXh~t the~active ~urface components m2y be "snapped" on. ~ter-
20~ n~vely,~the:a~vesi~ ce~componcntsm~ybe.positiorled~n;~ shapedpin.fol-
lowed ~y moun~n~ ~the pin in ~ .suppor~n~ arr~y, wher~ the "hcad" of the pin is
h~ped to re~in all ;lCtiVe .~url~ e components ~the: ~iye~ surface com~onen~ rnay:
be l.lter removed~ by either removinQ the pin from ItS SllppM~ or by clea~ring or
r~movmg~the he~d of the pin)~
5 ~ ~ ~ :: The~suppon rod.~ may be ~rouped in an array to facilît~te pa~llel pro-
essing, botù durin~ ynthesl~ ot the modul~r polymers and durin~ ~hei3 ~ssay. ~ ln a
pre~ently preferred embodlment. ~he .~upports ~"pins"~ ~re mounted in an 8 x 12 ~r~y
on .l ~bloek whi~h ~n~che~ ~the~ ~;p.~ ot weil:~ in ~ lT~ierowell assay pla~e. Preferably
the~ rod.~ ~re mounted by pre~q-fi~t or fri~:tion-fit into holes drilled or~molded in the
8UIBSTITUTE SIHE~E~T
. ~ ~
W~ g4/11388 21 ~ 8 ~ ~ 1 PCI/AU93/00S46
- X -
suppor~ block. The rods m;ly Inelude retainin~ fl~n~es or projeetions to insure that
eaeh rod proj~s,ts the same dis~n~e ~rom the block surface. Alternative mountinYme;lns inelude thre~din~ the rocts ;Ind holes~ ~dhe~ives~ one-pieL:e moldin~ m~neti~
couplin~ and the like. Other arrav formats ~re also considered within the seope of
this invention. For ex;lmpie. the ~upport rods may be positioned on a ~on~nuou~;belt~ or mav be ~npped individu~llv bv robotic manipulators~ to sirnplify autom;lted
h~nctlin~n
Pre~erablv. the ;l~uve .~uri;lees ~re provided in ~ fonn which may e~sily
be attached to ~nd removed from) the inert support by f~ic~on fi~inQ or snappin~1~) in~o plae. ~owever~ other forms of a~chment may be used, sllch as adhesive
(which may or may not be perrniment~ heat fusin~, threading (~ like ;3 nut and
bolt)~ Velcro~ slot and key. and m~ne~ism. For example~ one may emp~oy iner~
supports and ~e~ve sur~ce components h~ving ~rrous cores, ~nd retain the ac~ve
su~ac~ ~:omponents by elec~om~ne~xm. ln thi~ embodisnent. coupling re~ons to
15 the active surfaces m~y be a~elernLted by modula~ing the ~lec~nc current~ ~hereby
inducing vibration of the pins~ Effe~tive vibr~tion frequencies range ~rom about 4(~
~z to fi() ~Az or more. pr~ferably about SO-fiO Hz~ Altema~vely, the ~uppomng rods
mav be fashioned from~ or ~l~ed to, pie20ele~lrie ~ansducers which vi~rate at the
desired fre~uency whcn acuY~ted. .The ;lc~ve suFface comporlents are prefcrably
2() ;mnular in shape. but m;ly be ~ormed in any sh;~pe which o~ be re~ined on the iner~
suppon. For e:c~mple. active su~:ace components may be semiannul;~ (L'.~ omplet-in~ ;lbout 27()) ~nd ~till be re~ined by pre~;s-fie methods. A~ve surf~oe eomponents
which are re~ined by m;l~ne~ism need not be completely annul~r. The ~c~ve sur~aee
~:ornpollents ;lre prete~bly ~haped to in~,rease their s~f~ee ~ea. so th~ they have a
?S ,!re~ler .~urfaee ;Ire:l th;m ;~ eylinder o~` the .~me di;lme~er. In one embodiment of the
invention ~he aetive surta~e ~omponent~ re vaned or "gear" .~h~ped. in orde~ to m~
mize their ~;ur~e area. The inner sur~ee (faein~ the inert support~ m~y be provided
with fl;ln~ led~e.~ and/or other su e~ whieh may eng;lL?e ~:orre~ponding ~ ces
on the iner~ .~upport. The ~ermin;ll aetive sur~`a~:e component m;ly h~ve ;l different
: -
. __
9U~mUTE 9~1E9T
WO 94tll388 2 14 8 a 9 1 pcr/Aug3/oo~46
shape. be~ use the inerl ~uppor~ need nol pa.~s enurely through the ~omponent. For
ex~rnple~ the termin~l ~e~ive sur~ e ~omponent may be hernisphen~
Fi~ure I illusuates one pre~;ently preferred embodiment of the ~cave
sur~a~e s,omponent (1). Fl~. IA depi~t.~ ;3 plao view of the componen~ havin~ a
thhknes.~ t of '~.5 mm. a m~ imum ouler di~n~eter of 5.5 mm~ a min~num internal
diameter ~be~ween opposed inlern;ll proie~:~ions ~2)) of 2.() mrn~ wlth a maximum
internal di~meter of ~.(1 mm. Fi~Ture IB depic~s a ~oss section of the componenaThe eornponent Is molded from poivethylene ~ith 16 external "teethl' (3) and 4 inter-
nal teeth (2) arran~ed ~vmmetri~
I~J Figure ~ illus~les one presenùy preferred embodiment of the support
rod (11))~ designed for use with the ~omponents (I) of Fi&. 1. Rod 10 ~om~ises aprimary ~haft h~vin~ a diameler ot about 3.X ~ and a length of about 32.3 mm.
End 12 i.s rounded ~o f~eilit~te in~ertion in~o ~he support~ block~ and is provided wi~h
fl~n~e 13 tO insure uniform in~ertion. Flallg~ 13 is posi~oned abou~ 9.8 mm firom
1~ en~ 12, and measures I.l mrn in thi~kness by f~.0 mm in outer di~neter. The sec-
ondar~ sh~ft ~14) is co;~i~l with pnm~ shaft 11, ~nd~ extends about 2~1.0 mm there-
rom. Seconda~r sh~t 14 hns a diameta of about 2.0 nun, and is pr~vided ~ a
- ~lurality of projec~ns 1~ ~of gener~lly ~yl~dric;ll shape, havi:n~ a~diameter of about
~; ~ ().5 rr~n ~nd extend .lbout ll.X5 mm from the su~ce o~ the seconda~y shaft 14.
2() ~ ~ The~e pro~ections ~15 serve to io~ ee ~nd posibon compollents I on ~secondary shaft
/~. Second~ haft /i~ is~ herprovided with ~ generally frustro~:ol~ical end ca~p
! Ih h;lvln~ ~ minimum di~meter of ;lbout~l.l) mm~nd a m~c~mum jdl~net~r a~ ~out?,~ mm, slopin~ t~ ~bout 3(). ~ End ~ p 16 ~eilita2es addidon of ~e componon~s 1
to the ~;upport 11~. ~nd is rexpon~;ible for~re~inin~ ~he bo~tom-most~coarlp~nont.
5 ` ~ ~ T he u~iv~ ~;urt;~ m~v be of iden~ic~l or different ~.:omposi~ions~
d~pendln~ upon the~-he~ ;try to whlch Ihey~ will~bo subJ~ted. The coa~ng c~e
m;lde ot ;my of the porous re~;in'~ whiL~h ;Ire~u~ed~f~ convenaon~ solid phase p~lide
nd/or nucleic~ id .~yn~he~i~. Bec;lu~;e these res~ns ~ro porous. the su~ce ar:Ga of
th~ ti~e re~ion i.~ in~re;l~;eti dr;lm~ti~ lly~ ;md !;o allow.~ a muL:h ~ ter yield of
~ , :
r~
: ;'
WO 94J11388 2 14 g 3 9 ~ PCI/AU93/00~46 _;
!
- lt) -
modular polymer. In ~dctition~ ~he u.~e ot` this embodiment of the inYention makes it
particul~rlv eonvenien~ to ch;mPe the chemistry used in the synthe.sis. ;~nd indeed.
ch;ln~e the e,lass of poivmeri~ eornpounds to be ~ynthesized by selet:tin~ the
appropriate resin wl~h whieh IO eoat the ;lctive surfa~:e eomponent. Example.s of
porous re~in!i whi~,h m~y be u~;ed in sueh co;llinYs inelude benzhydryla~nine-polysty-
rene resin and polyacrvlamide gel inside kiesel uhr. Other suitable surface materi~
ins:lude. wi~hout limitarion. polvethylene ~ly-ol. celllllose and other n~rur~l polymer~.
I~er~ eld res~rl. Rink restn ;md poiymers of acryli~: aeid~ methylacrylate. metha~lic
acid~ methyl methacrvlate. dimethvla~rvlarnide. styrene. hydroxyethylacryl~te.
1~) hvttroxyethylmethacrvla~e. hyctrox,vpropylmeth~t:rylate. hyclroxyethylme~acrylalI~ide,
methylmethacrylate. ;md polyethylene~lyool monometh~cryla~e. and the like. ~nd
combintl~ions thereof. The c,omponent be~nn~ the ~:tive surface need not be homo-
~eneous. It is presently preferred to employ struc~ural supports f~shioned from not
just polyethylene~ polypropylene ~nd lts copolymers but ~Iso Teflon@~) (polyte~a-
15 flu~roethyiene) or ~ny other .stable ~nert sur~ace. The acnYe surf~ce m~y then be;lttached to the supporting surf~ce by any available means, in~:ludin~ sinlering,
dhesive. he~t fusing. and ~he like. The presently pre~erred method is graf~ng, by
pl;lcin~ the support surf~ce in ;l soluuoll of solvent and ~ve surf~ce m~ l and
iITadiat2ng ~he n~ixhlre with g~ radia~on. Prefe~Ted solvents are water. methanol
2(1 tMeOH). H,O/MeOH mixture~;~ dime~ylfom~ ~ide (DMF). ~d dir,nethylsuJfoxide
(DMSO). The mixmre is preplred ~nd iIr~i~ted as descri~ed in P 138855: see
;llso D. Mulle~-Sehulte ~t ~ Polvmer Bull ~1~X2) 7:77-X1.
The ~ ee~ re then modi~1ed. if desired. ~or the ~ele~:led ~:ouplin~
ehemistry. The modul;lr polymer.s m~y be "perm~nent" (i.~., not easily removed from
~5 the ~ ee) or "~le~vslble" (de~si~ned for f~leile ~:le;lv~L~e ~nd remov~l from the !iUp-
port). Cle;lv~bie modul;lF polymen~ will L~ener~lly~ h~ve ~ linka~e to ~he ;letive ~ ;e
whieh f;l-,ilit;ltes ~:le;lv.l !e lrom the surtaee under eondi~on~; no~ exper~enced durin~
~;ynthesi~s o~ the modul;~r polymer. I~ifferent linking chemis~ie.s m~y ;ll~o provide ~r
different N-termin.~ nd C-tennin.~ roup~ ~ in the ~ e ot pepticte~ Cle;lvable mod-
;
: : : _ _ .
SU~T nJTr s~rrT
2118~91
WO 94tll388 PCrlAU93/û0546
,
- I I
ul?r polymer~ may be provided.wilh l;lbeling ~roups ~or detection~ or binding iigand~
for purposes of separacion ;lnd purificauon. For example. cleavable modular poly-
mers may be bio~invlated lor ~ ed with another similar lig~nd) tO facilitate purifi-
~ ation (~ .. usin~ a streptavidirl column~. or labeled with fluoresceM or radioactive
S atom.~i tt' .~implify detection in ~ ~Inclln~ a~sav. Thus. one rnight simul~aneou.sly pre-
pare a colleetioD or libr~ry ot mociui;lr polvmers wherein one or more se~s are per-
manent. ;Ind other ~ets are t)ionnvi;l~ed. ~luoro!iceinated. radioactive. cle~vable to
~eidi~ termin~l ~roups. cle;lv;lble lo ;lmide terrr~inal groups, and cleavab~e to neutral
termInal ~?roups.
IU One forrn of cle;lv~ble linlc;l~le was described by ~ieysen. ~O~U/lW3~5
(inco~porated horein by re~erence)~ in; which the link cyclizes to a dil~etopiperazine
moiety with concomitant ~:leava~e of the pep~ide ;;hain f,rom the suppor~ ln thc pre-
.
ferred embodiment. lysine h~vin~ a protected oc-asI~ino group (e.g., with BC~C) is
oupled to the support throu~h the E-amino ~roup. Pro, whose c~boxy =us is
esterified with ~ suitably:re~ctive sp~or t"X") haviAg an orthogonally protected ~ 8-.
Fmoe) ~mino ~roup is then eoupled to the Lys c~rboxylate function. Synthesis of the
modular polymer then proceeds on the ~nino ~oup` on d~pro~c~on:
: Boc--Lys--OH , Pro~X--N~P~t
:~ 20 ~ ~ Pin~
Bo~lLys--Pr~ X~ Pro~
Bo~ lLys--Pro~X--~NHzn
: :Pin ~ ~
3()
:~ Alternauvely~ one m~v ~ynthe~ize the group firs~ and eouple~it to the
- ` pin~
.
,
: ~ ~ __
~: ~ : : : : ~
: ~ :
W094/11388 2148~3 1 PCI/AU93/~546
- 12 -
Pir~OOH - B~tYS~Pr~X~NH~Pr
"NH~
1()
: :
Cleava~e is effected by removins~ the BOC (or olher ~roup) protecnn~ the Ly~ a-
;Imino ~roup. and neutr~liz~n~ Ine re~u}~nP ~ . When the Ly~ a-an~ino -NH3'
: i~ neutr~ized to -NH,. ~he ~n~ine ~ ks the:Pro carbonyl aad displaces dle Pro. and
with it~ the mod~ar polymer. A di~etopiper~zine moiety is lef~ ~ttached to the active
. ~ surf~ce. Altern~ively~:one;may coupie the spacing group to the ac~ve surf~ce, to
form ;m ester link. Coup!ing of a suitably proteceed Lys to Pro will form ~he same
b~sic lin~er. However~ this con~ïEuration le~ves ~ diketopip3azine moiety a~ched
. : - ;
~ ~ tO the modul~r polymsr ~ter cle;lvage from the suppo~.
: 20 We~ ha~re ~lso developed a new linlcer, whieh facili~es clc~vage of a
modular polymer to provide ;m ~mide func~ion ~t th~ point of cleavage. l'he modul~ ::
~ polvmer is sy~lthesized link~d: to the. ~ve surf~ce~ through a protec~d;~ a-aminogly-
: ~ ine. After tbe modul;lr polyrner~ ~ynthesls i.s completed,~ the pror~c~ing g~up is
~: ~ ` : removed ~rom the a-all~ino ~roup. l~unersin~ the ~ive surfa~e in an ;lqucou~ solu~
oon ;lt pH 7-1 () results in ~le;lvl~e. Ieavm~ ;ln amide fune~on at the site of cle~vag~e :
on ~he modular polymer. .Is shown In the Scheme below: ~
..
- -) WO 94/11388 2 1 4 ~ 5 9 1 PCI~/~U93/00~46
- 13 -
' aOc--N~ '
Frnoc~H~Cit~Support
¦ Synthesize p~pbde
. ` _oc--INH
(pro~ected peptide~--~0--NH~H~C~Support
1()
I Side chain depratection (H+)
N H
~ pep~lae--50--~i~CH~Ct)~Support
: : 1 ) wash (pH ~ 5)
2) pH 7-10
. .
2() ~ ~H2
p~ptla~co~C~support
: ' ~ ' "/ \~ : ,
: 25 :
pep~e~Ct~NH2 : . NH2~Ci.~CC)--SL~pport
: ~ '~
:
This lirLkn~e :m~y be used for ~ny type of modular polysner~ The
gener~l formul~ for ~ bound rnodul;lr polymer is
, ~ ~Z
¦: 35 (M)n ~ (spacer~ Support
: ~ H ~ Q
4() : ~ ~
SU~F117UI. SRFF7
WO 94/11388 21 1~ ~ 9 PCr/AU93/00546 - !
- 14-
wherein M i~ ;1 monomer~ n Is an inteYer (preferably 2-3~)~ in~:lusive).
;md Z is a protectin~ t~roup. The ~pa~er 1~ optional.
Other cle~vable link;l~e~ m~y be employed. For example~ there ;lre
known photoeleavable linka~e~i~ whi~h ~:le~ve upon expo~ure to li~ht a~ a seleL:te~ fre-
S ~uency. The~;e link;lYe.~ nd the linku!e~ d~ieribecl ~bove~ ;~re ~lenerally mo~ uit-
;Ible for use with peptide- .md peptoid-b~ed modular polymers~ but may also be u~;ed
~ith nueleie a~;ids. Where the modui;lr polvmer i~ ~ nucleie a~id. it rnav be conven-
ien~ly removed by providin~ a cle;lv~bie linkaPe in the form of ~ res~ic~on enzyme
reco~nition site. I~ucleic aeid modul;lr polvmer~ may also inelude polvmerase pro-
]() moter/bindin~ sites and ~mplifi~:~tion primer hybridiz;3tion site~i~ to tacili~te duplexform;luon and amplifica~on (f~ .. by PCR~.
The ~ctive sur~e~ may also ~ry identifyin~ fearures tO f~cilita~e dif-
ferentia~ion between differcnt types of .surface. For e~cample. aeove surfaces of di~-
~erent types m~y have different colors ~nd/or pa~erns~ di~ferent si~es and/or shapes.
15 di~erent de~rees of adheren~:e tO the pin or rod (~.g., re4~ a di~feren~ amount of
force to remove ;l surfa~:e from a pin)~ differ~nt de~rees of ma~ne~ on~ ~c. Sur-
f~ces that dffler in color ~nd/or pa~ern may be ~eparated by h~nd on inspec~;ion.
(: olors may be ~hieved by in~ludin~ dyes in ~he ;lcave surface ~ornponent. either in
the a~ tive ~ ce la~er or ~n the suppor~in~ s~rucrure underneath~ or bo~h. P~teerns
20 ~hould generally be simple. ~nd m;ly be ob~ned~ ,n, by dyin~ only h~li of each
~:om~onerl~ Surfaces that di~fN in~de~ree of ~heren~;e may be separated from ~hepin or support by ~ me.lsured .~hnke or impact (removin~ first those s~f~:es thut ~re
loosely ;lttached~ followed by ;l more vi~orous sh~ke or imp~et to remove surf~ces
th~t ~re rnore a~htly bound). Surhce.~ th~t differ in ~ize ~nd/oF sh~pe rn~y be sep-
ur;lted by u simpie s~reen th~t ;Illow~ smull component~s to p~. while re~ining lar~er
componen~. Obviou!;lv~ urf;lce.s th~t ;lre m~ne~ized m;ly be ~ep;~ed from non-
m;l~ne~ized ~iurt`~ces usin~ ;l m;l~net. ele~:~om;la!ne~, or felToLI~ met;ll. The l~st ~wo
sy~lem.s ;lre more ~ui~ble tn ;lu~om~tion. Thus. one may prep~re ;~ eolles~on or se~
,
:
___
- :.) Wo 9~/113~8 2 ~ 4 ~ ~ 9 1 PCr/AU93/0~46
of modul;lr polymers as desLribe~i aboYe. In ~ever~l di~ferent forms. in one operation,
The dif~`eren~ fonn!i may Ihen be e;lsilY ~epar~ted for further rese~rch.
The use of modul;~r ;~tl~ e surf~ee components and inert components has
several advantaS7e.~. First. it permies one to prepare ea~h componerlt under op~imal
S eonditions. usinY the materi~is moxt ~iult~ble ror the function of each componenl. For
e,Y~mple. the ;ICtiYe .~ e ~om~onel1ls need not display the rigidity desired in the
supporl rods or .~upport b}o~ , onul;lr onsFuc~ion. the rnds m~y be made of
.
ri~id m~eri~ i. while the a~:~ive surra~e ~omponen~s may be made of softer ma~rials
which are optirnized for use a~ .~,vnthesl~ surf~ces.
IU , Another m~jor adv~nt;l~7e of m~nufacturing the por~on providing the
ac~ive region as a sep~rare en~ity IS the m~nin~izatiorl of ~ross-con~a~on of solu-
tions. l he polymer layer Qra~ed on~o the rods durin~ ehe rad~ahon process as des-
cribed in EP I ~XX55 is readily .~;olvated by nany solvents and as a eonses~uencet sol-
ven~s will uni~rate upw~rds throu!h ~his l~yer by c~pill2~r action. This results in
15 deple~on of the reservoir of re;l~ent ~md conseqllently, as described above. a larger
zone of uncert;~in synthesls yu~lity is ereated. Also~ unless ex~eme care is ~ken in
extensively washing thcse rods. the polymer layer acts as a reser~oir of the solven~s
used in synthesi~. Ie~din~ to the con~n~in~on of subseyuent soluaons. Whese the
portion provid~n~ the ~ctive region is m~nufac~red sepaxately ~s described here~,
2() this mi$r~ion of solvents ~nd re;l~ents cannot take place.
Another m~jor ~dv~n~ye of manuf~cturing tbe ac~ve r~gion as a sep-
te en~ty ~:ome$ nbout beL~;Iu~e the~ tive re~ion will typically be m~h smaLler
thnn the complete unie. 7herefore. more of the ac~ve regioll components c~n be
~;l~ed simult~neou~ly to cre;~te the ~tive re~lon wi~h consequen~ savings in ma~Iials
'~S and ~ime.
For the purpo~e of Illu~ tion only, the po~nn providillg ~e ~c~ve
re ~ion of the rod. th;lt i.s~ the re~ion o~` the rod on which the pcp~ide or other poly- -
merie mole~ul~ to b~ ~ynthe~iized. i~ vIinder with ;1 r~dius of 2 mm ~nd ~ height
-o~ ~-mm. the .~ur~ce ;Ire~ of whieh ;l~tive re!ion i~ 61.X mm~ (~ssulr~in~ th~L~ only
____ :
5UE~STITUTE SHEET
__
WO 94/11388 21 4 ~ ~ 9 I P~/AU93/00546
;
1 ~
one end of this eylinder is available for synthesis). However, if a slit 1 mm wide i.
made aeross the di~meter of this portion of the rod~ the surface are~ be~;omes X1.
mm~ ( 1.3 times the ;~re~ ot` the solid evlindrical portion ~. The sur~ace ;lrea of the por-
tion providiny the a~tlve re~ion c;m be inereased even f~her by modifieation of the
5 ~hape ot this portion or` the rod. For ins~nee. if ei~ht slits~ eaeh ().4 nun wide and
I mm deep ~re made inlo the .~ e of ~1 eylindrical poruon~ the surface area av;lil-
:lble tor .svnthesi.s is inereased to 1~1 mm~ Itwiee the area of the unmodified cylindri-
e;ll portion~. It will be ;lpp~rem ~ha~ Ihe surf~ce area of the ~c~ve re~ion of the rod
can be fllrther increa.sed by fur~her modificauon of i~s ~eome~ic shape. and equally
I() that such modifications can be made by moldinP the rod in its fin~l desiled shape or
by machil~}nY a molded rod into ItS final desired sh~pe.
However~ in ~ preferred emDodiment of the invemi~n. the por~on pro-
vidin~ the ~ctive re~ion of the rod is tn~de by joinin~ small pa~cles of solid mater-
ials to~ether, for instance~ by sinteTin~ using pressure or he~t or both. l~is can be
15 p~r~icul~rly u:;eful where it Is desireù to~ use p~r~cular ha;rsh chemistries or corrosive
~olvents. For inst~nce. ~ ss is re~ist~nt to most solvents which would m;lke rno.st
conven~ional plas~ic ma~i~lls~ unstable. Thus ~n ~c~ve region could be m~de by sin-
` teriny to~ether sm;lll spherical beads of ~lass. This cou~d then be ~2ted~ forinstance, by funcnonalizin~ the sur~ce with ~n ;~nino-silane. to make it suitable as
2() a base on which the~ pep~de or other polymeric compound could be synihesized. ln
this ex~rnple. the in~ctive re~ion would be made from a pamcul~ly resistant plasuc
u~h .1~ ~olyte~afluoroethylene. In thi~ way. ;l matenalIsllch as gl~s. which would
be ;m un~uit~ble m~teri;ll for the i~ae~ive re~ion. L:~n be u~ed with adv~n~ge in the
.
. ~c~ve re~1on
.'~5 .A turther ~;ldv.mt~e of thi.~; p;lr~ieul;~r emb~diment of the inven~:ion i~
the l,u~e inL~re:~e in the .~;urt`;~ee ;lre;l/volume ra~io of the ;lutive re~ion ~:~n be
;l~hieved. U.~ing~the e.~;lmple ~iven 3bove~ X~X5 ~iphen~ 1 rigid p;lr~iele~ e~:h with
r;lcliu~ ot` ().1 mm would oe~:upv the volwne o~ the por~ion providin the ~tive
.
re~ion of the-rod if ~:lo~ p;l~:kect to~ether. The ~a~e ~reu of the~e ~phere~; would
~ ~ '
: _--
SUE~S~TUTE SHEET
.~.
~ ~ WO 94/11388 2 1 4 ~ ~ 9 ~ PCr/AU93/00546
be 111~.4 Inm-~ 17.X timex the .~;uri;lce are;l of a solid ~ylindric~l por~on. De~reasinP
the si~e of the partieles to be .~iintered to~ether~ would provide ~ corre~pondin~
incre;l.~e in the surfaLe ;lrea a~ bl~ tor ~iynthesis. For ex~mple~ decreaxing the
r~diu.~ of the rivid .~iphere.~ co be ~Inlereci ~o ~).()Sj rnm increases the surfa~:e are~ tO
5 ''33 mm~. about 35.~ ~ime.~ tnat or t.~le .~olid evlinder. In practiee. because of the
. pro~ess of joinin~ the p;Lrti~:le~i to~etner~ ;md the fact that the p~cles are neither uni-
~orm in size n~r ri ~id~ the thenrer~ nerease in ~urfaoe are~ would not be aehieved.
However. very ~i~nifie;ml ~a~ hl ~ ace ~rea available for synthesis can be
achieved by makin~ the solid support bv .~in~erinY small pa~cles of maten~l together.
1() , ~
F~ther fe~tures of the prese!lt inven~on are illustrated~ by way of
ex~rnple only~ in the following ex;lmple.
c. ~m~
The examp!es presented below 3re provided as a further guide to the
pr~c~itloner of ordin~ry skill in ~he ;~ nd ~re not to be construed ~s limi~ing the
inven~on In any way. .
. ' :
EXAMPLE I
2l) (Synthesis of Pepndes)
Polyetl ylene pin~ were molded as depicted in Fig. 2, ~nd mouTlted in
X x 12 bloeks ;ls de~cribed in WO'jl/042fi~. ;
Remov~ble ~iyn~he~ ces for cleavable pep~ides ("cleavable
orown~"~ were molded ~s depieted in Fi~. I. usin~ polyeghylene. and w~:re ~hen radia-
25 uon-~?ra~ted u~ deseribed in EP l')XX55 ~i~h meth~ylic a~id/dimc~hy~acrylamide
(1()% MA. ~()'Y~ DMA in meth~nol). Cle~v~ble crowns were then denva~ized with
Bo~-hex~methylenedi;lmine ~ ordin~ to WO~1/04266 by imsnersin~ in a 60 ~
~olu~on of t-butoxy~:~rbonvlhe,c~me~hylenedi~mine (Boc-HMD), l:riethyl~am~ne ~TEA),
rti~:y~ he~cyle;lrbodiill~id~ (DCC) ;In(1 hydroxybenzo~iazole (H(:)BT) ~ 1:1.2) in
__ .
S~ 7lrrE 8H:'T
wo 94~ll3g8 2 1 4 ~ . PCr/AU93/005~6
.~ , ; .
- lX -
DMF. After Boe deproteenon with trifluoro~eeu~ id, the ;leuve surf~:e w~;
neutralized by washin ~n MeOH ~ mini~ 59c TEA in MeOH (2 x 5 min~ and MeOH
(5 rnin). The preformed diketopiperazine linker (Bo~-Lys(Fmoc)-Pro-O-HMB) was
~:oupled at fi() mM con~:en~tion with DCC/HOBT (1:1:1.2) for a set time tO ob~inS the de.~ired lo~din~ (H~IB = hvdroxvl-nethvlbenzoi~ id). On il surfaL:e area of 13()
mm~. ;lpproximauly 1.5 umole~ o~` linker ~;ln be eoupled within ~I) min. Unreae~ed
;lmino ~Yroups on the surt~ee ;lre e:lDped bv ;leetYl~tion.
Removable ~yn~he~ urface~ ~`or non-cle~vable peptides ("non-ele~vable
erowns") were molded ;lS depieled in Fi~ usinY polyet~ylene. and were then radi;l-
,
I() tion-urafted as deseri~ed in EP l')XX55 with 3()% hydroxypropylme~cryl~te inMeOH. The surfat:e w~s then derlva~zed with Fmoe-Gly (30 ITM). DCC ~nd di-
:~ methylarl~inopyridine (DMAP\ (1~ in DMF/dichloromelhane (1:3) for 15 min.
The unreacted hydroxyl ~roups on the sur~ce ;~e c~pped by acetyladon. Af~er Fmo~:
depro~ection in 20% piperidinelDMF ~nd washin~ with DMF (2 n~in) and MeOH (3
15 x 2 min)~ a eontrol Fmo~-~-Ala couplin~ is pe~fonned to obtain a to~l loading of Sû
;; : to l()V nmoles. Fmo~ Ala. DC ;lnd HOBT (1:}:1.2. 30 mM) in DMF is coupled
: for appro~umately 10 rnin nnd the remailling amino groups of Gly eapped by acetyl-
~: adon.
Esch pin was loaded with four non-cleav~ble;crowns~ fol1Owed by one
7V ~le;lvable :rown. The re~ul~n~ynthe~is stru~:ture was then used to synthesize a set
ot overl;lppin~ oct~pepodes derived from ~ne N~iss~ria ~onorrheu C30 srrain pilin
protein. .lm~no acids ~30-5~ (RAQ'YSEALLAEGQKSAVYYYL~HGKWP). Thus~
;lny ~iven pin e;lrried l~ve ~rowns h~vin~ iden~eal o~pep~des~ wieh eas:h pin carry-
.
in~ ;ln o~ pep~ide~different from tho:o~ pepndes c~Tied~on other pins.
.
(Ass~y ~ot Pep~des)
bl~ Pe~ud~
: ~ ~The: non--le;lv~ble erown~ were removed ~rom the 5-posiuon pins ~ndw~re pl;leed in ~orTespondln~ po~ieions on bloeks holdin~ l-position pins tsee WO~I/
: : - __ .
~: : SlJil~;TlTlJT SHE6T ~
__
~VO ~4~1138~ 2 1 4 ~ ~ 9 1 PCI`/~U93/0~
(J42fif)) to provide four iclenti~;ll epilope libr~ries. Each blo~:k was tes~ed for antibody
bindin~ by ELISA usin~ ;l l/. (h(i()() dilution of a rabbit ~nti-pillin antiserum. The
result~ (shown in Fi. i) demonstr~e mal ;lII four sets of pep~des ~re essentially
identi~
5 ~9~:
Cleav~ble ~rown~i were men removed from the pins, and the pepides
removed. E;leh ~rown ~V;~ reat~d ~dlh X()() ~1. of eIe~vage solution (0.1 M
NH~HCO~" ~1)% CH,,C~I. pH X.~ uol~; were submi~ed tO amino acid an~lysis.
HPLC demonstrated hi~h levei.~i o~ puri~e The typical yield of pep~de was about
1() ~()~) nmole/~rown.
E~ch pep~ide w~s then used ~t three concentsa~ons ~o illus~ate solu~on-
ph~se compe~iuon for bindin~ of the ;mtibody to ~he non-cleaved pepade set. Fourserum prep~ ons were tested: .
1)` . 1/21.()00 serum dilution~ wlth no added peptide (Fig. 4A),
2) 1/21.000 serum diludon~ peptide ~dded to 3~ nmole/mL (Fig. 4B~;
3) IJ21,000 serum dilu~on~ pepude ~dded to 3.5 nmole/mL (~g. 4C);
4~ l/21,000 ~emm di}u~onq pepnde added to 0.35 nmoIe/mL ~Pig. 4[3).
The ~n~iserum ~nd peptides we~ n~ixed and i3lcubated for 1 h~ a$ room
temper~ture before tesnng with the set of ~ound ~noncleavable) pep~des. The ELI5A
2(~ resuIts ;lre shown in Fi~ conceml~on of 35 nmole/mL, of thc cleaved pep~des
(Fi~. 4B) WL~ SUf~lCient tO inhibit ~n~ibody binding to the noncle~v~bIe pe~idesessena;lIly ~ompIeteIy~
:
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SUIIIrrllllTE lihEI: r
