WO 94/22053 ~ 21 S 8 S S o PCT/US94102982
C~ lTCAL FUNCTIONALIZATION OF POLYMERS
ACKNOWLEDGEMENT
This invention was made with U.S. go-~",.".~nl support under grant number
GM 27137 from the National Institute of General Medical Sc.iences and grant number
S N00014-92-J-1412(R&T code 413tOl i) from the Of fice of Naval Research. The U.S.
go~,."",cnt has certain rights in the invention.
FIELD OF THE INVENTION
This i..~e.ltiOIl pertains to chemical ~ 1 of polymers.
BACKGROUND OF THE INVENTION
F.~ i7P~l polymers have been the subject of intensive research, owing
to their wide potential h~ --- in biology, cl,~,.lu~l-y, ,-~A;r:--P, and in t~ - c involving
ion-P~r'~ resins, immnhili7PA. ~ "~ S, and el~ctnr~Ally c~lldu~
pol~,u~ .. Akelah et al.,Fuu: ' ' r~l~"-~.~ and Their A~vlicr~lions~ ~, and Ha11,
London (1990). ~ of polymer films or fflm surfaces with ~
15 i~t-udu~,lion of fimrtir,n~l groups is i".~vrt,t for the d~,~, lo~- . --t of new '~ such as
novel cc ~ t c Baum et al.,Chem. Mater. 3:714-720(1991); resist l~t ~rn<~ et
al.,Chem. Mater. 3:435-442(1991);1 ~ Pantano et al.,J. Am. Chem. Soc. 113:1832-
1833 (1991); and l~if ' ;alS~ Allcock et a!., Chem. Ma~er. ~:450-454(1991).
FY ' of existing methods for modifying polymer films include
sulfonation of pol~;.t.y,_.le, Gibson et al., Mac-l l~,l ' 13.34 (1980); r - of
poly(aryloxy)l,ho~ , Allcock et al., Chem. Mater. 3:1120(1991); plasma l~ of
polyester, Porta et al., Chem. Mater. 3::293 (1991); base hydrolysis of polyimide, Lee et al.,
Mac.u".<ol~~ s ~3:2097(1990);base hydrolysisofpol~ l.h-~ Allcocket al.,Chem.
Mater. 3:1441(1991); and base t~dt".~.~t of poly(vinylidene fluoride), Dias et al.,
Macromrle 1P,C 17:2529 (1984).
Another conventional method for .,.odilj ;"g polymers CG~ -g
the surface of a llydlu_al~ polymer such as pol~ll,yl~,.,c with nitrene or carbene
hlt~,.lll~dirt~,s ~ .dt~,d in the gas phase. Breslow, in Scriven (ed.), Azides and Nitrenes.
chapter 10, Academic Press, NY (1984). Also, dilluG~ocr~l~nc ~. ~ in solution has been
reported to modify 1,4-polybut~dierlec Siddiqui et al.,Macromolecules 19:595(1986).
Perfluo,opl,~,.,yl azides (PFPAs) have been shown to exhibit improved CH-
insertion efficiency over their non-nuu,;nat~cl analogues when the PFPAs were pl.ol~ ~d in
hydlucall)ùll solvents such as cyclQheY~:e or toluene. Keana et al.,J. Fluorine Chem. 43:151
(1989); Keana et al.,J. Oro. Chem. 55:3640(1990); Leyvaet al.,J. Or~e. Chem. 54:5938(1989);
and Soundalalajà-) et al.,J. Oro. Chem. 55:2034(1990). PFPAs were initiallydu~ul~ as
efficient phot~ elino reagents. Cai et al., Bioconiuoate Chem. 2:38 (1991); Pinney et al., J.
Oro. Chem. 56:3125(1991); and Crocker et al.,Bioconiuoate Chem. 1:419(1990). Recently,
bis-(PFPA)s have been shown to be efficient cross-linking a ,ents for pol~ ,.,ne, Cai et al.,
WO 94/22053 PCT/US94/02982 ~
2 ~ 2-
Chem. Mater. 2:631(1990); and poly(3-octy!thiophPnP), Cai et al.,J. Molec. Electron. 7:63
(1991).
In view o f the present state of the art in chemical ~..n ~ ;nn of pol~ u~ t
there remains a need for other methods for filnrticnqli7in~ polymers, p~ ,ul&~ly methods that
S are easier to perform and more readily ~lqpfrLt~ for filnrtio..~ ..g polymers with a wide
range of fi~nrtinn~l groups.
There is also a need for methods for funrtinn~li7ing polymers that have
h~,,etorul~ been resistant to being ch~Pmir~lly modified.
There is also an ongoing need for new types of chPmir~lly .- ~I;r.~
10 poly,..~.s for use in any of a wide variety of cpeci~li7Pd applic~ ...c such as, but not limited to,
~;ir' . ;I~'~ polymer films and other ~L.u.,lu.~,s, &JL~ , and - '' ~
cc--.~ matrices"- ~ , insulators for ~ J cini materials, fibers, foams, and
films.
SUMMARY OF THE INVENTION
The foregoing needs are met by the present i.. ~entioll which provides
methods for cu.dlcull~ modifying (i.e.,filnrtir~n~li7ing) various puly u~C ~ ~ ~~ and
provides various r....~ioA~li7P~ polymers.
rOIy~ e~ that can be fu ~ i7Pd ac~..l.n to the present
..ti.... include any of various ~ co...~ ;..g synthetic and/or natural polymer
20 -l~ _'- having chemical moieties each capable of u...ie.~o;ng an addition reaction with a
nitrene.
According to the ptesent invention, a polymeric ~ .~ is fi. - I jo al;~ -i
by adding to the poly---~.-;c ~--l-,l~ - e a fi-----l;n-~li7ino reagent. The fu--- I;n~ i7ing reagent
c(~ ;se~ m~l~ '~~ each having a nilt~..-GOenic group snd a filnr~jnnqli7ing group. The
~5 mnlPclllPs of the ru~ i7ing reagent are brought into reactive ~lùxhluly to the polymer
c such as by, but not limited to, fonning a solution Of the filnrtinnqli7;ng reagent and
the polymer mel~ ' The solution can be formed into a film or other suitable shape, then
dried.
While the ~ of the ft!nrti(~n~li7ing reagent and the polymer
30 mnle~lllrs are in reactive proximity, the mnl~ lrc are exposed to A reactiû~. e..~ y source such
as photons, electrons, or heat. In the presence of the reactiu.. e,._.~y source, the ~ mC
groups on ~l~ lr-c of the fim~ tinr li7ing reagent form nitrene i.~t~,. -' that c~ tly
react with -CH, -NH, -OH, -C=C-, C-C and other groups on the polymer mrl ' so as to
cause "nitrene addition" or "nitrene insertion" of the fimctinn~li7ing groups to the polymer
35 mtle~ulto5. The nitrene addition or nitrene insertion results in the filnrtjnn~l groups l~- --.g
covalently bonded to the polymer molecules.
The nil-~.~ogenic groups on molecules of the ru li~ i7in" reagent are
azide groups or analogous chemical oroups capable of forming a reactive nitrene when exposed
WO 94/22053 _ 3 PCT/US94/02982
to a reactio.~ c...,.Oy source.
According to the present invention, the polymers can be fi~nr~ noli7P~I via
either a single-stage or A multi-stage process. In a multi-stage process, each stage typically
involves different functionDli7ino reagents. In both single- and multi-stage IIIU~_S, at least
5 one stage involvesa r.il.cnog_.lic funrti., ~l;,;,.g reagent.
In a single-stage process, each ~ '- of the r~ i7inco, agent
coll,l..ises, in addition to the r.;l.~.los_.lic group, a funrticnsli7ino group covalently coupled to
the nilrc.l~O_.lic group. The fi-nr~i.mAli7i-l,0 group can be virtually any desired chemical group
that does not cross-react with the nitrenogenic group or is ge.~,.. ~;r~lly pl~ ' from
10 reacting with the nitrene hllc....ediatc. E.g.,the fi~nrti~nDli7ing~ group can be selected from,
but is not necessarily limited to, l ' -q t;~e labels, lluul~..lt labels, en_ymes,
~,l",",-=~oloo,ir~o-lly active groups, .lj~,.-o~ y active groups, ~nti~ipc~ nucleic acids,
sulr.~ ulls, and any of a wide variety of other groups.
F~nrtinnDli7ino reagents adapted to L-.~ li7P s ~ in multi-stage
15 reactions can be configured in several ways. According to one method, a first r~ g
reagent is reacted with the polymer mr.~ ~~~ SO as to achieve covalent of the first
r~ r~ i7ing~-reagent mrlP~ lPc to ~he polymer -'q ~ rt~.~.al-i, a ~second r. -~ g
reagent is added so as to react with, and lh~.Grulc covalently bond to, the attached first
r.---- ti.~..cl;,;--g reagent ~ In such a method, the first ru~ li7in,o reagent
20 COI~p(;if-~ - each colllpti~ing. in addition to the llill~...oO_.lic group, a first
~,..,. ~j~,..-1i7in~,o, group adapted to p~-lic.i~,alc in duwll~llwlll ~.L_~II;DI.~ after -' ' of the
first r ~ jo~DIi7in;g reagent have been covalently bonded to the polymer -' ' via nitrene
o~ n For ~ . 'a, the first fi-nrtjcn~-li7ino group can be an active ester that is reactive
with -NH groups, -OH groups, or other lluclco~ ilic proups on ~1O 1PQ of a second
25 r.. ~ Ali7in~,0, reagent. The second fi-nrtjon~li7ino reagent, then, can provide a second
r.. I;~"Ali7ing group ultimately desired to be attached to the polymer ~'~ ~ . such as an
en_yme, antibody. .I;~ lic agent, or ll-~ ,.-lic agent.
An alternative multi-stage process CO~ liS~ first reacting the second
fi~nrtjrr~li7ing reaOgent (cOlll~Jl; Ih~g the second, or ultimately desired, ru--- l;-~n~li7in,. group)
30 with the first functionAli7ing reagent (inrh-ding a nill~GO_~l;c group); then, in a second
reaction, reacting the product of the first reaction with the polymer m-~'~ 1PC in the presence
of a reaction-energy source so as to covalently attach the product of the first reaction to the
polymer '~ IPS vianitrene addition.
A class of preferred functic n~li7ing reagents for single- and multi-stage
35 pl~:.~.S accoldillg to the present invention consists of N-hydlv~ ie active ester-
funrtinnAli7P~ pe.nuclu~ ,.lyl azides (NHS-PFPAs). The NHS active ester groups become
covalently attached to the polymer m~le les via Oe--~ -r~ during the reaction of highly
reactive nitrene h~te,lll~idt~_s derived from the PFPA portion of the reagent '- lPc, (Irhe
WO 94/220~3 215 8 S ~ O PCT/US94/02982 ~
reactive nitrene portion of the hltt;llllcdiAtes are preferably c<,~ ;n-~1 structurally such that
the nitrene portion cannot react intrPmrlac~ rly with the NHS active ester portion.) Thus, the
polymer ~ become n modified n (i .e., n filnrtion~l i7Pd ") . Afterward, the active esters can
participate in further reactions with a variety of reagents CO~ g primary amines or
5 hydroxyls (such as ~ ec) by way of amidre or ester formation, respectivdy.According to arother asp~ect 1Of the present invention, a mixture ~
1~ s of a ..il.~nc forming filrlrtionqi~7 ng reagent and polymer ~ e-S can be applied,
such as in the form of a film, to the surface of a s~lbstr~e. Then, the coating or film is
e~posed to a reaction e..~ / source (such as photons or a beam of particles such as an
10 electron beam) in a spatially selective way to fi--rtinnsli7P certain regions of the surface and
not others, thereby creating a filrlrtil~n~1i7PA pattem on the surface. Such patterns can have
.n.... ,.- ~..c ...c6~.. -~ in l~u~,~u~ t~ and smaller, due to the highly resolved manner in which
the coated surface can be exposed to the l~lio.i C.l~ source. Thus, the present i..~t;o~
has wide applir~hility in micIoelcullù--ics and in the Col-~lruulion of novel u~ ~dle
15 ~ ~
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. I shows IR spectra of pol~Ol~-e.-e ;~. ]...l;ng 8 wt-q~ NHS-PPPA
(co---l~ol-~ 1 in Scherne 10), wherein plot "a"was obtained before ILululy~i~, plot "b~was
obtained after phcloly~ plot "crwas obtained after t.~llu~..t with the amine 3 (Scherne 10);
20 and the peaks at 2300 cm~l are from CO~.
PIG. ~ ôhOws IR spectra of poly(3-oclylllliopll~lc) ;--- l~ l: ng 10 wt-% NHS-
PFPA (c.,~ u~ 1 in Scherne 10), wherein plot "a"was obtained before photul~ , plot "b"
was obtained after photolysis; and plot ~c"(shown offset from plot "b")was obtained after
l.~t,.... ...~nl with the arnine 3 (Scheme 10).
PIG. 3A is a phot~ lugl~ ll obtained using an optical u~,
g linear and circular filnr~jon-~li7~A patterns produced on a film of pol~ e and 8
wt-% NHS-PPPA by el~l.(,n beam lithography.
FIG. 3B is a pl,ct ùgn~ph obtained using a IIUUI~ICG u~u~
fitted with a n..O.~i;.. filter set, d, : g the funrti~tn~li7~ patterns of PIG. 3A after
30 l.~l,~nt with amino-nuo.-,s~;.l.
FIG. 4A is a photomicrograph obtained using a fluo~ ~ce /JScû~ of
circular patterns produced vn a film of poly(3~ctyl~hioph~onp) and 7 wt-% of NHS-PPPA by
exposing the film to electron-beam lithûg-~ hy con<~ n~ and ~ ly treating the film
with amino-lll,o.e~e.~, wherein the ~r.ic,ùscol~c was fitted with a Ih~l~---;--r filter set.
FIG. 4B is a l.h~tu~ og--~ph obtained using a lluu~.~c~ c,s~pc of
circular patterns produced on a film of poly(3-octyl~hi< ph~ne) and 7 wt-% of NHS-PFPA by
exposing the film to electron-beam litho~raphy conditions and sullae ~ ,ntly treating the film
with amino-fluorescein, wherein the Ul;Clus iulJe was fitted with a fluorescein filter set.
~ WO 94/22053 21 58S5 ~ PCT/US94102982
FIG. 4C is a ph~to ~ rpl~ obtained using a nuG.~..cc .,u~.uscu~ of
circular patterns produced on a film of poly(3 oclyl~ oph ~-) by eYllQcing the film to d~ll~
beam lithography cnnriitil~nC and s~ l-sc~lu .~lly treating the film with aminc-lluu~ wherein
the ~lu~;luSCOpG was fitted with a .I.nrl_...;nf filter set.
S FIG. 4D is a photoll~icluglnl~h obtained using a fluo.es~.. ,e usco~ of
circular patterns produced on A film of poly(3-octylthiophene) by exposing the film to el~;lro~
beam lilllG~nphy cr~nriitionc and ,--~ u ~ly treating the film with amino-lluGl~ wherein
the IIUCIUDC(JI)C was fitted with a lluu~;D~h~ filter set.
~ETAILI~D DESCR~PTION
lû The following terms are used herein:
A "polymeric material" is a material ~ ;ng polymer ~' ' or a
network of polymer mrk 1 ~
A ~polymer r--le '^" is a relatively large -'o '~ formed by the covalent
linking together of smaller mrl '~,r termed "~ "~". .~ ~ The .--- ~ . present in a
lS polymer ~'- '- can be the same or different. Polymer -Ir ~~ can be natural, such as
(but not limited to) any of various pol~c~harides and pol~J~tid~, or ~.llh.t;c such as (but
not limited to) nylon and pol~_lllyl~,nc. In a pG~ , material, polymer ~'~ ' can be
~- ~ with each other in any of several ways, ;..- h,-l; .g r.ull cu...l.,.~ r (as a i' r ~
or 8 covalently cross-linked network (as a 11~ u ~et).
2û A '~functinnsli7pfi polymer" can pertain to either a r. -- ~;o~ pol~ ,.ic
material or a molecule of a filnctil~nsli7-~d pûlymeric material. Fu - ~ i polymer
mr~~ _lr$ cGIl~ ,e one or more fimrtinnnl groups covalently bonded thereto accGI.lh.g to the
present invention.
A "fi~nrtionsl groupn is a group of one or more atorns bonded together in an
25 o- ~;2~ way so as to have a desired chemical property. Certain L .- li- ~ l groups can, when
covalently bonded to a pûlymer 1 ~h~ le according to the present invention, p... . in one
or more o~itiitionol bonding reactions with either a similar fi~nrtinnal group or a different type
of fimrtion~l group. Such bonding reactions can result in: (a) Lll- 1--- ` l to the r---- ~;n..~l
groups of any of a variety of f-l~litinnsl funr,tinrol groups; or (b) coupling together (cross-
3û linking) of the fimrtinnsli7~d polymer mnl~frlllec Many other fi7nrtjon91 groups r~ ~ to
polymer molecl~lpc according to the present invention can confer altered rhlon irsl ~lU~ Lcs to
the polymer molpcnl~os such as, but not limited to, making them labeled or tagged with a
lluur~cc,lt, .~Loa~ /e~ immlmologic, ~ n~1ic, or 11. ~ ic rnarker.
A "funrtinn~li7ing reagent" acco..li..g to the present i..~e..l;o.. is a reagent35 adapted for fimrtjnn~li7ing a polymer accolding to the present invention. Molecules of
filnrtjnn~li7ing agents have at least one nitrenogenic group (as a first fi~nrtionsl group) coupled
to a second functional group, wherein the nitrenogo-nic group is preferably C~ ln;..~l by the
fimrtion~li7ino-reagent molecular structure between the nitreno~enic group and the fi-nr~inn~
215 8 ~i ~ O -6- PCT/US94/02982 ~
group The nil~,.,o~;~uic groups are capable under reaction conditions of fimr~ n~ j7jng
polymer m~
A "nitrenogenic group" on a filnrti~n-li7ing reagent is a chemical moiety
thst, when exposed to a reaction-energy sourceh becomes a nitrene group.
A "nitrene group" (also generally termed nitrene~ or "nitrene ;.~t............. ,~ .t.,")
is a I li.,ul_r forrn of nitrogen group that can be depicted as a singlet by the ;.l.u~;l.. ;; R-N
and as a triplet by the sllu~,Lul~; R-N-. Nitrenes are regarded by persons skilled in the art as
the nitrogen analogs of carbenes. Like carbenes, nitrenes are generally regarded as
:..t~ F1:~t s Nitrenes are highly reactive and generally cannot be isolated under ordinary
10 co~ ..C. However, certain chemical reactions such as .ea~,liolLs P~co~ to the present
h-~ lioll would not oll.~"~.;se be e, ' ' 's by known reaction - ' without the
p~wu~cd c ~ -e of nitrenes. Ir.-.portant nitrene reactions can be ~ 1 by the
following:
(a) Nitrenes, inrh~1ino aryl nitrenes, can undergo addition .~-lio~s at -CH
15 sites and at -NH sites; e.g.:
Ar-N + R3C-H --Ar-NHCR 3
Ar-N + R~N-H--Ar-NHNR2
25(b) Nitrenes can also undergo addition at -C-C- and -C=C- bonds; e.g.:
R-N + R2C=CR~--R2C\--/CR2
30 R
As used herein, the term "addition reaction" when used in the conte~t of
35 .~ n~ of the nitrene group of the functirn-1i7ino, reagent with polymer I 1--~, generally
refers to any of the various addition and insertion reactions that nitrenes can undergo with
polymer mr'- '~s according to the present invention.
According to the present invention, a filnrtinn-~li7inv reaction occurs when a
filnrti-~n--li7ing reagent co",i),;sil~g a nitrenogenic group is exposed to a reactic,.. e..~,.gy source,
40 which converts the nitrenogenic group to a nitrene i~lG~"~ediate~ The filnr~ n-li7ing reaction
proceeds by reaction of the nitrene intermediate with a polymer .1~
A "reactio" c..c.~y source" is an energy source that drives a funrtil n~li7ing
reaction accoldillg to the present invention by, in particular, c~ ,.li.-g ~ ,..ogc..ic groups on
~ W0 ~4/22053 215 85 5 û PCT/US94/02982
function~li7ing reagent mrl~ules to nitrenes which react with the polymer ~ Suitable
reaction-energy sources include (but are not limited to): photons (such as ultraviolet (UV)
light, deep-UV light, laser light, X-rays, and heat in the form of infrared . " or
con~]u~live heating), energi_ed el~t~ui)s (such as an electron beam), snd e~ ;i~d ions (such
5 as an ion beam). These reaction-energy sources are conventionally used for such tasks as
litllG~Iayhy~ scanning l~ ,loScopy, and, in the case of UV and visible photons, effecting
ph~-tv.~ LI reactions and excitation of fluorescent mol~ l~c
A ~r . ~ li7ingreaction" is a reaction in which polymer I _' are
funrtir,nali7~d according to the present invention. A funrticnDli7in,s, reaction can consist of one
10 or more stages. At least one stage involves the reaction in the presence of a l~;li.
source of the polymer ~' l~s with mrll5 l~s of a filr~rtj~" ~ g reagent c~
.I;tl~,no6~,.lic groups.
According to the present invention, a polymer ~ 1P is L--t l;n-~ 7r~ by
a cl.~.l..,h~ whereby funrtir,nDI groups on fi~nctionDli7ing reagent ~ become c(J.aL~Illy
15 bonded to the polymer ~1~ le Such covalent bonding is achieved by c~-..~ ;on of
n;l-~ -o~,e..ic groups on the r---- ;j.~rl~li7ing reagent mnl~ S (the filnrtjl~u~l;,;.~,~ reagent
also each co...~ ;ng a desired fi~nrtion~l group as set forth below) to a rlitrene
;~.t. .. ~ tn highly reactive with the polymer -' _' - by e>.~u.G of the r
reagent mr~ ' to a reaction-energy source.
The lu-,-~lio-~li7ing reagent is pl~fe~alll.y selected from a group cn~ &
generally of: aryl azides, alkyl azides, alkenyl azides, alkynyl azides, acyl azides, and r- ~- - t~l
derivatives, all capable of carrying a variety of s--l,.lil~ Most ylef~"ably~ fluorine (and/or
chlorine) atoms are present to the ~ ;.------- extent possible in the po~ --C on the
fimrtionDli7ins reagent mrJle Ir adjacent the azide group.
25 Each of the r.,.ego;.. g azides may also contain within the same ~'~ '^ any
of the following filr~rtil~n~l groups, c~ ;nP~ structurally from reacting with the nitrene
moiety after the nitrene moiety is ,2ene...t~;
(a) carboxyl groups and various derivatives thereof such as (but not
fiC~.ily limited to): N-hydroxy-~-- c;-- -- ~1~ esters; N-l.yd.~ ;a~ole wters; acid halidw
30 co..~ ,o.,~l;--~ to the carboxyl group; acyl imirlD7rl thioesters; ~-nilr ,yLenyl esters; alkyl,
alkenyl, alkynyl and aromatic esters, i..cludi,-g esters of biologically active (and optically active)
alcohols such as cholesterol and glucose; various amide derivativw such as amides derived
from &----..~ primary, and ~cond~ amines and inchl~lin~s, biologically active (and optically
active) amines such as c~,i"eph,h~e, dopa, enzymes, ~ntibo~ oc, and fluorescent l--r~
(b) alcohol groups. either free or ~t.,~;rled to a suitable c&~ ylicacid
which could be, for eY~ . lo, a fatty acid, a steroid acid, or a drug such as naprosin or aspirin;
(c) haloalkyl groups wherein the halide can be later rlicpl~ed with a
nncle~philic group such as a carboxylate anion, thiol anion, carbanion, or alkoxide ion, thereby
WO 94/22053 2158 5 ~ ~ PCT/US94tO2982
--8--
resulting in the covalent ~ of n new group at the site of the halogen atom;(d) mQIPimido groups or other ~iennrhi1ir groups such that the group may
serve as a ~ion~lrhil~ in a Diels-Alder cy~loQd~liti-~n reaction with a 1,3-diene~ : .8
r'- _'- such as, for example, an e"o~
(e) aldehyde or ketone groups such that ~.,I,se~ deriv is
possible via r~ ~n of well-kno,vn carbonyl derivatives such as hy~ 5. b~-LVn~
or oximes, or via such ~".~rl,~ ",~ as Grigriard addition or alkyllithium ~ ion; and
(f) sulfonyl halide groups for ~u~ lu ( reactions with amines, for
G . Ir, to form ~ 1 r..~ P c
The foregoing functional groups are particularly adapted for i ~ m in
dov.~.sl.Gc.~ L~ u~tl~(i.e.,cl.~.l,i~t.yp~.rul--l~ aftertheru~ohlgr. ~ 1groupsare
attached to the polymer I -'- IPC) whereby yet other ru l;~ ~l groups can be covalently
attached to the polymer ~ - by reaction with the already e ~ - ~ fi-nrtin~l groups.
A general reaction by which a .e~.~.,,ltdti~e r ~ reagent is
15 co ~_.t-,d to a nitrene h~te.lll3d;dt, is:
X-R-N3 ~ X-R-N + N2
photons or e~ beam
where X is the rl cli . ~' group, M3 is the nil-e.-og_..ic group (an azide in this instance), and R
is an aromatic ring, h~,t~ ullldtiC ring, or other carbon~., ~-;; g r. ,,
A reaction-energy source Colll~ ;llg UV light can be supplied to the
reaction by, for example, one of the following lu~lG.~ hli~e l,-~cdu.-,~. (a) A sample
25 co.ll~ ;ng fiu tio~ g reagent, -le les and polymer mnl~ ' is placed in a well of a
Rayonet Ph(J~ l Reactor fitted with either 350-nm, 300-nm, or :254-nm lamps and
i..~.J;atcd at ambient t.,...l,c,alu.~ for several minutes under air. The duration of the
irradiation can be adjusted to change the exposure dose. (b) The sample is ' ~ through
a high-resoh~tion ~hcl ~ ', for example, by (but not limited to) ~,.u; UV l;~ .l,y.
30 (c) Photolysis is carried out in a KSM Karl Suss deep-UV contact aligner using a contact
high-recol~ltion 1 l ot~ It will be readily ap~,l~;ated by persons skilled in the art that such
~,l.,ccdu.ei, can also be generally used to provide the fun- fionQli7ing reaction with photons of
wav~ hs other than UV.
A reaction-energy source co..-~ ;--g electrons can be supplied to the
35 reaction by the following ~c~ e.lhl;~/e ~Jlu~ ule A film sample co---~,-.,..lg filnrti--nQli7ing
reagent mnle _'^ and polymer molecules is h. Ii --! under vacuum by an electron or particle
beam with an energy selected within the range 1-40 kV. (A lepl~s~,,.l~ti~e electron-beam
source is a JEOL 840A electron Ill;~ JSCOIJC modified for electron-beam lilhogl~llJlly.) The
beam is stepped across the film surface to expose certain areas and not others. A dwell time
~ WO 94/22053 21 585 !; O PCT/US94/02982
at each step can be adjusted to change the exposu~e dose.
Particularly effective fUnrti( n~li7ing reagents are selected from the group of
lluo~u~h.,.lyl azides (PFPAs) derived from 4-azido-2,3,5,6 tet.~rluGrùb~,..~. acid in which
the carbonyl group is further activated through reactive ester, amide, acid halide, or rni~ed
5 anhydride rU. ' nn
For G~.alllrl~, and not intended to be limiting, le},l. ~e L l;n~ j7~d
UUIV~JL~I.YI a_ides have the general ~hUUlUI~;;
X
F ~ F
F ~3
wherein X can be any of the following: CN, CONH2, CHO, CO2Me, COMe, NO2, CO2H,
COCI, CO-T ' 'o. CONHS, CH2OH, CH2NH2, COCH2Br, ~ o, NH-biotinyl,
15 CONH-R (where R is a polypeptide moiety), CONH-X-S-S-Y-NH-biotinyl (where X and Y
are spacer ato~ns and the S-S bond is reductively cl~ e at a later stage), and
CONHS-SO 3Na.
Rc~ t61ive activated PFPAs include ~but are not limited to) the N-
hydluA~ _ le (NHS) ester A (also do~i~r ~ ~ "NHS-PFPA"), the ~n;l,~.~)l.e~l ester B,
20 the 1-hY~IIUA~ UII- le ester C, the acyl ' le D, the acid chloride E, the mi~ced
anhydride F and the 2,2,2-trichloroethyl ester G:
N,~gO-N~ N~NOZ
F F
C D E
N3~LO ~ OEt N3~OCH2CCb
F F F
WO 94/22053 PCT/US94/02982 ~
2ls8ss Q ~o
In addition to the foregoing c~ z filnrt~ g reagents, it is possible
tG utilize other PFPAs having "spacers" situated between the reactive filn~t;. n~l group and the
PFPA moiety, such as:
N3~ 11 ~0--N~ N3~R 11 0--N~
F F ~0 F F O
Other . ~ aryl szides useful as r~ 7in~ reagents are similar to the above ~ , '
10 except that another aryl~moiety replaces the PFPA, such as:
~C ~R
~'o~ c.~ that can be ~ 7~ ac~.ding to the present
,.,tion include virtually any polymer co...l).isi..g polymer ~ - po~ -.g -CH groups, -
NH groups, -OH groups, C-C sites, and/or -C=C- sites. Such polymers include, but are not
limited to:
(a) 1 pol~ - as e~ ;r~1 by pol~_ll.yl~".c, poly~h~lchloride,
polyt~ n~o~ lc~ pol.~,.u~ ,..c, polyl,ut~".cs, and copol~u.e.~ thereof;
(b) acrylic resins such as pol~..~,~ and ~oly.uc.~ of acrylic acid,
n.cll-&c..~lic acid [poly(.--clhyl..-t;tl.&c.y' ~), poly~,A~ l.ac.y' )], and &c,yluûil,ile;
(c) pol~ n ,.e and its analogues such as poly(~-chlo.u~l~.c;nc) and poly(~-
25 lI ~ dIU~ G);
(d) ull~tulatcd polyolefins such as poly(isoprene) and poly(l ~ ..F);
(e) polyimides such as polyimide(b~ F~ f t~ aca.buA~lic
~id.~ Lll;de/l_t. .yl~ nFAi~ in~o.);
(f) pol~"t~,.s such as poly(l-;---.,ll-~l~,..c adipate) and poly(h~ u, Ihylene
30 sebacate);
(g) colljuadled and con~luc~ing polymers such as poly(3-alkyl~l.;ul.l.. nr),
poly(3-alk~l~.j..o!-), and polyaniline;
(h) inu.~;~....c polymers such as poly(a~ylùx~ h~ -F)~
poly~tlil1uo.u~,11.oxy)pl~o~ c3, polysilanes, and polycarbocil -, siloxane polymers, and
35 other silicon~o-~ polymers;
(i) organic metals (i.e.,organic polymers with metallic properties) such as
poly~luc~ ;nF~ and pol~;,.lu~..,.;u~s, as described in Chemical and EnYiu~,c.;-,~ News (August
31, 1992),p.8.
WO 94/22053 _ 2158 5$ o PCT/US94/02982
(j) o~ n.... ~ lic polymers such as p~lllar~ m poly-yneand r~.~u~.~c
c~-..t-; .;..g polyamides; and
(k) pol~cchalides such as cellulose fibers, chitin, and starch.
F~nrtinna-li7ation of polymer m-)hP~lllPs &cco,J;..g to the present invention
S requires that ~ PS of the r" ..~iu"~li7ing reagent and the polymer mnl '-- be brought
into "reactive p.u~ . ilyn;i.e.,brought together s~lffiriPn~ly closely so as to undergo a
ru...~ li7i-~g reaction when exposed to the reaction-energy source. One way in which this
can be done is to prepare a solution co..,l..i~ing the polymer ml~l IPC and the r ~ Cl;,: ,g
reagent. Another way is to prepare a ~lCpPn~inn or mixture cGllll,lisi..g the fun~tinn~li7i~
10 reagent and polymer particles or aggk ~ ~ of the polymer. Yet another way is to spply
the fi~nrtionali7in~ reagent (such as a solution of the r- li-~ inp reagent in a solvent
capable of abso.l.;.)g into the polymeric material) to a surface of the polymer, then allow the
r,J-.- li.~l~CIi7ing reagent to absorb into the polymeric material.
F~ in~Cli7ation of a polymer can occur in one or more stages, dflW ~ g
15 upon various factors such as the particular polymer to be fi~nrtin-ali7PA: the form of the
polymer (i.e.,solution, particulate, ~ n~, non-fluid mass); the ru .- iin.~Cl group(s) to be
attached to the polymer -'- '-~ the ne~.;ly to protect the fUnrtinnal groups from
u..des;~l reactions during reaction of the r .itio~ g reagent with the polymer -'
and on other matters.
For ~i rl~ in a onc ~t~ filnrtif--~ , polymer l ~ and
-'~ ' of a fimrtiQnali~in~ rPagent each having a nit.~ oge,.i-~ group and a desired
funrtinnal group are brought into reactive proximity. Upon exposure to a reactio.. c..,,~
source, the l.iL.~inGg_..;C groups are converted to nitrenes which react with -CH, -NH, -OH,
-C=C-, C-C, and other groups on the polymer mrl~ IPS reactive with nitrenec, thereby
25 covalently bonding the fi . Ii.- ~l groups to the polymer 1~ IPC The fi -.,~in~.~l groups
typicallydo not require additional che...;;.l.y p.,~ru~.cd on them to confer the desired useful
property to the resulting fi~nrticnali7P~ polymers.
In a two-stage filnrtinnali7ation protocol, each stage involvesa different
filnrtjnn~li7ing reagent. In many ;.. ~ , the first stage can be pe.rc.. ~d by ;~te.~ ;"g
3û mnla lPc of a first functionali~ing reagent depthwise into the polymer mass, such as by first
forming a fluid solution or ~U~lJ~l.ioll comprising the polymer and the first fiunrtin~--l;,;..g
reagent; forming the fluid into a desired shape; then converting the fluid into a product having
a rigid form. The reactio.. ene.~;y source is then applied to the rigid product to co~ ..lly
bond the first fimrtjnnsli7ing reagent to the polymer -' '-- S~ ly, during the
35 second stage, the second r.... ~ n~li7ing reagent is applied to a surface of the rigid product.
As an example of a two-stage funrtinn~li7Otinn reaction, the first stage
involves a first functionoli7ing reagent such as an NHS-PFPA cc..-q o~ Upon exposure to a
reaction e.)e-~;y source, the azide group of the PFPA portion is converted to a nitrene
WO 941220~3 PCT/US94/02982
2 1 5 8 5 5 0 I r~ --
;nt~ le that reacts with polymer m~ Q Thus, the NHS active-ester groups on the
NHS-PFPA m~ c become covalently attached to the polymer m~!e l~os by a reaction that
can be generally indicated as shown in Scherne 1 (wherein a polymer ,/-l~ lo is .ci}.re d
by a c;.-,u...sc. ibed P):
~) + N3~c--R ~ ' (~'t~c--o-/,/~
Scheme 1
As can be seen, the NHS-ester portions of the PFPAs do not ~~ in
this first-stage chc,. ~,t~y. Rather, the NHS-esters, after being I r .~d to the polymer
If '-s, are utili_ed in second-stage ,L~ y, d;~ below.
In the second stage, the NHS-esters readily react with m~ of a second
15 fiu ~ li7ino reagent. The second functi~n~li7ino reagent is selected from a group c~ ; ,o,
of lr'~ ; o primary or s~ond6.~/ amines and/or h~ldluAyls. Reaction of
NHS-esters with primary amines proceeds via amide formation as shown in Scheme 2.
2 ~ C~
Scheme 2
wherein ~, ~ 2 is as shown in Scheme 1. Reaction of NHS-esters with l-~.l-v~ls
25 proceeds via ester formation, as shown in Scherne 3.
2 ~'Y ~ ~c~- o~
Scheme 3
wherein co..~ u~d 2 is as shown in Scheme 1.
Since many types of biological molecules possess amine nnd/or hydroxyl
groups, these mt~lm~les can serve as fi~nrtionqli~ino reagents adapted for reaction in a second-
35 stage fill~rtion~li7~ticn reaction with NHS-esters covalently bonded to the polymer '- l~c in
a first-stage filn~ tion~li7~ion reaction. Thus, it is possible to attach any of a wide variety of
m--leculec, in~lurlina l.~-.u..,olecules such as proteins, nucleic acids, ca-l,olly~ t~,~" and various
other molecules, to polymers using methods according to the present invention.
WO 94/22053 215 8 5 5 O PCT/US94/02982
-13-
It is also possibls &cco..l;,.g to ~he present invention to first prepare
log~,~lic derivatives of mnlec-llçs (such as biomolecules, drugs, analytes, catalysts [;...'l,.,l;.~g
transition metals], and .l;qg.~ ic agents) to be attached to the polymers, brin~ the derivatives
into reactive pro~i-- ily with the polymer '~ lçs, then expose to a .ca~,lion _n~ source to
S cause the n;l,enG~,_..;c derivatives to covalently bond to the polymer -' '- - via nitrene
' c~ A;~ . It is r.~.~d.~ that the ~ ...o~,enic moiety be structurally c~ ".; P~ to
prevent the nitrene from readily reacting with another part of the same ~'~ '~ Thus, with
NHS-PFPA r~ ti~ g reagents, the 4-position of the phenyl ring is the p.e~..~ position
for the a_ide group.
To convey the scope of the present invention without " ~g in any way to
be limiting, the following.~ ,ntali~/e r~ tin ~li7~ nc accc,.l;,lg to the present i..~tio"
are provided:
(a) Ca,.,;..ogenic or IllulA~,_n;c pol~ licaromatic h~.l.~.l~u.~s can be
attached to polymer r'~ IPS to render the polymers "c~ " r polycyclic
1~ h.~.l.oc~l~.ls include ethidium co...ps ~lc and various pyrene co...l o~A~l~ (such as 1-
L~lalllinc and 6-~...;--o~ ,nc). It is also possible, when ~ l.;..g such cQ~ tk
to polymer -hP,~ '-s, to employ "spacer groups~ serving to ~lift~the l,~J,~,lran from the
polymer ~'- '- A .~ L.li~e spacer~ lg hyd.oca.l,o.l is the primary amine
derived from 1 ,~.e,,~ul~.;c acid. Such reactions can be depicted generally as shown in
20 Scheme 4.
C H2N H2 (cH2)3co2H (cH2)4N H2
~ F
~ rcnc-Z/~/f~ ~ )=~
~ C--N~--Z--f~y~cn c
Scherne 4
wherein 2 is as shown in Scheme 1 and Z represents a spacer group.
WO 94/22053 PCT/US94/02982 ~
21 5855~ -14-
(b) The hydrophobiçity of a polymeric material can be altered, after
of NHS-ester groups to the polymer m~ ' ~ 9 in a first-stage reaction (via a
nitrene ;~ " .~ e), by s ~bcfy~ nt reaction of the NHS-ester groups with long-chain
aliphatic amines such as 1--s-minr~hf~Y~df~csnf~ in a second-stage reaction. Such a reaction can
5 be generally depicted as shown in Schern~S:
. . ~
f ~
~C ~
Scherne S
wherein R is a chain of hjd.upl-ol .c atoms such as, for ~ l, ', C~2H,s-, oleyl, G~.~d~l,
3-~ J c1f ~, or hexyl-l.-..~th~l~;lyl;and 2 is as shown in Scheme 1.
(c) The hydrophilicity of the polymer can be altered, after ~ ' of
NHS-ester groups to the polymer ~1 '~ in a first-stage reaction (via a nitrene
- ' ), by ,~ reaction of the NHS-ester groups with .. h c p~ g highly
polar ~'~ '~9 inssecond-stage reaction. Sucha..,...epo~ g polar r'~ '~~ include
(but are not e- ly Iimited to): 2 lV~C~-..;.-~" ~lominf, pol~_t~'~
20 at pH 7), polyl~ e (also p~utu~t~ at pH 7), glycerol, and other polyLJLu.~y c~
Such reactions can be generally depicted as shown ir. Scheme 5 but wherein R is HOCH 2CH2-,
or NH2(CH2CH2NH-)n-CH2CH2-; and 2a and 2b are as shown in Scheme 1. For pol~..l ohols,
such reactions can be generally depicted as shown in Scherne 6:
Scheme 6
30 wherein R is, for exsmrle, CH-CHOH-CH .~OH; and 2 is as shown in Scheme 1.
(d) The polymer can be made ~ulrhce-a~-tive by first ~fl~- l.;"g NHS-ester
groups to polymer n~ in a first-stage reaction. The reaction to make the polymer
mf~ ulr~oe _ ~, proceeds by a second-stage reaction employing any of various
gmins~ or hydroxylated ~deter ent~ mr~lecllles such as, for example, l-amino~loAf~sn-~is acid.
35 At pH 7 and after I ' of this coll,l,uu-,d to a polymer mrlf~--lf~, the carbo~yl group is
ionized and the cv~ ruU ~1 extends away from the polymer molecule as a long hJdIO~hC~ ic tail
t~ g in a polar ca,l~oAylate anion. Such reactions can be generally depicted as shown in
Scheme 7.
W094/220~3 ~ ~158~o PCT/US94/02982
,~ ,c
s
Scheme 7
wherein R is -(CH2)n-CO~H; and 2 is as shown ir. Scherne 1.
(e) Enzymes and other polypeptides can be attached to polymer ~ 1
10 p~ iously fim~tionsli7r~ in a first-stage reaction with, for e , h~., an NHS active ester. The
, nt second-stage reaction proceeds by, foT example, a reaction of a Iysine amino group
present on the polypeptide ,ole lr~-C with the NHS active ester. A .~ I~,.t~ti-~e reaction is
depicted as shown in Scherne 8:
~ (~ ~,. ~f/~C--N~t~)
Scheme 8
wherein the ~ ;ull-,clil,~d E with attached NH2 group .~.~ ,,.t~ a polypeptide c~ g a
20 Iysine residue. F , ' 9 of such a pol~ t.dc include (but not limited to) an enzyme (e.g.,
peroxidase), lectin, or antibody. Comroun~ 2 is as shown in Scheme 1.
(f) ~n~ibor~irc, Iectins, and other proteins can also be attached to polymer
5 by filnr~tin~ ;,;.-g reactions similar to such l~a li-,--s for Yn~ g enzymes. Such
attached mn' ~ can then be used, for example, as highly selective sensing agents in
25 ~ ~
(g) SPRCi~l;7~ e _lf can be attached to polymer -'- l~s to control
the wettability of the polymer or alter the ability of living cells to adhere to the polymer.
(h) Polymer -~e _l~C can be bioli--ylated in a one or twofftage reaction,
followed by l.eal.~ t of the biotinylated m~ $ with, for eY~mrlR. a d~_~ivaliz~ avidin or
30 streptavidin. The avidin or streptavidin are thus used as bridging units for ~b~
1,,.. n~ of other h:omr~l~cllls5 to the polymer. Re~ .Gsc..t~ti~e reactions are as follows:
.
Two-sta~ereact;on (Scheme 9)
~. R~ C~
Scheme 9
WO 94/220~;3 PCT/US94/02982 ~
21 58~0 -16-
wherein 2 is as shown in Scherne 1 and RNH2 ,vpresv..l:, the amino group of N-
S~NH
>~ ~
NH2(CH2~6NHCO(CH2)4 NH
A gne-stas~e reaction is e~emrlifi~ by bringing the polymer ~ and --If l~o,S of the
PFPA dv.;~ali~,vs of biotin:
~ (cH2)~coNH(cH2)5co NHCH2~N3
HN~NH F F
O
15 into reactive p.u,.i~,u~, followed by exposure to pl,ulul~;.;s or an electron beam.
To further illustrate and describe the present i..~ liu.., the following
, ' ~ are ~u~ idv~:
F~mnle 1
1n this FYI . 'e, we fiilnrtit~n~ I the Ityd-~.l~.. polymer pol~ ,I~,. -
20 (PS) by -CH insertion of Fh~t~' - 'ly g~ ~ - d nitrene i~lt~,. - " -
Referring to Scheme 10, the &vli~v estvr azide 1 was formed by ~,t ;rif-~ n
of N-hyd~uA~ e (NHS) with 4-azido-~,3,5,6 ~- n~n~ acid. The &vli`ilv~azide 1 was selected for study as a ,vl,.esv~lt~live rl -liu~ ing agent because NHS esters
react readily with amine~o"lS i..i.~g reagents to form amides (Rl-NH-COR).
~ WO 94/22053 21 58 55 D PCT/US94/02982
-17-
~n F~F ~ 1. spin coating
~3 ~ N3~ ~i æ iT~ 254 nm
F F 1 electron-boam
H--~--gw~ H2NCH2~
~j ~N~
H2N~CON~ 3 ' H2N~CHz~N~
2 ~ 5 ' ~ ~~~~~ lr --_
--N~
8 OH
Scheme 10
A solution co~ ing 50.2mg PS (mean l l~r weight 125,000to
250,000daltons) and 4.0 mg NHS ester l in 1.0 mL xylene was prepared, yielding an 8 % w/w
solution of 1. The solution was spin-coated on a NaCI disc using a photoresist spinner
~Headway Research, Inc., Garland, Texas) set at 1000 rpm. After drying the disc at 50 C for
one hour, the film .~ ;l.;..g on the disc had a thi~kn~s~ of about 0.7 ~n, as measured using
WO 94t22053 PCTIUS94102982
215855 ~ -18-
sn e~ (Rudolph Research, Inc., Flanders, New Jersey). The film was photolyzed for
1.5 minutes using a Rayonet photù.~ lo. (Southern New England Ultraviolet Co., Branford,
C~ n~); . ' ,;;,.g 2~4-nm lamps as photon sources.
The photolysis resulted in the smooth d~ollllJo~ilion of the azide group with
S C~ C~ ~jf~ forTno~ n of the r-~ ;o- Ali7~1 PS 2 derived from a CH-insertion reaction. The
photolysis was ;-u..;tun,d by the di~p~ of the azide &bso.l,tion at 2124 cm-l, ss
;...I;r-f~d in FIG. 1 by co--:p~.-i--g icu~ves ~a"(beFore pl-olc~ly:.;s) and ~b~(after pl~olul~ ;s). The
active ester csrbonyl absorption around 1750 cm~l was not affected by the ~Lololy~;4 resction.
Next, the fi~n~tjonsli7~1 PS film 2 was further f l~ ~" ' by ;~ .r~
10 st room t~ ---r ' c; for over two hours in a solution of 5.4 mg 4-azido-2,3,5,6-
ht~..lluulul~,.L~' - (3) (i.e., the hydrochloride salt of 3) snd lO mg Et3N in nil-.
(Nil--- ~ ,c is a solvent that does not dissolve PS.) The film was then removed from the
solution and ;.. -- .~1 in 40 mL n;l-l). ' ^ for 10 minutes, rinsed using h;llu---- ll - ~r~ then
air dried. The coupling reaction that occurred during said imm.o~i~n of the iiln~tif~n0li7Arl PS
15 film 2 in the solution of 3 was -loi.itu.cd by IR ~I,~t,u .~; using a Nicolet Model SDXB
FTIR ,~l-u,,wtu, (M~' , WisCUII:~;II).
As the coupling reaction p.uccedcd, an a_idc a' ~ t;on pealc at 2121 cm~
.c~.~,cd because, as the amine 3 attached to the fiLInrfic~noii7~l PS 2, the azide group of 3
lc~lkd~ d attached and ulll~a~t~ A CGIl~ decrease in absu.~,tion at 1750cm~1 was
20 atl-;b.-t~ to loss of the carbonyl groups (~C=O) of the active ester, as also seen in FIG. 1 by
COII-IJ~-;-. curve ~c"with curve "b". The IR spectra confirrned that amine 3 reacted with the
NHS active esters of the film 2, resulting in the further Tnn~-C- ~n of the PS by h~G~ -nn
of the ~.lluul~L~ LidG groups along the PS polymer chain to yield a r~ i7P~l PS
polymer 4.
IR intensity CGIll~JafiSlOn of the azide absorptions (C~ of curve "c~
with curve ~a~of FIG. 1) in-lir~ed that about 40 percent of the original number of azide
groups became hl~ll Uldt~ into the PS chain of polymer 4 as a result of l.~ of 2 with
3. This was probably due to the fact that photolysis of azide 1 in the presence of PS resulted
in less than a 100-percent yield of CH insertion. It is also possible that some of the NHS
30 groups may have been cleaved by adventitious hydrolysis during the tl~tlll~lt with the solution
of amine 3 in l~ Q~
~ WOg4/22053 _ 215~SSO PCT/US94/02982
-19-
Examr)les 2 and 3
These FY~mple5 co.. ~.. ;se control c,~ .-b for Example 1. ~o~ c
are as shown in Scher,ne 10.
In Example 2, A solution of PS was prepared as in Example 1 but without
5 NHS active ester 1. The PS solution was formed into a film and photolyzed as in Example 1,
then treated with a solution of the amine 3 in nil.u~ r. Afterward, no azide a' ~JLon
was observed in the IR a~eullulll of the film.
In Example 3, a film of PS r~ ~-;..;..g active ester 1 was prepared as in
Example 1. The FY~--Fle 3 film was not photolyzed but rather treated directly with a solution
10 of the amine 3 in r.;l.~ - ~ ~ IR a~l~pllulu... l. y revealed the ~ . r -e ofal~ lion at 2124and 1750cm~1, showing that the nil.~ " ~ had ~ P~enti~lly a!l
of the active ester 1 or the cc..~.l-u.,~ g amide out of the polymer.
F . '- 2 and 3 showed that both the NHS active ester 1 and photolysis
are needed for the ...r~J;I;- A~ " of the PS film with NHS active ester groups.
Example 4
In this F~ ~ . ' ~, referring further to Scherne 10, N ~ ~yl 4-amino-
t~ n~ o~ul)~ !, (5) was used as a model for the polymer 2. To prepare 5, a mixture of
214 mg (1.00 mmol) 4-amino t~ uulubc~lzùic acid, 119 mg (1.00 mmol)
N-L~ :l.o~ r, and 21 1 mg ( 1.00 mmol) di~ _lohcx~lc&.; ~ ' ~e in 10 mL CH2C12 was
stirred for 24 hours. The mixture was filtered and the solid was dried. The solid was then
stirred with 6 mL acetone and the mixture was filtered. The filtrate was ~ to leave
262 mg (83 percent yield) of 5 as n white solid having a melting point of 200-201 C. IH
NMR: ~2.899(s, 4), 4.665(s, 2). IR: 3522,3418,1779,1749,1683,1530,1507,1317cm~l. MS:
306 (M+, 2), 192 (100), 164 (30).
A mixture of 11 mg (0.036 mrnol) of the active ester 5 and 6.9 mg (0.031
mmol) of amine 3 in CDCI3 was prepared and allowed to react (not all the yield of 5, prepared
above, dissolved in CDC13). Progress of the reaction was ~u~ilulcd by IH NMR ~ lroa~
at room t~ c. As the reaction plu~ aed~ new signals at ~4.7 (d) were observed.
After 24 hours, a clear solution was ob~in~l When the reaction mixture was assayed by !H
30 NMR :.~J~I.U.. ~ t.~, no greater amount of signal was seen at ~3.941 for 3 and ~2.899 for 5.
The mixture was separated by l~c~ lati~/e thin-layer ch.u...atography (hexane-THF 1:1) to give
12 mg (94 percent yield) of the amide 6 as a white solid having a melting point of 155-156 C
(actually a dccc,lul,oailion te~ clalulc). lH NMR: ~4.286(s, 2), 4.701(d, 2), 6.402(m, 1). IR:
3411,2122, 1686, 1668, 1497, 1314, 1239 cm~l. MS: 411 (M+, 1), 383 (20), 192 (100), 164 (18).
35 The IR alJ~tlull~ of the amide 6 showed an azide absorption peak at 2124 cm~l, which was also
observed in the polymer film of Example I after photolysis and reaction with amine 3.
WO 94/22053 215 85 5 ~ PCT/US94/02982 ~
-~0-
Examr)le S
In this FY~mrle, shown generally in Scheme 11, we investigated the
funrti--n~li7~inn of the co.-Ju~ e polymer poly(3-octylthiophene) (al)bl.,~i~t~ P30T). P30T
can be pho~och~n~ic~lly cross-linked by bis-PFPA and can be used for the direct pr~ du~li~ of
S conductive ~Iru~lul~ via cross-linkinp, under electron-beam lithogrnphic c~nrli~ionc~ Cai et al.,
J. Mol. Electron. 7:63 (1991).
0 ~ 3~ æh 7 Oom
~ --NH~C--R
F F o
-- 9 R ---O--N~
3,
McNO2 F F
__10 R ---HCH2~Ns
F F
HCH2 ~0~
Scheme 11
For this FY~mple, the P30T was prepared from 3-octyl~hioFhAn- as
reported in Cai et al.,_.
Referring to Scheme 11, a solution of 25.8mg P30T and 2.6mg (10 %
35 w/w) of the NHS ester I in 0.8 mL xylene was spin-coated on a NaCI disc, dried, photolyzed,
and developed as describe~ in Example 1. The photolysis reaction yielded a fun-~ion~li7P<i
polymeric film 9. The film 9 was treated with the amino azide 3 (structure shown in Scheme
10) in nil~ ctlldne under conditions as described in Example I for treating PS. A
WO 94/22053 PCT/US94/02982
2,1Ss~o
funrtirn~li7PiA polymeric film 10 formed which involved an amide-fu---k.liol. reaction between 3
and the NHS active esters with COnCOn~ covalent ~ t of a new set of a_ide groupsto the P30T polymer. (In FIG. 2, compare curve "bwith curve "c~.) The IR ~ hUIII of the
film 10 showed a m~A~Pr~tRly strong absu,ytiu~l at 2121 cm~l for the azide group (FIG. 2).
S It is believed thflt the C-H insertion reaction yielding the rl~
polymer 9 occurred along the octyl side chains without involvement of the tl,;opl~ r ring. This
is based upon the ob~,vation that photolysis of the simple PFPA ester methyl 4-a_ido-
t~tl~n.--.,ub~ c in ~ 'thiophene yielded methyl (N-cyclohexyl q :.. ,.o)-
tetlhfl.-~.ulJf ..~ t~ as the only CH-insertion product that could be isolated.
Example 6
This Example is a control e,.y~filll~ for Example 5.
A solution of 23.2 mg P30T in 0.8 mL xylene (in the absence of 1) was
treated with the amine 3 as d~ibcd in Example 5. No azide au~ulytiull was observed in the
IR syC~llulll of the resulting film, indicating that no hl.-olyolalion of the amine 3 occurred.
Therefore, a first filnrtjon~li7~inn of P30T with a ~u~ A such ais 1 is
n: y in order to perform a second funrtinn~li7~ion with the amine 3.
Example 7
In this F~---F'n. we investigate the use of electron-beam 1;11.~,.~,.1.,~ to
.Q~c-....l.l;~l. both cross-linking of a polymer (i.e.,PS) and the h~tlud--.,lion of NHS active ester
20 groups in the polymer in a single step. General reactions are ill ~ in Scheme 10.
A solution of 50.2 mg of PS and 4.0 mg of NHS ester 1 (8 % w/w) in 1.0
mL xylene was spin-coated on a silicon wafer as de~..lil-.,d generally in Example 1. The film
was dried for 35 minutes at 90C and exposed to an electron beam using a scanning electron
~..ic.uscoye (SEM) (....~r~t.--~,d by JOEL-SEM, Peabody, Maryland)""oA;I;-~ for el.~:~.on
25 beam lithography. Nabity et al., Rev. Sci. Instrum. 60:27 (1989). The electron beam was used
to "draw" micron-sized patterns (in the form of eight five-line patterns and a pattern of five
circles of different J;a.-l~,te.s) on the film. The exposed film was ~d~ ,lo~l" by dipping in
xylene for 35 seconds, rinsing in isoyluyyl alcohol for 10 seconds, then drying with a stream of
nitrogen, thereby yielding a "developed" film 2. The film 2 was phutuglal,hcd using an optical
30 Il.i. loscol,e, yielding results shown in FIG. 3A.
In FIG. 3A, the widths of the lines in each five-line set were 0.1,0.2,0.5,1.0,
and 2.0 ~m. Each successive five-line set was obtained with an hl~l~s~ electron-beam
intensity relative to the yl~dh~g set. In the top row of sets, the elc~l.un beam were 50, 60, 70, and 80 ~/cm 2. In the bottom row of sets, the cl~l-un beam ;-~ were
35 90,100,110,and 120 ~Icm2. The line width of each of the circles was the same: 0.5 ~m.
The electron-beam intensity used to "draw"the circles was 60 p~/cm2.
The lines and circles shown in FIG. 3A are c0l~.l~5~1 of filn~tjon~li
polystyrene 2 (i.e.,polystyrene m~lec~ sc having active esters covalently bonded thereto).
WO 94/22053 PCT/US94/02982 ~
21~85~
,
Referring now to Scheme 10, the film 2 (after ob~ the pl~otv~ c
shown in FIG. 3A) WflS immersed in a solution of 2.5 mg of amino-lluo.~,sc~;l. (co. .l u~ l n
and 8.3 mg of Et3N in 1.5 mL of EtOH for 4 hours so as to introduce an easily visible
IIUOI~ "t marker at the active-ester sites on ~he film. Afterward, the film was washed with
5 EtOH, i...--.~ in EtOH for 2 hours, rinsed with EtOH, then air-dried to yield the film 8.
The film 8 was observed under a fluo,~ ,ncO Ill;CIUD~,Op6 (Carl Zeiss, Germany) ~Iu;l.~,r~t
with e~;lluû.O~.~ optics. The --.c-vscopc was fitted with a lluol~;ll filter set (e~rit~ n
wavelength 450-490nm, emission wavelength 515-565 nm). The [luo-~nce patterns shown in
FIG. 3B were observed.
Since the lluolO;,~onl patterns ~.h~ t~J by film 8 (FIG. 3B) were
c~ k..t.l to the patterns observed of the r. ~. ti~ i7~i pol~ .ne. 2 shown in FIG. 3A, we
ei that coupling of the lluol~,3ce,~cG marker in film 8 occurred only at sites (film 2) on
the polymer to which active esters had been previously coupled.
In FIG. 3B, filnrti-n~li7~ n of PS had occurred at a dosage of about 50
15 ~/cm2 in the film. We also found that cr~cclinkin~ of PS alone required about 90 ~C/cm2.
ExamPle 8
This Example is an exl,er;l..G..tal control for Example 7. Compolln~s are as
shown in Scherne 10.
A PS film was prepared without the NHS ester 1 but otl..,.~.;~ treated as
20 d ,;l~d in Example 7. The film was exposed to an electron beam and d~ ,lop~ as
rl~ r I ;l~d in E~cample 7 and photog~ LGd using an optical ...,.,.~,~opc. The PS film was then
treated with amino-nuG,wn,.,;-- 7 and observed under a nuo,w~nce l usco,~. No
nuu.~..cG pattern was observed. Therefore, prior ~ lirl-.-~ l of the NHS active ester 1 to
the PS -'~ '-- was required for the ~ f ~ 1 of the amino-nuol~cc... label 7
25 to the polymer.
Example 9
This Example is similar to Example 3 e~ccept that, in this F - , 'e, we
"drewrmicron-sized patterns on a P30T film c~"~-;-.;.-~ NHS active ester using an electron
beam. The general reactions are shown in Scherne 11.
A solution of '~5.7 mg of P30T and 1.8 mg of NHS ester 1 (7 % w/w) in 0.6
mL of xylene was spin-coated on a silicon disc and dried at 60C for 30 minutes. The resulting
film was exposed to an electron beam as described in Example 3 so as to "draw"micron-sized
patterns on the film (line width 0.5 ~lm; beam intensity 20 ~/cm ). The film was then
"d~ ,lo,u~" by dipping in xylene for 10 seconds, rinsing in isûplu~,yl alcohol for 10 seconds and
35 drying under a stream of nitrogen gas to yield the film 9. The film was then ;~ he~l in a
solution of 1.5 mg of amino-fluorescein 7 and 6 mg of Et3N in 1 mL of EtOH for 4 hours.
The film was then washed with EtOH, immersed in EtOH for 1 hour, washed again with
EtOH, then air-dried to produce the sample film 11.
_ WO 94t22053 PCT/US94/02982
~3~ 21 58SS O
The sample film 11 was observed and pholo~;la~ ed using a lluo.~nce
llli.,lUsco~,c e~ ,ed with a ~ L~ ;..r filter set (excit~tir~n wavelength 510-560nm, emission
w~ ,lc,l~lh ~ 590 nm), yielding the results shown in FIG. 4A. The same sample film w~s
- observed and rho~ng.~ 1 using the lluol~c~.lc~ u~co~ e ~ ,e;l with a lluo.~.
filter set (eY~ wavelength 450-490 nm, emission wavelength 515-565 nm) yielding the
results shown in FM. 4B. As can be seen, ~ lly identical patterns were observed
having strong lluul~.l~c at both the .1.~1~...;..r. PYS '-~ .~;uL,-Iglll (FIG. 4A) and the
nuo-G~ excitP~ n wavelength (FIG. 4B).
P30T alone is strongly llucilG~.lt at the ,l.nlc",;t~r P~ritP~ n ~
10 but only weakly lluol~.~l at the lluoles~;ll ercit~tinn ~ le.lgll-. (This is why the films in
this Example were observed using a ll,~~ r filter set and a lluol~;l. filter set; strong
lluo~ucc observed at the nuole;lce~ll PYl`itsl~ n ~ UI~,U~ 1ll would nccG~Iily be due to the
presence of other ~ Pc than just P30T.) In FIGS. 4A and 4B, the observed strong
nuOl~.l~C at both the .I.o~l~...;.~r. and nuolG~,hl eYrits~ n wavelcn~;llls indicates that
15 nuul~cc;n became attached to the regions exposed to the electron beam (FIGS. 4A and 4B).
Examl~le lO
This Example is a control for Example 9.
A P30T film (without the active ester 1) was exposed to an electron beam
(intensity 30 ~Clcm~, line width 0.5 ~an), de~ulv~,~, then treated with amino-lluu~... 7 as
20 desrrihed in Example 9. The micronffized patterns ~drawn" on me control P30T film were
identical to the patterns in Example 9. When the control film was ~ ..~i using alluo~G~nce lll._luscol,c, strong nuol~c~,.,.,e was observed at the .I ~I-...:..r. G " ' ~n
~ .Iglll(FlG. 4C), but only weak lluol~ c was observed at the nu~l~v~. e~
wavelength (FIG. 4D).
The results indicate that ~ lly no lluol~;ll 7 became attached to
P30T in the âbsence of activated ester groups. Therefore, the presence of NHS active ester is
required in order to obtain any ~ l covalent coupling of the nuOl~;ll 7 to P30T.Examnle l l
In this FY- r'~. we filn~ti~nsl;7~1 poly(3-octylll.io~hv..e) (P30T) as shown
30 in Scheme 12.
WO 94/22053 PCTIUS94/02982
~ 1~ 8~ 4
S F F 2. hY 254 nrn
or ~lectron
~ N~b~ ,0
poly~3-octylthloph~n~ N--o~
NH2CH2~N~ ~ F F
F F 3 5 ~NHCH2~N~
~ F F
~ ~--NH
an^~noac~amldo- ~ 5. ~, ~
nuorGscGl~ 3 ~ ~
OH
Scheme lZ
In this scheme, a solution of NHS-PFPA (2) and P30T (1) was spin-coated
onto the surface of a silicone substMte in a manner as generally ,I;c.,..~,~l above, then e~posed
30 to a reaction-energy source such as 254-nm photons or an electron beam to yield the
fi~nrtion~li7pfl P30T (3). S~lbs~qnPnt reaction of the filnrtjon~li7PA P30T 3 with the PFPA
co. lvu ~1 4,."vduc~d 5. Reaction of 5with s- olluo~.,h~ yieldedll-,v.~lu-
labeled P30T (6).
While the invention has been described in c~nnP~tirn with p.~,f~ ,d
35 c~..l,o~ and multiple examples, it will he understood that it is not limited to those
embo~limPntc On the contrary, it is intended to cover all alternatives, ...od r- ' ~mC~ and
equivalents as may be included within the spirit and scope of the invention as defined by the
~ ,.ded clsims.
