CA2158550A1 - Chemical functionalization of polymers - Google Patents

Abstract

Methods for covalently modifying polymeric substances, and various so modified polymers, are disclosed. The methods require a functionalizing reagent comprising molecules each having a nitrenogenic group and a functionalizing group. The functionalizing reagent molecules arc brought into reactive proximity to the polymer molecules and exposed to a reaction-energy source such as photons, electrons, or heat, which converts the nitrenogenic groups to nitrene intermediates that covalently react with -CH, -NH, -OH, -C=C-, C-C, and other groups to cause nitrene addition or insertion of the funtionalizing groups to the polymer molecules. Functionalization can be via a one- or multiple-stage process.

Description

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 3 _ 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 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 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.

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 ~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~

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 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

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.

~ 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.

Claims (23)

1. A method for funtionalizing a substance comprising polymer molecules, the method comprising the steps:
(a) providing a substance comprising polymer molecules, the polymer molecules having chemical moieties each capable of undergoing an addition reaction with a nitrene;
(b) providing a first functionalizing reagent comprising molecules each having a nitrenogenic group and a first functional group;
(c) adding the first functionalizing reagent to the polymeric substance so as to bring the molecules of the first functionalizing reagent into reactive proximity to the polymer molecules; and (d) while the polymer molecules and the molecules of the first functionalizing reagent are in reactive proximity, exposing the molecules to a reaction-energy source so as to convert the nitrenogenic groups to nitrenes that undergo addition reactions with the chemical moieties on the polymer molecules, thereby covalently bonding the first functional groups to the polymer molecules.

2. A method as recited in claim 1 wherein step (a) comprises providing a substance comprising polymer molecules possessing chemical moieties selected from a group consisting of-CH, -NH, -OH, -C=C-,-C-C-, SiO-H, Si-OH, and Si-OSi moieties.

3. A method as recited in claim 1 wherein, in step (a), the polymer molecules are selected from a group consisting of saturated polylefins, acrylics, polystyene, polystyrene. analogs, unsaturated polyolefins, polyimides, polyesters, conjugated polymers, conducting polymers, inorganic polymers, organic metals, organometallic polymers, polysaccharides, and polypeptides.

4. A method as recited in claim 1 wherein the reaction-energy source is selected from a group consisting of energized electrons, energized ions, photons, and heat.

5. A method as recited in claim 1 wherein step (d) comprises exposing preselected portions of the substance to the reaction-energy source so as to functionalize only said portions of the substance.

6. A method as recited in claim 5 wherein exposing preselected portions of the substance to the reaction-energy source is performed by impinging a beam of electrons on the preselected portions of the substance.

7. A method as recited in claim 1 wherein, in step (b), the nitrenogenic groups on the molecules of the first functionalizing reagent are azide groups.

8. A method as recited in claim 7 wherein step (b) comprises providing a first functionalizing reagent selected from a group consisting of aryl azides, alkyl azides, alkenyl azides, alkynyl azides, acyl azides, and azidoacetyl compounds.

9. A method as recited in claim 8 wherein step (b) comprises providing a first functionalizing reagent from a group consisting of functionalized perfluorophenyl azides having the structure;

wherein X is selected from a group consisting of CN; CONH2; CHO; CO2CH3; COCH3; NO2;
CO2H; COCl; CO-imidazole; CONHS; CH2OH; CH2NH2; COCH2Br; N-maleimido; NH-biotinyl; CONH-R, wherein R is a polypeptide; CONH-X-S-S-Y-NH-biotinyl, wherein X and Y
are spacer atoms; and CONHS-SO3Na.

10. A method as recited in claim 9 wherein step (b) comprises providing a first functionalizing reagent selected from a group consisting of perfluorophenyl azides derived from 4-azido-2,3 5,6-tetrafluorobenzoic acid.

11. A method as recited in claim 10 wherein step (b) comprises providing a first functionalizing reagent selected from a group consisting of N-hydroxysuccinimide-functionalized perfluorophenyl azides.

12. A method as recited in claim 1 wherein step (b) comprises providing a first functionalizing reagent comprising molecules in which the first functional group is selected from a group consisting of carboxyl groups, acid halides, acyl imidizoles, thioesters, p-nitrophenyl esters, alkyl esters, alkenyl esters, alkynyl esters, aromatic esters, amides, free alcohol groups, alcohol groups esterified to carboxylic acids, haloalkyl groups, maleimido and other dienophilic groups, aldehydes, ketones, and sulfonyl halide groups.

13. A method as recited in claim 12 wherein step (b) comprises providing a first functionalizing reagent on each molecule of which the first functional group is constrained from reacting with the nitrenogenic group.

14. A method as recited in claim 1 further comprising the steps, after step (d), of providing a second functionalizing reagent comprising molecules each having a second functional group reactive with the first functional group; and exposing the first functional groups bonded to the polymer molecules to the second functionalizing reagent under conditions conducive for causing the second functional groups to undergo chemical reactions with the first functional groups, thereby covalently bonding molecules of the second functionalizing reagent to the polymer molecules.

15. A method as recited in claim 14 wherein the first functional group is an ester and the second functional group is selected from a group consisting of hydroxyls,primary amines, and secondary amines.

16. A method as recited in claim 14 wherein the molecules of the second functionalizing reagent each further comprises a third functional group.

17. A method as recited in claim 16 wherein the third functional group comprises a polypeptide.

18. A method as recited in claim 17 wherein the third functional group comprises an enzyme.

19. A method as recited in claim 18 wherein the third functional group comprises horseradish peroxidase.

20. A method as recited in claim 16 wherein the third functional group comprises a moiety selected from a group consisting of hydrophilic, hydrophobic, surface-active, carconigenic, mutagenic, diagnostic, therapeutic, fluorescent, and radiolabeled moieties.

21. A method for functionalizing a substance comprising polymer molecules, the method comprising the steps:
(a) providing a substance comprising polymer molecules, the polymer molecules having chemical moieties each capable of undergoing an addition reaction with a nitrene;
(b) providing a first functionalizing reagent comprising molecules each having a nitrenogenic group and a first functional group;
(c) providing a second functionalizing reagent comprising molecules each having a second functional group reactive with the first functional group;
(d) adding the first functionalizing reagent to the second functionalizing reagent under conditions conducive for an addition reaction of the first functional group with the second functional group so as to cause molecules of the second functionalizing reagent to bond to molecules of the first functionalizing reagent, thereby forming a reaction product of the first functionalizing reagent bonded to the second functionalizing reagent;
(e) adding the reaction product to the substance so as to bring the molecules of the reaction product into reactive proximity to the polymer molecules of the substance; and (f) either subsequent to or simultaneously with step (e), exposing the substance to a reaction energy source so as to convert the nitrenogenic groups, on molecules of the reaction product in reactive proximity to the polymer molecules, to nitrenes that undergo addition reactions with the chemical moieties of the polymer molecules, thereby covalently bonding molecules of the reaction product to the polymer molecules.

22. A substance having polymer molecules functionalized according to the method recited in claim 1.

23. A substance having polymer molecules functionalized according to the method recited in claim 21.