Effective Inhibitors of L{Emagglutination by Influenza Virus Synthesized Ftom Polymers Having Active Ester Groups

Reodnted from Journal of Mediciaal Chemistry' 1995'38' Cop''righr @1995 by rh" A;;;;; til;i.J3*i"ii-..a-np.i"t"a by iermidion of the cop5night owner. 4179

Effective Inhibitors of l{emagglutination by Influenza virus synthesized ftom Polymers Having Active Ester Groups. Insight into Mechanism of Inhibition

Mathai Ma:nmen, Georg Dahnann, and George M. Whitesides* Departmentof Cfumistry,Harvard lJnioersity,12 Orford.Street, Cambridge, Mossothusetts 02138

ReceiuedJune 23, 19956

Highly effective sialic acid-containing inhibitors of influettzavirus X-31 were synthesized using p.f"vflf-t"cryoyloxy)succinimiael (pNfu), a polyper preactivatedby incorporation of active ester gt;;;r.' poiymers containing two and threL different componentswere prepared by sequential i"actioo of pNAS with twJ and three amines, respectively. This preparation of co- and terpolymers was synthetically more efficient than rnethods involvin_g_copolymerization of different monomersand gave polymers that were more easily comparedthal tlosgsgnerat-ed of pNAS) had a by copolymerization. Poiymeir i" this study (pre_pat"-atory a_sin-gleb-atct-r .onrturf degreeof polymerization (DP * 2000) indprobably h-ada distribution of comp_onents that was more random than analogous polymets prepared by copolym-erization. Use of g-jtycosides of sialic acid made it poisiblsto investigate inhibition by diffeplt polymers at without artifacts due to the actioll-of temperatures ranging from 4 to'gg "C oC_hydrolytic neuiaminidase. The inhibitors were, in general, more effective at 36 than at 4 "C. The hemagglutination (HAI) assaywas used to measure the value of the inhibition constant d{AI eachpolymer. The value of^(H for the two-componentpolymer containi,ng2|% sialic acid on p"fv.."ylamide backboneui 4 'C was 4 nM (in terms of the sialic acid moieties present in "solution) and was approximately 50-fold more effective than the best inhibitors previously described and 2b-fold^*or. effective than the best naturally occurring inhibitor. The most effective inhibifor sy"ttresized in this work contained LTVobenzyl amine and 20Vosialic acid on a polyacrylamidebackbone, and its value of {il was 600 pM at 36 "C. Approximately 100 pofytir*r tft"t differed in one or two componenis were assayed to distinguish between two ii*itittg mechanisms for inhibition of the interaction between the surfaces of virus and L"ytftt*ytes' high-;ffinity binding through polyvalency, and steric stabilization- The results suilest itr"t bolh mechanismspLv rn impottant role. The system comprising polyvalent infriUitors of agglutination of earttrrocytes by influenza provides a-system that may b-euseful as a model for-Inhibitors of other patLogen-host interictions, a large number of which are themselvespolyvalent.

-600 per particle) Introduction (2-3 copies/100nm2; or copies virus on the surface of influenza virus.l2 SA is a nine-carbon designed We describe a series of polymeric inhibitors sugar that occurs with high density (22-25 molecules/ vin s X-31"1-4to to inhibit the attachment of influenza 10b nm2) on the surface of the mammalian cell.15-17SA potency of the best mammalian cells. The inhibitory terminates many of the extracellular saccharidic por- (/f,N : inhibitor described here 0.6 nM; calculated on tions of mammalian glycoproteins and glycolipids' the basis of sialic acid groups present in solution) is We assayed our inhibitors using the hemagglutination about 2 orders of magnitude better than that of other inhibition (HAI) assay.l8 Interaction between virus and synthetic inhibitors (of which the best are liposomes erythrocytes in solution leads to the formation of an presenting sialic acid on their exterior surface, I{N : extended, cross-linked gel (hemagglutination)' TrheIIAI 200 nM)s or natural inhibitors (of which the best is assay enabled us to measure the ability of an inhibitor Itj* : 100 nM)6 (Figure 1)- equine crz-rn&croglobulin, to prevent hemagglutination by preventing the interac- Nature uses polyvalency-the cooperative association tion between the surfaces of virus and erythrocyte; we of a receptor (or aggregate of receptors) containing quantitated the effectiveness of the inhibitor with an containing multiple recognition sites $rith a molecule inhibition constant, Iti*. We defin. ffN as the low- ligands-in a number of bio- multiple compiementary est concentration of ligand that successfully inhibits logical contexts, including many interactions between hemagglutination. All values of XfN reported here pathogen andhost.?'8 The attachment of influenzavims are in terms of the concentration of SA groups in to its target cell is a model system for such pollrualent solution (whether free in solution as monomeric units, interactions; this attachment is mediated by multiple attached covalently to the backbone of a soluble poly- simultaneous interactions between viral hemagglu- mer, or associated noncovalently with a structure such tinine-11 (IIA) and cellular sialic acid2'12'13(Neu5Ac,la as a liposome). SA). HA is a glycoprotein that occurs with high density We used SA-containing polymeric inhibitors (glyco- polymers)ls that are based on polyacrylamide (pA) or * addressed. Author to whom correspondence should be (pAA). this strat- @Abstract published inAduance ACS Abstratts, September 15, 1995. poly(acrylic acid) We have described 0022-2623/95/1838-41?9$09.00/0@ 1995 American Chemical Societv 4f80 Journal of Medicinal Chemistry,7995, Vol. 38, No.21 Mammen et al.

ery previously,5'20-22and it has also been used explicitly Scheme 1. Two strategies: (A) Copolymerizationo and by others including Matrosovi ch,23,z+Rny,2,-zt Paulson,28 (B) Preactivationb Bednarski,2eand Lee.30 The monomeric a-2-methyl-1tr- NeuAc (a-MeSA)l4 inhibits virally induced hemagglu- A. COPOLYMERIZATION tination at 2 mM 0tT* - 2 mM). The value of ^4N for the most effective polymer reported in the present work oX*r*,1 is 600 pM. This highly effrcient inhibition of hemagglutination reflects the ability of these polymers to \-i*r"rl"# prevent interaction of two biological surfaces: those of the virus and the erythrocyte. We believe that this \rl rux, mechanism of action reflects a pharmacologically rel- J r; n1rux, evant property,3l but we have not poly- assayed these 2) R2NH2 mers for activity in uiuo. 3) NH3 Polymeric versus Monomeric tnhibitors. A sig- B. PREACTIVATION nificant body of work has developed around the syn- oa thetic modification of monomeric SA in order to improve Von\ hv its ability to inhibit virally induced hemaggluti- t( AIBN o ttu1ion.20'32-34The most successful of these attempts gave derivatives of SA w"ith I4 equal to 3.7 pM,323 o pNAS orders of magnitude better than cr-MeSA. \ ONHS ON Both natural polymeric (in- Y and synthetic inhibitors In1 ruH, o^ottHs hibitors containing multiple copies of SA) are much more successful at inhibiting hemagglutination R'NH2- other organic Aroup than are their (hydrophobrc, bulky, monomeric counterparts. A number of polymeric charged, etc.) systems have yielded values of .fN less than 1 o (medium-pressure ptM.20,22.2s'37The mechanism by which a polymer acts Irradiation Hg arc lamp through a c.uar:z vessel for 6 h) of a mixture of substituted acrylamides and a rairca. - as an inhibitor of hemaggiutination, and the reasons initiator (AIBN) yields a substituted poiy'acrylamide Hea:ir: a that it is more successful than its monomeric counter- solution ofN-(acryloyloxy)succinimide (NAS.r and AIB\ ir: be::zer= part, is understood in broad outline,2l but not in detail. to 60 "C yields the preactivated "parent" pollrner p\AS surs.- The monomeric inhibitor must compete with SA groups quent sequential treatment with different, amj.nes 1.e.,:-- d:. N-substituted polyacryiamide. on the surface of the erythrocytes for SA-binding sites of IIA on the surface of the virus; it is at an intrinsic (iv) 1efrnd inhibitors of hemagglutination that are more disadvantage in this competition since the interaction effective than those already known. between virus and erythrocyte is polyvalent. Using Activated Polymers to Construct Copoll'- The polymer can inhibit hemagglutination by at least mers. Various physical properties of the pollrcer two conceptually independent mechanisms (Figure 2).21 (conformation in solution, conformation on the surface First, it may act by high-affinity interactions based on of the virus, conformational flexibility, net charse. size. the possible entropic advantage of many SA groups on degree of branching, degree of hydration, and h1'circ' the same polymer binding to the surface of the virus; phobicity) might, in principle, influence the successof these potentially cooperative interactions may lead to inhibition by polymers. We have previously attempted efficient competitive inhibition of viral attachment by to correlate some physical characteristics of the competing with the polyvalent cooperative interactions polymer-especially the structures of the side charn-.of between virus and cell. Second, the polymers may act the polymer and of the group linking the SA to the by steric stabiiization:3s SA groups on the same polymer backbone-with their effectiveness in inhibiting hemag- may bind noncooperatively (with similar affinity to that glutination.22 This previous work was based on a of monomeric SA) to the surface of the virus, but bring copolymer of two or more differently N-substituted with them a large, water-swollen polymer that may acrylamides: one was l/-substituted with a spacer- make sites on the surface of the virus adjacent to those group terminated with SA, 2; the other was.l/-substi- bound to SAphysically inaccessible to SA on the surface tuted w.ith the test-group, R2 (Scheme 1A). We defrne of the cell. This second type of inhibition is a type of mixed inhibition (a combination of competitive and noncompetitive inhibition). The relative importance S:.iNHso and detailed mechanisms of these two types of inhibition are currently not understood in sufiicient detail to design new polymers that are more effective inhibitors than o those already known. tl We had four goals in this work: (i) to develop a /v synthesis for polymeric inhibitors based on pA and pAA H that is of more use than that used previouslV (copolymerization of a mixture of acrylamide monomers); (ii) to lf,'ut the ratio of side chains containing Ri to the explore the role of temperature in inhibition of hemag- total number of side chains in the polymer (eq 1); for glutination by SA-containing polymers; (iii) to under- example,.XsAis the proportion of side chains with linkers stand the relative importance of the two major mech- terminated in SA. The "standard polymer" is one in anisms of inhibition for these polymers through a guided which /SA : 0.20. All comparisons between polymers choice of different side chains on the pA backbone; and are ultimateiy referenced to the standard polymer. Effectiue Inhibitors of Hemagglutinatr'on Journal of Medicinal Chemistry, 7995,Vol. 38, No- 21 4181

-r-d> IncreasingEtfectiveness

pA pA(10%SA)-C-glycoside pA(20%SA)-C-glycoside 4'%benzvl) o n1atr0%sA)-c-slvcoside-lo1,ro*sA)-o-srvcosidelI pA(eo%sA' I a-rure-siarosioe I r^'fr?:'iH;:lv,'.11i?'jf)"' 0) I r divarentl I ilonou"rentInhibitorr t;bil;;l Lioosomes(SA) I c | I I I I a wmada vz r rr .10-s 1O-3'[rrHn 1O-4 10-6 rc-7 1O-B 10-9 10-10, lrr*rrl llr+rlrtlrrrnrrr lrrlrrrr lxfAr tnl fuurrrriirlrrrr 0 1O-3 10-4 1O-5 10-6 10-7 10-B 10-9 10-1 T ma aa a m 6 = .iJtvttu.,or"o",n. (u lu.;inl I | bovine-l czMr. .irin. o2rnl z I fetuin humano,2M guineapig o2M Figure l' Values of Klul for natural and synthetic inhibitors of hemagglutilation based on SA.-Monomeric inhibitors (opeD (solid boies), previously studii)d polymeric inhibito." (h.t"hed bote"), and the blst polymeric inhibitors ftom the present study lor".j'i"" slown. Syntnetic polymeric inhibitors are based on liposomes, polyacrylic acid) (pAA) or_polyacrylauride(pA). The width of the boxes ripresent ihe uncertainty in the values (generally + a factor of 2; that is, + I well iD serial dilution). o characterizing different acrylamide monomers (most of J*R' _ tRNH:l the amines used were commercially available). lR'NHzl + IR'NHd+ NH, Exploring the Role of Temperature Using C- Glycosides. Changes in the temperature at which the (Copolymerization) lPreactivation) HAI assay is performed might, in principle, change the of lffN. Temperature influences sev- Copolymerization (Scheme 1A) possibly introduces measured value of the HAI assay: the preferred distribu- two uncontrolled variables into the experiment (Figure eral variables of the polymer in solution;the rate 3): (i) unknown differences in rate constants for copo- tion of conformations of the polymer to the surface of the virus; lymerization among differently N-substituted acryla- of adsorption formation and the preferred mides might result in differences in the distribution of the rate of structure of conformations of the polymer on the SA along the backbone of the polymer; (ii) the length, distribution the ability of a layer of polymer polydispersity, and tacticity of the polymer might surface of the virus; and of a particle to stabilize that particle change as the side chain was altered, due to differences on the surface It has been difficult to examine the influence in rates and stereochemistry of polymerization. sterically. on hemagglutination using substrates In the present study, we used a different strategy to of temperature of SA, because neuramini- prepare polyrneric inhibitors based on addition of amines containing O-glycosides (NA) cleaves these groups rapidly. Assays of to polymers having activated esters of carborylic acid dase38-40 contain SA connected to the backbone groups as side chains (Scheme 1B); this strategy has inhibitors that polymer by O-glycosidic linkages could only be been used previously,36'37but with different motivations. of the performed 4 'C (degradation of the inhibitor occurred We refer to the synthesis of different polymers by this at acceptably slowly at 4'C). The physiologically more stratery as "preactivation". Polymerizing N-(acryloyl- 'C- temperature is approximately 36-37 In oxy)succinimide yielded the preactivated polyrner poly- relevant this study we used C-glycosidesof SA (1) becausethey lN-(acryloyloxy)succinimidel (pNAS). We then con- to the action of NA. We collected parallel verted the activated esters on pNAS to amides by are resistant and 36 oC for each polymer. sequential reaction with two amines: frrst with 1, a data at 4,19, molecule containing a SA group connected to a flexible The Hypotheses Guiding the Suryey of Side spacer (1 : R1NH2,Scheme 1); then with R2NH2,where Chains. There are at least two independent mecha- we varied R2 depending on the hypothesis being tested. nisms that might contribute to the inhibitory action of Preactivation was synthetically and mechanistically these polymers: high affinity polyvalent binding and (Figure polymers more attractive than copolymerization for several rea- steric stabilization 2). We synthesized sons: (i) the characteristics of the polymer backbone- incorporating different side chains from different chemi- length, polydispersity, tacticity-were kept constant (the cal classes(charged, polar-uncharged, and nonpolar), to same batch of pNAS was used for all experiments); (ii) try to distinguish between these two mechanisms of us the distribution of SA groups along the backbone, inhibition. No single structural modification allowed although not controlled, was the same for all pollnners, to identify clearly which of the two mechanisms is more poly- since introduction of the SA moiety was the first step important for inhibition of hemagglutination by in modifoing pNAS; (iii) the distribution of SA groups mers. We expected that the results in aggregafe would, along the backbone was probably more uniform than however, be informative. A guided survey of side chains from copolymerization (addition of the sterically bulky allowed us both to explore mechanisms of inhibition by (R2NHz, molecule I to adjacent sites along the backbone of the polymers and to determine the side chain polymer was less likely than addition to nonadjacent Scheme 1) on a pA backbone that promoted the stron- sites); (iv) preactivation was more convenient syntheti- gest inhibition. cally than copolymerization, since constructing many We had five hypotheses: (i) Incorporating a number different polymers did not require synthesizing and of negatively charged groups along the backbone might 4182 Journal of Medicinal Chemistry, 7995, Vol. 38, No. 21 Marnmen et aI.

A. ATTACHMENT A. DIFFERENT RATES OF CHAIN PROPAGATION SA

fi w o ( ui.r. $ fficen+ OV, Virus-Cell lAHxz nxn-.zoa \9 Oa, Association €> \/ GrowingChain "y' HzN B. INIHIBITIONOF ATTACHMENT Propagation '.:r[:r*:GW+GMonovarenta\ W &{/\- Termination A-

POSSIBLECONSEOUENCES ffdfl",:,,,:'FG& d,>6ff\ i S\ "\" Potyvarentft ;f*i[:'G&4 rbr,r"lhg OESIRED UNOESIRED Uniform distribution on a i) Non-uniform ii) Short polymer long polymer backbone distribution backbone Figure 3. (A)Acrylamidesubstituted ri'rth a stencalil'bulky Figure 2. (A) Influenza virus attachesto its host cell through molecule(for example,2)might add more slou'I1'to the growing polywalentinteraction of HA with SA. (B) Inhibition: mono- polymericchain that does unsubsittutedacr-r'lamide. tB) meric moleculescontaining SA inhibit attachmentby competi- Consequently,at leasttu'o deviations frorn the desiredpolyner tive inhibition, whereaspolymeric molecules inhibit attach- (a long one,with SA distributedevenll. alons the backbone) ment by either (i) high-afftnity binding through polyvalency are possible:(i) the bulk1' substt:utedacrl'larnldes may (competitiveinhibition); andor (ii) steric stabilization of a poiymerizeinto blocksafter ail the ursubstttutedacrylamide surface(mixed inhibition). is reacted;(ii) the bulkl' subsirtutei acn'ianrdesmay form short polymerswhen pol5menzedrr the absenceof the change the conformation of the poiymers by favoring unsubstitutedacrylamides. long persistance lengths (thus favoring an extended, rigid-rod geometry).3s We expected extended polymers molecular weight of the hydroll-zed sample rvas 146 kD to be less effective than random-coil polymers in steric (M*) (DP t 2000). The distnbutron of moiecular stabilization because these polymers have less confor- weights was namow (Mr: 75-ir-Dand ll/z = 279 kD).42 mational entropy. (ii) Placing bulky substituents on or Sequentialaddition of tw'odifferent amines to pNAS near the backbone might also lead to longer persistance yreldedthe finai functionalizedpollmer rScheme1B). lengths for the polymer and decrease steric stabiliza- We first acided0.20 equiv per activated ester of 1. In tion.35 In addition, bulky substituents on sites adjacent the amine. R:NH:. in 'crowd" most instances.we added second to SA might the sialic acid and thus hinder excess. In some instances, Rr\TI: s'as not added in accessto the IIA on the viral surface. (iii) Adsorbing a excess,and we quenchedthe residual. unreacted active negatively charged polymer onto the surface of a virus esters of the polymer with ammonia. might result in increased stabilization by electrostatic The number of equivalents of 1 determtned 7s'r in the repulsion of the negatively charged surface of the final polymer. To establish the relationship between the erythrocyte. Conversely, adsorbing a positively charged number of equivalent of I and /sA, w€ performed a set inhibitor on the surface of the virus might promote of experiments in which pNA,S was reacted w-ith quanti- eiectrostatic attraction between the polymer-coated ties of I that varied from 0.05 to 1.2 equiv per activated virus and the erythrocyte. (iv) Adding hydrophobic side ester, then was quenched with ammonia. We used two chains might increase the aftinity of the polymer for the sorts of analysis on the resulting polymers (Figure 4): virus by inducing secondary hydrophobic binding inter- (i) lH NMR spectroscopycompared the integrated area actions. (v) Using cross-linking agents in small propor- under the peaks characteristic of the SA (e.g., the tions might increase steric stabilization. These agents protons of the acetylated nitrogen) to the area under might lead to branching of the polymer, or gel formation, the peaks characteristic of the polymer (e-g., the and thus increase the effective degree of polymerization, -(CHCH2)- protons); and (ii) combustion analysis DP. We have recently found that increasing DP gener- provided estimates of relative values oflsA directly from ally increases the effectiveness of the polymers.4l analysis of sulfur.a3 For a small number of equivalents, was generally smaller than predicted theoretically; Results and Discussion XsA as the number of equivalents was increased, there was Synthesis of Polymers. We prepared all polymers stronger agreement between the measured value of UsA used in this study from a single batch of pNAS (Scheme and that predicted theoretically. For most of the 1B). We characterized the molecular weight of this polymers used in this study, we used 0.20 equiv per "parent" polymer by complete hydrolysis to pAAfollowed activated ester; our analyses suggested that 1sA was by gel filtration chromatography (GFC): the average equal to -0.17-0.18 in these polymers. Effectiue Inhibitors of Hemagglutination Journal of Medicinal Chemistry, 1995, VoI. 38, No. 21 4183

9 10- I - - rl 1.0 ,,o'- 1.0 a , rltf a a rl a 0.8 *1at1u) ? 0.8 t I neofellcal O I \^ d' Vo 0.6 to'8 I 0.6 a xflfi,tor I z!f;"ror .' O- 0.4 I a'6 I I 0.2 t6 0.2 7 to- I r Illllll | | rrrr.rl,,,,",.1 0.0 e60 1 10 n2 rO3 rO4 Timeof Pre-incubation(s) 0 0.2 0.4 0.6 0.8 1 1.2 Figure 6. Using pA with /sA : 0.2, the value for /f,A {o), H,ri,:Hl,sA per deireased with increasing time of preincubation (the time for activated ester which virus and inhibitor mix, prior to addition of erythro- lH in this study (shown Figure 4. We estimated zsAin the polymers using NMR cytes). The time of preincubation used analysis (O) and combustion analysis for sulfur (O) as a by a dashed line) was 1800 s (30 min). function of the number of equivalents of SA added per activated ester in the polymer (see text for details). SA need to bind approximately half the HA on the surface of the virus in order to inhibit hemagglutination. Measuring 4N requires three components in PBS (at varying SideView buffer: erythrocytes, virus, and inhibitor concentrations). As we performed the assay, virus and allowed to preincubate for 30 \'/ inhibitor were mixed and ,/ min at a specified temperature. Erythrocytes were then was allowed to Top View added and mixed, and the system @@@,@@@ incubate for t h. The order and timing of addition of Incubatefor t hr the three components strongly influenced the results: ser\ t pell^1 if we added the inhibitor last, all inhibitors were ineffective; if we added the erythrocytes last, the time o@oo@@ that the polymer and virus were allowed to preincubate strongly influenced AfAI Gigure 6). We have not protocols which the virus was added last. ,Y"'nii,,",',' 't - I I i,, i,o. ria examined in \ rl The erythrocytes usually required at least 30 min of \ oC ' | 'l 'l \ 'l '! lxr:rar '| 'l \ ' incubation at 36 (or t h at 4'C) to form stable pellets or gels; we allowed experiments at all temperatures to l-{-,a f.--7 f{2 N:/l ' oC, | | f%:^\l ifu*-a: F {i''^' incubate for t h before reading the plates. At 4 the I rlffit l\usKl trFJl patterns of inhibition did not change as \Mevaried the time of incubation from 1-12 h. At higher tempera- K2/

Table 1. Inhibition of Hemagglutination by Polymers That Do these differences to the different methods of preparing Not Contain SA the polymer, we can only guess the precise mechanistic polymer afN qrur origin of these results: the highly effective inhibition (for pAA 450b 100 by polymers prepared by preactivation all values pAA 750b 210 of XsA) rnay have been because of a more uniform pAA 1200b 230 distribution of SA along the backbone of the polymer pAA 30006 400 (that is, fewer wasted SA moieties) than was possible pAA 40006 1000 pA 2006 > 13000 using copolymeization; the rapid decreasein effective- pA 200,6l\Va acid 300 ness of the polymers prepared by copolymerization at MeO-PEG(2000)-OH' > 100000 high values of Xsa rnay have been because the DP for > MeO-PEG(5000)-OH" 100000 these polymers decreased with increasing /sA (Figure MeO-PEG(5000)-NH2' > 100000 .)9\). '14I"I is given in terms of total concentrationof monomeric The polymers prepared previously by copolymenza- units. o MW (kD); pAA: poly(acrylicacid); pA = polyacrylamide. 'Number of monomericunits shown in parantheses;MeO-PEG tion had values of .(u that decreased linearly with = o-methoxypoly(ethyleneglycoi). DP;+i a doubling of DP resulted in a halvrng of the value of ^(ru. The average value of DP for these copolymers t o-1 was 4000,2r,22where the average value of DP for the polymers in the present work was 2000. If all else was o 36oc,12 h constant (especially the temperature and the value of 10-8 we the polymers from the present study a 36oc,1h .fsA), expected to have values of ^(H 2-fold higher (worse) than the ! 19oc,rh polymers prepared previously. All data indicate sig- 10-6 tr aoc,rh nificantly more effective inhibition (values of KfN xfrrtrvrl were 100-fold lower) than obtained previously. between the values of .(il obtained 1o-4 The differences at different temperatures increased with increasingXsA. The lowest value of.fa we observed in this study was 200 pM (at : 0.50 at 36 'C with al2-h incubation). lo-2 fa 0.4 These two observations suggest that kinetically slow

L processes of some sort (probably equilibration of the Figure 7. I{I is plotted a functionof the proportionof SA polymer over the biological surfaces involved) increase in the polymers,t'A. Data were collectedfor polymers(pre- effectiveness of the polymers in inhibiting hemaggluti- paredby preactivation;Scheme 1) at three differenttemper- nation. (tr), (o), (a). atures: 4'C 19"C and36 "C Datafrom previous Effectiveness of the Polymers Decreases with studiesfor polymers(prepared by copolymerization)at 4 "C Charge. We performed a number of are also shown (l). Incubation times were generally t h. Increasing Longer incubation times (12 h, O) yielded lower values of experiments in which we changed the side chains of SA- 14I"I. containing polymers (in all cases,XsA equals 0.20) and correlated these changes to differences in .ft The - SA-containing polymers (seebelow). We found, as have standard polyrner (7s't 0.20, R2NHz- NHs) provided others,22that pA does not inhibit hemagglutination at a point of reference. We divided the changes into groups any concentration up to 100 mM. The pAA, perhaps based on the chemical nature of the side chain and the surprisingly, inhibits hemagglutination: pAA with lower hypothesis we were testing. In the first experiments, molecular weights were more effective than pAA with we varied the charge of the side chain group (R2NHz, higher molecular weights. These polymers may interact Scheme 1) from *1 to -3 using the molecules shown in -3, nonspecifically vrith the surface of both the vims and Figure 8. As the charge changed from 0 to the the erythrocyte. fhey may then inhibit hemagglutina- effectiveness of the polymers decreased;a change from tion mainly through steric stabilization. 0 to *1 resulted in an even sharper decrease. Since the groups introduced charge welg relatively large, the Dependence of KI- on t'A: Qualitative Differ- that ences from Previous Studies. We found previously effects of charge and size ot ^4' were not cleanly that SA-containing polymers varied in effectiveness decoupled by this one experiment. The different influ- -1 (4{AI) as a function of /sA in the polymer (Figure 7). encesof the *1 and side groups, which are of similar The shape of the curve that describes the relationship size, suggested, however, that size alone is not fully for the between these between /sA and AT* is characteristic of many of the responsible observed differences polymeric inhibitors of hemagglutination that we have differently charged polymers. previously examined: as fsA increases from 0, the In an attempt to distinguish the effects of steric bulk effectiveness increases rapidly, forms a plateau, then from those of charge, we sJrnthesized three sets of decreasesrapidly as.fsA exceeded-0.5. In this work, polymers in which the charge on the side chain was we varied the value of Xsa from 0.0 to 1.0 (Fieure 7). varied relatively independently of the size of the side The pattern of inhibition was qualitatively different chain: (i) Using 0.10 and 0.20 equiv of charged side than before: the initial increase in effectiveness with chain per activated ester, we obtained polymers that increasing XsAwas not as sharp; the plateau was less were of similar size to the standard polymer, but still pronounced; and the decreasein effectiveness occurred substantially charged. (ii) Hydrolyzing the polymer at highervalues offl and was less pronounced(in some rather than quenching it with ammonia (Scheme 1A) cases, it did not occur at all). Although we attribute yielded polymers with carboxylates as side chains Effectiue Inhibitors of Hemagglutination Journal of Medicinal Chemistry, 1995, Vol. 38, No. 21 4185

10-9 XRz o.0.1 -tr-- -tr- - r- 0.2 fi-7 \ linear t- -CONH,^O-MOx x,Haltrul 1 1.0 ,/'n^\ ) ()oa 10-5 XrjAt (nn) - >ov -CONHJ LJ

/-a I \ v-] ,o) \o 1o-3 _ao^"_ro.*o_/ 10-1-2 sidechain "n"rn",on Na+ rich PBS K* rich / / I.p \\ o- buffer buffer -ruH / I *t{o- Figure 10. Polymerscontaining 18-crown-6 (selective for K+, - _NH ?( *i, *"jo- :^tt1 o- O), 15-crown-5(selective for Na+, O), or linear triethylene ".fiHrur", glycol (l), polymer (tr) o^o- Yo and the standard were assayedin o^o- phosphatebuffered saline (PBS) or in buffersenriched in either Na* or K*. PBScontained 8 g of NaCl, 0-2g of KCl, 1.1g of Figure 8. groups (CONHR2, Charged as side chains where Na2HPO4,and 0.2 g of KHzPOa.Na+-rich buffer was madeby R2 is charged) (Scheme proportions 1). Various of charged side replacingall K* in PBS with Na+; K+-rich buffer was made chains, were (O), (a),1.0 (O). XR', assayed: 0.1 0.2 by replacingall Na+ in PBS with K+.

over pA; the negatively charged, isosteric polyrnerswere rc-7 less effective than the standard polymer. In another experiment relating charge to .(ru, we rfnl 0vr) incorporated Na+- and K+-selective crown ethers in the side chains (Figure 10), and compared the values of 10-5 Aj* to that of a polymer containing linear triethylene glycol (not selective for a specific cation). We used buffers enriched in either Na+ or K+. These buffers 0 0.2 0.4 0.6 0.8 1 were equivalent to phosphate-buffered saline (PBS) in Numberof equivalentsof 1 ionic strength and pH, but only a single type of cation was used: Na+-rich buffer contained only Na+, and K+- Figure (MW 9. EDC and NHS are usedto couple1 to pAA rich buffer contained only K*. By examining the values 750kD). EDC,NHS and I wereused in a 1:1:1ratio. The value of for these polymers in the three buffers (PBS, for ^4w (o), is plotted a function of the number of equiva- 4w lents of 1 per free carboxylicacid of pAA. Na+-rich, and K+-rich), we hoped to detect a change in the inhibition that could be correlated with the selectiv- (primary amides and carboxylates are approximately ity of the crown ether. We observed that in all instances isosteric). Alternatively, coupling pAA to I using EDC in which the polymer selective for Na+ or K+ was and NHS yielded similar SA-containing polymers \,\rith assayed in Na+-rich or K+-rich buffer, respectively (conditions carboxylates as side chains. (iii) Incorporating crown in which the polymer presumably had a net ethers yielded polymers whose charge could be titrated positive charge), the polymers became less effective than by varying the concentration of the cation that the side when assayedin PBS. Furthermore, when the polymer chain could specifically complex. The results of these selective for Na- or K+ was assayed in K+-rich or Na+- three experiments follow. rich buffer, respectively, the polymer became more effective than when assayed in PBS . Since we do not Incorporation of lower proportions of the charged side krrow the dissociation constants for the complexes chains resulted in a less pronounced but similar rela- between the crown ethers and the cations, we do not tionship between the charge and the value ^f;N of know the magnitude of the positive charge on the (Figure 9). polymer in the different buffers. These observations are The parent polymer pNAS was reacted with 0.20 consistent qualitatively, however, with those from ex- equiv of I and then hydrolyzed to give a negatively periments in which positively charged side chains are charged, isosteric analog of the standard polymer. The incorporated into the polymer (Figure 10): positively value of ^(u of this charged analog was g00 nM, charged polymers are much less effective inhibitors of which was approximately 300-fold less effective than the hemagglutination than neutral polymers. standard polymer. In an analogous experiment, we Effectiveness of the Polymers Decreases with coupled I to pAA (IVIW 750 kD) using EDC and .l/- Increasing Size of the Side Chain. We varied the hydroxysuccinimide. We did not characterize the actual size of uncharged side chains using four different extent of incorporation of I into the polymer. We varied families of molecules: primary amides, linear ethylene the equiv of I from 0.0 to 1.0 (Figure 9); the form of the glycols, linear polyols, and cyclic ethers (Table 2). The plot relating the number of equiv of 1 and the value of trends for all four families were the same: the effective- 4 was similar to that observed for the pA polymers ness of the polymers decreasedwith increasing size of (Figure 8). In this experiment, the introduction of the side chain (Figure 11). We represent the size of the charge introduced zo increase in size of the side chains side chain by the number of atoms (C, H, N, O) 4f86 Journal of Medicinal Chemistry, lgg1, VoI. 58, No. 21 Mammen et al.

Table 2. Inhibition of Hemagglutination by Polymers with Polar Side Chains, CONHRz, that Differ in Size aa Segmentor Porymer Molecule Number of Atomsd Identification ,*+4f. ) 1,,^, )A (Figure 12) ffLftr -coNH2 o Sizeof Side-Chain Small Medium -coNH_,Ar.,rJ i\r12 l2 Extentof Steric Low Medium Crowding -CONH -,Ôn T2 Figure 12. Extent of steric crowding around the sialic acid (cylinders) increases with size of the side chain (represented -CONH-,Ô.^--OH r9 u by curves of different length). Large side chains may introduce sufficient steric crowding to hinder accessto the HA receptors -CONH ,-* -.,r. O O -.ÔH 26 on the surface ofthe virus. OH -coNH_A.orl 16 f

. ^-q .= lu " - OH OH H l H -coNH.-v\rôH "l*.--^,O.-,,-.-^ O-et Z 28 OH OH :-;" "^ rt o 1que KHA| (M) A ,Qn i -coNLJo . vl 16 .^-E i-- rt ,l- .CONH i rl ) o l9 ?? rôr I \JI A1 (o TI \) 0 0.05 -CoNli-/ or----o- 01 01s c.2 l' ao b--) Figure 13. The value of /(la increased as the proportion of ,b 48 cross-linking agent 2 ,7'\ was increased. We assayed the \o 'C -6g1r111-,2ôa,o polymers at various temperatures: 4 "C (O), 19 "C (O), 36 J (tr).

" The totai number of atoms in -CONHRT. experiments, the effectivenessof the polymers decreased as the size of the side chain increased,we 10's tentatively conclude that steric stabilization plays a role in inhibi- -ra tion of hemagglutination b1-polymers. \ro 10-8 Another possibly relevant consequenceof increasing the size of the side chains was to crowd the sialic acid 1o-7 stericaiiy and therebl'tunder accessto the IIA receptor j (Figure K|Ar(M) .-'+l 12). We do not know if steric crowding was the l 1 - major mechanism by' which the effectiveness of the 10-6 c t=__\l polymers was decreased \___\_"- as a function of the size of the Jl ug side chain. In an experiment reported previously,22the oj\ 10-s3 iength of the linker connecting the SA to the backbone of the polymer was increased. This experiment attempted to decreaseany effect of steric crowding. The 10-4 observation was, however, that polymers 01020304050 the containing long linkers were less effective than polymers qrith Number of Aloms in CONHR2 linkers of intermediate length. Figure 11. As the sizeof the sidechain CONHR2 (CON(R2)z) Low Levels of Cross-Linking Increase the Ef- was increasedthe value of .dil increased.See Table 2 for identifrcationof the labels.Foui familiesof neutral sidechains fectiveness of Pol5rmers. We added various propor- are shown: primary amides(O), cyclic ethers (C), polyethylene tions (0.02-0.20) of diamine 2 to cross-link the esters glycols(l), and linear polyols (D). With all families of side within the same polymer and between polymers (Figure chains,the sizeis roughly proportionalto the number of atoms 13). While low levels of cross-linking increased the in CONHR2. effectiveness of the polymers, high levels had the opposite effect. We reported previously that the ef- contained in CONHR2: we did not calculate molecular fectiveness of polymers correlates positively with DP. volumes. As the number of atoms in the side chain was Introducing low levels of cross-linking may be equiva- increased, the value of KIN decreased. The backbone lent to increasing the effective DP. Interestingly, as the of polymers containing bulky side chains may be less temperature increased, the differences between the (fewer flexible available conformations) than those with polymers differing in X'also increased. At this time, small side chains. Furthermore, such polymers may we do not know the precise mechanistic origin of these adopt more extended (rigid-rod) conformations. In results. either case, the ability of such poiymers to stabilize a Ilydrophobic Side Chains Increase the Effec- surface sterically probably decreases. Since in these tiveness of the Pol5rmers. Different nonpolar (hy- Effectiue Inhibitors of Hemagglutination Journal of Medicinal Chemistry,1995, Vol. 38, No.21 4187

Table 3. Inhibition of Hemagglutination by Polymers with performed at a single temperatr:re). As the temperature HydrophobicSide Chains, CONHR2 of the assay was increased, the effectiveness of the Kl"t (nM) polymer generally increased to varying extents. These aa. observations can be rationalized in two ways: (i) If the Hydrophobic side chain Equivo A tg oc 360C HAI assay measured equilibria between the virus, polymer and erythrocybe (that is, the systern was at -coNHz 0.8 38 3.8 1.9 thermodynamic equilibrium), then the dependence on -coNH^---* temperature suggeststhat there is a positive change in 0.1 r6 entropy during the process of inhibition. The value of I{ varied, however, as a function of time of incuba- -coNHo 0.1 10 1n 5 tion and time of preincubation, suggesting that the system was not at thermodynamic equilibrium during -coNHCH2o 0.05 10 2 2 the assay. (ii) If the system was not at thermodynamic 0.1 l0 I 0.6 equilibrium, then we expected that the effectiveness of 0.2 /4 t0 2 inhibition should increase with increasing temperature 0.4 10 o A (the rate of most processes increase with increasing -coNHs temperature). The dependenceof the value of .IfN on 0.i l0 2 temperature did not follow similar trends with all polymers. For example, the value of^44 decreasedby -coNHcHzq a 0.1 10 L a factor of two for polymer containing the 20VoSA (ys^ - 0.2) as the temperature of the assay was increased o Number 'C oC, of equivaientsof R2NH2per activatedester used from 4 to 36 compared with a factor of 60 for the in the synthesis(Scheme 1). polymer containing LA\VoSA 12rsn- 1.0). These differences drophobic) side chains were incorporated into the poly- in the temperature dependence of the values of mer in an attempt to increase the affinity of the polymer 4* for polymers containing different side chains for the o'ir',rc.32'34'a5We expected that interactions imply that there are differences in the enthalpy of between the hydrophobic groups and hydrophobic sites activation for inhibition using different polymers. on the surface of the virus (including the lipid mem- Conclusions brane) would enhance binding of the polymer to the viral surface. Due to decreasing solubility with increasing This work confirms that incorporation of SA groups extent of incorporation, we were limited generally into the side chain of polyacrylamide strongiy enhances (except in the case where R2NHz - benzyl amine, BA) its ability to inhibit hemagglutination mediated by to values of ,KfN less than -0.1. In the synthesis of Influenza A X-31. Polymers prepared using the strategy the polymers (Scheme 1), addition of 0.1 equiv of the of preactivation reproducibly gave values of ^4N ttrat hydrophobic R2NH2 w&s followed by quenching w-ith were more than 2 orders of magnitude better than ammonia to convert all unreacted activated esters to polymers prepared previously by copolymerization. We primary amides. have suggested possible reasons for this enhancement Adding certain hydrophobic side chains yielded poly- (Figure 3), but we have not explored these hypotheses mers that were more effective than the standard further in this work. polymer (Tabie 3). With BA the solubility of the The effectiveness of the polymers generally increased polymer remained satisfactorily high with extents of with increasing temperature. Since the system was incorporation as high as 0.4: we varied 7BAfrom 0.0b probably not at therrnodynamic equilibrium (the periods to 0.40 and found that asXBAincreased, the effectiveness of preincubation and incubation both influenced the -- of the polymer increased. Even polymers with XBA measured value of ,fN), this observation is consi.stent 0.05 improved the effectiveness of the polymer by a w'ith the increase in rates that are expected with factor of 2 atroom temperature. This increase was low increasing temperature. Differences in the relation- but reproducible. The hydrophobic groups added bulk ships between temperature and the value of ^(w for to the polymer and, in the absenceof a balancing effect, different polymers imply that there are differences in were expected to decrease the effectiveness of the the enthalpy of activation for inhibition by these poly- polymer. The case of the BA group provided an example mers. positive of a effect that more than balanced the negative Steric Stabilization Is an Important Mechanism effect of bulk. in Preventing Interaction of Surfaces. Side chains The effectiveness of the polymers increased by a factor that we expected would increase the likelihood oC of steric of 4-20 as the temperature was increased from 4 to stabilization generally increased the effectiveness of the 36 "C. In contrast, the effectiveness of the standard polymer; side chains that we expected would decrease polymer increased by a factor of 2. As temperature the likelihood of steric stabilization generally decreased increases, the. importance of hydrophobic interactions the effectiveness of the polymer. Taking clues from the (which are ofrben entropically driven) generally in- area of steric stabilization (also known as polymeric creases: that the effectiveness of the polyrners increased stabilization) of coiloidal suspensioDS,3stwo character- significantly with increasing temperature was therefore istics may be responsible for changing the effectiveness reasonable.a6 of a polymer in stabilizing a surface sterically (Figure Measurements of Klw at Different Tempera- L4). First, if the conformation that a polymer adopts tures. We took parallel data for all side chains at 4, in solution becomes less 'C random-coil and more rigld- 19, and 36 (the entire assay-preparation of virus, rod (that is, if the ratio of its persistance length to blood and buffer, preincubation, and incubation-was contour length approaches 1), then the polymer is less 4188 Journal of Medicinal Chemistry, 1995, VoL 38, No. 21 Mammen et aI.

effective at inhibiting interaction between virus and cell. Methods of increasing the affinity of the polymer for the surface of the virus beyond that of the standard polymer may include: increasing the affinity of a single SA- HA interaction by using tighter binding derivatives of Virus/ >a- SA; incorporating hydrophobic groups that bind adven- titiousiy, or by design, to secondary sites on the surface of the virus; incorporating tight-binding inhibitors of NA. Methods of increasing the ability of the poiy'rner Rigid Rod Random Coil Collapsed ffi@ of the virus stericaliy may to stabilize the surface include: increasing DP, and incorporating a feu very water-swollen side chains. Polyva- Flexibility in large, flexible and Solution- + +++ +++ tent inhibitors of the interaction between influenza and erythrocyte provide a system that may be useful as a Number of Sites model for inhibitors of other pathogen-host interac- of Attachment + + +++ tions.

Steric Stabilization + +++ + Experimental Section Figure 14. The conformationof the polymer on the surface Materials and Methods. All solvents and reagents were of the colloidalparticle (the virus is modeledhere as a colloidal purchased from Aldrich and used t'ithout further purification particle) partly determineshow effectivelythe polymer stabi- uniess otherwise noted. Reaction mrxtures were stirred lizesa suspensionof theseparticles. The polymersshown here magnetically and monitored by thin-iayer chromatography on haveequal length. Relative to a random-coilpolymer with an gel precoatedglass plates (Merck). Flash column chro- intermediatenumber of sites of attachment,changing either silica was performed on silica gel60rzs+(230-400 mesh, the conformationto a rigrd rod (and keeping the sites of matography Merck) using the solvents indicated. Size-exclusion chro- attachment constant),or increasingthe number of sites of E. was performed on Biogel P2 resin or Sephadex attachment(and keepingflexibility constant),decreases the matography water as the eluent. Ion-exchange chro- effectiveness'ofthe polymerin G10 using distilled stabiiizinga colloidalsuspen- performed (H* form) sion. matography was on Dowex 50W-X8 cation-exchange resin. Vortexing of copolymer samples was performed with a Fisher vortex Genie II. Dialysis was able to prevent a colloidal suspension from flocculating. performed using Spectra/Por Moiecularporous Membrane Second,if the polymer binds to the surface of the colloid (28.6-mm cylinder diameter, 2-mL volume, molecualr weight cutoff -12 kD). The protected dipeptide GIU(OtBu)Glu(OtBu)- by a very large number of attachment points, then the oC) polymer is collapsed onto the surface and is again not OtBuaT and compound 120 (mp 240 were prepared as previously able to prevent effectively a colloidal suspension from described. points were obtained using a Mei-temp ap- flocculating. The polymers that are effective at steric All melting paratus and were uncorrected. Proton (lH) and carbon (13C) (a stabiiization of a colloidal suspension are long high NMR spectra were measured on a Bruker AII-400 MHz or AIV[- value of DP), well solvated, have a large number of 500 MHz NMR spectrometer. Chemical shifts are reported available conformations, and are random-coils. in ppm relative to TMS for the lH NMR spectra, and relative High-AfEnity Binding Is an Important Mecha- to DMSO-de at 39.5 ppm for the 13Cspectra. nism in Preventing Interaction of Sur{aces. Side N-Acetylneuramintc acid was obtained from extraction of chains that we expected would decrease the affinity of edible Chinese swiftlet's nest. Erlthrocytes from 2-week-old used within the polymer for the surface of the virus generally chickens were purchased from Spafas Inc., and 48 h of shipment. Influenza virus (X-31) was obtained from decreased polymers; the effectivenessof the side chains the laboratory of Professor John Skehel. The PBS used in the that we expected would increase the afiinity of the IIAI assays was prepared from 80 g of NaCl, 2 g of KCI, 11 g polymer for the surface of the virus generally increased of NazHPOa, and 2 g of KHzPOT in 1 L of distilled HzO. This the effectiveness. As discussedin the previous section, 10x stock solution was diluted as needed with distilled HzO we expect that increasing the affinity of the pol5nner and then adjusted to pH 7.2 with 1 N NaOH. for the surface of the virus by increasing the number of N-(Acryloyloxy)succinimide. Acryloyl chloride (33.4 g, attachment points DBy, at some critical number of 30 mL, 369 mmol) was added dropwise to a stirred solution of .l/-hydroxysuccinimide (42.5 g, 369 mmol) and triethylamine attachment points, becomes an ineffective in stratery (4I.0 g,56.5 mL,409 mmol, 1.1equiv) in CHCIg(300 mL, 1.23 oC, building better inhibitors of hemagglutination" We M) at 0 "C. The solution was aliowed to stir for 3 h at 0 suspect that between two polymers of the same length then washed with water (2 x 300 mL) and brine (300 mL), and having the same affinity for the surface of the vi:ms, then dried over MgSOa, and recrystalized from a solution of the one with a lower proportion of tighter binding ethyl acetate/hexane(1:1) to give 46.1 g (273 mmol) colorless 1H monomers will be more effective; that is we suspect that crystals in 72Vcyield (mp 69 "C; lit.36mp 69.5 "C). NMR (400 d 6.78 (:CHz, dd, 1H. ,J2s"^= 17-2 Hz, incorporating small proportions of very tight binding MHz, CDCIg): lr,",," = 0.78 Hz),6.40 (:CH2, dd, lH, .JBg"-= 17.2H2, #.i" = analogs of SA will be an effective strategy of building 10.88 Hz),6.24 (CH:, dd, lH, J8.i, : 10.88 Hz, ,Fso,,.: 0.78 highly effective inhibitors of hemagglutination. Hz), 2.94 (CHzCHz,s, 4H). Some of the polymers from this study are the best PolyfN-(acryloylory)succini'nidel (pNN). A mixture inhibitors of hernagglutination that have been reported. of N-(acryloyloxy)succinimide (3.15 g, 18.6 mmol) and AIBN Of the polymers whose side chains are 20VoSA, our most (20 mg,0.007 equiv) in benzene(150 mL) was heated at 60'C effective inhibitor at 36 "C is one where l\Vo of the side for 24 h. After the solution was cooled to room temperature (rt), precipitate This precipitate was frltered chains are benzyl groups (Ri : CHzPh, 1; a white formed. Scheme (THF, mL). - and washed four times with tetrahydrofuran 30 4AI 600 pM). In conclusion, we suspect that poly- Dryrng in uacuo afforded poly[N{acryloyloxy)succinimide] mers that interact strongly with the surface of the virus, (3.08 g, 18.2 mmol,98Vo)as a white fluffy solid. The polymer or those that effectively stabilize that surface, will be was taken up in dry THF (300 mL), vigorously stirred for 3 Effectiue Inhibitors of Hemagglutination Journal of Medicinal Chetnistry, 1995, Vol. 38, No- 21 4189

days, filtered, and dried in uacuo. IR36 (Nujol mull): 3340, (6) Pritchett, T. J.; Paulson, J. C. Basis for the Potent Inhibition of 3200,1730, 1660,1210, 1070 cm-l. Influenza Infection by Equine and Guinea Pig a2-Macroglobulin. 264. Deter:nination of Molecular Weight of pNAS. A solu- J. Biol. Chem. L989. 9850-9858. (7) Cohen, J. A.; Williams, W. V. Molecular Aspects of Reovirus- tion of pNAS (2 mg) in 6 N HCI (aq, 1 mL) was heated at 105 oC Host Cell Interaction. Microbiol. Sci. 1988, 5, 265-270. for 24 h in a sealed tube. After cooling to rt, the pH was (8) Collono, R. J. Virus Receptors: the Achilles' Heel of Human adjusted to 7.2 with 1 N NaOH and water (1 mL) was added. Rhinoviruses.Adu. Exp. Med. Biol. 1992,312,6I-70. The solution was exhaustively dialyzed against buffer (pH (9) Boycott,R.;Klenk, H.D.;Ohuchi, M. Cell Tropism of Influenza 7.2: L50 mM NazSOa,10 mM NazHPO+). The solution of pAA Virus Mediated by Hemagglutinin Activation at the Stage of Virus ViroLogyLgg4, 203,313-319. thus obtained was (Waters Entry. analyzed by HPLC flltrahydrogel (10) Mazanec, M. B.; Coudret, C. L.; Fletcher, D. R. Intracellular Linear) using the following standards: pAA of MW 130 kD, - Neutraiization of Influenza Virus by Immunoglobuiin A Anti- 390 kD, and 1100 kD. Found: Mp 129 kD, Mr.i :75 kD, Hemagglutinin Monoclonal Antibodies. J. Virol. 1995, 69, 1339- Mw - 146 kD, Mz: 279 kD. 1343. Preparation of Mixed Polymers Containing SA. A (11) Wiley, D.C.; Skehel, J. J. The Structure and Function of the Membrane Glycoprotein of Influenza Virus. solution of | (243 in triethylamine (TEA, 0.5 mL) was Hemagglutinin 4mol) Annu. Reu. Biochem. 1987, 56, 365-394. (6.78 added to a stirred solution of pNAS g, 1.22 mmol NHS (12) Weis,W.;Brown, J. H.; Cusack,S.; Paulson, J. C.; Skehel,J. J.; ester) in dimethylformamide (DMF, 20 mL). The solution was Wiley, D. C. Structure of the influenza Virus Hemagglutinin 'C stirred at rt for 20 h, heated at 65 for 6 h, and then stirred Complexedwith its ReceptorSialic Acid. Nature 1988,333, 426- at rt for an additional 48 h. This procedure yielded tlrrestoch 43r. (13) solution of preactivated polymer with : 0.20 (the stock Paulson, J. C.; Sadler, J. E.; Hill, R. L. Restoration of Specific ZsA Myxovirus Receptors to Asialoerythrocytes by Incorporation of solution contained pmol pmol 2.0 SA/mL and 8.0 NHS/mL). Sialic Acid with Pure Sialyltransferases. J. Biol. Chem. L979, AII other polymers that contained different proportions of SA 254.2120-2124. were prepared analogously by changing the amount of 1. The (14) The correct IUPAC name for this compound is 5-acetamido-2,6- next step varied depending on the polymer. anhydro-3, 5-dideoxy-2-C-[3-[(2-aminoethyl )thio Jpropyl]-o< ry thro - For Polymers Containing SA on a pA Backbone manno -nonulosonic acid. (15) V. F.; Knox, R. J.; Nordt, F. I.; Regan, D. H. Red (R2NHz : NHs). The stock solution (600 4.8 pmol Seaman, G. aL. NHS) Cell Aging. I. Surface Charge Density and Sialic Acid Content (concentrated was added dropwise to NHaOH aqueous,1.5 mL) of Density-Fractionated Human Erythrocytes. Blood L977, 50, and stirred at rt for 12 h. 1001- 1011. For Pol5rmers Containing SA and One Other Compo- (16t Van, B. W. P.: Smets, L. A.; Emmelot, P. Increased Sialic Acid nent (R2NH2 = NHs). R2NHz (48 7.rmol,10 equiv) and TEA Density in Surface Glycoprotein of Transformed and Malignant (48 pmol, 10 equiv) were added to a stirred stock soiution (600 Cells. General Phenomenon. Cancer Res. 1973, 33,2913-2922. (17) Levine. S.; Levine, M.; Sharp, K. A.; Brooks, D. E. Theory of the pL,4.8 /mol NHS, 1 equiv). The resulting solution was heated Electrokinetic Behavior of Human Erythrocytes. Biophys. J. at 65 "C for 6 h. Any remaining NHS ester was quenched by 1983.42, r27-r35. addition of NFIaOH (concentrated aqueous, 1.0 mL) follwed by (18) Rogers, G. N.; Pritchett, T. J.; Laue, J. L.; Paulson, J. C. stirring at rt for 12 h. Differential Sensitivity of Human, Avian and Equine Influenza For Polymers Containing SA and TV*o Other Compo- A Viruses to a Glycoprotein Inhibitor of Infection: Selection of Receptor-SpecificVariants. Virology 1983, I 31, 394 - 408; W. H. nents. R2NHz (0.48 pmol for yBz, :0.1) (0.48 XR' and TEA O. Tech.Rep. Ser. L959,64.7-32. prmol)were added to a stirred stock solution (600 4L, 4.8 pmol (19) Roy, R.; Tropper, F. D.; Romanowska, A. New Stratery in 'C NHS). The resulting solution was heated at 65 for 6 h. Glycopolymer Syntheses. Preparation of Antigenic Water-Soluble NH+OH (concentrated aqueous, 2 mL) was added and the Poly(Acrylamide-co-p-Acrylamidophenyl B-lactoside). Bioconl u - oC solution heated at 65 for 6 h and then stirred at rt for an gate Chem. 1992, 3, 256-261. (20) additional 12 h. Sparks, M.A.; Williams, Ii W.; Whitesides, G. M. Neuramini- dase-Resistant Hemagglutination Inhibitors: Acrylamide Co- For all three classes of polymers above, the resulting polymers Containing a C-glycosideof N-Acetylneuraminic Acid. mixture was then dialyzed exhaustively against distilled HzO, J. Med. Chem. 1993.36, 778-783. then NHaCI (1 M), again with distilled HzO, and then lyoph- (21) Lees,W.J.; Spaltenstein,A.; Kingery, W. J. E.; Whitesides, G. ilized to yield a white powder. Tlpical recovery of SA was > M. Polyacrylamides Bearing Pendant a-Siaioside Groups Strongly 80Vo. Inhibit Agglutination of Erythrocltes by Influenza A Virus: Multivalency and Steric Stabilization of Particulate Biological Systems.J. Med. Chem. 1994,37,3419-3433. Acknowledgment. The authors gratefully acknowl- (22) Spaltenstein, A.; Whitesides, G. M. Polyacrylamides Bearing edge many fruitful discussions with George Sigal. We Pendant a-Sialoside Groups Strongly Inhibit Agglutination of thank Professor J. J. Skehel for kindly providing us with Erythrocytes by Influenza Virus. J. Am- Chem. Soc. 1991, 113, 686-687. Influenza virus X-31. G.D. was supported by Fors- (23) Matrosovich, M. N.; Mochalova, L. V.; Marinina, V. P.; Byra- chungsstipendium of the Deutsche Forschungsgemein- mova, N. E.; Bovin. N. V. Sl.nthetic Polymeric Sialoside Inhibi- (DFG) tors of Influenza Virus Receptor-Binding Activity. FEBS Lett. schaft and M.M. by an Eli Lilly predoctoral 1990. 272. 209-212. fellowship (1994). This work was supported by the NIH (24) Matrosovich, M. N.; Gambaryan, A. S.; T\rzikov, A. B.; Byramova, Grant GM30367 and GM 39589. The NMR facilities at N. E.; Mochalova,L. V.; Golbraikh, A. A.; Shenderovich,M. D.; Finne, J.; Bovin, N. V. Probing of the Receptor-Binding Sites of Harvard were supported by NIH grant 1-S10-RR04870- the Hl and H3 Influenza A and Influenza B Virus Hemagglu- 01 and NSF grant CHE88-14019. tinins by Synthetic and Natural Sialosides.Virology 1993,196, 111-121. (25) Supporting Information Available: A table of values of Gamian, A.; Chomik, M.; Laferriere, C. A.; Roy, R. Inhibition of oC Influenza A Virus Hemagglutinin and Induction of Interferon IfN at 4,19, and 36 for all 100 polymers is available on by Synthetic Sialylated Glycoconjugales. Can. J. MicrobioL 1991, request (6 pages). Ordering information is given on any 37, 233-237. current masthead page. (26) Roy, R.;Zanini, D.; Meunier, S.J.; Romanowska,A. Solid-Phase Synthesis of Dendritic Sialoside Inhibitors of Influenza A Virus References Hemagglutinin. J. Chem. Soc.,C hem. Commun. 1993, 213- 2\7 . (27) Roy, R-; Andersson, F. O.; Harms, G.; KeIm, S.; Schauer, R. (1) Krug, R. M. TheInfluenza Viruses; Plenum Press: New York, Synthesis of Esterase-Resistant9-0-Acetylated Polysialosidesas 1989. Inhibitors of Influenza C Virus Hemaggiutinins. Angew. Chem. (2) Paulson,J. C. Interactionsof Animal Viruseswith Cell Surface t992, 104, 1551-1554. Receptors.lnThe Receptors;Conn, P.M., Ed.;Academic Press: (28) Unverzagt, C.; Kelm, S.; Paulson,J. C. Chemical and Enzymic New York, 1985;pp 131-219. Synthesis of Multivalent Sialoglycopeptides. Carbohydr. Res. (3) Kilbourne,E. D. BuIl.W. H. O. 1969,41,643. t994, 163, 167-170. (4) Kilbourne, E. D. Influenza; Plenum MedicalBook Co.: New (29) Spevak,W.;Nary. J. O.;Charych,D. H.; Schaefer,M.E.;Gilbert, York,1987. J. H.; Bednarski, M. D. Polymerized Liposomes Containing (5) Kingery-Wood,J. E.;Williams,K W.; Sigal,G. B.;Whitesides, C-glycosidesof Sialic Acid: Potent Inhibitors of Influenza Virus G. M. The Agglutination of Erythrocytesby Influenza Virus is in uitro Infectivity. J. Am. Chem. Soc. 1993, 115, 1146-1147. Strongly Inhibited by LiposomesIncorporating an Analog of (30) Lee, Y. C. From X-Ray Crystallography to Cure of Flu? Trends Sialyl Gangliosides.J. Am. Chem.Soc. 1992, 114,7303-7305. Glycosci. 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(31) Mochalova, L. V.; Tuzikov, A. B.; Marinina, V. P.; Gambaryan, (41) Sigal, G.; Whitesides, G. M. Manuscript in preparation, 1995. : : A. S.; Byramova, N. E.; Bovin, N. V.; Matrosovich, M. IV. @D M; = number-average molecular weight IA',M,,4lNi Mw : Synthetic Polymeric Inhibitors of Influenza Virus Receptor- weight-average molecular weight : LN',M,2,E.N',M; M2 l- Binding Activity Suppress Virus Replication. Antiuiral. Res. averagemolecular weight : 2l'l,M,3DI'tr,M,2. 1994,23, 179-190. (43) The amount of water in the sample was calculated from the (32) Knowles, J. R.;Weinhold, E. Design and Evaluation of a Tightly ratios of carbon to hydrogen and nitrogen. Using the corrected Binding Fluorescent Ligand for Infleunza A Hemagglutinin. J. mass for the polymers, we established a relation betrveen the Am. Chem. Soc. 1992. 114, 9270-9275. percentageof sulfur and fA. A similar relationship between the (33) DeNinno, M. P. The Synthesis and Glycosidation of N-Acetyl- number of equiv of 1 and XsAwas obtained using the ratio of S neuraminic Acid. Synfiesis 1991, 483-493. to N from combustion analYsis. (34) Toogood, P. L.; Galliker, P. K.; Glick, G. D.; Knowles, J. R. (44) In the HAI assay a dilute suspension of chicken erythroclles rs Monovalent Sialosides that Bind Tightly to Influenza A Virus. added to the wells of a 96-well microtiter plate. These n'ells have J. Med. Chem. 199L.34,3140-3143. a volume of 2504L and a conically shaped baserthe erythrocltes (35) Everett, D. H. Basic Principles of Colloid Sciznce;Royal Society settle by gravity and concentrate to a point ta'pellet". easily of Chemistry: London, 1988; Chapter 3. identified visually). If influenza virus is present rn suspenslon (36) Pollak, A.;Blumenfeld, H.;Wax, M.;Baughn, R. L.;Whitesides, with the erythrocltes, the virus cross-links the erythrocl-tes bv G. M. Enzyme Immobilization by Condensation Copolymeriza- interaction of viral HA with cellular SA groups and forms a loose tion into Cross-Linked Polyacrylamide Gels. J. Am. Chem. Soc. gel. The erythrocycesin such a gel do not settle to a pellet:- they 1980,102,6324-6336. ire "hemagglutinated" and appear homogeneously red. An (37) with Schnaar, R. L.; Y. C., L. Polyacrylamide Gels Copolymerized inhibitor that binas to the virus prevents formation of the gel; Active Esters. A New Medium for AIfinity Systems. Biochemistry it inhibits hemagglutination. 1975.14.1535-1541. (45) Sparks, M.A.;Williams, K. W.; Lukacs, C.; Schrell, A.; Priebe, (38) F. Rollins, B. Madren, L. I{- Anti-Influenza Virus Hayden, G.; S.; G.; Spaltenstein, A.; Whitesides, G. M. Synthesis of^Potential the Neuraminidase Inhibitor 4-guanidinoNeu5Ac2en Activity of Inhibitors of Hemagglutination by Influenza Virus: Chemoen- in Cell Culture and in Human Respiratory Epithelium. Antiuiral zymic Preparation of N-S Analogs of N-acetylneuraminic Acid' Res. 1994, 25,123-137. Tetrahedron 1993, 49, I-12. (39) Janakiraman,M.N.;White, C. L.;Laver, W.G.;Air, G. M.;Luo, (46) Privalov, P. L.; Gill, S. J. Stability of Protein Structure and M. Structure of Influenza Virus NeuraminidaseBlLeel4O Com- Hydrophobic Interaction. Adu- Prot. Chem. 1988, 39,79I-234' plexed with Sialic Acid and a Dehydro Analog at 1.8-A (4?) M.; Gomez, F. A.; Whitesides, G. M. Analysis of the Resolution: Implications for the Catalytic Mechanism. Biochem- Mammen, of Ligands Containing the N-2,4-diqitrophenyl (DNP) istry 1994, 33, 8I72-8L79. Binding Monoclonal Rat anti-DNP Antibody Using (40) NA is a tetrameric glycoprotein found on the surface of influenza Group to Bivilent Electrophoresis. Azal. Chem.1995, in press' at a density 1O-foldlower than that of HA (102-103 copies/4M2). Affinity Capillary Its natural enzymatic action is to cleave the glycosidic linkage connecting SA to lactose. NA is, however, tolerant of many non- lactosyl groups. JM9504666