Synthesis and conformational studies of poly(l-lysine) based branched polypeptides with Ser and...

Journal of Controlled Release 63 (2000) 81–95www.elsevier.com/ locate / jconrel

Synthesis and conformational studies of poly(L-lysine) basedbranched polypeptides with Ser and Glu/Leu in the side chains

a , a b c c*´ U ´ ´ U ´Gabor Mezo , Judit Remenyi , Judit Kajtar , Krisztina Barna , Dezso Gaal ,aFerenc Hudecz

aResearch Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest 112, POB 32 Hungaryb ¨ ¨Department of Organic Chemistry, Eotvos L. University, Budapest, Hungary

cNational Institute of Oncology, Budapest, Hungary

Received 10 March 1999; accepted 23 July 1999

Abstract

In a new group of polypeptides, the branches were composed of DL-Ala oligopeptide, L-serine and L-leucine or L-glutamicacid residues. The synthesis of eight different side-chain combinations is described. In the first group, Ser was attacheddirectly to the ´-amino groups of polylysine, and Leu or Glu was situated at the side chain end (poly[Lys(X –DL-Ala –i m

Ser )]). Alternatively, Leu or Glu was positioned next to the polylysine backbone (poly[Lys(Ser –DL-Ala –X )], wherej j m i

X5L-Leu or L-Glu and m|3–6, i#1 and j#1). The second group of polymers was synthesised by grafting oligo(DL-alanine)chains to the e-amino groups of polylysine, followed by coupling of Ser and Leu or Glu consecutively to the chain ends,however, in a different order, resulting in the polymers (poly[Lys(X –Ser –DL-Ala )] and poly[Lys(Ser –X –DL-Ala )],i j m j i m

where X5L-Leu or L-Glu and m|3–6, i#1 and j#1). The effect of amino-acid composition and sequence of side chains inbranched polypeptides on solution conformation was studied by CD spectroscopy. CD spectra recorded in aqueous solutionsof various pH (2–11) and ionic strengths (0.02–2.0 M NaCl) suggest that leucine- and serine-containing polypeptides havemore ordered (a-helical) structure than the polymers with glutamic acid and serine residues in the same position. Theinfluence of serine residues on ordered structure (helical or b-sheet) formation depends on its position in the side chain aswell as on the nature of amino acid X (Leu or Glu). The incorporation of Ser into the branches resulted in polypeptidespossessing prolonged shelf stability and high water-solubility. No toxic effect of this new class of polymers was observed onmouse spleen cells, even after 4 h of incubation. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Macromolecular carriers; Synthesis of polyamino acids; Solution conformation; Cytotoxicity; CD studies

1. Introduction isotopes. These constructs are also considered for thedesign of long-acting conjugates [1]. Synthetic an-

Appropriate macromolecular carriers are often tigens for vaccination and/or for production ofused for the development of drug delivery systems to conventional or monoclonal hapten-specific antibo-target various bioactive compounds or radioactive dies also contain natural proteins (KLH, bovine

serum albumin) or synthetic macromolecules [2].In our laboratory, two types of branched chain*Corresponding author. Tel.: 136-1-209-0555; fax: 136-1-372-

2620. polymeric polypeptides based on a polylysine back-

0168-3659/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 99 )00175-3

U82 G. Mezo et al. / Journal of Controlled Release 63 (2000) 81 –95

bone have been synthesised to study the correlation These latter compounds were eliminated faster frombetween structural and functional properties. In the the blood stream than EAK or SAK. However, theirXAK type of polypeptide, short oligo(DL-alanine) blood circulation time was still high compared to thatchains were grafted to the polylysine backbone of polycationic polymers.followed by coupling of a single optically active We have also demonstrated the high accumulationamino acid to the end of the branches (poly[Lys(X – of polycationic poly[Lys(DL-Ala –Leu )] (ALK) ini m i

DL-Ala )], where i#1 and m|3) [3]. In the second spleen, while LAK, which has a reversed side chainm

version (AXK type), a reverse sequence of side sequence, was eliminated rapidly from the body bychains was built up. First, an amino acid, X, was kidney and liver [7]. LAK has considerable immuno-attached directly to the ´-amino groups of poly- modulatory activity, while ALK exerts a muchlysine, then the side chains were elongated with weaker effect on SRBC antigen-induced immuneoligo(DL-alanine) obtained by polymerisation of N- responses [11]. EAK and its acetylated derivative,carboxy-DL-alanine anhydride, resulting in the anionic Ac-EAK, showed some accumulation inpoly[Lys(DL-Ala –X )], where i#1 and m|3 [4]. tumour cells [12,13].m i

The synthetic branched polypeptides were char- The branched polypeptides AK, LAK, EAK andacterised by size and physicochemical (primary Ac-EAK were used successfully as carriers forstructure, conformation) [3–5] and biological (toxici- peptide epitopes [14–16] or antitumor agents [5,17–ty, pyrogenicity, biodegradation, immunoreactivity, 19]. However, the solubility of some hydrophobicbiodistribution) [5–7] properties. Correlation has conjugates decreases with time, restricting theirbeen demonstrated between the chemical structure of potential therapeutic use. Based on the observationpolymers and in vitro cytotoxicity [6] or biodistribu- that the presence of hydroxyl groups in serine /tion [7]. The side chain sequence and charge of threonine-containing polymers (SAK, TAK, ASK)branched polypeptides have a pronounced influence [8] and considering the biological importance of Gluon their tissue distribution and, especially, on their or Leu in the side chains [5,7], we have invented ablood clearance. The glutamic-acid-containing new group of polymeric branched polypeptides. Thepoly[Lys(Glu –DL-Ala )] (EAK) has an amphoteric common features of these compounds are the pres-i m

character and showed prolonged survival in blood, ence of Ser residue in the branches.while most of the cationic polymers are rapidly For this reason, we have synthesised branchedeliminated from the circulation [7]. polypeptides with eight side chain combinations.

Recently, polypeptides with hydroxyl groups in Side chain elongation of the previously publishedthe side chains were prepared with high water SAK and ASK [8] by glutamic acid or leucinesolubility and long-term shelf stability [8]. We found resulted in poly[Lys(X –Ser –DL-Ala )] (XSAK)i j m

that the introduction of serine residues into poly- (Fig. 1) and poly[Lys(X –DL-Ala –Ser )] (XASK)i m j

cationic branched polypeptides increases their blood (Fig. 2) polymers, where X5L-Glu, L-Leu; i, j#1,circulation time and reduces in vitro cytotoxicity [9]. m|6–7. We also added Ser residue to the EAK,Poly[Lys(Ser –DL-Ala )] (SAK) approximates the AEK, LAK and ALK polymers, giving rise toi m

blood clearance profile of EAK [10]. This property beneficial carrier properties. Poly[Lys(Ser –X –DL-j i

of EAK and SAK can be explained by similarities in Ala )] (SXAK) (Fig. 1) and poly[Lys(Ser –DL-m j

their solution conformation [5,8]. Namely, the for- Ala –X )] (SAXK) (Fig. 2), where X5L-Glu, L-m i

mation of an ionic bond between the a-amino group Leu; i, j#1, m|3–4, were also prepared.and the g-carboxyl group of the terminal glutamic Due to the important role of solution conformationacid in EAK or an H-bond between the a-amino of polymeric polypeptide in biodistribution [7] and ingroup and the hydroxyl group of the terminal serine vitro cytotoxicity [6], we have performed a detailedin SAK. This postulation was based on CD measure- circular dichroism investigation. CD spectra of thements under various pH (2–12) and ionic strength newly prepared polymers were recorded not only(0.02–2.0 M NaCl) conditions and was supported by under nearly physiological conditions (pH 7.4, 0.2 Mdata obtained with the polymers containing reversed NaCl) but also in solutions with a wide range of pHsequences in the side chains (AEK, ASK) [5,8]. values and ionic strengths. These studies shed light

UG. Mezo et al. / Journal of Controlled Release 63 (2000) 81 –95 83

Fig. 1. Outline of the synthesis of the branched polypeptides, LASK, EASK, SALK and SAEK.


Fig. 2. Outline of the synthesis of the branched polypeptides, LSAK, ESAK, SLAK and SEAK.


on the correlation between side-chain structure (se- used in accordance with the recommended nomen-quence, charge) and solution conformation and might clature of graft polymers [21]. For the sake ofbe important for the further development of macro- brevity, codes for branched polypeptides were con-molecular carriers for therapeutic applications. In structed using the one-letter symbols for amino acidsthis communication, we also report on the results of (Table 1).an in vitro cytotoxicity study of this new set ofcompounds.

2.3. Poly[Lys]?HBr

aThe synthesis was carried out from N -carboxy-2. Materials and methods´N -benzyloxycarbonyl-lysine anhydride, as reported

previously [22]. Conditions for polymerisation were2.1. Materialschosen to obtain an average degree of polymerisationof approximately 100 (monomer–diethylamine molarAmino acids used for this study were purchasedratio550:1) or approximately 300 (monomer–di-from REANAL, Hungary. Benzyloxycarbonyl chlo-ethylamine molar ratio5150:1). Protecting groupsride, pentachlorophenol, N,N9-dicyclohexylcar-were cleaved by HBr using acetic acid containingbodiimide, 1-hydroxybenzotriazole and 4-35% HBr. Complete removal of blocking groupsmethylmorpholine were from Fluka (Buchs, Switzer-from polypeptides was controlled by UV spectra atland).l5254 nm [23]. Diethylether was used to precipitatepoly[Lys]?HBr. The sample was dissolved in water,2.2. Intermediatespurified by exhaustive dialysis against distilled waterusing visking casing and was isolated by freeze-Pentachlorophenyl ester of Z-protected amino-acid

¯ ¯drying. The average relative molar masses (M , M )´derivatives was prepared according to Kovacs et al. w z

were determined by sedimentation equilibrium using[20]. Amino-acid derivatives were checked for puritya short column technique at 258C (MOM 3180 typeby elemental analysis and by ascending thin-layeranalytical ultracentrifuge, Hungary), and calculatedchromatography using Merck precoated plates, DC-by the van Holde and Baldwin method [24]. TheAlufolien Kieselgel 60, and an ethyl acetate–

¯number average of the relative molar mass (M ), thepyridine–acetic acid–water (333:20:6:11 v/v) sol- n¯ ¯polydispersity factor (M /M , and the averagevent system. Melting points were determined using a z w]

hot plate apparatus (VED-NAGEMA, GDR). degree of polymerisation (DP ) values were com-n

The nomenclature of branched polypeptides was puted from data described above [24].

Table 1Characterisation of branched chain polypeptides

]a b c¯Compounds Abbreviation DP Mn w

poly[Lys(Leu –Ser –DL-Ala )] LSAK 280 227 9000.95 0.9 7.0

poly[Lys(Leu –DL-Ala –Ser )] LASK 280 219 7001.0 6.4 1.0

poly[Lys(Ser –Leu –DL-Ala )] SLAK 110 58 6000.9 1.0 3.0

poly[Lys(Ser –DL-Ala –Leu )] SALK 110 58 6000.9 3.0 1.0

poly[Lys(Glu –Ser –DL-Ala )] ESAK 280 233 8001.0 0.9 7.0

poly[Lys(Glu –DL-Ala –Ser )] EASK 280 235 5001.0 7.0 1.0

poly[Lys(Ser –Glu –DL-Ala )] SEAK 110 60 4000.9 1.0 3.0

poly[Lys(Ser –DL-Ala –Glu )] SAEK 110 60 4000.9 3.0 1.0

a Amino acid composition was determined by amino acid analysis as described in Materials and Methods.b Number of average degrees of polymerization.c Average molecular mass of polymers; calculated from the average degree of polymerization of poly[Lys] and of the side chain

composition determined by amino acid analysis.


2.4. Attachment of the side-chain terminal amino to 260 nm. The dichrograph was calibrated withacids (AA) to polymers D-(2)-pantoyl-lactone at 220 nm and with epiandros-

terone at 304 nm [25].Poly[Lys(X )] (X K) where X5Glu, Ser or Leu The samples were dissolved at concentrations ofi i

[4,8], and AXK- and XAK-type polypeptides where 0.5–1.0 mg/ml in 0.02 M NaCl and the pH wasX5Glu, Ser or Leu [5,8,22] were prepared using adjusted by the addition of 1.0 M HCl (acidicpolylysine with the relative molar mass distribution solution) or 1.0 M NaOH (alkaline solution). The

¯ ¯(M /M 51.3) reported earlier. ionic strength of the solution was adjusted by addingz w

In the new polymers, the side-chain terminal 5.0 M NaCl. CD band intensities in the tables and2amino acids (AA) were attached to the above-de- figures are expressed as [u ] values, in deg?cm /M

scribed branched polypeptides as Z–AA–OPcp de- dmol. The [u ] values were calculated for one lysineM

rivatives. Briefly, 4 mmol of the protected and residue of the main chain carrying a whole sideactivated amino-acid derivative and an equivalent chain.amount (4 mmol) of 1-hydroxybenzotriazole (HOBt) The interpretation of CD spectra of polypeptidesdissolved in 10 ml dimethylformamide (DMF) were in aqueous solutions of various pH values (2–12)added to the polymer (2.67 mmol) dissolved in 10 and ionic strengths (0.02–2.0 M) was based on theml of a water–DMF (2:8, v /v) solvent mixture. A CD spectra of poly[L-Lys], from which all the50% molar excess of both Z–AA–OPcp derivative branched polypeptides were derived. Conformationsand HOBt, calculated for the side chains of the whose CD spectra were similar to that of unchargedpolymer, was used. The pH of the solution was poly[Lys] [26] (in the uncharged state the CDadjusted to eight by adding N-methylmorpholine spectra were characterised by negative maxima at(NMM) to the reaction mixture. Stirring was con- 221 and 208 nm, of about the same intensity) weretinued at room temperature overnight. The solvent considered helical, whereas CD spectra similar towas removed in vacuo and the residue was triturated that of charged poly[Lys] [26] (in the charged state,several times with diethylether containing 10% di- the CD spectrum is characterised by a weak negativechloromethane, for removal of excess active ester maximum at 234 nm, a positive maximum at 218 nmand pentachlorophenol. Z-protecting groups were and a negative maximum at 199 nm) indicate ancleaved with HBr using acetic acid containing 35% unordered solution conformation.HBr and the polymers were worked up as describedabove.

2.7. In vitro cytotoxicity assay

2.5. Amino acid analysisThe spleens of BDF mice were removed and1

placed, ice-cold, in RPMI-1640 medium containingThe amino acid composition of polymeric poly-

10% fetal calf serum, then homogenised [27]. Spleenpeptides was determined from amino acid analysis

cells were filtered through sterilised gauze, and theusing a Beckmann (Fullerton, USA) model 6300 6concentration of the cells was adjusted to 1.5310analyzer. Prior to analysis, samples were hydrolysed

cells /ml. Polypeptides were dissolved in RPMI-1640in 6 M HCl in sealed and evacuated tubes at 1058C

medium. Cells were incubated with varying amountsfor 24 h.

(1.5, 15, 50 mg/ml) of polypeptide in a shaker for 1or 4 h at 378C. Control cells were incubated with

2.6. Circular dichroism medium alone. The viability of spleen cells wasmeasured using a trypan blue exclusion assay [28].

CD spectra were recorded using a Roussel-Jobin Three parallel experiments were performed withdichrograph model VI (Jobin-Yvon, Longjumeau, every polypeptide at each concentration and at leastFrance) in quartz cells with an optical path length of 200 cells were counted for staining with trypan blue.0.02 cm at room temperature, under a constant The percentage viability6SD was calculated fromnitrogen flush. CD spectra were registered from 195 the mean of three measurements [27].


Table 2Characteristic values of CD spectra of branched polypeptides containing Lys, Leu, Ser and Ala amino acids

23pH NaCl l (nm), [u ]?1021(mol l )

Max Cross Max Cross Max Cross Min Max Cross

Poly[Lys(Leu –Ser –DL-Ala )]0.95 0.9 7.0

2.1 0.02 ,190 198.5 215 223.5(225.2) (25.0) (25.5)

2.00 ,198 201 209.0 214.5 222.0 247(.4.0) (210.5) (29.6) (211.2)

11.2 0.02 ,190 199 205.0 214 223.5 243(.29.7) (211.3) (27.3) (29.3)

2.00 ,198 202 210.0 214.5 221.5 244(.10.0) (222.2) (221.5) (223.2)

Poly[Lys(Leu –DL-Ala –Ser )]1.0 6.4 1.0

2.0 0.02 ,190 198 203.0 214.5 223.5 248(213.4) (25.9) (26.4)

2.00 ,198 202 209.5 214.5 222.5 248(14.2) (216.9) (216.3) (219.2)

11.1 0.02 ,190 198 207.0 214.5 222.0 248(39.6) (227) (220.5) (220.6)

2.00 ,198 206.5 215.0 222.0 249(225.2) (219.0) (219.2)

Poly[Lys(Ser –Leu –DL-Ala )]*0.9 1.0 3,0

11.2 0.20 195 199 207.0 215.5 222.0(14.9) (235.5) (228.6) (230.0)

Poly[Lys(Ser –DL-Ala –Leu )]0.9 3,0 1.0

1.9 0.20 195.0 201 209.0 215.5 221.5(6.3) (29.5) (28.5) (28.6)

2.00 ,200 208.5 216.0 221.5(29.4) (28.1) (28.3)

11.4 0.02 190.0 199.5 208.0 215.5 222.5(13.8) (27.8) (27.5) (27.6)

0.20 195.0 200 208.0 215.0 221.5(4.2) (28.9) (27.5) (27.6)

*Samples dissolved in 2.0 M NaCl were turbid.

3. Results and discussion sation of N-carboxy-DL-alanine anhydride either tothe e-amino groups of polylysine, as in the case of

3.1. Synthesis SXAK and XSAK polymers (Fig. 1), or to thea-amino group of the single amino acid attached

In the present series of experiments, new types of directly to the polylysine (SAXK, XASK), whereserine-containing branched polypeptides were pro- X5Glu or Leu (Fig. 2). The Ser residue as well asduced (Figs. 1 and 2). The side chains connected to amino acid X (Leu or Glu) were introduced by thethe ´-amino groups of polylysine were built up from HOBt-catalysed active ester method using penta-oligo(DL-alanine) chains (three–seven DL-alanine res- chlorophenyl ester derivatives [5,8]. Reaction withidues) containing Ser and Glu or Leu residues. The 1.5 M Z–AA–OPcp combined with equimolarrelative position of Ser versus Glu or Ser versus Leu HOBt, which were calculated for each side chain,was different and resulted in eight side-chain se- resulted in a blocking efficiency of over 90%,quence variations of branched polypeptides. according to amino acid analysis. It should be noted

DL-Alanine oligomers were grafted by polymeri- that this coupling strategy was originally developed


for the production of XAK or AXK type poly- teristics of branched chain polypeptides, togetherpeptides [5,8], but the present set of data clearly with abbreviations, are presented in Table 1.indicate its efficacy for the synthesis of more com-plex SXAK and XASK polymers. Removal of Z- 3.2. Conformation of branched polypeptides inprotecting groups and benzyl groups, in the case of solutionglutamic-acid-containing versions, was carried outby treatment with 35% HBr–glacial acetic acid and The conformations of the new polypeptides inverified by UV spectroscopy in all cases. The charac- aqueous solution were studied under acidic (pH|2),

Table 3Characteristic values of CD spectra of branched polypeptide chains containing Lys, Ser, Ala and Glu amino acids

23pH NaCl l (nm), [u]?1021(mol l )

Max Cross Max Cross Max Cross Min Max Cross

Poly[Lys(Glu –Ser –DL-Ala )]1.0 0.9 7.0

2.1 0.02 ,190 198.0 210 216.0 225 233.5 249(217.8) (2.5) (21.7)

2.00 ,198 201 207.5 214.5 224.5 238(2.7) (28.0) (24.8) (27.9)

11.3 0.02 ,190 197.5 218.0 228.0 247(229.2) (21.7) (23.0)

2.00 ,200 202.5 215.5 223.0 248(219.8) (27.1) (27.8)

Poly[Lys(Glu –DL-Ala –Ser )]1.0 7.0 1.0

2.2 0.02 ,190 193 205.0 216 222.5 249(18.2) (216.8) (23.6) (24.4)

2.00 ,200 209.0 215,5 221.0 247(223.2) (220.8) (221.6)

10.9 0.02 190 199.0 218.0 226.0 244(228.2) (22.0) (22.3)

2.00 ,195 198.0 220.0 227.0 248(227.1) (23.5) (23.8)

Poly[Lys(Ser – Glu –DL-Ala )]0.9 1.0 3.0

1.9 0.02 190 197 206.5 217.0 225.0(7.7) (27.2) (24.8) (25.8)

2.00 ,200 208.0 212.0 219.5(213.2) (212.9) (214.4)

11.4 0.02 ,190 198.0 217.0 225.5(217.2) (22.9) (24.0)

2.00 ,200 207.5 215.5 222.5(213.0) (211.2) (211.6)

poly[Lys(Ser –DL-Ala – Glu )]*0.9 3.0 1.0

2.0 0.02 190 202 208.0 225.0(12.2) (21.6) (24.6)

2.00 ,200 208.0 222.5(24.0) (27.3)

11.6 0.02 190 193.5 200.0 215 220 225 229.5(5.1) (27.0) (0.4) (20.9)

2.00 ,200 200.0 217 224.5(24.8) (22.5) (22.9)

*All samples were slightly turbid, but no precipitation was observed.


neutral (pH|7) and alkaline (pH|11) conditionsusing solvents with different ionic strengths (0.02,0.2 and 2.0 M NaCl) (Tables 2 and 3, Figs. 3–8).The first conclusion of these experiments is thatpolymeric polypeptides with Leu and Ser have ahigher tendency to form ordered (helical) structuresthan the same types of polymer with Glu and Ser(Tables 2 and 3, Figs. 3 and 4). This is in line withour previous studies since amino acids with bulkyalkyl side chains in the branches show a highertendency to produce polypeptides with an a-helicalconformation. Polymers SAK or ASK showed ratherunordered structures under identical conditions [8],while the elongation of the side chains by leucine(LSAK, LASK) increased the ordered character ofthe compounds (Fig. 5). The CD spectrum of LSAKcorresponds to a random coil conformation in a0.02-M NaCl solution at pH 2.1 (negative maximum,200 nm, a weak positive maximum at l5215 nmand a negative maximum at l5227 nm) (Table 2).

Fig. 4. CD spectra of branched polypeptides in 0.2 M NaCl atneutral pH (7.0–7.3) and room temperature. Results are expressedas mean residue ellipticity values [u ] . SALK (- - -- - -), SLAKM

(??????), SAEK (———) and SEAK (– ? – ? –).

Increasing the pH and/or the NaCl concentration inthe solution resulted in a higher amount of a-helicalconformer (Table 2, Fig. 5A). Helical secondarystructure was observed in 2.0 M NaCl solution at allpH values studied. The intensity of the respectivemaxima in the spectra increased on increasing the pH(Table 2, Fig. 5A).

In contrast to LSAK, the spectrum of LASKreflected a partially ordered conformation in a 0.02-M NaCl solution, even at acidic pH. Similarly,LASK adopted a more ordered (helical) structurecompared to LSAK in solutions with elevated pHand/or ionic strength (Table 2, Fig. 5B). It should benoted that ASK was not able to form an orderedstructure under comparable conditions [8].

The relative positions of Ser and Leu residues inthe side chains seemed to significantly influence thesolution conformation of the polymeric polypeptides.

Fig. 3. CD spectra of branched polypeptides in 0.2 M NaCl atThe presence of serine at the side-chain end andneutral pH (7.0–7.3) and at room temperature. Results areleucine next to the polylysine backbone (SALK)expressed as mean residue ellipticity values [u ] . LSAK (- - - - -),M

LASK (??????), ESAK (———) and EASK (– ? – ? –). produced a polymer with a highly ordered structure


Fig. 5. CD spectra of LSAK (A) and LASK (B) in different NaCl solutions at various pH values and at room temperature. Results areexpressed as mean residue ellipticity values [u ] . (A) pH52.1, 0.2 M NaCl (- - - - -); pH57.0, 0.02 M NaCl (– ? – ? –); pH57.0, 2.0 MM

NaCl (??????); pH511.2, 0.2 M NaCl (———); (B) pH52.0, 0.2 M NaCl (- - -- - -); pH57.2, 0.02 M NaCl (– ? – ? –); pH57.2, 2.0 MNaCl (??????); pH511.2, 0.2 M NaCl (———).

under all conditions studied (Table 2, Figs. 4 and [22], while the serine residue attached to Leu at the6B). The CD spectrum at pH 1.9 in 0.02 M NaCl end of the branches increased the capability to formexhibited an a-helical conformation of the poly- an a-helical structure.peptide (Fig. 6B). Increasing the pH and the salt These data indicate that the simultaneous presenceconcentration of the solution had no significant of Ser and Leu residues in the side chains results ininfluence on the pattern of the spectra (Fig. 6B). This polymers with a pronounced tendency to formobservation was similar to that detected previously ordered structure under appropriate conditions. Infor ALK, although the intensity of the maxima was this, the position of the Ser residue seems to be thelower [5]. predominant factor. Polypeptides with side-chain

The highest degree of ordered structure was terminal Ser are able to adopt more ordered struc-observed in the CD spectra of SLAK, containing tures than those containing Ser inside the branches.Ser–Leu dipeptide elements at the end of the branch- This difference might be explained by the presence /es (Figs. 4 and 6A). In 0.02 and 0.2 M NaCl absence of H-bonds formed between the -NH group2

solutions, the spectra revealed a large positive maxi- of the side-chain terminal amino acid residue (Ser inmum below l5200 nm and two negative maxima at the case of SLAK and SALK; Leu in the case ofaround 207 and 222 nm, with almost identical LSAK and LASK) and the -OH group of Ser. Inintensities. The [u ] values of these spectra were the SLAK and SALK polymers, Ser residues are situatedM

highest among the Leu- and Ser-containing poly- at the end of the branches and, therefore, are freelymers. The intensity of maxima increased slightly on available for H-bond formation between neigh-increasing the pH (Fig. 6A). The polymer precipi- bouring residues. This might lead to the stabilisationtated at 2.0 M NaCl solutions, independently of the of an ordered solution conformation. In the other pairpH value. It is interesting to note that LAK pos- of polymers (LSAK and LASK), the side-chainsessed an unordered conformation in acidic solution terminal amino group and the -OH group of the Ser


Fig. 6. CD spectra of SLAK (A) and SALK (B) in different NaCl solutions at various pH values and at room temperature. Results areexpressed as mean residue ellipticity values [u ] . (A) pH52.1, 0.02 M NaCl (- - - - -); pH52.1, 0.2 M NaCl (– ? – ? –); pH57.0, 0.02 MM

NaCl (??????); pH511.2, 0.02 M NaCl (———); (B) pH51.9, 0.02 M NaCl (- - - - -); pH57.1, 0.02 M NaCl (– ? – ? –); pH57.1, 2.0 MNaCl (??????); pH511.4, 2.0 M NaCl (———).

residue are not close enough to develop a H-bond most ordered structure at neutral pH, which wasinteraction. Consequently, these polymers could dis- independent of the ionic strength of the solutionplay CD spectra of less ordered conformation. (Figs. 4 and 8B). The shape of these spectra suggest

The explanation of the conformational features of that a-helical and b-sheet conformational elementsGlu- and Ser-containing polymers is more difficult. It are present and are mixed with a random coilis due to the presence of three functional groups structure. The b-sheet content increased on increas-(a-amino of the side-chain terminal amino acid, ing the NaCl concentration (Fig. 8B). EASK showedg-carboxy of Glu and hydroxyl of Ser) in the side a limited tendency to form an a-helical structure inchains. neutral solution and this capability was not influ-

The common feature of the CD spectra of all four enced significantly by the increase in the ionicpolymers (ESAK, EASK, SAEK and SEAK) in strength (Fig. 7B). Under similar conditions, un-alkaline solution is that these CD curves correspond ordered steric arrangements were observed in theto a random coil conformation (Table 3, Figs. 7 and case of ESAK and SEAK in 0.02 M NaCl solution8). Elevation of the ionic strength of the solution (Figs. 7A and 8A). An increase in ionic strength (toslightly changed the pattern of the CD spectra, 2.0 M NaCl) induced a helical transition, which wasindicating the appearance of an ordered conformer more impressive in the case of SEAK (Figs. 7A andpopulation of the polymers. However, only SEAK 8A).showed a tendency to adopt a helical conformation in Under acidic conditions, all Glu-containing poly-2.0 M NaCl solution at high pH (Table 3). mers showed increased helix-forming ability (Table

The four different Glu-containing polypeptides 3, Figs. 7 and 8). The tendency to form a helicalshowed the highest conformational variety under structure at constant ionic strength decreased in theneutral conditions (Figs. 7 and 8). SAEK had the order SEAK.EASK.ESAK. The helix content of


Fig. 7. CD spectra of ESAK (A) and EASK (B) in different NaCl solutions at various pH values and at room temperature. Results areexpressed as mean residue ellipticity values [u ] . (A) pH52.1, 0.2 M NaCl (- - - - -); pH57.05, 0.02 M NaCl (– ? – ? –); pH57.05, 2.0 MM

NaCl (??????); pH511.3, 0.2 M NaCl (———); (B) pH52.2, 0.2 M NaCl (- - - - -); pH57.25, 0.02 M NaCl (– ? – ? –); pH57.25, 2.0 MNaCl (??????); pH510.9, 0.2 M NaCl (———);

polymers was highly influenced by the ionic strength However, a serine residue at the end of the sideof the solution (Table 3, Figs. 7 and 8). SAEK chains, as in the case of the Leu-containing poly-adopted an ordered conformation, in which the a- mers, might contribute to the stabilisation of thehelix elements were mixed with b-sheet conformers, ordered structure by preferred H-bond formation.as demonstrated by the CD spectra (positive maxi-mum ,200 nm, negative maximum .220 nm and a 3.3. In vitro cytotoxicity of polypeptidesshoulder ,210 nm) (Table 3, Fig. 8B). The poorsolubility of this compound indicates the aggregation The effects of the polypeptides, at various con-of the polymer chains. centrations, on the viability of isolated spleen cells,

Previous studies revealed that AEK polymer pos- which are considered to be a highly sensitive in-sesses b-sheet-forming ability [4], but the presence dicator of in vitro cytotoxicity, are summarised inof serine at the end of the branches (SAEK) further Table 4. The data showed no toxic effects of theincreased this tendency. Elongation of the ASK serine-containing branched polypeptides on mousepolymer with a glutamic acid residue increased the spleen cells, either after incubation for 1 or 4 h.capability of polypeptides to form ordered structure(Fig. 7B) [8], while the same change of the SAKpolymer decreased its ability to adopt an ordered 4. Conclusionconformation in solution (Fig. 7A) [8].

Based on the CD measurements described above, Taken together, these studies suggest that thewe concluded that the conformation of Glu-con- conformational properties of polymeric polypeptidestaining polypeptides is determined by the presence can be altered by the rational combination of Ser andand position of glutamic acid in the branches. Glu or Leu residues in the branches. Our data


Fig. 8. CD spectra of SEAK (A) and SAEK (B) in different NaCl solutions at various pH values and at room temperature. Results areexpressed as mean residue ellipticity values [u ] . (A) pH51.9, 0.2 M NaCl (- - - - -); pH56.9, 0.02 M NaCl (– ? – ? –); pH56.9, 2.0 MM

NaCl (??????); pH511.4, 0.2 M NaCl (———); (B) pH52.0, 0.2 M NaCl (- - - - -); pH57.0, 0.02 M NaCl (– ? – ? –); pH57.0, 2.0 MNaCl (??????); pH511.6, 0.2 M NaCl (———).

Table 4Effects of polypeptides on the viability of mouse spleen cells

Polypeptide Treatment Control Peptide concentration (mg/ml)Incubation time (h)

0.0015 0.015 0.05aLSAK 1 98.261.0 98.160.4 97.960.7 97.460.7

4 96.360.5 94.960.8 95.060.7 93.861.2LASK 1 94.461.7 97.761.8 95.561.2 95.762.7

4 94.762.5 92.0608 92.064.6 90.563.0SLAK 1 98.860.1 98.661.7 96.561.5 95.963.1

4 95.362.1 90.261.1 90.762.5 87.462.8SALK 1 98.261.0 98.060.9 98.260.3 97.160.8

4 96.360.5 96.260.6 95.160.3 94.061.2ESAK 1 98.861.1 94.863.9 97.261.1 94.465.1

4 95.561.0 91.063.0 91.161.5 90.062.0EASK 1 95.563.3 97.760.5 97.762.1 95.663.9

4 94.762.5 91.162.5 91.761.5 87.764.1SEAK 1 98.861.1 95.961.6 97.661.8 96.663.3

4 95.561.0 95.960.5 93.862.1 91.662.0SAEK 1 94.461.7 97.962.4 96.362.3 96.061.8

4 94.762.5 94.361.6 94.062.5 91.164.0a Results are expressed in % as the mean6SD for three parallel measurements.


lysine) backbone: synthesis, conformation and immuno-indicate that incorporation of Ser residues into themodulation, in: Ch.G. Gebelein, Ch.E. Carraher (Eds.),branches with Leu/Glu markedly increases the or-Polymeric Materials in Medication, Plenum Press, New-dered-structure-forming capability of the polymerYork, 1985, pp. 263–289.

and significantly improves the water solubility. Poly- U ´ ´ ´[4] G. Mezo, F. Hudecz, J. Kajtar, Gy. Szokan, M. Szekerke,mers with side chains containing Ser and Leu were The influence of the side chain sequence on the structure–

activity correlations of immunomodulatory branched poly-capable of adopting a mainly helical secondarypeptides, Biopolymers 28 (1989) 1801–1826.structure. However, in case of the simultaneous

[5] F. Hudecz, Design of synthetic branched-chain polypeptidespresence of Ser and Glu, CD spectra suggest that,as carriers for bioactive molecules, Anti-Cancer Drugs 6

under appropriate conditions, a different type of(1995) 171–193.

ordered structure (b-sheet) could be formed. In ´ ´ ´[6] F. Hudecz, I. Kurucz, A. Lanyi, D. Gaal, A.L. Kovacs, G.´summary, the position of the Ser residue both in U ¨Mezo, E. Rajnavolgyi, M. Szekerke, Carrier design: cytotox-

icity and immunogenicity of synthetic branched polypeptidesLeu- and Glu-containing polymers predominates thewith poly(L-lysine) backbone, J. Control. Release 19 (1992)extent of the ordered conformation. The incorpora-231–243.tion of Ser at the side-chain terminal favours the

[7] J.A. Clegg, F. Hudecz, M.V. Pimm, R.W. Baldwin, Carrierformation of an ordered (helical or b-sheet) struc- design: biodistribution of branched polypeptides with poly(L-ture, while its presence inside the branches results in lysine) backbone, Bioconjugate Chem. 2 (1990) 425–430.a less ordered solution conformation. U ´[8] G. Mezo, J. Kajtar, I. Nagy, M. Szekerke, F. Hudecz, Carrier

design: synthesis and conformational studies of poly(L-Data presented in this communication might belysine) based branched polypeptides with hydroxyl groups inuseful for the design of Leu-containing macromole-the side chains, Biopolymers 42 (1997) 719–730.cules with improved water solubility for therapeutic ´ ¨ U[9] F. Hudecz, M.V. Pimm, E. Rajnavolgyi, G. Mezo, A. Fabra,

application [29] or for the development of Glu- ´ ´D. Gaal, A.L. Kovacs, M. Szekerke, Carrier design: newcontaining polymers for self-assembly systems [30]. generation of polycationic branched polypeptides containingA further benefit of the polypeptides presented in this OH groups with prolonged blood survival and diminished in

vitro cytotoxicity, Bioconjugate Chem. 10 (1999).communication is their lack of in vitro spleen cellU[10] M.V. Pimm, S.J. Gribben, G. Mezo, F. Hudecz, Strategies fortoxicity.

labelling branched polypeptides with a poly(L-lysine) back-123 125 131 111bone with radioiodines ( I, I, I) and radiometals ( In,

51Cr) for biodistribution studies and radiopharmaceuticaldevelopment, J. Label. Compd. Radiopharm. 36 (1995) 157–Acknowledgements172.

´ U ´[11] D. Gaal, F. Hudecz, G. Mezo, F. Gal, M. Szekerke,This work was supported partly by grants from the Modulation of drug and ionizing radiation activities by a new

Ministry of Health and from the Hungarian National group of branched polypeptides, J. Cancer Res. Clin. Oncol.116 (S) (1990) 622–630.Science Fund (OTKA) No. T014964 and T030838.

´[12] F. Hudecz, Y. Kojima, Y. Miyamoto, J. Kajtar, H. Maeda,The authors are grateful to Professor Maria SzekerkeEffect of charge on the biodistribution of synthetic branched

for her advice and careful reading of the manuscript. polypeptides, J. Control. Release (1994) 301–302.[13] M.V. Pimm, F. Hudecz, Biodistribution in tumour-bearing

mice of polycationic, amphoteric and polyanionic branched125polypeptides with poly(L-lysine) backbone labelled with I

References 111and In: tumour accumulation less than of labelled serumproteins, J. Cancer Res. Clin. Oncol. 122 (1996) 45–54.

U[1] W.R. Gombotz, D.K. Pettit, Biodegradable polymers for [14] G. Mezo, M. Szekerke, I. Kurucz, F. Hudecz, Synthesis andprotein and peptide drug delivery, Bioconjugate Chem. 6 conjugation with carriers of herpes simplex virus glycop-(1995) 332–351. rotein-D peptides of predicted antigenicity, in: E. Bayer, G.

´ ´[2] F. Hudecz, H. Votavova, D. Gaal, J. Sponar, J. Kajtar, K. Jung (Eds.), Peptides 1988, Walter de Gruyter, Berlin, 1989,Blaha, M. Szekerke, Branched polypeptides with a poly(L- pp. 701–703.lysine) backbone: synthesis, conformation and immuno- [15] F. Hudecz, Alteration of immunogenicity and antibodymodulation, in: Ch.G. Gebelein, Ch.E. Carraher (Eds.), recognition of B-cell epitope by synthetic branched chainPolymeric Materials in Medication, Plenum Press, New- polypeptide carriers with poly[L-Lys] backbone, Biomed.York, 1985, pp. 263–289. Peptides, Protein and Nucleic Acids 1 (1995) 213–220.

´ ´ ´[3] F. Hudecz, H. Votavova, D. Gaal, J. Sponar, J. Kajtar, K. [16] K. Wilkinson, M. Vordermeier, R.J. Wilkinson, J. Ivanyi, F.Blaha, M. Szekerke, Branched polypeptides with a poly(L- Hudecz, Synthesis and in vitro T-cell immunogenicity of


conjugates with dual specificity, Bioconjugate Chem. 9 characterization of the model peptides, Coll. Czech. Chem.(1998) 539–547. Commun. 45 (1980) 933–940.

U U U ´ ´[17] G. Mezo, I. Mezo, A. Seprodi, I. Teplan, M. Kovacs, B. [23] A. Yaron, A. Berger, Multi-chain polyamino acids containing´ ´Vincze, I. Palyi, J. Kajtar, M. Szekerke, F. Hudecz, Syn- glutamic acid, aspartic acid and proline, Biochim. Biophys.

thesis, conformation, biodistribution and hormone related in Acta 107 (1965) 307–332.´ ´vitro antitumor effect of a GnRH antagonist-branched poly- [24] F. Hudecz, S. Kutassi-Kovacs, J. Kajtar, GPC, CD and

peptide conjugate, Bioconjugate Chem. 7 (1996) 642–650. sedimentation analysis of polylysine and branched chain´ ´ U[18] B. Vincze, I. Palyi, D. Daubner, A. Kalnay, G. Mezo, F. poly-Lys–poly-DL-Ala polypeptides, Colloid Polym. Sci. 262

´ UHudecz, M. Szekerke, I. Teplan, I. Mezo, Antitumour effect (1984) 208–212.of a GnRH antagonist and its conjugate on human breast [25] P.H. Schippers, H.P.J. Dekkers, Direct determination ofcancer cells and their xenografts, J. Cancer Res. Clin. Oncol. absolute circular dichroism data and calibration of commer-120 (1994) 578–584. cial instruments, Anal. Chem. 53 (1981) 778–782.

U ´ ´[19] G. Mezo, G. Sarmay, F. Hudecz, J. Kajtar, Zs. Nagy, J. [26] N.J. Greenfield, G.D. Fasman, Computed circular dichroismGergely, M. Szekerke, Synthesis and characterization of spectra for the evaluation of protein conformation, Biochem-p-bromo-Phe-branched polypeptide-monoclonal antibody ter- istry 8 (1969) 4108–4116.

´ ´nary systems for potential use in boron neutron capture [27] D. Gaal, F. Hudecz, A.L. Kovacs, M. Szekerke, Immuno-therapy (BNCT), J. Bioact. Compat. Polym. 11 (1996) 263– modulatory effect of synthetic branched polypeptides II, J.285. Biol. Response Mod. 5 (1986) 148–159.

´[20] J. Kovacs, R. Giannotti, A. Kapoor, Polypeptides with [28] P.O. Seglen, Preparation of isolated rat liver cells, Methodsknown repeating sequence of amino acids. Synthesis of Cell. Biol. 13 (1976) 29–83.poly-L-glutamyl–L-alanyl–L-glutamic acid and poly- [29] D. Teitelbaum, R. Arnon, M. Sela, Copolymer 1: from basicglycylglycyl–L-phenylalanine through pentachlorophenyl ac- research to clinical application, Cell. Mol. Life Sci. 53tive ester, J. Am. Chem. Soc 8 (1966) 2282–2292. (1997) 24–28.

[21] IUPAC–IUB Commission on Biochemical Nomenclature, [30] D.W. Urry, Five axioms for the functional design of peptide-Biochem. J. 127 (1972) 753–756. based polymers as molecular machines and materials: Princi-

[22] F. Hudecz, M. Szekerke, Investigation of drug–protein ple for macromolecular assemblies, Biopolymers (Peptideinteractions and the drug–carrier concept by the use of Science) 47 (1998) 167–178.branched polypeptides as model systems. Synthesis and

Synthesis and conformational studies of poly(l-lysine) based branched polypeptides with Ser and...

Documents

Transcript of Synthesis and conformational studies of poly(l-lysine) based branched polypeptides with Ser and...