Poly(ethylene glycol) grafted polystyrene dendrimer resins: Novel class of supports for solid phase peptide synthesis

Polymer 53 (2012) 4076 e 4090

Contents lists available at SciVerse ScienceDirect

Polymer

journal homepage: www.elsevier.com/locate/polymer

Poly(ethylene glycol) grafted polystyrene dendrimer resins: Novel class of

supports for solid phase peptide synthesis

M.A. Siyad, G.S. Vinod Kumar^*

Chemical Biology, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram-695 014, Kerala, India

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 2 April 2012

Received in revised form

Poly(propyleneglycol)dimethacrylate cross-linked support using monomers Styrene and 4-chloromethyl

styrene (PS-PPGDMA-VBC resin) was prepared by suspension polymerization method. Dendritic

templates were synthesized from Schiff base and trimesic acid molecules and assembled independently

on PS-PPGDMA-VBC support to the second generation. N,N-bischloro ethyl amine dendrimer was

generated from Schiff base units and introduced to the chloromethyl groups uniquely through secondary

amine. It was followed by acidolytic cleavage, diazotization and thionyl chloride reactions leading to ﬁrst

generation dendrimer. O-benzyl ether dendrimer was created using 1,3,5-tris(hydroxyl methyl)benzene

unit which was produced by the LiAlH reduction of carbonyl groups of trimesic acid molecule. The

4

hydroxyl methyl groups formed were converted to chloro methyl groups by adding thionyl chloride that

leads to ﬁrst generation dendrimer. Similar synthetic routes were followed to produce second generation

28 June 2012

Accepted 6 July 2012

Available online 25 July 2012

Keywords:

PS-PPGDMA-VBC resin

Dendrimer

Solid phase synthesis

n

dendrimers too. Poly(ethylene glycol), M z 1500) has been grafted to chlorine termini of both den-

drimers and used to check various physical and chemical properties of different peptide synthetic

conditions. Reactions were qualitatively analyzed by FTIR, ¹³C NMR, SEM and quantitatively analyzed by

UV measurements and CHN analysis. Classically difﬁcult ACP sequence was synthesized on both PEGy-

lated dendrimer supports and compared to Tenta Gel. Biologically signiﬁcant disulﬁde bonded endo-

thelin class of peptides were also synthesized using novel supports using various literature reported

methods.

Ó 2012 Published by Elsevier Ltd.

1. Introduction

organic synthesis should have an optimum hydrophobic/hydro-

philic balance in addition to the compatibility with the organic

The concept of solid phase organic chemistry has evolved since

the introduction of styrene cross-linked with divinylbenzene (DVB)

by Merriﬁeld [1] and drawn much attention during the past few

decades principally because of enormous applications of peptides

and non-peptides in the ﬁeld of medicinal chemistry and chemical

biology [2e4]. Polymer supported approaches have found partic-

ular favor in organic synthesis because they simplify product

isolation and allow the use of excess reagents help to force the

reactions to completion. Initially it was believed that the solid

matrix would act only as an inert carrier, but many chemical

reactions in the past few decades conﬁrmed that physical as well as

chemical nature of support have a crucial role in the success of

polymer supported reactions. An efﬁcient polymeric support for

molecules being synthesized. Experimental studies show that solid

phase reactions verily depend on the swelling characteristics of the

polymer and solvation of peptidyl resin in different solvents [5]. In

peptide synthesis, the conformational changes in the growing

peptide after each amino acid attachment are also to be considered

since the changing conformations can have a dynamic inﬂuence on

the physical as well as chemical properties of the growing peptide.

Polystyrene cross-linked with DVB (PS-DVB) having pendant chloro

methyl groups as points of attachment, has come to be known

simply as Merriﬁeld resin and the most commonly employed resin

for solid phase reactions. This is because of the advantages such as

good mechanical and chemical stabilities and the facility of deri-

vations with a wide variety of functional groups for substrate

attachment. But the use of Merriﬁeld resin in many cases, however,

is accompanied by difﬁculty during synthesis. This difﬁculty is

primarily due to the high hydrophobic character of the matrix, the

short and rigid cross-linker DVB connecting the PS backbone and

the presence of local higher cross-link density regions because of

higher reactivity of DVB relative to styrene in the free radical

*

Corresponding author. Chemical Biology, Molecular Medicine Division, Rajiv

Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram-695 014,

Kerala, India. Tel.: þ 91 471 2529526; fax: þ91 471 2348096.

E-mail address: gsvinod@rgcb.res.in (G.S.V. Kumar).

0

032-3861/$ e see front matter Ó 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.polymer.2012.07.011

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4077

0

copolymerization [6,7]. Different strategies have been employed to

modify the hydrophobicity associated with PS-DVB resin such as

replacement of DVB with relatively more ﬂexible cross-linking

units and incorporation of linker units. The new cross-linkers

(DMSO), anisol, 2-mercaptoethanol, 2,2 -dithiobis(5-nitropydine)

and 1-methylimidazole (MeI) were purchased from Aldrich Chem-

ical Company, USA. Infrared (IR) spectra of simple molecules as

well as polymer samples at different stages of reactions were

recorded with a Shimadzu IR 470 spectrometer using KBr pellets.

Optical density (OD) values were measured with a Shimadzu

ultravioletevisible spectrophotometer at 290 nm. Carbon-13

[8e16] have been designed both to increase the ﬂexibility of the

polymer backbone to allow for better diffusion through the

matrices and also to impart a variety of solvent-like properties to

the resins [17]. Solid supports based on using poly (ethylene glycol)

13

nuclear magnetic resonance ( C NMR; cross-polarization magic-

angle spin (CP-MAS)) spectra of the samples were taken using a dsx

300 (75.47 MHz). High performance liquid chromatography (HPLC)

analysis was conducted using a Pharmacia Akta puriﬁer system

using C-18 reverse phase semi preparative HPLC column and binary

gradient system water and acetonitrile containing 0.1% TFA as the

solvents. The ﬂow rate was 1 mL/min, and detection was at 214 nm.

The HPLC conditions used for all synthetic peptides were same: C-18

column; buffer (A) 0.1% TFA in water:acetonitrile (19:1, v/v) and

buffer (B) 0.08% TFA in acetonitrile:water (4:1, v/v). Flow rate 1 mL/

min: gradient used 0% B in 5 min. 100% B in 30 min and 100% B in

35 min. SEM pictures were taken using Hitachi SS 2000 scanning

electron microscopy. Mass spectra of peptides were obtained with

a Kratos MALDI-TOF MS instrument.

(

PEG) as the major component [18e21] can be more compatible

with aqueous solution as ﬁrst demonstrated with the poly(ethylene

glycol)-poly(acrylamide) (PEGA) resin [22]. The hydrophobic char-

acter of PS can be effectively modiﬁed by grafting large linker units

such as PEG to the hydrophobic PS core and due to the unique

conformational ﬂexibility of PEG chains, PEG-PS resins are

compatible with both polar and non-polar solvents [23e28].

The majority of dendrimeric structures were developed using

orthodox solution chemistry [29e31], increasing attention has

recently been diverted to dendrimeric preparation on solid

supports [32,33]. Instead of convergent route adopted in solution

chemistry; on solid support a divergent route must be adopted. In

this paper, poly (ethylene glycol) grafted poly(N,N-bisethylamine)

and poly(O-benzyl ether) dendrimers were synthesized to the

second generation (G

2

) on poly(propylene glycol) dimethacrylate

2.2. Synthesis of PS-PPGDMA-VBC resin

cross-linked polystyrene support and used for comparing the yield

and efﬁciencies of peptide synthesized. As a replacement for

grafting PEG directly to the PS backbone which reduces the grafting

efﬁciency due to steric hindrance, dendrimer sites have been

created ﬁrst and subjected to PEGylation so that more number of

PEG molecules can be incorporated with minimum steric

hindrances. Dimethacrylates rather than diacrylates were chosen

mainly because of high chemical stability [34] of methacrylates and

have more favorable copolymerization parameters with styrene

Styrene (20 mL) and poly(propylene glycol)dimethacrylate

(5 mL) were destabilized using 1% NaOH solution (3 ꢁ 15 mL). It was

thoroughly washed with water (3 ꢁ 10 mL) and dried well using

2

anhydrous CaCl . 1% PVA solution (PVA, Mn w 70,000) in water

(110 mL) was prepared and stirred well in a polymerization vessel

ꢀ

at 85 C under a constant stream of N

cross-linked PS-PPGDMA-VBC resin,

2

gas. For preparing 2 mol%

mixture of PPGDMA

a

(1.08 mL, 2 mol%), styrene (10.88 mL, 95 mol%), VBC (0.502 mL,

3 mol%), toluene (2.28 mL, 20 vol% of monomer ratio, as diluents)

and dibenzoyl peroxide (500 mg, as free radical initiator) were

mixed and added to the PVA solution. The stirring was continued

for 8 h. Different mol percentage of cross-linked resins (4, 6 and

8 mol%) were prepared by varying styrene and PPGDMA compo-

sition by ﬁxing VBC as 3 mol%. The polymer beads were collected by

ﬁltration through a sintered disc (G3) and successively washed

with hot water (20 mL ꢁ 3, 30 min) to remove PVA, followed by

acetone (20 mL ꢁ 3, 3 min) and methanol (20 mL ꢁ 3, 3 min) to

remove unreacted monomers. The formed polymer was then

successively solvent extracted with toluene, DCM, acetone and

methanol. The yield of 2 mol% PS-PPGDMA-VBC resin obtained was

7 g. The polymer beads were sieved and those of 200e400 mesh

sizes were chosen for entire study of dendrimer developments and

PEG grafting.

[35] and hence the cross-linking will be more uniformly distributed

throughout the resin. So the present paper describes the synthesis

of novel class of PEGylated dendrimeric polystyrene supports, its

characterization and evaluation as ideal polymeric supports

towards various stages of solid phase peptide synthesis. In this

regard, we have kept all the advantages of styrene based support

and tried to incorporate the ﬂexibility of PPGDMA cross-linker as

well as amphiphilicity of grafted PEG units through dendritic sites

to achieve the desirable features of ideal solid supports.

2

. Experimental section

2.1. Materials and methods

All reactions involving moisture sensitive reagents were con-

ꢀ

ducted in oven dried (110 C) glassware. All solvents were

commercial grade and puriﬁed before use. Dichloromethane (DCM)

2.3. Synthesis of bis[2-(benzaldeneamino)ethyl]amine Schiff base

and trimethylamine were distilled from anhydrous CaCl

2

and

tetrahydrofuran (THF) from metallic sodium. 1,3,5-tris(hydroxyl

methyl)benzene and PEG were dried well by lyophilization

before use. All side chain protected F-moc amino acids (L)

with different cysteine derivatives and 2-(1H-benzotriazol-1-yl)

The Schiff base was prepared by mixing one molecular equiva-

lent of dien (1.061 mL, 10 mmol) with two molecular equivalent of

benzaldehyde (2.031 mL, 20 mmol) at room temperature. Stirring of

mixture was continued until only oil remained and was used

for reaction without further puriﬁcation. The Schiff base formation

1

,1,3,3 tetramethyluraniumhexaﬂuorophosphate (HBTU) were

purchased from Peptide international company (USA).

-Hydroxybenzotriazol (HOBt) and 1-(2-mesitylenesulfonyl)-3-

1

was conﬁrmed via H NMR. H NMR data (400 MHz, CDCl

(s, 1H, eNH), , J ¼ 7.1 MHz) ¼ 3.6 (t, 4H, eCH

¼ 2.91 (t, 4H, eCH

J ¼ 7.1 MHz), ¼ 7.5 (m, 4H, J ¼ 7.5 MHz), ¼ 7.8 (m, 6H,

J ¼ 7.5 MHz), ¼ 8.65(s, 2H, ]CH).

3

)

d

¼ 2.0

1

d

2

d

2

,

nitro-1,2,4-triazol (MSNT) were purchased from Novabiochem

Ltd., UK. Styrene, poly(propylene glycol)dimethacrylate (PPGDMA)

d

(M

n

z 560), 4-chloromethyl styrene (VBC), polyvinyl alcohol (PVA)

(

Mn w 70000), benzoyl peroxide, thionyl chloride, diethylenetri-

2.4. Synthesis of 1,3,5-tris(hydroxyl methyl) benzene

amine (dien), benzaldehyde, trimethylamine, Poly(ethylene glycol)

PEG), sodium hydride, 1,3,5-benzenetricarboxylic acid, diisopro-

(

Trimesic acid (5 g, 23.8 mmol) was suspended in methanol

pylethylamine (DIEA), triﬂouroacetic acid (TFA), thioanisol, triiso-

(15 mL) and heated to reﬂux. When a clear solution obtained,1.6 mL

propylsilane (TIS), 1,2-Ethanedithiol (EDT), dimethylsulfoxide

2 4

of con.H SO was added slowly and the mixture was reﬂuxed for

4078

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

6

h. After 3 h, white solid get precipitated from the solution. The

mixture was cooled to room temperature, slowly neutralized with

saturated NaHCO solution, ﬁltered and washed with excess water

reactions using Fmoc-Gly-OH/HoBt/HBtU/DIEA and piper-

idine:DMF (1:4 v/v) mixtures respectively. The amino loading

capacity obtained was 0.207 mmol/g. The yield of amino ethyl resin

3

until the aqueous layer turned neutral. It was then dried to yield

a white solid of trimethyl-1,3,5-benzenetricarboxylate (5.72 g).

1

(G (a)) was 1.83 g.

LiAlH

4

tablets (2.5 g, 66 mmol) were suspended and reﬂuxed in dry

atmosphere. Trimethyl-1,3,5-

2.7. Diazotization reactions

THF (50 mL) under an inert N

2

benzenetricarboxylate (5 g, 20 mmol) was dissolved in 100 mL

dry THF and added to the suspension in 1 h. The solution was then

reﬂuxed for 24 h, cooled to 0 C and acidiﬁed with 1 M HCl. THF was

2 M HCl (30 mL) was added to the amino ethyl resin (1.5 g,

0.207 mmol/g) taken in a R.B ﬂask and kept at 0 C for 15 min. To

ꢀ

this ice-cold resin, add 5 M chilled sodium nitrite solution (50 mL)

in drop wise manner with constant stirring. After the addition of

sodium nitrite solution, the reaction was allowed to continue for 1 h

in ice chilled condition and brought back the reaction to room

temperature. The diazotized resin beads were washed with excess

of hot water (5 ꢁ 10 mL), 2 M NaOH solution (3 ꢁ 10 mL), ethanol

(5 ꢁ 10 mL), methanol (5 ꢁ 10 mL) and ether (5 ꢁ 10 mL) and

qualitatively veriﬁed using ninhydrin. The formed hydroxy ethyl

removed under reduced pressure and the aqueous suspension

(

100 mL) thus obtained was extracted with ethyl acetate for 48 h in

a liquideliquid extractor to yield 1,3,5-trimethylolbenzene which

appeared as colorless crystals in room temperature. The mother

liquor was concentrated to yield second fraction. Both the fractions

were NMR pure and the combined yield was 4.96 g. The formation

1

of ligand was conﬁrmed via H NMR. H NMR (300 MHz, DMSO-d6,

ꢀ

2

5

C):

d

¼ 7.096 (s, 3 H), 5.21 (t, J(H,H) ¼ 5.6 Hz, 3 H), 4.45

1

resin (G (a)) was solvent extracted with ethanol and methanol and

(

d, J ¼ 5.6 Hz, 6 H).

washed with ether and kept under vacuum. The yield of resin

obtained was 1.5 g. The hydroxyl loading value was determined by

method explained in 2.5(b) and obtained as 0.206 mmol/g.

2.5. Incorporation of (a) Schiff base and (b) 1,3,5-tris(hyroxy

methyl)benzene to PS-PPGDMA-VBC resin

2.8. Conversion of hydroxyl to chloro groups

(

a) To the pre-swelled PS-PPGDMA-VBC resin (2 g, 0.104 mmol/g)

in 1,4-dioxane (25 mL), suspension of Schiff base (4.64 mL,

Hydroxy ethyl Schiff base (G

1

(a)) (1 g, 0.206 mmol) and hydroxy

methyl benzyl ether (G (b)) (1 g, 0.206 mmol) dendrimer resins

1

8

0 mmol) has been added in presence of trimethylamine

ꢀ

were suspended in double distilled DCM (25 mL each) taken in two

RB vessels and allowed to swell for 1 h each. The excess solvents

were ﬁltered off and thionyl chloride (302

0 mmol for hydroxyl ethyl Schiff base and hydroxyl methyl benzyl

ether dendrimers respectively) has been added drop wise to the

(

500

mL) and heated at 100 C for 48 h with occasional stirring.

Excess ligand present in the reaction medium will act as an

acceptor of eliminated HCl resulting in formation of yellow

crystals of ligandeHCl complex. The solid materials were

ﬁltered off, washed well with water to remove the crystalline

compound and with dioxane and transferred to a solvent

extraction apparatus. After extraction with dioxane for 48 h,

the resin was collected, washed with ether and dried under

mL, 20 mmol and 306 mL,

2

ꢀ

corresponding vessels kept at 55 C with occasional swirling. The

vapor mixtures of thionyl chloride and DCM formed were

condensed back to the reaction vessels with the aid of continuous

circulation of water. The reactions were monitored up to 12 h,

brought back to room temperature and unreacted thionyl chlorides

were removed by the slow addition of distilled ethanol. Both

reaction mixtures were washed with DCM (5 ꢁ 15 mL), THF

1

vacuum. The yield of Schiff base resin (G (a)) formed was

2

.53 g.

(

b) O-benzyl ether dendrimer was synthesized by dissolving 1,3,5-

tris(hydroxyl methyl)benzene (2 g, 13 mmol) in dry THF

(

5 ꢁ 15 mL), THF/H O (1:1, 5 ꢁ 10 mL), methanol (5 ꢁ 15 mL),

2

(

4

30 mL) and weighed amount of dried sodium hydride (0.936 g,

0 mmol) was added to this solution with occasional stirring.

ethanol (5 ꢁ 15 mL) and ether (5 ꢁ 15 mL) and dried under vacuum.

The chlorine loading values of chloro ethyl (G (a)) and chloro

methyl (G (b)) dendrimer resins were calculated as 0.206 mmol/g

and 0.204 mmol/g respectively by Volhard’s estimation method

10].

The reaction mixture was allowed to keep for 30 min to form

sodium salt of 1,3,5-tris(hydroxyl methyl)-benzene. Well dried

PS-PPGDMA-VBC (2 g, 0.104 mmol/g) polymer and 1 g excess of

1

[

1

,3,5-tri(hydroxyl methyl)benzene was added to this solution

ꢀ

2

and heated to reﬂux at 80 C for 12 h under inert N atmo-

2

.9. Development of G generation dendrimers

sphere. The polymer beads were washed successively with

dilute HCl (3 ꢁ 15 mL), methanol (5 ꢁ 15 mL), ethanol

The experimental conditions used to create G

2

generation

(

5 ꢁ 15 mL) and ether (5 ꢁ 15 mL). Solvent extracted with THF,

dendrimers were same as that described for G dendrimers. The

1

washed with DCM and ether and dried at room temperature in

vacuum. The yield of hydroxy methyl polymer (G (b)) formed

quantities of polymer supports used and reagents added at

different stages of reactions were as follows; Schiff base resin

1

was 2.49 g. The hydroxyl capacity of resin was determined [15]

and obtained as 0.206 mmol/g.

[

Schiff base treatment (2 g resin, 9.28 mL), imine bond cleavage

using 6 M HCl (2 g resin, 90 mL), diazotization using NaNO /HCl

(100 mL/60 mL) mixture (2 g resin), thionyl chloride reaction (1.5 g

resin, 960 L)]; O-benzyl ether resin [1,3,5-tris(hydroxymethyl)

benzene (2 g resin, 4 g), sodium hydride (1.872 g), thionyl chloride

reaction (1.5 g resin, 906.8 L)].

2

.6. Schiff base cleavage

m

The removal of bulky phenyl groups of Schiff base resin (2 g) was

ꢀ

achieved by stirring with 6 M HCl (90 mL) at 60 C for 12 h.

Benzaldehyde molecules liberated as an oily emulsion during

hydrolysis and yellow polymer beads formed were ﬁltered off,

washed with 1 M sodium hydroxide solution (5 ꢁ 15 mL) and with

water (5 ꢁ 15 mL) until the solution was turned neutral. The N,N-

bisethylamine polymer beads formed were successively washed

with ethanol, methanol and ether and dried under vacuum.

Lyophilize the sample and the free amino groups formed were

quantiﬁed by UV method by acylation followed by deprotection

m

2

2.10. Grafting of polyether 1500 to G dendrimers

Solution of sodium polyethyleneglycoltae (PEG mol.wt.1500)

was prepared by dissolving calculated amounts of sodium hydride

(98 mg, 4.08 mmol, for both chlorine terminated G dendrimers) to

2

poly(ethylene glycol) (6.15 g, 4.1 mmol, for bisethyl and 6.06 g,

4.04 mmol for chloromethyl benzyl ether dendrimers) in dry THF

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4079

(

100 mL each) till a green color persisted. The reaction mixtures

spectrophotometric

measurement

of

dibenzofulvene-

were allowed to stand for 30 min with occasional stirring under N

2

piperidine adduct at 290 nm. Further coupling reactions were

carried out using 1.5 equiv excess (with respect to initial

hydroxyl capacity of resin) of Fmoc amino acids, HBTU, HOBt,

and DIEA in DMF. The cleavage cocktail used was reagent K

atmosphere and

1 g each of chlorinated dendrimer resins

(

0.410 mmol/g for Schiff base and 0.404 mmol/g for O-benzyl ether)

were quantitatively transferred to corresponding reaction media

ꢀ

followed by 2 g excess of PEG and heated at 80 C to reﬂux for

2

comprising of TFA (4.125 mL), H O (0.25 mL), Phenol (0.25 mL),

overnight reaction. The reaction vessels were brought back to room

temperature, ﬁltered off and unreacted sodium hydride was

washed away with ethanol. The polymer beads were successively

washed with dilute HCl (5 ꢁ 15 mL), THF(5 ꢁ 15 mL) and ethanol

thioanisol (0.25 mL) and ethanedithiol (0.125 mL) in volume

ratio of 82.5:5:5:5:2.5 and has been used for cleaving all the

three ACP peptides for a duration of 4 h under identical

synthetic conditions. The peptides were ﬁnally precipitated

using chilled ether and washed thoroughly with cold ether to

remove the scavengers. Peptides were lyophilized and used for

analysis.

(

5 ꢁ 15 mL)followed by solvent extracted with THF and methanol

and washed with ether (5 ꢁ 15 mL). The yields of PEG grafted

polymer beads collected were 1.424 g and 1.419 g for Schiff base and

O-benzyl ether dendrimers respectively. The eOH loading values of

grafted dendrimer resins were determined by method explained in

b. Endothelin-1 human peptide having amino acid sequence

NH -Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-

2

0

.5 (b) and the value obtained was same for both resins as

.246 mmol/g.

Phe-Cys-His-Leu-Asn-Ile-Ile-Trp-OH was synthesized on

weighed (300 mg, 0.037 mmol) amount of PEGylated

poly(N,N-bisethylamine) dendrimer resin. A pre-formed acti-

vated ester method using MSNT/F-moc-Trp(Boc)-OH/MeI

2.11. Swelling studies

(1:1:0.75) was used for anchoring ﬁrst amino acid to the free

The resin was placed in a syringe ﬁxed with a Teﬂon ﬁlter at the

resin and the amino capacity was quantiﬁed by UV method.

Subsequent amino acids were assembled in a stepwise manner

to form the desired peptide chain by HoBt: HBtU: DIEA coupling

method. In all the acylation couplings, 2 fold molar excess of

Fmoc amino acids were used and the progress of acylation and

deprotection reactions at each stage was detected by Kaiser test.

The ﬁrst disulﬁde bond formation between 1st and 15th cysteine

was carried out by on bead method [46] in which tert-Butylthio

(S-t-Bu) and 4-methoxytrityl (Mmt) groups were used as thiol

protecting groups and second disulﬁde bond formation was

carried out between 3rd and 11th residues by TFA/DMSO/Ani-

sole method [47] in which sulfur atom was protected with

t-butyl groups. In the ﬁrst step, S-t-Bu was removed by reduc-

tion to liberate free thiol by treating the peptidyl resin with 20%

mercaptoethanol in DMF (2 mL) for 3 h. The resin was then

bottom. The solvent was sucked into the syringe and after 3 h;

excess solvent was removed by applying force on the piston. The

extent of swelling of the resin in each solvent was determined from

the volume of the resin placed after the solvent incubation. The

experiment was repeated to ensure reproducible values. The

experiments were continued with other solvents, with different

cross-linking mole percentages of PPGDMA as well as with PEG

grafted and non-grafted resins and compared with commercially

available Tenta Gel. All the measurements were carried out at room

ꢀ

temperature (30 C). The swelling data was expressed in volume/

gram (mL/g) of resin.

2

.12. Chemical stability studies

0

Both PEGylated dendrimer resins (100 g each) were treated with

reacted with 5 fold excess of 2,2 -dithiobis(5-nitropyridine)

different reagents used for various peptide synthetic conditions.

The different reagents and conditions used were; 100% TFA (10 mL,

(DTNB) in DCM (12 mL) for 1 h and the free thiol thus repro-

tected and activated with the 5-nitropyridinesufeny(5-Npys)

group. This was followed by the cyclization step in which the

resin was treated with 1% TFA in DCM (4 mL) in the presence of

1

2

(

2 h), 30% TFA in DCM (10 mL, 4 h), 20% piperidine in DMF (10 mL,

h), 2 M aqueous ammonium hydroxide (10 mL, 4 h) and 2 M NaOH

10 mL, 4 h) at room temperature. After the treated time, the resins

were ﬁltered properly and washed with DCM (10 ꢁ 10 mL), DMF

TIS (40 mL) as scavenger. The reaction was monitored by

measuring the absorbance of 5-nitropyridine-2-thione at

386 nm. The resin washed with DMF (3 ꢁ 15 mL), DCM

(3 ꢁ 15 mL) and ether (5 ꢁ 5 mL) was dried well. Mono disulﬁde

bonded resin was suspended in a mixture of cleavage cocktail

(

10 ꢁ 10 mL), ethanol (10 ꢁ 10 mL) and ether (10 ꢁ 10 mL) and dried

in vacuum. The preliminary idea regarding chemical stability of

support was obtained by weighing solution before and after

treatments. The IR spectra recorded after treatments were

compared to the original. The stabilities of resins were conﬁrmed

by monitoring the identical IR spectra of TFA treated and untreated

resins.

having TFA (4.7 mL), thioanisol (125

(50 L) and double distilled water (125

kept at room temperature for 4 h. The resin was ﬁltered off,

washed with fresh TFA (2 mL), DCM (3 ꢁ 500 L) and vacuum

mL), triisopropylsilane

m

mL). The mixture was

m

evaporated to obtain a thick oily residue. The peptide was

precipitated as white powder by addition of ice-cold ether, and

washed thoroughly with cold ether (8 ꢁ 10 mL) to remove the

scavengers. Freeze dried and fractionally collected for second

disulﬁde bond formation. 10 mg of peptide was dissolved in

50 mL of TFA: DMSO: Anisole (97.9:2:0.1) in a 100 mL round

bottom ﬂask and the mixture was allowed to stir for 1 h at room

temperature. The excess TFA then was removed under reduced

pressure and the peptide was precipitated by adding ice-cold

ether. Repetitive washing with cold ether (5 mL ꢁ 10) fol-

lowed by scratching will remove the DMSO and anisole. Dried

the peptide by lyophilization and used for HPLC and MALDI

analysis.

2

.13. Peptide synthesis

a. Acyl carrier protein (ACP) synthesis was carried out on well

dried PEG grafted N,N-bisethylamine and O-benzyl ether

(

500 mg each, 0.123 mmol) dendrimers as well as on linker

attached (HMPA) Tenta Gel resin (500 mg, 0.130 mmol) and

compared. The synthesis was carried out by standard Fmoc

strategy. PEGylated dendrimers and HMPA-Tenta Gel resins

were used for esteriﬁcation with Fmoc-Gly-OH in dry DCM. The

C-terminal attachment was carried out by MSNT coupling

under an inert condition and using the molar ratio (1:1:0.75) of

Fmoc-Gly-OH/MSNT/MeI (0.073 g/0.073 g/15

mL, 2 mmol

excess each) in minimum quantity of dry DCM. Removal of

Fmoc group was achieved using 20% piperidine:DMF (1:4 v/v)

mixture. Quantitative couplings were measured by

c. Endothelin-2 human peptide H

Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-

Trp-OH having two intra molecular disulﬁde bonds Cys

2

N-Cys-Ser-Cys-Ser-Ser-Trp-

1

ꢂ15

and

4080

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

Cys³

ꢂ11

and C-terminal tryptophan residue was synthesized on

The direct introduction of dien into polymer beads will lead to

a number of products as a result of N-alkylation at different amine

groups in the ligand. But the primary amine readily forms 1:2 Schiff

base with aromatic aldehydes and can be easily regenerated by

mineral acid hydrolysis. So dien can be directly introduced into

chloro methyl groups of cross-linked polymer uniquely through

secondary amine of Schiff base ligand moieties. This was achieved

by protecting the primary amino groups of ligand by forming Schiff

base prior to immobilization without causing undesirable bridging

reactions. Schiff base was formed by mixing 2 equiv of benzalde-

hyde with 1 equiv of dien at room temperature and the mixture can

be directly added without prior puriﬁcations to the pre-swelled

resin swelled in 1,4-dioxane. The integrity of initial polymer

PS-PPGDMA-VBC and each stage of dendrimer developments were

qualitatively analyzed by FTIR spectra. Initial resin showed sharp

PEG grafted poly(O-benzyl ether) dendrimer resin. The sulfur

atom of 1st and 15th cysteine residues were protected with

tert-butylthio (S-t-Bu) and 4-methoxytrityl (Mmt) groups for

on bead disulﬁde bond formation and 3rd and 11th positions

were protected with 4-Acetamidomethyl (Acm) groups which

are kinetically stable in TFA and labile in iodine method. The

amount of resin taken, methods used for ﬁrst amino acid

substitution and on bead disulﬁde bond formation were same

as the procedure used for peptide b. All the cyclization was

ﬁnished within 30 min. The peptidyl support was washed with

DMF (5 ꢁ 15 mL) and DCM (5 ꢁ 15 mL) and dried well under

vacuum desiccator. Peptidyl support was treated with cleavage

2

cocktail contains TFA: TIS: H O (95:2.5:2.5) for 4 h. The

peptides formed were precipitated with ether and washed

with cold ether (15 ꢁ 15 mL) to remove cation scavengers.

Lyophilized and puriﬁed the main fraction by preparative HPLC

and used for iodine oxidation. Dissolve the Cys(Acm) peptide in

0% aqueous methanol (10 mg/mL) under a blanket of N

M HCl (0.1 mL/mg) followed by immediate addition of 0.1 M I

in 50% aqueous methanol (1 mL). After 45 min of vigorous

stirring, quench the iodine by adding 1 M aqueous ascorbic acid

200 mL) drop wise until the mixture was colorless, and

concentrate by evaporation under reduced pressure to

approximately one third of the original volume. Freeze dried

and used for analysis.

ꢂ1

bands at 1718 cm and 1122 cm correspond to the ester carbonyl

as well as the CeO stretching of cross-linker in addition to the usual

peaks of polystyrene. The Schiff base resin showed peak around

ꢂ1

5

1

2

. Add

1630 cm corresponds to imine eCH]Ne linkage of Schiff base

2

incorporated to the chloro methyl groups of styrene backbone. The

inclusion of bulky phenyl groups will further increase the hydro-

phobic character of polystyrene backbone as well as steric

hindrance for further reactions. The imine linkage eCH]Ne con-

necting between dien and benzaldehyde units was also not found

stable enough to withstand the various chemical transformations

such as acid and ester hydrolysis. So we have selectively removed

(

the phenyl groups of Schiff base by reacting the resin with 6 M

ꢀ

3

. Results and discussion

hydrochloric acid at 60 C for 12 h (Shown as B

1

in Scheme 1a).

ꢂ1

After acid hydrolysis the peak at 1630 cm disappeared due to the

3.1. Synthesis of PEGylated dendrimer resins

breakage of eC]Ne linkage and a new broad spectrum emerged

around 3412 cm due to the regeneration of primary amines of

ꢂ1

Beaded PS-PPGDM-VBC resin was synthesized by free radical

aqueous suspension polymerization of styrene and 4-chloromethyl

styrene (VBC) using various cross-linking degrees of PPGDMA (2, 4,

dien incorporated. The removal of phenyl rings were qualitatively

analyzed by positive ninhydrin test also.

The quantiﬁcation of dibenzofulvene:piperidine adduct formed

from pyridine and resin bound F-moc-Gly-OH gave twice

(0.207 mmol/g) the initial chlorine functional loading proved the

quantitative introduction of dien to the polystyrene backbone. The

6

and 8 mol%). In all cases of suspension polymerizations, the

percentage of VBC was ﬁxed as 3 mol% and quantities of styrene and

cross-linker varied accordingly. In suspension polymerization,

monomers such as styrene and 4-chloromethyl styrene (VBC) and

cross-linker PPGDMA dissolved in diluent toluene were dispersed as

droplets in hot water. When toluene used as the diluent instead of

cyclohexane and carbon tetrachloride, the main fractions of the

polymer collected were uniform spherical beads having mesh size in

between 200 and 400 and the resulted polymer used for further

reactions. The spherical identity of polymer bead was analyzed using

scanning electron microscopy as shown Fig. 4a. Beads >400 mesh

size have less total surface area and beads <200 mesh size tend to

clog the ﬁltering disk of the synthesizer. PVA used as protective

colloid provided adequate stabilization to the copolymer droplets.

PVA with an average molecular weight of 70,000 Da was found to be

effective in the present polymerization. The solubility of high-

molecular-weight PVA was not satisfactory and low-molecular-

weight PVA did not give proper stabilization to the droplets,

resulting in coalescence of the copolymer nuclei. Reproducible

results were obtained by adjusting the amount of stabilizer PVA,

geometry of the vessel, and stirrer and the stirring rate. The chlorine

loading of PS-PPGDMA-VBC resin was estimated by Volhard’s esti-

mation method and obtained as 0.104 mmol/g. We have chosen

resulted amino ethyl (G

1

(a)) resin was subjected to diazotization

in

reaction using NaNO /HCl in an ice-cold condition (Shown as C

2

1

Scheme 1a). As a result of diazotisation reaction, free amino groups

get converted to hydroxyl groups resulted in hydroxy ethyl resin

(G (a)) showed negative ninhydrin test. Hydroxyl loading was

1

quantiﬁed by esteriﬁcation with Fmoc-Gly-OH in dry DCM using

MSNT and MeI and the loading value obtained was 0.206 mmol/g.

The hydroxy terminal groups were further converted to chlorine by

the drop wise addition of thionyl chloride in dry DCM (Shown as D

1

in Scheme 1a). The chlorine capacity was quantiﬁed by Volhard’s

estimation method and obtained as 0.206 mmol/g. After thionyl

chloride treatment, the disappearance of broad peak around

ꢂ1

3500 cm indicated the alteration of hydroxyl groups to chlorine

groups and resulted in chloro ethyl resin (G

initial PS-PPGDMA-VBC resin and various stages of Schiff base

reactions are depicted in Fig. 1.The ﬁrst generation (G (a) in Scheme

1

(a)). FTIR spectra of

1

1a) chloro ethyl dendrimer resins were further subjected to same

series of reactions such as grouping of Schiff base, acidolytic

cleavage, diazotization reaction and thionyl chloride treatment

(Shown as A

conditions to get the second generation G

2

, B

2

, C

2

and D

2

in Scheme 1a) under identical synthetic

(a) dendrimer. The

2

mol% PS-PPGDMA-VBC support for dendrimer synthesis and PEG

2

grafting due to its high swelling characteristics compared to higher

cross-linking densities of PPGDMA. Poly(N,N-bisethylamine) den-

drimer was synthesized on PS-PPGDMA-VBC resin by assembling

various stages of reactions were quantitatively and qualitatively

analyzed by above described methods. Different stages of den-

drimer developments to the G (a) generation using Schiff base

2

Schiff base moiety (Shown as A

1

in Scheme 1a) which was prepared

integration is depicted in Scheme 1a.

by reaction between diethylenetriamine (dien) and benzaldehyde

molecules. The formation of Schiff base ligand was identiﬁed via H

NMR analysis (Data given in experimental Sec. 2.3).

Poly(O-benzyl ether) dendrimer in solution was ﬁrst introduced

by Frechet [36] and widely exploited [37e39]. Such dendrimers

were assembled through a convergent route, but on solid support

1

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4081

a

H

N

3 3

N(CH )

Cl

N

Schiff base resin

(G (a))

1

+

HN

0

1,4-dioxane, 100

C

N

H

0

N

A₁

H

PS-PPGDMA-VBC resin

B

1

6M HCl, 60 C, 12h

Schiff base

NH

2

Cl Cl

Cl

N

Ethyl amine resin (G (a))

1

N

NH

2

0

2

/ HCl, 0 C

N

C₁NaNO

Cl

OH

N

Cl

N

Hydroxy ethyl resin (G (a))

1

N

Cl

OH

N

Cl

D₁SOCl

2

, DCM, 12h

N

N,N-bisethyl amine (G (a))

2

dendrimer resin

Cl

A B C D

2

N

Chloro ethyl resin (G (a))

1

Cl

Cl Cl

b

O

OH

HO

O

OCH₃

+

CH OH / H / Reflux

LiAlH / Dry T.H.F

4

O

3

O

OH

^O^C^H3 ^O^C^H3

1,3,5-benzene tricarboxylic acid

1,3,5-Tris(hydroxymethyl)benzene

Trimethyl-1,3,5-benzene tricarboxylate

HO

+

HO

NaH / THF

Wash with dil.HCl

OH

Cl

O

E₁

HO

PS-PPGDMA-VBC resin

Hydroxy methyl resin (G (b))

1

Cl

Cl Cl

Cl

0

F₁SOCl , 55 C, DCM

2

O

Cl

E F

2

O

Cl

O

Cl

O

Cl

O

Chloro methyl resin (G (b))

1

O

Cl

O

O-benzyl ether G₂(b)dendrimer

O

Cl

Cl Cl

Cl

2 2

Scheme1. a. Synthesis of poly(N,N-bisethylamine) G (a) dendrimer on PS-PPGDMA-VBC resin. b. Synthesis of poly(O-benzyl ether) G (b) dendrimer on PS-PPGDMA-VBC resin.

4082

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

a divergent route must be adopted and used to follow inverse

Frechet type dendrons on polymer resin. The on resin reduction of

diesters of dendrons [40,41] to diol using metal hydrides such as

building unit of dendron. Although the phenolic group of

3,5-bis(hydroxymethyl)phenol can be selectively attached to the

solid support, the undesired bond formation with benzylic diol

groups can sometimes be happened.

4 4

LiBH or LiAlH results in deterioration of resin quality by the

deposition of metal hydrides or metals itself in to the pores. It will

obviously affect the diffusion of reagents and hence results in the

retardation of reaction rate. So we have used an alternative path in

which reduction of diester bonds of dendrons to diol groups

done prior to immobilization to the solid supports. Initially we have

used triol derivative 3,5-bis(hydroxymethyl)phenol obtained

by the reduction of dimethyl-5-hydroxyisophthalate as the

So instead of using phenol derivative, a more facile and

symmetric 1,3,5-tris(hydroxyl methyl)benzene was employed

prepared from 1,3,5-benzene tricarboxylic acid commonly known

as trimesic acid. Initially trimesic acid was esteriﬁed with methanol

by adding catalytic amount of sulfuric acid and heated to reﬂux for

6 h. It was followed by neutralization with NaHCO

3

resulted in

triethyl-1,3,5-benzenetricarboxylate. LiAlH reduction followed by

4

Fig. 1. FTIR spectra of Schiff base reactions (a) Initial PS-PPGDMA-VBC resin (b) Schiff base resin (G

) (f) PEGylated resin (G ).

1 1 1

) (c) ethyl amine resin (G ) (d) Hydroxyl ethyl resin (G ) (e) Chloro ethyl resin

(

G

1

2

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4083

extraction with ethyl acetate in a liquideliquid extractor at room

temperature forms colorless crystals of 1,3,5-tris(hydroxyl methyl)

benzene. Because of the symmetric nature of dendron, the bridging

through each hydroxy methyl groups resulted in identical resins in

all respects and avoided problems generally associated with

ether dendrimers were found to be comparatively less than that

calculated from N,N-bisethylamine dendrimers and it might be due

to the bridging formation occurred at the time of O-benzylation

reaction between resin bound chloro methyl groups and hydroxyl

methyl groups of dendritic units.

3

,5-bis(hydroxymethyl)phenol. The reduction reaction of carbonyl

Since poly(ethylene glycol) (PEG) molecule has an amphipathic

nature and is solvated well by both polar and non-polar solvents

[28], optimal support properties can be achieved by grafting PEG

into the polystyrene matrix resulting in an inclusive change in the

solvation behavior. At the same time the mobility and reorganiza-

tion of PEG molecules in the reaction medium create a highly

dynamic process during the course of the reaction. It was observed

that the presence of polar groups in the support will noticeably

affect the performance of resin such as swelling nature, compati-

bility between reaction medium and growing molecules as well as

rate of reaction. So PEG has been grafted on both chlorinated

group was observed from FTIR and formation of dendron was

conﬁrmed from H NMR (Data given in experimental Section 2.4).

After LiAlH

acid carbonyl group of trimesic acid disappeared proving the

hydrogenation reaction. The dendron 1,3,5-tri(methylol)benzene

was assembled to the PS-PPGDMA-VBC resin by Williamsons

etheriﬁcation (Shown as E

resulted in hydroxy methyl resin (G

rination reaction using thionyl chloride (Shown as F

1

ꢂ1

4

reduction, the peak near 1740 cm corresponding to

1

in Scheme 1b) using NaH in dry THF

(b)). It was followed by chlo-

in Scheme 1b)

1

resulted in chloro methyl dendrimer (G

1

(b) in Scheme 1b). After the

ꢂ1

dendron addition a broad peak around 3490 cm appeared in FTIR

spectrum corresponding to free hydroxyl groups of hydroxy methyl

resin and disappeared by the addition of thionyl chloride proving

the conversion of hydroxy methyl groups to chloro methyl groups.

The qualitative interpretation using FTIR for different stages of

dendron grouping reactions are summarized in Fig. 2.

2

dendrimer resins (G ) and the method of grafting is shown in

Scheme 2. solution of sodium polyethyleneglycolate was

A

prepared by dissolving sodium hydride in dry THF containing PEG,

and the chlorinated resins were quantitatively transferred to

respective reaction media and reﬂuxed for 12 h each in an atmo-

2

sphere of N . PEG grafting has been qualitatively analyzed by FTIR

The method of preparation of O-benzyl ether dendrimer was

summarized in Scheme 1b. The introduction of benzyl ether den-

drimer was quantiﬁed by esteriﬁcation of hydroxy methyl resin

with carboxylic acids of Fmoc-Gly-OH. The absorbance of diben-

zofulvene:piperidine adduct formed was measured at 290 by UV

method. The hydroxyl loading value was volumetrically conﬁrmed

by acetic anhydride method and both quantiﬁcations gave

concordant value of 0.206 mmol/g. The amount of chloro methyl

groups formed by thionyl chloride reaction was 0.204 mmol/g and

and the spectra of both the supports showed an intense broad

ꢂ1

absorption peak around 3500 cm

corresponding to the free

ꢂ1

hydroxyl groups and slight broadening of peaks around 1100 cm

corresponding to ethereal bonds. After PEG grafting, the hydroxyl

capacities of PEGylated resins were measured by UV absorbance of

dibenzofulvene:piperidine adduct formed with mixture Fmoc-Gly-

OH/MSNT/MeI. The values obtained were same as 0.246 mmol/g for

both PEGylated poly(N,N-bisethylamine) and poly(O-benzyl ether)

dendrimer resins.

was estimated by Volhard’s method. The chloro methyl G

dendrimer was again subjected to grouping of dendron units and

thionyl chloride reactions (Shown as E and F in Scheme 1b)

respectively led to the formation of G (b) generations dendrimers.

Assemblage of dendron and thionyl chloride reactions was quali-

tatively and quantitatively veriﬁed. The % conversion of chlorine

atoms of each generation by ligands were calculated using the

1

(b)

As expected, after PEG grafting, the functional loading values of

resins were found to decrease remarkably. This is because of the fact

that the number of chemically reactive sites within the resin remains

constant and mass of resin increased considerably due to PEGyla-

tion. This will reduce the polymer functional loading capacity

signiﬁcantly. As preliminary results, decrease in capacity values and

increase in masses supported the PEG grafting. The PEGylation is

2

13

following expression (shown for the case of G

1

) and summarized in

further evidenced from

C NMR analysis. C NMR of initial

Tables 1 and 2

PS-PPGDMA-VBC and different stages of reactions are summarized

in Fig.3. The NMR spectrum of the initial PS-PEGDMA-VBC resin

shows an intense peak at 130 ppm, which is assigned to all aromatic

carbon atom (C-1) except the one that is linked to the backbone

chain. The low intense peak around 145 ppm attributed to the

benzene carbon (C-2) is linked to the backbone chain (ipso carbon

atom). The value at 40 ppm is due to C-3 carbon which connects the

phenyl rings to the backbone chain. Because this peak is somewhat

broad (35e55 ppm), due to methylene of the backbone (which is

expected to appear at 35e40 ppm) as well as methylene carbon of

chloro methyl groups (usually expected to appear 40e50 ppm) are

not separately visible. An intense sharp peak around 70 ppm

corresponds to the CeOeC carbon atom of poly(propylene glycol)

dimethacrylate cross-linker. After dendron grouping, a slight

broadening of peak corresponding to cross-linker CeOeC happened.

PEGylation is evidenced by sharp intense peak around 70 ppm

in both grafted resins. Surface morphological studies showed that

after PEGylation the clean and smooth surface morphology of

PS-PPGDMA-VBC support has been changed to rough and irregular

due to the tethering effect of PEG molecules, as shown in Fig. 4.

Chlorinecapacityof G ꢂChlorinecapacityof G

2

1

%Conversion ¼

Chlorinecapacity of G₁

ꢁ

100

The extent of incorporation using both kind of dendron mole-

cules were quantitatively found by CHN analysis in which G

generation amino ethyl Schiff base and O-benzyl ether dendrimers

for getting amino methyl O-benzyl ether dendrimer resin, chloro

methyl G (b) and G (b) resins were subjected to Gabriel pthalimide

reaction) were used for CHN analysis. The amino capacity values

mmol/g) of both kind of dendrimer resins were found out by using

2

(

1

2

(

an expression, N/14n, where ‘N’ is the % of N obtained from CHN data

and ‘n’ is the number of nitrogen atoms present in ligand which is 3

for Schiff base and 2 for tri(methylol)benzene ligands. The functional

loading values calculated by different methods for both dendrimers

at various stages of reactions were summarized in Tables 1 and 2.

The % conversion has also been calculated from amino loading values

obtained from CHN analysis using the same expression used for %

conversion and found to be in concord with chlorine loadings

calculated. The PS-PPGDMA-VBC resin was also subjected to Gabriel

3.2. Swelling studies

2

pthalimide reaction to get amino resin and eNH groups formed

were quantiﬁed by UV method using Fmoc-Gly-OH and the loading

value obtained was 0.103 mmol/g. It has been observed that func-

tional loading values and % conversions calculated for O-benzyl

Swelling of a cross-linked polymer is equivalent to solvation of

a linear polymer [42]. Instead of dissolving and forming a solution,

the cross-linker act as anchors to prevent excessive motion of the

4084

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

Fig. 2. FTIR spectra of trimesic acid reactions (a) Trimesic acid (b) Trimesic acid reduced (c) Initial PS-PPGDMA-VBC (d) Hydroxy methyl resin (G

PEGylated resin (G ).

1 1

) (e) Chloro methyl resin (G ) (f)

2

polymer chains required to form a solution. As a result, solvent is

taken up by the cross-linked polymer resulting in an increase in

volume as solvent molecules occupy positions between polymer

chains. The swelling of a resin in a given solvent is an indication of

site accessibility and thus resin functionalization. Since polymer

supported reactions are heterogeneous in nature and solvent/

substrate molecules have to diffuse from bulk phase to the resin

matrix, swelling parameter is an important criterion for a given

Table 1

Functional loading values calculated for poly(N,N-bisethylamine) dendrimers.

Gen.

eNH

mmol/g

2

(UV)

eOH (UV)

mmol/g

eCl (Volumetric)

mmol/g

% conversion

(Volumetric data)

% N (CHN)

eNH

mmol/g

2

(CHN)

% conversion

(CHN data)

G

1

2

0.207

0.412

0.206

0.411

0.206

0.410

98.08

99.03

0.856

1.709

0.204

0.407

98.05

99.51

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4085

Table 2

Functional loading values calculated for poly(O-benzyl ether) dendrimers.

Gen.

eOH (UV)

eCl (Volumetric)

% conversion

% N

eNH

2

(CHN)

% conversion

mmol/g

(Volumetric data)

(CHN)

mmol/g

(CHN data)

G

1

2

0.206

0.405

0.204

0.404

96.15

98.04

0.566

1.117

0.202

0.399

96.12

97.53

polymer. So we have measured the solvent imbibition character-

istics of PEGylated dendrimer supports at different stage of reac-

tions. Many methods have been reported for determination of

degree of swelling [43e45] and we have chosen the syringe

method. A delicate balance exists between the degree of cross-

linking and swelling. Reducing cross-link density causes increase

in swelling, but at a cost of mechanical stability, beads broken by

agitation can be difﬁcult to ﬁlter. Conversely, the increasing cross-

link density results in greater mechanical strength but at a cost of

swelling ability. If reagents cannot interact with each other in

a swollen medium, no reaction will occur. In order to check the

inﬂuence of cross-linking density on swelling ability, we have

synthesized PS-PPGDMA-VBC supports in different mol percent-

ages (2, 4, 6 and 8 mol%) of PPGDMA and measured the swelling

volume in different solvent polarities. Results are summarized in

Fig. 5(a). It was observed that as the cross-linking density increases,

the solvation ability of support gradually decreases resulting in

poor solvation and hence reduction in swelling nature. It is because

of the reason that increase in cross-linker density restricts the

mobility of polymer chains that allows the inﬁltration of solvent

molecules through solid matrices resulting in less rate of diffusion

and hence swelling. When the swelling nature of the different

cross-linked PPGDMA solid matrix compared, the 2 mol% cross-

linked system showed better swelling character, and was used for

developing dendrimers and PEG grafting.

resins verily depend on PEG grafting and showed better swelling

results in all solvents used for comparison and the extent of swelling

was found higher in polar media. The development of dendrimer

sites alone not marginally affects the swelling character, but in the

case of chlorinated O-benzyl ether dendrimer resin, swelling nature

has slightly reduced in polar solvents and it might be because of the

increase in hydrophobicity due to increase in number of phenyl

rings in the polystyrene backbone. But swelling volumes of PEGy-

lated resins were high compared to 2 mol% cross-linked, dendrimer

coupled and commercially available Tenta Gel resins which proved

the aptness, compatibility and solvent imbibition ability of newly

developed PEG grafted resins for solid phase organic reactions. The

swelling results of different stages of dendrimer resins are

summarized in Fig. 5(b). The high swelling results might be due to

the presence of amphiphilic nature of PEG units which can adopt

different helical structures in reaction medium leading to high

mobility and compatibility with solvent molecules [28]. The grafted

PEG molecules can act as spacer molecules which can take the

substrate molecules from the reaction medium in a more superﬁcial

mode and will be get solvated easily compared to rigid sterically

crowded hydrophobic macromolecular network. So in both polar

and non-polar media, newly developed supports can be used

effectively for organic reactions due to appreciably high swelling

characteristics.

The swelling nature of chlorinated G

2

dendrimers and corre-

3.3. Chemical stability studies

sponding PEG grafted resins were measured by syringe method and

compared with commercially available Tenta Gel. The swelling

behaviors of PEGylated resins at different stage of dendrimer

synthesis were measured using various polar and non-polar organic

solvents. Our results showed that the swelling characteristics of the

A rigorous situation for choosing a support for solid phase

organic synthesis is that the resin should be chemically stable until

the substrate is detached at the end of synthesis. An ideal support

should have comparable physical and mechanical properties so that

O

H

n

N

Cl

HO

O

n

N

O

Cl

H

N

n

N

+

Cl

O

N

O

H

O

n

1

. NaH + PEG in dry THF

Cl

O

PEGylated (N,N-bisethylamine) dendrimer

(

55 C , 30 min )

O

N,N-bisethylamine (G (a))

2

n

O

2

. Mixture added to resin

in dry THF (80 C overnight)

Cl

O

H

0

n

O

Cl

O

H

+

n

O

HO

O

Cl

O

H

Cl

n

O

PEG

O-benzyl ether (G (b))

O

H

2

n

PEGylated (O-benzyl ether) dendrimer

Scheme 2. PEGylation of poly(N,N-bisethylamine) G

2

(a) and poly(O-benzyl ether) G (b) dendrimers.

4086

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

Fig. 3. ¹³C NMR spectra of (a) PS-PPGDMA-VBC resin (b) Schiff base resin (G

benzyl ether resin (G ) (d) PEGylated N,N-bis(ethylamine) (G ) (e) PEGylated (O-benzyl

ether) (G ).

1

) (c) O-

1

2

it permits identical manipulations such as shaking and ﬁltration in

various solid supported reactions. So we have tested the chemical

stability of PEGylated dendrimer resins in various peptide synthetic

conditions. The resin treated solutions were weighed before and

after treatments as well as recorded the IR spectra of treated

samples and compared to the original. Dendrimer resins were

treated with TFA for 12 h, the reagent generally used to cleave the

peptide from support, and checked at different time intervals (1 h

each). The PEGylated poly(N,N,ebisethylamine) dendrimer was

found chemically stable throughout the period of treatment, but

poly(O-benzyl ether) resin was not. Poly(O-benzyl ether) den-

drimer was found to be stable up to 5 h and starts to show

degradation which was evidenced from FTIR analysis. This is due to

the formation of highly stable benzyl carbocation formed by the

attack of hydronium ion generated from TFA. The resins were

treated with 30% TFA/DCM mixture for 4 h, the reagent usually used

for Boc removal, and found to be stable as evidenced by recording

identical IR spectra. The stability of supports in various basic

environments were also veriﬁed by treating resins with 20%

Fig. 4. Scanning electron micrographs of (a) PS-PPGDMA-VBC resin (b) PEGylated

poly(N,N-bisethylamine) G₂dendrimer (c) PEGylated poly(O-benzyl ether) G₂

dendrimer.

to withstand the various nucleophilic attack generated by these

basic reagents.

3.4. Comparative peptide synthesis

4

piperidine:DMF mixture (2 h each), 2 M NH OH (4 h each) and 2 M

NaOH (4 h each) and recorded identical IR spectra. The spectral

studies showed that newly synthesized resins were stable enough

The nature of polymer/peptide interaction can be revealed by

synthesizing difﬁcult peptide sequences on newly synthesized

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4087

Fig. 5. Swelling comparison of (a) PS-PPGDMA-VBC resin in various cross-linking densities (b) PEG grafted and non-grafted dendrimer resins with Tenta Gel.

supports. So we have checked the efﬁciency of PEGylated den-

drimer resins by synthesizing classically difﬁcult ACP as well as

biologically potent endothelin classes of disulﬁde bonded peptides.

Due to slow Fmoc deprotection and poor coupling reactions in

various stages of its synthesis, a difﬁcult peptide now used as

a standard to evaluate the efﬁciency and various chemistry used

during the synthesis is Acyl Carrier Protein fragment (ACP). The

peptide ACP (peptide a) is highly hydrophobic in nature and is

reported as difﬁcult sequence for synthesis due to bulky side chains

All the coupling reactions were completed in one coupling itself,

however a second coupling was given in all stages of acylation to

ensure the reaction completion. Since the reactions are diffusion

controlled and because of the presence of hydrophilic cross-linker

as well as ﬂexible PEG molecules in newly developed dendrimer

supports, within short period the reaction sites can be fully exposed

to the reaction medium, hence short reaction time and less quantity

of reagents were sufﬁcient for reaction completion. After the entire

synthesis, the peptides were removed from the supports using the

cleavage cocktail comprising of TFA and cation scavengers. The

b

and high <SP > values which is a measure of the peptide tendency

to aggregate. This sequence has been widely accepted as model

peptide to illustrate the synthetic efﬁciency of a reagent when

a polymer or synthetic condition is changed. The difﬁcult peptide

universal cocktail reagent K [TFA/H

2

O/Phenol/Thioanisol/EDT

(82.5:5:5:5:2.5)] has been used for cleaving all the peptides under

similar cleavage conditions. The peptides were ﬁnally precipitated

using ice-cold ether and washed thoroughly with excess of ether to

remove the scavengers and dried well. The yields of crude peptides

before and after cleavage were noted for all the three resins and

compared. The % crude yields (calculated by weighing resins before

and after synthesis) of peptides before cleavage for poly(N,N-

bisethylamine), poly(O-benzyl ether) and Tenta Gel resins were

96.6%, 92.6% and 84.7% respectively. The % yields of crude ACP

peptides obtained after cleavage (calculated from ﬁrst amino acid

substitution values) were calculated as 93.4%, 88.6% and 80.02% for

poly(N,N-bisethylamine), poly(O-benzyl ether) and Tenta Gel resins

respectively. The crude fractions were puriﬁed, collected and

lyophilized and the % of pure peptides collected were 84.8%, 79.3%

and 71.9% for poly(N,N-bisethylamine), poly(O-benzyl ether) and

Tenta Gel resins respectively. The HPLC proﬁles of ACP crude

peptides synthesized and corresponding MALDI-TOF (using puriﬁed

fractions) obtained are shown in Fig. 6aec and Fig. 6def for N,N-

bis(ethylamine), O-(benzyl ether) and Tenta Gel resins respectively.

sequence of ACP (V⁶⁵

Q

66

A

67 68 69 70 71 72 73 74

A

I

D

Y

I

N

G ) fragment

was synthesized on newly developed PEGylated dendrimers as well

as on Tenta Gel resins and compared the yields and efﬁciencies.

PEGylated poly(N,N-bisethylamine) and poly(O-benzyl ether)

dendrimer resins were used for synthesis without terminal modi-

ﬁcation and linker attachment because PEG itself proved as linker

for esteriﬁcation reaction. The remaining amino acids were incor-

porated to dendrimer resins using 1.5 mmol excess with respect to

ﬁrst amino acid loading values and used for further calculations.

Using PEGylated dendrimer resins, deprotection of Fmoc groups

using 20% piperidine:DMF mixture was completed in less than

15 min for most of amino acids (except for Arg and Asp which took

2

0 min) and acylation reactions at each stage of amino acid

coupling was ﬁnished within 30 min. Using Tenta Gel, the time

required for all amino acid coupling as well as deprotection reac-

tions were found to be 45 min and 30 min respectively, but with Tyr

and Ile the time needed for 100% reactions were found to be less.

4088

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

3

.5. Synthesis of disulﬁde bonded peptides

with DMF (5 ꢁ 15 mL), DCM (5 ꢁ 15 mL) and ether (5 ꢁ 15 mL) and

dried well before detaching the peptide from the support. The

peptide was removed from the support using cleavage solution

Endothelin-1 is a class of highly potent vasoconstrictor peptides

ﬁrst isolated from porcine endothelial cell supernatant. Three

structural isoforms of the hormone have been identiﬁed, ET-1, ET-2

and ET-3 encoded by three distinctive genes expressed indepen-

dently in the human genome. Endothelins act to stimulate

contraction of many smooth muscle tissues including blood

vessels, uterus bladder and intestine. In addition to the linear

forms, a number of single and double disulﬁde bonded peptides

have been cloned and synthesized. Disulﬁde bridges play a pivotal

role in maintaining biologically active conformations of several and

natural synthetic peptides. Here, endothelin-1 human peptide

having 21 amino acid residues with disulﬁde bonds between 1st

2

having TFA:H O:EDT:TIS in volume ratio of 94:2.5:2.5:1 for 4 h. The

peptide was precipitated by adding cold ether and washed using

excess ether to remove the scavengers. The HPLC chromatogram of

the single disulﬁde bonded peptide is shown in Fig. 7a. The peptide

was further puriﬁed by fractional collection method and used for

second disulﬁde bond formation in which 10 mg of peptide was

dissolved in 50 mL mixture of TFA: DMSO: Anisole 97.9:2:0.1 (v/v)

in a R.B ﬂask and the mixture was stirred for 1 h at room

temperature. The volume of mixture was concentrated under

vacuum and precipitated using chilled ether. This was followed by

continuous washing with cold ether to remove scavengers and

used for lyophilization and HPLC analysis. The HPLC proﬁle of

peptide after second disulﬁde bond formation is shown in Fig. 7b.

The formation of ﬁrst and second disulﬁde formation was

conﬁrmed from MALDI-TOF analysis in which the theoretical mass

values did match with the data obtained from analysis and are

shown in Fig. 7c and d. The yield of crude peptide obtained after on

bead disulﬁde bond formation was 83% which is calculated on the

basis of ﬁrst amino acid substitution. The yield of peptide obtained

after puriﬁcation was 73%. The yields of crude and puriﬁed

peptides obtained after second disulﬁde bond formation was 69%

and 61% respectively.

and 15th residue [Cys¹] and 3rd and 11th residue [Cys

ꢂ15

3ꢂ11

] was

synthesized using PEGylated poly(N,N-bisethylamine) dendrimer

by the combination of on bead disulﬁde [46] and TFA/DMSO/Ani-

sole [47] methods. The peptide having amino acid sequence is

2

NH -Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-

Cys-His-Leu-Asn-Ile-Ile-Trp-OH (Peptide b). The ﬁrst C-terminal

attachment was carried out by MSNT coupling under dry condi-

tions using mixture of Fmoc-Trp(Boc)-OH/MSNT/MeI in molar ratio

of 1:1:0.75 in dry DCM. Quantitative incorporation of ﬁrst amino

acid was veriﬁed by spectrophotometric measurement of adduct

dibenzofulvene-piperidine formed. Further coupling reactions

were carried out using 2 equiv excess of Fmoc amino acids, HBTU,

HOBt and DIEA in DMF. The sulfur atom of 3rd and 11th Cystine

residues were protected with tert-butyl (t-Bu) groups which are

stable in TFA and kinetically labile in TFA/DMSO/Anisole mixture.

Tert-Butylthio (S-t-Bu) and 4-methoxytrityl (Mmt) groups were

used as orthogonal thiol protecting groups of 1st and 15th cysteine

residues undergoing on bead disulﬁde bond formation. On bead

disulﬁde bond formation was carried out prior to cleavage from

support followed by second disulﬁde formation by TFA/DMSO/

Anisole method which was performed after detaching peptide

from resin. The method followed for on bead disulﬁde bond

formation was explained in experimental Section 2.13(b). The

cyclization reaction was monitored by measuring the absorbance

of 5-nitropyridine-2-thine at 386 nm. All the cyclizations were

completed in less than 35 min. The resin was repeatedly washed

Endothelin-2, human peptide having primary structure

H N-Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-

2

Cys-His-Leu-Asp-Ile-Ile-Trp-OH (peptide c), was synthesized on

PEGylated poly(O-benzyl ether) dendrimer. The ﬁrst amino acid

attachment and peptide synthesis were same as that described for

peptide b. The disulﬁde linkages that connect the cysteine amino

1

ꢂ15

3ꢂ11

acids are Cys

and Cys

. The disulﬁde bond formation was

achieved by combination of on bead as well as iodine oxidation [48]

methods. The sulfur atom of 1st and 15th cysteine residues were

protected with S-t-Bu and Mmt groups respectively for on bead

disulﬁde bond formation and 3rd and 11th positions were pro-

tected with 4-acetamidomethyl (Acm) groups which are kinetically

stable in TFA. Initially on bead disulﬁde bond formation was carried

out on peptidyl resin and the protocol followed was exactly same as

described for peptide b. The cyclization was completed in less than

Fig. 6. HPLC and MALDI-TOF of ACP synthesized on (a) PEGylated poly(N,N-bisethylamine) (d) corresponding MALDI-TOF (b) PEGylated poly(O-benzyl ether) (e) corresponding

MALDI-TOF (c) Tenta Gel (f) corresponding MALDI-TOF.

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

4089

Fig. 7. HPLC and MALDI-TOF of endothelin-1 synthesized on poly(N,N-bisethylamine) (a) After on bead formation (b) After TFA/DMSO/Anisole oxidation. MALDI-TOF of peptide (c)

After on bead reaction (d) After TFA/DMSO/Anisole oxidation.

3

(

0 min. The peptidyl supports thoroughly washed with DMF

5 ꢁ 15 mL) and DCM (5 ꢁ 15 mL) and well dried single disulﬁde

bonded peptidyl support was suspended in cleavage cocktail

comprising of TFA: TIS: H O (95:2.5:2.5) for 4 h. The peptide

2

of N gas followed by the addition of 1M HCl preceded by imme-

diate addition of 0.1 M iodine in 50% aqueous methanol. The

reaction mixture was allowed to stir for 45 min, quench the iodine

by adding 1 M ascorbic acid in drop wise manner until the mixture

turned colorless and concentrated by evaporation under reduced

pressure. The crude peptide has been subjected to freeze-drying

and analyzed by HPLC. The HPLC chromatogram of crude product

is shown in Fig. 8b. The pure fraction has been collected by HPLC

(Fig. 8c) and checked the masses by MALDI-TOF analysis. The

MALDI-TOF results of pure fractions of on bead and iodine oxidized

peptides are shown in Fig. 8d & e respectively.

2

cleaved was precipitated with chilled ether followed by excessive

washing with cold ether to remove all scavengers. The HPLC

chromatogram of the mono disulﬁde bonded peptide is shown in

Fig. 8a. The peptide is further puriﬁed by fractional collection

method, lyophilized and collected for second disulﬁde bond

formation by iodine oxidation method. The Cys(Acm) peptide

(

10 mg/mL) was dissolved in 50% aqueous methanol under a stream

Fig. 8. HPLC and MALDI-TOF of endothelin-2 synthesized on poly(O-benzyl ether) (a) After on resin formation (b) After iodine oxidation(crude) (c) After iodine oxidation (puriﬁed)

and corresponding MALDI-TOF of peptide (d) After on bead reaction (e) After iodine oxidation.

4

090

M.A. Siyad, G.S.V. Kumar / Polymer 53 (2012) 4076e4090

[3] Thomson LA, Ellman JA. Chem Rev 1996;96:555e600.

4

. Conclusions

[

4] Gallo MA, Barrett RW, Dower WJ, Fodor SPA, Gordon EM. J Med Chem 1994;

7:1233e51.

3

In conclusion, poly(ethylene glycol) grafted poly(N,N-bisethyla

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to evaluate its performance in various peptide synthetic conditions.

The dendrimeric templates were shaped from Schiff base and tri-

mesic acid molecules. The Schiff base synthesized from diethyle-

triamine and benzaldehyde has been uniquely bonded to the chloro

methyl groups of resin speciﬁcally through secondary amine groups

followed by acidolytic cleavage, diazotization and thionyl chloride

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hydrophilic balanced system and showed excellent physical as well

as chemical characteristics throughout the different stages of

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properties of ideal solid supports such as mechanical and

thermal stabilities of styrene backbone, ﬂexibility of poly(propylene

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poly(ethylene glycol) molecules. Both PEG grafted dendrimer resins

swells extremely well in most of common solvents used for study

and is, therefore, suitable for a broad range of organic chemistries.

The newly synthesized PEGylated dendrimeric supports can be

effectively used for different peptide synthetic conditions which are

proved by synthesizing classically difﬁcult ACP sequence and

disulﬁde bonded peptides in high yields and purities. Here we look

forward that the described properties will transform well for future

applications to solid phase syntheses of other types of organic

molecules and of combinatorial libraries also.

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Acknowledgments

The authors acknowledge the CSIR, New Delhi, for providing

funding and RGCB for research facilities. The help rendered by IISc,

Banglore, for NMR and MALDI facilities is gratefully acknowledged.

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Article Doi

Poly(ethylene glycol) grafted polystyrene dendrimer resins: Novel class of supports for solid phase peptide synthesis

DOI: 10.1016/j.polymer.2012.07.011

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