Poly(ethylene glycol)-Containing Supports for Solid-Phase Synthesis

Chapter 17

Poly(ethylene glycol)-Containing Supports for Solid-Phase Synthesis of Peptides and Combinatorial Organic Libraries 1

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Downloaded by PURDUE UNIV on August 31, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch017

George Barany , Fernando Albericio , Steven A. Kates , and Maria Kempe 1

Department of Chemistry, University of Minnesota, Minneapolis, MN 55455 Department of Organic Chemistry, University of Barcelona, E-08028 Barcelona, Spain PerSeptive Biosystems, Inc., 500 Old Connecticut Path, Framingham, MA 01701 Department of Pure and Applied Biochemistry, University of Lund, S-221 00 Lund, Sweden 2

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4

The choice of a polymeric support is a key factor for the success of solid-phase methods for syntheses of organic compounds and biomolecules such as peptides and oligonucleotides. Classical Merrifield solid-phase peptide synthesis, performed on low cross-linked hydrophobic polystyrene (PS) beads, sometimes suffers from sequence-dependent coupling difficulties. The concept of incorporating PEG into supports for solid-phase synthesis represents a successful approach to alleviating such problems. This chapter reviews the preparation of families of poly(ethylene glycol)-polystyrene (PEG-PS) graft as well as (highly) Cross-Linked Ethoxylate Acrylate Resin (CLEAR) supports developed in our laboratories, and demonstrates their applications to the syntheses of a wide range of targets in connection with numerous research objectives. Solid-phase synthesis (SPS) is a powerful tool for the preparation of a wide range of molecules (1-5). The methodologies were first applied to the assemblies of biomolecules such as peptides and oligonucleotides (2-8), and the past few years have witnessed a great proliferation of work extending the principles to the synthesis of organic compounds, particularly in conjunction with combinatorial libraries (9-22). The efficiency of solid-phase methods depends on a number of parameters, of which the choice of polymeric support is a key factor for achieving success. For solid-phase peptide synthesis (SPPS), the majority of work through the 1980's has been carried out with the low divinylbenzene-cross-linked polystyrene (PS) beads that were introduced to the area in Merrifield's original studies (1, 2). Sequence-dependent coupling difficulties were ascribed by Sheppard and others to the hydrophobic nature of PS, and spurred the development of more hydrophilic supports (5). While it is not certain that this reasoning to move away from PS was entirely valid, there can be no doubt that a wide range of materials, many of them now commercially available, have since been established as useful for SPPS. These include polyamide supports (regular or encapsulated in rigid materials such as kieselguhr or highly cross-linked polystyrene) (23-26), polystyrene-Kel F (PS-Kel F) grafts (27-29), polyethylene© 1997 American Chemical Society

In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

239


240

POLY(ETHYLENE GLYCOL)

polystyrene (PE-PS) films (30), membranes (31-33), cotton and other carbohydrates (34, 35), and chemically modified polyolefins ("ASPECT') (36). Solid-phase synthesis of oligonucleotides (7) has been carried out mainly on controlled pore glass (CPG). Much solid-phase synthesis of organic compounds is carried out on various functionalized PS resins, and some of the other materials mentioned in this paragraph have been used as well. Arguably, the most significant set of practical advances in materials for solidphase synthesis has come from the recognition that properties of supports are often improved by the incorporation of multiple hydrophilic ethylene oxide units (Table 1). The present chapter focuses on families of poly(ethylene glycol)-polystyrene (PEG-PS) graft and Cross-Linked Zsthoxylate Acrylate flesin (CLEAR) supports developed and established from our own laboratories. Grafting of PEG onto PS, for other applications, was carried out first in the 1970's and early-1980's by Inman (37), Regen (38), Warshawsky and Patchornik (39), Sherrington (40), and Mutter (41,42). Our initial studies and contemporaneous pioneering independent work of Bayer with Rapp (50-54) were reported in the mid-1980's; the commercial viability and advantages of PEG-containing supports [including Meldal's PEGA (56,57)] were realized in the 1990's. Scanning electron micrographs of four such supports are shown in Figure 1, and should be contrasted with respect to the size, spherical nature, and uniformity of beads. Herein, we review methods for the preparation of PEG-PS and CLEAR supports, and provide examples for their applications to synthesis. Goals for Solid-Phase Supports Criteria for what constitutes an optimal set of features for a suitable SPS support are controversial, and much of the conventional wisdom in the field is based on empirical observations. While the term "solid-phase" might imply a static resin support, it turns out that this is not at all the case with those materials which serve best for SPPS (64-66). Reactions commonly occur on mobile, well-solvated, and reagent-accessible polymer strands throughout the interiors of the supports; relatively few of the sites occur in surface regions (67). The conventional wisdom in the field has therefore been that supports should have the minimal level of cross-linking consistent with stability, in order to allow the material to swell. PEG-PS fits this model, since the parent hydrophobic PS is low-crosslinked and the hydrophilic pendant PEG chains enhance the swelling to cover a broad range of solvents (Table 2, middle columns). Nevertheless, we have shown that CLEAR supports, which are similar to PEG-PS in terms of the presence of ethylene oxide units but differ insofar as CLEAR's are highly cross-linked (^ 95% by weight of cross-linker), also demonstrate substantial swelling in many solvents (Table 2,rightcolumns). The mechanical and physico-chemical properties of PEG-PS and CLEAR translate into excellent performance characteristics for stepwise SPPS, both continuous-flow and batch wise, in all cases in conjunction with Fmoc/iBu chemistry and in some cases with the Boc/Bzl strategy. Information is also accumulating for solid-phase oligonucleotide synthesis and solid-phase organic synthesis (SPOS) with these materials. PEG-PS Supports The defining idea behind grafting PEG onto PS was to combine in the same support a hydrophobic core of PS with hydrophilic PEG chains. A further concept was that PEG might act as a "spacer" separating the starting point of solid-phase synthesis from the PS core (Figure 2). Our general approach for the preparation of PEG-PS supports relies on the covalent attachment via amide linkages of PEG of defined molecular weight onto suitable amino-functionalized microporous PS. Bayer and Rapp have championed an alternative way to prepare a version of PEG-PS, which they refer to as POE-PS (known commercially as TentaGel or NovaSyn), by means of


17.

BARANY E T A L .

241

PEG-Containing Solid Supports

Table 1. Poly(ethylene glycol)-containing synthetic polymers used as supports in solid-phase synthesis a


Support

Description

Reference(s) PEG-PS

poly (ethylene glycol) grafted covalently onto 1% 43-49 cross-linked microporous poly(styrene-codivinylbenzene)

POE-PS (TentaGel or NovaSyn)

poly(ethylene glycol) polymerized onto poly(styrene^>^\rinylbenzene)

PEG/PS (ArgoGel)

55 poly(ethylene glycol) polymerized onto a malonate-derived l,3-dihydroxy-2-methylpropane "branching" unit bound at the 2 position to benzyl group from poly(styrene-C6>-divinylbenzene)

PEGA

poly^^-^memylaCTylamide-cobisacrylamido poly(ethylene glycol)-C0monoacrylamido poly(ethylene glycol))

56, 57

TEGDA-PS

rx)ly(styrene-Ci>-tetra(ethyleneglycol) diacrylate)

58

PEO-PEPS

3,6,9-trioxadecanoic acid coupled to polyethylene-polystyrene (PE-PS) films

59

CLEAR

poly(trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate^i^^ylarnine)

60-63

a

50-54

Abbreviations and trademarks for PEG-containing polymers are literally those of the referenced inventors and/or companies commerciaHzing the appropriate supports, and are listed without any judgement by the authors of this review on the similarities and differences among the materials.




242

Figure 1. Scanning electron micrographs [accelerating voltage: 10 kV] showing the shape and texture of (a) PEG-PS, (b) TentaGel, (c) PEGA, and (d) CLEAR-IV.


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BARANY E T A L .

243


Table 2. Swelling of PS, PEG-PS, and CLEAR Supports


Solvent

Bed volume (mL) of 1 g polymer

PS

b

CH Cl2 2

MeOH DMF

CH3CN H2O

nd nd nd 2 5 nd 5.5 5 5.5 2 2

CLEAR-I*

PEG-PS high-load

PEG-PS low-load

CLEAR-IV

f

0

0

Hexane Toluene /BuOMe EtOAc THF TFA

a

3 5 3.5 5 6.5 12 10 7 8 6.5 8

nd nd nd 4 4.5 nd 6 5 5 3.5 3.5

nd nd nd 4 4 nd 5.5 4.5 5 4 3

3 5 3 4.5 5 6.5 5.5 5 5.5 5 4

a

Solvents listed in order of increasing dielectric constant MBHA-PS: 1.0mmol/g Low load PEG-PS: 0.15 mmol/g High load PEG-PS: 0.55 mmol/g C L E A R I,: 0.26 mmol/g CLEAR-IV: 0.17 mmol/g nd = not determined

b

c

d

e

f

X

[

PEG

)

(PS)

Figure 2. General structure for PEG-PS supports ("spacer" model). PS = low cross-linked polystyrene, PEG = poly(ethylene glycol), typically of average molecular weight 2000 (~ 44 repeating units); X = starting point for peptide (or other) synthesis.



244


anionic copolymerization of ethylene oxide onto a PS-bound initiator. While we are not in a position to contrast the relative merits of these two ways of accessing PEG-PS, it should be noted that our methodology allows the final weight ratio of PEG:PS, as well as the final loading, to be controlled by both the starting loading of amino groups on PS and the molecular weight of PEG. Final loadings (0.1 to 0.6 mmol/g) can be readily tailored: lower loadings appear to be better for large macromolecular targets and higher loadings are preferred for combinatorial SPOS. Our original "first generation" approach (43, 47) started from commercially available homobifunctional PEG (diol). This was converted in six chemical steps and one key chromatographic step to the pure heterobifunctional Λ^-Boc-protected PEG-ωamino acid (Figure 3). Thefirstchemical step, reaction with thionyl chloride in the presence of pyridine, was intentionally prevented from reaching completion, so as to partially derivatize PEG. The resultant statistical mixture of dichloride, monochloride, and unreacted diol was reacted further with ethyl isocyanatoacetate to convert PEG hydroxyls to the corresponding urethanes, sodium azide m DMF to convert PEG chlorides to azides, and hydroxide to saponify the pendant esters. At this stage of the overall process, the desired derivative of defined structure 4 [boxed in Figure 3] was isolated efficiently by ion-exchange chromatography with a stepwise ammonium bicarbonate gradient. Catalytic hydrogenolysis men smoothly converted the azide to the amine, which wasfinallyprotected as its Boc derivative. It should be stressed that all chemical transformations other than the first step proceed quantitatively, hence avoiding potential problems should extraneous functional groups be carried over to the final product. Polymeric intermediates and thefinalderivative were purified based on the physical properties of PEG, yet they were amenable to accurate characterization by analytical and spectroscopic techniques commonly applied to low molecular weight organic compounds. The next step in preparing PEG-PS suitable for evaluation in peptide synthesis was to carry out oxidation-reduction coupling of the heterobifunctional protected PEG 5 onto aminomethyl-PS, followed by deprotection/coupling cycles to introduce an "internal reference" amino acid (IRAA), usually Nle, and a handle such as 5-(4-Fmoc-aminomethyl-3,5dimethoxyphenoxy)valeric acid (Fmoc-PAL-OH) (68, 69). Typically loadings were 0.25-0.30 mmol/g (loadings for starting PS was 0.6 mmol/g), which implies a ratio of PEG:PS of 0.55:0.45. Although the original route (Figure 3) to PEG-PS is certainly unambiguous, we sought simpler alternatives that might avoid thetime-consumingprocess — particularly the chromatography step — to obtain the required PEG intermediates. Particularly attractive was to couple onto p-methylbenzhydrylamine (MBHA) polystyrene resins, already containing an IRAA, homobifunctional PEG-diacids [Figure 4, ref. 46], The PEG-diacids (9) were obtained easily by reaction of the corresponding inexpensive PEG-mamines (8) with succinic anhydride. This approach accepts a modest level of accompanying cross-linking (e.g., PS with 0.6 mmol/g substitution typically gave grafts with PEG:PS = 0.45:0.55, from which it can be calculated that ~ 2/3 of the PEG remains available to act as a spacer). The pendant carboxyl groups of PEG-PS derived from the PEG-acids were next converted to amino groups (final loading typically 0.15-0.25 mmol/g) by a coupling reaction with excess emylene&amine (EDA). Through a fortuitous set of observations described elsewhere (45,70) we were led to consider the possibility that environmental effects (Figure 5) contribute to the efficacious properties of PEG-PS, and to devise an unambiguous formulation to test this idea, as follows: N -Fmoc, ^-Boc-ornithine was coupled onto aminomethyl-PS (0.95 mmol/g), the Fmoc-group was removed, and a monofunctional methoxy-PEGacid was added. The resultant graft comprised PEG:PS = 0.60:0.40, with a loading of 0.31 mmol/g; Boc removal exposed the ornithine side-chain for handle incorporation and further peptide chain assembly (44, 45, 70). Such supports were successful when applied to challenging target sequences, allowing us to conclude that the PEG environment was much more critical than any spacer effects. a


17. B A R A N Y E T A L .

245


HO(CH CH 0) H = PEG(OH) 2

2

n

2

1.SOCl2/C5H5N/C H CH3 2. OCNCH C0 Et / cat. Et N / CH Cl2 6

2

5

2

3

2

PEG(CI) ( Ο - C - NHCH C02Et) 2

3. NaN / DMF 4. O H / H 0 3

2

PEG(N )P-C-NHCH C0 H)


3

2

2

DEAE-Sephadex remove corresponding diacid and diazide

N -(CH CH 0) —C-NHCH C02H 3

2

2

n

2

5. cat. H (Pd/C)/ EtOH 2

6. B0C2O/DIEA/DMF

Boc-NH-(CH CH 0) —C-NHCH C0 H 2

2

n

2

2

H N —(PS) 2

^Vs-^,

(n-Bu) P 3

BOC—NH—I P £ G r-C-NH—(PS 1. TFA-CH Cl2(1:1) 2. DIEA-CH Cl2 (1:19) 3. Boc-IRAA-OH, DCC/HOBt 4. TFA-CH CI (1:1) 5. DIEA-CH Cl2 (1:19) 6. Handle introduction 2

2

2

2

2

X-{

HANDLE

J - C - I R A A — N H - | PEG

NH—(PS)

Figure 3. Preparation of heterobifunctional PEG (5) in six steps, and subsequent use to generate "first generation" PEG-PS supports. Compounds 2 and 3 are statistical mixtures, whereas the boxed compound 4 is the compound with the shown defined structure as isolated after ion-exchange chromatography. The Figure shows the grafting of 5 onto amino-functionalized supports, introduction of an "internal reference" amino acid (IRAA), and introduction of a carboxyl group-containing handle. X = starting point for peptide (or other) synthesis. The indicated chemistry was carried out both for η = 45 (PEG-2000) and η = 90 (PEG-4000). Adapted from Zalipsky et al. (réf. 47). In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

246


H N-( 2

PEG } - N H

Ο


8

2

in DMF

H N—(PS), 2

TBTU-DIEA, in DMF

10 (i) /BuCOCkDIEA in DMF (ii) EDA (iii) HCI in MeOH

HCI. H

2

N ^ —

N

Figure 4. Preparation of "second generation" PEG-PS supports from homobifunctional PEG-diacids. Adapted from Barany et al (réf. 46).

Figure 5. General structure for PEG-PS supports ("environmental" model). PS = low cross-linked polystyrene, PEG = poly(ethylene glycol), typically of average molecular weight 2000 (-44 repeating units); X = starting point for peptide (or other) synthesis.


17. BARANY ETAL.

247


A "high load" PEG-PS (Figure 6, ref. 49) was prepared following a combination of both the "spacer" and "environmental" strategies. N -Fmoc, ^-Bocornithine was coupled onto MBHA-PS, the Fmoc group was removed, and homobifunctional PEG-diacids were added as already described. The Λ^-Boc group was then removed, and PAL handle (1 equiv) was coupled for optimal results. Remaining free amino groups were capped with Ac2Û, to achieve typical final loadings of 0.35-0.6 mmol/g. PEG-PS supports were first commercialized on the basis of MBHA-PS as the parent resin. As a consequence, the linkage between PEG and PS was labile to HF, and such PEG-PS's were compatible only with the Fmoc/rBu strategy for SPPS. However, by starting with aminomethyl-PS, HF-stable supports compatible with Boc/Bzl chemistry are obtained readily (48). For such cases, handles such as Bw-arninobenzhydryloxyacetic acid (BHA-linker) (71) are used in place of PAL. Downloaded by PURDUE UNIV on August 31, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch017

a

C L E A R Supports We described recently (60-62) a novel approach to PEG-containing supports, the CLEAR family (Table 1), which is very much complementary to the chemistry of PEG-PS discussed above. Rather than derivatizing and combining preformed polymers as is the case with PEG-PS, we opted to create supports de novo by copolymerizing appropriate monomers and cross-linkers that might lead to biocompatible structures with good mechanical stabilities. For a number of reasons, our work focused on branched PEG-containing cross-linkers such as trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate [structure 12 in Figure 7]. Each branch of 12 contains a polymerizable vinyl endgroup as well as a chain with on average four to five ethylene oxide units. A high molar ratio of 12 was corx)lymerized with amino-functionalized monomers, such as allylamine (13) or 2-aminoethyl methacrylate (14), which were chosen in anticipation of the later need of starting points for the solid-phase synthesis. The fact that amino groups could be introduced directly rather than by transformation of another functional group or by deprotection of a protected amino monomer, was an unexpected yet advantageous discovery in the CLEAR family. Incorporation of amines into synthetic polymers has been reported to be difficult due to (i) addition of the amine to activated vinylic double bonds (72), and (ii) Ο -*N acyl migration resulting in hydroxylated acrylamides when starting with amino acrylates (73). In addition, various non-functionalized monomers and crosslinkers (15-17) have been copolymerized successfully with 12. Five different CLEARS will be discussed in the following: CLEAR-I and CLEAR-V (made from 12 and 13); CLEAR-II (from 12,14, and 15); CLEAR-III (from 12,14, and 16); and CLEAR-IV (from 12, 13, and 17). All of these polymers have hydrophilic PEG-like character, even though individual oligo(ethylene oxide) chains are quite short compared to chains in PEG-PS (Figures 8 and 9). CLEAR particles of irregular shape are prepared by bulk polymerization followed by grinding and sieving, whereas the more preferred spherical beads are obtained by a suspension polymerization procedure. The loadings of the resins are affected by the amount of the amino-functionalized monomer used during the polymerization. Typical loadings are in the range of 0.1-0.3 mmol/g. As with PEG-PS, thefreeamino groups are usually acylated with an IRAA and extended with PAL or another handle.


POLY(ETHYLENE G L Y C O L )

248

Fmoc-PAL-[ (CH2) (ÇH ) 3

2

PEG)—

NH—CH-C-fps)—

C-CH-NH-{

NH I Fmoc-PAL

)—PAL—Fmoc

NH i Ac

65-75%


PEG

3

25-35%

Figure 6. General structure for "high load" PEG-PS supports. PS = low cross-linked polystyrene, PEG = poly(ethylene glycol), typically of average molecular weight 2000 (-44 repeating units); Fmoc-PAL = tris(alkoxy)benzylamide handle. Adapted from McGuinness et ah (ref. 49).

CH (O—CH — CH2)/—O—C —CH — CH 2

I

2

2

ι

C^Hs— C-CH -(0-CH - CH ) —O— C —CH — CH 2

2

2

2

IQ CH

(O—

2

CH — 2

ΟΗ ) —

O—C

2 Λ

—CH

—

CH

2

12 / + m+n~14 ÇH 3

CH -CH-CH -NH

CH - C— C—O—CH2—CH — NH

13

14

2

2

2

2

2

CH

(j> Ç H

3

II II

3

CH - C— C-(0-CHz—CH^—O—C — C- CH 2

2

15 p~9 CH30

CH = C — C —Ο — (CH — CH — O),—C^Hs 2

2

2

16

Poly(ethylene glycol)-Containing Supports for Solid-Phase Synthesis

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