Resins for Solid Phase Peptide Synthesis - Core Resins HEADING_TITLE

Core Resins

Polystyrene
Polystyrene is the most common core resin in solid phase peptide synthesis, but other core matrices include polyacrylate, polyacrylamide, and polyethylene glycol.   These other core resins have been utilized, often with impressive results, in the preparation of “difficult” peptides prone to aggregation but they have not gained widespread popularity.  
Uncrosslinked, or linear, polystyrene will dissolve in hydrophobic solvents and precipitate in protic solvents.  This property has been exploited by Janda in an interesting synthesis of prostaglandin F2α   Most polystyrene supports used in solid phase chemistry, however, contain 1% or 2% divinylbenzene (DVB) as a crosslinking agent.  These cross-linked polystyrenes are insoluble in all common solvents.  Typically, these resins are prepared and utilized as small, spherical beads.  
Even though the cross-linked polystyrene resins are insoluble in organic solvents, they are solvated and swollen by aprotic solvents such as toluene, dimethylformamide (DMF), and dichloromethane (DCM).  One gram of 1% DVB cross-linked resin will swell 4 to 6 times its original volume in DCM.  In contrast, one gram of 2% DVB cross-linked resin swells only 2 to 4 times its original volume in DCM.  The swelling factor is important in solid phase synthesis, since reaction kinetics is diffusion controlled.  Consequently, resin that swells more will have a higher diffusion rate of reagents into the core of the matrix, resulting in shorter reaction times and more complete chemical conversions.  Typical swelling factors of 1% crosslinked polystyrene in a selection of common solvents is listed below.
Swelling factor of 1% crosslinked polystyrene in various solvents (mL/g of resin):Core Resins


Polystyrene


Polystyrene is the most common core resin in solid phase peptide synthesis, but other core matrices include polyacrylate, polyacrylamide, and polyethylene glycol.1  These other core resins have been utilized, often with impressive results, in the preparation of “difficult” peptides prone to aggregation but they have not gained widespread popularity.

 
Uncrosslinked, or linear, polystyrene will dissolve in hydrophobic solvents and precipitate in protic solvents.  This property has been exploited by Janda in an interesting synthesis of prostaglandin F2  Most polystyrene supports used in solid phase chemistry, however, contain 1% or 2% divinylbenzene (DVB) as a crosslinking agent.  These cross-linked polystyrenes are insoluble in all common solvents.  Typically, these resins are prepared and utilized as small, spherical beads.

 
Even though the cross-linked polystyrene resins are insoluble in organic solvents, they are solvated and swollen by aprotic solvents such as toluene, dimethylformamide (DMF), and dichloromethane (DCM).  One gram of 1% DVB cross-linked resin will swell 4 to 6 times its original volume in DCM.  In contrast, one gram of 2% DVB cross-linked resin swells only 2 to 4 times its original volume in DCM.  The swelling factor is important in solid phase synthesis, since reaction kinetics is diffusion controlled.  Consequently, resin that swells more will have a higher diffusion rate of reagents into the core of the matrix, resulting in shorter reaction times and more complete chemical conversions.  Typical swelling factors of 1% crosslinked polystyrene in a selection of common solvents is listed below.


Swelling factor of 1% crosslinked polystyrene in various solvents (mL/g of resin):

THF 5.5   Acetonitrile 3.5
Toluene 5.3   Et2O 3.2
DCM 5.2   EtOH 2.0
Dioxane 4.9   MeOH 1.8
DMF 4.7   Water 1.0 (no swelling)

 

Polystyrene beads are available in sizes ranging from less than a micron to 750 microns in diameter.  Reaction kinetics is generally faster using smaller beads due to the higher surface area to volume ratio.  In practice, however, too small a bead can lead to extended filtration times.  Beads in the range of 75 to 150 microns in diameter offer a good balance of reaction kinetics versus reliability.  Bead size is commonly reported in Tyler Mesh size, which is inversely proportional to the nominal diameter.  Two commonly used resin sizes are 100-200 mesh and 200-400 mesh (75-150 microns and 35-75 microns respectively).

MESH SIZE vs BEAD DIAMETER

Mesh Size Graph

 

 

Size distribution is a critical factor for high quality synthesis supports; especially those used to generate combinatorial libraries.  Since each bead is essentially a microreactor, large differences in size between beads will result in unequal quantities of product in the final mixture.  Such heterogeneity can lead to screening errors due to over or under representation of individual components of the mixture.3  Bead size is also an important element in libraries since the number of possible compounds is limited to the number of beads used in the synthesis.  Obviously larger libraries can be made from the same weight of resin by using smaller beads, but the quantity of each individual compound is sacrificed.  The approximate number of beads per gram of resin can be calculated with the following formula:

 

 

 

where D= the mean bead diameter in cm and density = density of the resin in g/ cm3.

Merrifield Resin

The most fundamental substituted polystyrene resin is chloromethylated polystyrene, commonly called Merrifield resin after the Nobel Laureate who pioneered its use in peptide synthesis.4  Substrates are attached to Merrifield resin by nucleophilic displacement of chlorine.  The resulting resin-substrate bond generally is acid stable and requires strong acid conditions for cleavage.  Although carboxylic acid substrates are not easily cleaved from Merrifield resin under acidic conditions, other cleavage methods including saponification,5 transesterification6 and cyclization-release7 have proven effective.  Since the Merrifield resin-substrate bond is stable to most reaction conditions in solid phase synthesis, a wide array of synthesis resins,8 scavenger resins,9 and polymer-supported reagents10 have been prepared by attaching appropriate linkers to Merrifield resin.
Merrifield resin, as well as many other substituted resins, is generated by one of two methods: direct incorporation of the substrate onto the polymer core through an electrophilic aromatic substitution reaction or co-polymerization of the substituted monomer with styrene.  Generally, substituted resins are prepared by direct incorporation of the substrate, which invariably results in a mixture of isomers.  Merrifield resin, for instance, typically has a 70:30 mixture of para-and meta-chloromethyl substitutents.  Co-polymerization, on the other hand, allows the use of purified monomers, enabling the preparation of resins with up to 98% para substituent. Additionally, adjusting the relative proportions of styrene and substituted monomer can precisely control the degree of substitution of the resin. 

Hydroxymethyl Resin

Although Merrifield resin is the foundation of many popular resins, incomplete coupling of the substrate can lead to unreacted chloromethyl sites on the resin.  If such sites will interfere with the proposed chemistry, it is possible to obtain chlorine free resin through the use of hydroxymethyl polystyrene.11  Substrates are attached to the resin by reaction of an electrophile, such as an activated carboxylic acid, with the resin or by Mitsunobu reactions.12 Unreacted core sites can be acetylated (end-capped) by reaction with excess acetic anhydride in pyridine.  

Amino Core Resins

Aminomethyl (AM) resin has long been used in solid phase peptide synthesis as a core resin to which various linkers could be attached through a stable amide bond.13 4-Methylbenzhydryl amine (MBHA)14 resins, however, were originally developed for the formation of peptide amides using the Boc-N protection/TFA deprotection strategy.  These resins form very stable amide or amine linkages to either carboxylic or electrophilic alkyl substrates.  Generally, strong acid conditions are required to cleave substrates from these resins; therefore they are also used as base resins for anchoring acid labile linkers such as Rink amide linker.15  These resins are generated through electrophilic aromatic substitution and the substitution ranges can be difficult to control.  As a result, despite optimized reaction protocols, variance may be seen when purchasing these resins.  Impurities on the resin can include unreacted carbonyl groups as a result of incomplete amination or leachant of substrates and byproducts during manufacture. AAPPTec has identified the factors leading to such impurities and has eliminated them through its proprietary manufacturing techniques, in process testing and final quality analysis. 

Footnotes

1 García-Martín, F.; Quintanar-Audelo, M.; García-Ramos, Y.; Cruz, L. J.; Gravel, C.; Furic, R.; Côté, S.; Tulla-Puche, J.; Albericio, F., J. Comb. Chem., 2006, 8, 213 –220.

2 Chen, S.; Janda, K. D., Tetrahedron Lett., 1998, 39, 3943-3946.

3 For a discussion of possible screening errors, see Thompson, L. A.; Ellman, J. A., Chem. Rev., 1996, 96, 555-600.

4 Merrifield, R. B., J. Am. Chem. Soc., 1963, 85, 2149-2154.

5 Chamoin, S.; Houldsworth, S.; Kruse, C. G.; Bakker, W. I.; Snieckus, V., Tetrahedron Lett., 1998, 39, 4179-4182; Frenette, R.; Friesen, R. W., Tetrahedron Lett., 1994, 35, 9177-9180.

6 Marquais, S.; Arlt, M., Tetrahedron Lett., 1996, 37, 5491-5494; Pulley, S. R.; Hegedus, L. S., J. Am. Chem. Soc., 1993, 115, 9037-9047; Tortolani, D. R.; Biller, S. A., Tetrahedron Lett., 1996, 37, 5687-5690.

7 Hanessian, S.; Yang, R.-Y., Tetrahedron Lett., 1996, 37, 5835-5838; Le Hetet, C.; David, M.; Carreaux, F.; Carboni, B.; Sauleau, A., Tetrahedron Lett., 1997, 38, 5153-5156; Park, K.-H.; Abbate, E.; Najdi, S.; Olmstead, M. M.; Kurth, M. J., Chem. Commun., 1998, 1679-1680.

8 Barco, A.; Benetti, S.; De Risi, C.; Marchetti, P.; Pollini, G.P.; Zanirato, V., Tetrahedron Lett., 1998, 39, 7591-7594; Bräse, S.; Köbberling, J.; Enders, D.; Lazny, R.; Wang, M.; Brandtner, S., Tetrahedron Lett., 1999, 40, 2105-2108; Breitenbucher, J.G.; Johnson, C.R.; Haight, M.; Phelan, J.C., Tetrahedron Lett., 1998, 39, 1295-1298; Brummond, K.M.; Lu, J., J. Org. Chem., 1999, 64, 1723-1726; Dressman, B.A.; Singh, U.; Kaldor, S.W., Tetrahedron Lett., 1998, 39 , 3631-3634; Hu, Y.; Porco, J.A., Jr.; Labadie, J.W.; Gooding, O.W.; Trost, B.M., J. Org. Chem., 1998, 63, 4518-4521; Kobayashi, S.; Aoki, Y., Tetrahedron Lett., 1998, 39, 7345-7348; Sylvain, C.; Wagner, A.; Mioskowski, C., Tetrahedron Lett., 1998, 39 , 9679-9680; Winkler, J.D.; McCoull, W., Tetrahedron Lett., 1998, 39, 4935-4936.

9 Coppola, G.M., Tetrahedron Lett., 1998, 39, 8233-8236.

10 Adamczyk, M.; Fishpaugh, J.R.; Mattingly, P.G., Tetrahedron Lett., 1999, 40, 463-466; Andersen, J.-A.M.; Karodia, N.; Miller, D.J.; Stones, D.; Gani, D., Tetrahedron Lett., 1998, 39, 7815-7818; Dodd, D.S.; Wallace, O.B., Tetrahedron Lett., 1998, 39, 5701-5704; Vidal-Ferran, A.; Bampos, N.; Moyano, A.; Pericàs, M.A.; Riera, A.; Sanders, J.K.M., J. Org. Chem., 1998, 63, 6309-6318.

11 Martin, G. E.; Sambhu, M.; Shakhshir, S. R.; Digens, G. A.; J. Org. Chem., 1978, 43, 4571-4574; Fréchet, J. M. J.; de Smet, M. D.; Farrall, M. J., Polymer 1979, 20, 675-680.

12 Dodd, D. S.; Wallace, O. B., Tetrahedron Lett., 1998, 39, 5701-5704; Nicolaou, K. C.; Watanabe, N.; Li, J.; Pastor, J.; Winssinger, N., Angew. Chem. Int. Ed. Engl., 1998, 37, 1559-1561.

13 Mitchell, A. R.; Erickson, B. W.; Ryabtsev, M. N.; Hodges, R. S.; Merrifield, R. B., J. Am. Chem. Soc., 1976, 98, 7357-7362; Mitchell, A. R.; Kent, S. B. H.; Engelhard, M.; Merrifield, R. B., J. Org. Chem., 1978, 43, 2845-2852; Giralt, E.; Andreu, D.; Pons, M.; Pedroso, E., Tetrahedron,  1981, 37, 2007-2010.

14 Christensen, M.; Schou, O.; Pedersen, V. S., Acta Chem. Scand. B, 1981,  35, 537-581; Matsueda, G. R.; Stewart, J. M., Peptides, 1981, 2, 45-50.

15 Story, S. C.; Aldrich, J. V., Int. J. Peptide Protein Res., 1992, 39, 87-92; Hutchins, S. M.; Chapman, K. T., Tetrahedron Lett., 1996, 37, 4869-4872.