0. C. Neckers Bowling Gwen State University Bowling Green, Ohio 43403
Solid Phase Synthesis
Immobilizing chemical reagents and catalysts on polymer supports has received wide application in recent years (1-5). Solid phase synthesis utilizes reactiue residues attached to insoluble polymeric supports as reagents in synthesis. The most extensive applications are in polypeptide synthesis as well as in the area of immobilized enzymes and catalysts. In principle, the separation of reactants from catalysts and ~ r o d u c t sis the s t e ~in a chemical svnthesis where many losses take place. Distillations, recryatallizations, and other ourification orocedures are never 100% efficient, and when isolated yields are less than quantitative, these processes are usually to blame for some product losses. Solid phase synthesis utilizes a solid resin, that is a polymer support, for a handle to which reagents are hound so that they can he easily separated from products after reaction is completed. Solid phase or immohilized catalysis involves the attachment of a catalyst for a chemical reaction on the backbone of an insoluble support.
NH,
I + RC--CM)H IH
COOH
-I
-NH
R
HsN--€-R'
I H
R'
I H
H
+
HP
(1)
Peptide Bond
The difficultv. of course. arises because everv amino acid has both an &no residue and a carhoxyl reskue, and, unless one or the other is converted to a derivative which cannot react in the sequence, several different products may he obtained. T o circumvent this problem Blocking Groups are used which prevent the functional group which one does not wish to react, from participating in the sequence ( 91~ ~ -,In solution, sequential polypeptide synthesis involves three steps. These are the coupling of the first two amino acids residues, eqn. (2)
Polypeptide Synthesis
The utilization of solid supports in polypeptide synthesis was develoned nearlv simultaneouslv hv Professor R. Rruce Merrifield'(6) a t ~ockefelleruniv&sGy and by Professor Rohert Letsinger (7) a t Northwestern University. Merrifield (8) immohilized an amino acid to the backbone of a polystyrene derivative and carried out sequential peptide bond formation with the original amino acid serving as an anchor to the polystyrene resin NHBlg
NHBlg 0
~ I c - II 4 ~ I H
COOBlg
+ NH,--C-R'I I H
Blg
-
NHBlg
o
COOBlg
I I H
I II RC-C-NH-*-Re IH
EOYPI~~B
- Blocking Group
(2)
followed by unblocking usually of the blocked amino group eqn. (3) and the next coupling step, eqn. (4).
BI$- Blocking group
$ = h l m e r bead Letsinger and Kornet (7) used another approach, converting a styrene-divinylhenzene copolymer to a hydroxymethyl derivative and then to a chloroformyl derivative which was then reacted with the terminal amino group of the N-terminus amino acid
Polypeptide synthesis, in general, requires the coupling of a specific amino acid to another through the formation of an amide, or peptide, bond, eqn. (1).
NHBlg
II
I
On polymer supports, two additional steps are necessary. The first step involves coupling of the original amino acid to the polymer support, eqn. (5). This is followed by sequential peptide forming reactions, after the duration of which, the grown polypeptide must he removed from the backbone of the polymer support, eqn. (6). Volums 52, Number 11, November
1975 / 895
These steps are repeated until the desired polypeptide chain length is obtained
Though there are many other polymeric derivatives used and coupling procedures outlined, those above represent typical processes. In practice, polystyrene is almost exclusively the polymer support used in peptide synthesis (1, 21, Figure 1. There are several reasons for this: First, polystyrene can he functionalized easily to a reactive vet selective styrene reagentLgenerally chloromethylpolystyrene. Second, Figure 1. A bead of styrene-divin~ e n ibe aonro~ri. ~-l ~ .s t v r can .. . YlbenZene copolymer magnified ately crosslinked and con700X when viewed under an elecstructed in an insoluble bead Iron micro~cope.(Taken by Pro. configuration. Polystyrene, tesSOr Richard Crang. Department beinga hydrocarbon, is hydroof Biology. Bowling Green Universit". phobic, yet compatible with organic reagents; therefore, the heads can he approached by the reagents and solvents which are used for peptide synthesis. Third, polystyrene is under ordinarv condinot deeraded bv chemical reagents " tions and withstands the many chemical operations necessarv for the seoluential svnthesis of long .chain Dolv~eotides. . .-. Attaching an amino acid to polystyrene (5) is often carried out by nucleophilic substitution of the carboxylate end of an N-block amino acid on chloromethylpolystyrene. This reaction is rapid in the appropriate solvents and can be carried out virtually to completion on a polymer support. For reasons of solubility, the cesium salt of the N-blocked amino acid, synthesized by neutralization with cesium bicarbonate (7), is used most efficiently in these reactions eqn. (8) (10).
-
Coupling of chloromethyl polystyrene and its derivatives to amino acids through nucIeophilic displacement is the preferred method (10). Wang (10, for example, developed a p-alkoxy-o-henzyl alcohol resin which is used to couple reactive carhoxyl group residues, like acid chlorides, to a resin backbone. This reagent is particularly useful for coupling peptide fragments with free carboxyl groups to a polymer support.
For oligosaccharide synthesis on a polymer support, Frechet and Schuerch developed an allyl alcohol functional group on a polystyrene (12).
For the synthesis of polynucleotides on polymer supports, Khorana and coworkers (13) developed a polystyrene supported p-methoxytrityl chloride.
696 / Journalof Chemical Education
As a result of this nucleophilic displacement, a covalent hond between the polymer support and the amino acid reagent is formed. The covalent ester linkage provides a very substantial anchor but one which can be removed, since the polymer support amino acid hond is that of a benzyl ester. The next stage in the sequence is the deblocking of the blocked amino acid of the attached amino acid residue. Blocking groups are chosen because they are stable under the conditions of peptide couplings, hut easily removed either with acids or base. Thus, esters of carbonic acids, called urethanes, are the most frequently used blocking groups. Blocking amino acids with tert-hutoxy carhonyl residues are common and many are commercially available, eqn. (9).
Coupling of the second amino acid to the amino group terminum of the first polystyrene anchored amino acid is accomplished, either with dehydrating agents like dicyclohexylcarhodiimide(DCC) eqn. (10) or with activated acid derivatives like acid chlorides, eqn. (11).
thesis in that reagents and products are more easily separated when one or the other or in some cases even both, are attached to an insoluble residue. Polymer based organic reagenta have been prepared and utilized by several research groups. The reactions in which the immobilized reagents are involved are single step rather than multiple step; however, this somewhat limits the utility of these reagents. Nevertheless, the concept of immobilized chemical reagents is provided added value if the reagent can he recovered after the sequence in which it is used and regenerated on the polymer support. A second useful feature of immobilized organic reagents involves reactions in which residues can he held apart from or close to one another during chemical reaction. An example of this advantage was provided by Patchornik and coworkers in the alkylation of ester derivatives on polymer supports (23). In solution, crossed alkylation of phenyl acetic acid esters produces low yields of the desired products and higher yields of the comparative alkylation of the starting ester (13). If the starting ester is held away from the anionic center by the intervention of a polymer suhstrate, alkylation can he carried out in lieu of the competing side reaction eqn. (14).
4 It is important in polypeptide synthesis that each successive reaction be driven to completion. Therefore, dicyclohexylcarhodiimide coupling has become almost the exclusive coupling system of choice in polypeptide synthesis. After the numher of amino acid residues required for the protein or polypeptide have been incorporated on the hackbone of the polymer, the grown polypeptide is usually removed either with hydrofluoric acid or hydrobromic acid in suitable solvents for the polypeptide derivative, eqn. (12).
in lieu of R 0
0
I
II
7
+ RC-I
4-C-CHCOOR
-
0
H4*-R
II
L o
I
i m reaction yields because of eqn. (13)
a
f
HW-CH-C-R
+. major pmduet
0
2 ~ H . o cI + H - ~ I -0 c I R
P = polystyrene-divinyl benzene copolymer Table 1.
The success of resin hound polypeptide synthesis is most striking. The technique has been automated (14) so that the successive steps of deblocking-coupling and the associated wash procedures can be carried out in the absence of an operator. Under these conditions the synthesis of insulin took eight days while that of rihonuclease A, with its 127 amino acid residues, took several months (15). The technique has some pitfalls since polymers incorporate small quantities of reagents and products and therefore the grown polypeptide may well contain small amounts of contaminants from the synthetic sequence (2, 16). A few examples of polypeptides synthesized by solid state methods are shown in Tahle 1. Many of the example proteins in Tahle 1have striking biological activity. Polymer Bound Organic Reagents
As a result of the impetus provided by Merrifield's work, many reports of organic reagents attached to polymeric substrates have also appeared. The useful features of these reagents and processes are like those in polypeptide syn-
Soma Polypeptides Synthesized by Solid State Mathcdr
Name of P~lvpeutide~
Structure
overall Yield, Ref(%) erence
Bradykinin
L-arginyl-L-prolyl-L-prolylglycinyiL-uhenylaianyi-L-seryl-Lprolyl-L-phenylaionyl-Larginine Alg.PrO.PrO.Gly.Phe.Ser Pro.Phe.Arg.
38
(171
~n ~ n g i o t e n r i n Hydrophobic
Arp.Arg.Val.Tyr.lleu.Pro.Phe. Cycle[-L-Val-D-Pro-D-Val-L-ProI.
56
(211
(19)
tide Ribonuclea6e A Soybean Tryusin Inhibitor Acyi Carrier Protein P2 Fragment of
St(loh~1ococ-
Volume 52, Number 11, November 1975 / 897
The attachment of the reacting residues to the polymer support has the effect of isolating the reactive anion on the polymer support. Thus the carhanion
is held enough away from the starting ester
so that addition to the carhonyl carbon to give the oxyanion
A oarticularlv obvious examole of the difference between bo~ndor~anometallie catalysts and their solution phase counterparts is demonstrated by Gmbbs' work with hydrogenation catalysts. The Grubbs' catalyst, made by. the followinr! sequence from chloromethyl~olystyrene-div-
does not occur. As a consequence, no produds attributable to the self-condensed products are observed and the anion was modeled after a homogeneous solution catalyst model, the so-called Wilkinson catalvst (36) RhCl(PhaPh . - .Both the free catalyst, R ~ c ~ ( P ~and ~ Pthe ) ~polymer supported one can he acylated directly. A second sequence utilizing a polymer support was reported by Crowley and Rapoport (24) when, in a Dieckmann condensation sequence, the undesired product of a pair of products produced competitively was left on the polymer, whereas the desired one was cleaved from the polymer in a reaction sequence, eqn. (15) 0
could be used to hydrogenate alkenes with 1 atm Hp. The specificity of the polymer supported catalyst, however, was much greater, Table 3. The laraer, more rieid olefin is reduced much more inefficiently the polymer head, than by the solution cataIvst. This dramatic result implies that the reactant and the hydrogen must penetrate into the bead in order for reduction to take place. The more rigid olefin cannot penetrate the head as well as the smaller olefins. An electron microscopic magnification (X 2500) of the surface of a polystyrene-divinylhenzene head (Figure 2) shows these pores in the bead. The same bead (X 250) is shown alongside for comparison. The efficiency of these catalysts, depends on the number of times an individual catalyst molecule can he reused for its catalytic activity (maximum molar turnover number). Sometimes molar turnover numbers are very high and in certain cases have been reported to he over 30,000 (35). Therefore, there are some commercial processes now in
b;
II
bar
R, desired pmduct cleaves from the
+
R'&L?* undesired substitution pmduct-held an the
All of the above researchers used polystyrene-diviuylbenzene supports and the reactions were carried out in much the same wav as Merrifield's oolwentide svntheses. . .. Some additional examples of organic reactions carried out using polymer substrates are given in Table 2.
.
lmmoblllzed Catalysts for Organic ProceDses
Potentially the most significant application of polymer supports in chemical reactions is as catalysts for simple processes. Many soluble catalysts, like some organometallic derivatives used in hydrocarbon dimerizations, in hydrogenations and carhonylations, can be used as effectively if they are attached to a polymer derivative. One advantage of basing a catalyst on a polymer support involves the ease with which the catalyst is separated from the reaction products and left over reactants. Since many organometallic complexes used as catalysts are very expensive, losses during their use are minimized if thev can he easilv recovered. A oolvmer suooort orovides that possibility. in certain cases based E a t a ~ ~ s t s react differently than those which are not bound. This feature can make anchored catalysts useful for other kinds of processes. Some sample uses are the reductions of hydrocnrhons with polymer based rhodium or titanium catalysts, eqn. (16) (31-34), dimerization of dienes with polymer based catalysts, eqn. (171, and hydroformylation reactions using a similar catalyst, eqn. (18). 698 / Journal of ChemicalEdmation
beads magnified (a) 250X and (b)
which polymer support catalysts are about to be used in the preparation of organic materials. Another major advantage of the methods is that the polymer imparta stability to the catalyst. Thus, polymer based rhodium catalysts ean he longer lived than their monomeric counterparts or can be handled more readily in the air. Many clear cut examples of the use of polymer supported catalysts in synthetic, catalytic processes other than hydrogenation now exist too. For example, Pittman and Evans Table 2.
Tabla 3. Hydrogenation of Alkener Uring Free and Polymer Supported Catalyrt +H,PP~, Olefin Cyclohexene
1-~exene cyclooctene A -cnoiertene
(rel. rate) RhCIL. 1 2.55
RhCI(Ph,P), (,el. rate)
1.0 1.4
0.40
1.0
0.03
0.8
Exampler of Organic Reartions Using Polymer Subatrater Other Polymer Bound Reagsnts
Reactlon
Ref.WncI1 (25, 261
Polystyrene wound Wittlg Reagents
cry,
Poly-NWS + Peroxide
+
1
Insoluble Polymeric Carbodimlde
(@-A+,
CHO
Polymer Acstalr as Protecting GIoum In Wanzoln Condenrations
( & A = ~ c H o )
Polymer Based Poroxidiring Agents
Volur~m52, Number 11. November 1975 / 699
(37) have reported the cyclization of ethyl pmpiolate, H r n C O O E t , using polymer hased nickel carbonyl catalysts.
Table 4.
Some Examples Udng Rhodium Catalyst
for b m m e t r i c Reduction
Ketone
Silsne
Acetophenme
HSi(C,Hs),
Acetophenone
HSi(OEt1,
Almhol (S)-(-I-PhsnyC methylcarbin01 (SI-(-I-Phenylmethylcar-
Chem- Optiicsl cal Yield Purity 47
60
3.8
10
The distribution of products differs somewhat from the homogeneoua catalyst. Evans and Pittman (37) have reported combination catalysts which can be used in sequence to carbonylate an alkene and then hydrogenate the product.
Recentlv Kaeen and coworkers (38) have re~ortedthe --u use of an insoluble polymer supported optically active rhodium comnlex to catalvze the assvmetric reduction of alkoptically active hydrocarbons. enes capa61e of
