Synthesis on soluble polymers: new reactions and the construction of small molecules

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Synthesis on soluble polymers: new reactions and the construction of small molecules Dennis J Gravert and Kim D Janda* To avoid the heterogeneous reaction conditions of solid-phase chemistry, liquid-phase synthesis provides homogeneity through the use of soluble polymer supports that can be selectively precipitated and filtered to achieve product purification. Peptides, oligonucleotides, and oligosaccharides have been synthesized by this method. Furthermore, combinatorial libraries of small molecules have been produced by liquid-phase synthesis to aid in the search for new pharmaceutical agents including compounds active against HIV. Soluble polymeric reagents have also been developed for greater ease and efficiency in organic synthesis.

Addresses

The Scripps Research Institute, Department of Chemistry and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, OA 92037, USA *e-mail: [email protected] Current Opinion in Chemical Biology 1997, 1:107-113

http://biomednet.com/elecref/1367593100100107 © Current Biology Ltd ISSN 1367-5931 Abbreviations

DOX GIcNAc HELP LPCS MeO-PEG NMR PEG RRE

~c~'dioxy-p-xylyl n-acetyI-D-glucosamine high efficiency liquid phase liquid-phase combinatorial synthesis polyethyleneglycol monomethyl ether nuclear magnetic resonance polyethyleneglycol HIV-1 Rev response element

To overcome the limitations intrinsic to solution- and solid-phase methods, the use of soluble polymer supports in liquid-phase synthesis reinstates the familiar reaction conditions of classical organic chemistry and yet facilitates product purification by allowing for the selective precipitation and filtration of the polymer-bound product. Polymer solubility in a variety of solvents not only provides homogeneous reaction conditions but also enables individual reaction steps to be monitored by UV-visible spectroscopy, infrared spectroscopy, and/or proton and 13C nuclear magnetic resonance (NMR) spectroscopy without requiring preliminary cleavage of the product(s) from the polymer support. Moreover, aliquots taken for characterization may be returned to the reaction flask upon recovery from these nondestructive analytical methods. By using soluble polymer supports in liquid-phase synthesis one avoids the difficulties of solution- and solidphase synthesis while retaining their advantages. Several different polymers have been investigated for use in liquid-phase synthetic schemes, and previous reviews have been written describing the incorporation of soluble polymers for peptide, oligosaccharide, and oligonucleotide synthesis, and, to a lesser extent, for small molecule synthesis [2-4,5",6°°1. This review summarizes very recent demonstrations of liquid-phase synthesis including small molecule combinatorial library synthesis and polymer-supported reactions.

Liquid-phase synthesis Introduction To speed the discovery and development of new pharmaceutical agents, laboratories are applying the techniques of combinatorial chemistry to synthesize small molecule libraries to probe for lead compounds using high-throughput screening assays. Since chemistry is currently the limiting step in the drug discovery process [1], improved methods of small molecule library synthesis are required. Solid-phase combinatorial methods have been pursued to facilitate product purification, the resin-bound product can simply be filtered and excess reagents and impurities rinsed away. However, these insoluble supports create heterogeneous reaction conditions that lead to nonlinear kinetic behavior, unequal distribution and/or access to the chemical reaction, solvation problems, and pure synthetic problems traditionally associated with solid-phase synthesis. Homogeneous reaction conditions can be achieved using classical methods of organic synthesis modified into a combinatorial format ('solution-phase' synthesis); however, attempts to drive reactions to completion may require the tedious purification of intermediates.

The first examples of liquid-phase synthesis were simple modifications of the Merrifield method of peptide synthesis [7] that employed linear, soluble polystyrene to achieve familiar homogeneous reaction conditions while attempting to preserve many aspects of solid-phase chemistry [8-11]. In order to improve reaction rates and yields, other soluble polymers were investigated, including cellulose, polyacrylamide, polyacrylic acid, polyethylene iminc, poly(N-isopropylacrylamide), polymethylene oxide, polypropylene oxide, pol3winyl alcohol, poly(N-vinyl-2pyrrolidinone) and their various copolymets [2-4,5°,6°°]. The properties of polyethylene glycol (PEG), however, proved to be the most suitable for peptide synthesis as well as for the preparation of other molecules. PEG is soluble in a wide variety of aqueous and organic solvents and exhibits high solubilizing power that maintains homogeneous reaction conditions even in cases where the attached molecule is normally insoluble in the reaction medium. Isolation of polymer-bound compounds simply requires concentrating reaction solutions and diluting with either diethyl ether or tert-butyl methyl ether to induce

108 Combinatorialchemistry

PEG precipitation. Careful precipitation conditions or cooling of polymer solutions in ethanol or methanol yields crystalline PEG, as a result of its helical structure, which results in a strong propensity to crystallize [12]. Thus, as long as the polymer backbone remains unaltered during liquid-phase synthesis then purification by crystallization can be utilized at each reaction step. The characterization of PEG-bound organic moieties is often straightforward, as the polymer does not interfere with spectroscopic or chemical methods of analysis; additionally, MeO-PEG (PEG monomethylether) contains a single methoxy group (IH NMR chemical shift (8)=3.38ppm; ethyl protons of PEG backbone: 8 = 3.64 ppm [13]) that provides an internal standard for easy monitoring of reactions by 1H NMR spectroscopy.

Synthesis and screening of a combinatorial peptide library Liquid-phase synthesis is amenable to combinatorial techniques; in fact, all manipulations, including portion-mixing or split synthesis, can be performed under homogeneous conditions. The integration of liquid-phase synthesis into a combinatorial format has resulted in 'liquid-phase combinatorial synthesis' (LPCS) [14"']. Using LPCS and a recursive deconvolution methodology, a combinatorial peptide library of 1024 pentapeptides was synthesized on MeO-PEG and screened for ligands to an antibody to 13-endorphin [14"',15]. In this protocol, the synthesis of the combinatorial libra," involved the simultaneous production of partially synthesized libraries by saving and cataloging aliquots during peptide synthesis. These partial libraries were later utilized in the recursive deconvolution screening method which incorporates an iterative synthesis and screening approach to identify novel library members that inhibited the binding of leucine enkephalin to its antibody. The native epitope Tyr-Gly-Gly-Phe-Leu and several other pentapcptides were identified as potent binding ligands. The diverse solubilizing power of MeO-PEG provided a direct method for screening polymer-bound peptide libraries in a homogeneous assay without preliminary cleavage as the presence of the polymer exerted little effect on measured binding affinities between MeO-PEG-peptide conjugates and free peptides.

Peptides containing non-natural amino acids Synthesis on PEG using chromium aminocarbene complexes led to the preparation of peptides containing non-natural amino acid residues to increase the scope of peptide libraries [16]. Initially attempted on solid-phase support, reactions under heterogeneous conditions were problematic. The use of PEG allowed the synthesis of several dipeptides and tripeptides. Transesterification allowed quantitative cleavage of peptides from the PEG support; however, overall yields were reduced because of incomplete diastereoselectivity and less than quantitative coupling yields. Nevertheless, after two photochemical

coupling cycles, a tetrapeptide was obtained in fair yield of 58% after purification to a single diastereoisomer, whereas under solid-phase conditions a poor yield of 18% was realized.

Peptidomimetic library of '~-aza-amino acids' or 'azatides' The construction of peptidomimetics consisting of oligomers of '{x-aza-amino acids' or 'azatides' has been accomplished through liquid-phase synthesis [17]. Azatides are peptidelike molecules made from building blocks resembling natural amino acids, except that the {x-carbon of each residue has been replaced by a nitrogen atom. Lacking the asymmetrical structure of normal peptides, azatides may serve to explore peptide secondary structure as well as to provide a source of bioactive molecules with improved pharmacokinetic properties. Upon development of general synthetic procedures aimed at generating an alphabet of c~-aza-amino acid monomers, coupling mediated by bis(pentafluorophenyl)carbonate enabled the synthesis of an azatide pentamer in 56.7% overall yield. Coupling reactions were monitored by IH NMR and ninhydrin analysis without preliminary cleavage from MeO-PEG.

Oligonucleotide synthesis In a manner analogous to liquid-phase peptide synthesis, the covalent attachment of the growing chain of nucleotides to a macromolecular support also simplifies the otherwise arduous task of purification during oligonucleotide synthesis. The different reaction conditions required for oligonucleotide synthesis compared to peptide synthesis imposes new constraints on the polymer support; however, the versatile solubility properties of PEG allow its use in efficient syntheses of both peptides and oligonucleotides. In fact, 100rag quantities of oligonucleotides from gram quantities of MeO-PEG are routinely obtained using the high efficiency liquid phase (HELP) method of synthesis [18-20]. Advantages of H E L P over solid-phase methods include the requirement of less excess of reagent (as a result of homogeneous reaction conditions), the ability to obtain large amounts of oligonucleotides in a single synthetic run, and easy monitoring of reactions by nondestructive methods. To address the anticipated need for kilogram quantities of synthetic oligonucleotides for therapeutic and diagnostic applications, a large scale H E L P method was developed [21"]. Instead of using phosphotriester or phosphoramidite chemistry, oligonucleotide synthesis in this case employed H-phosphonates to allow for the recovery and recycling of excess reagents in order to realize significant cost savings in large-scale applications. By performing only one oxidation step at the end of oligonucleotide synthesis, the new method eliminated a synthetic step during each coupling cycle and resulted in higher overall yields by reducing the number of precipitation steps in the total synthetic procedure.

Synthesis on soluble polymers Gravert and Janda

Poly(N-acryloylmorpholine) has been investigated as an alternative soluble support in the H E L P method of oligonucleotide synthesis [22]. Advantages over MeOPEG include direct attachment of the first nucleoside unit without the use of a linker and easier removal of a dimethoxytrityl protecting group with a single acid treatment instead of several. However, the initial attachment of nucleoside to poly(N-acryloylmorpholine) proceeded to only 50% of loading capacity, and IH NMR analyses were obscured by the multiple signals of the polymeric carrier. These disadvantages did not prohibit the synthesis of a octanucleotide with an overall yield of 83%, (based on amount of starting polymer with first nucleoside attached) close to the value obtained in the MeO-PEG-supported H E L P procedure.

Oligosaccharide synthesis The development of efficient methodologies for oligosaccharide synthesis has been driven by the recognition of the essential role these molecules play in biological processes and by the pursuit of a novel class of therapeutics based on these entities [23]. During development of a liquid-phase method of oligosaccharide synthesis, the direct linkage of MeO-PEG to the anomeric carbon (simply defined as one carbon of the sugar bonded to two oxygen atoms) was found be labile under certain glycosylation reaction conditions [24]. Stable attachment was made to other sugar hydroxyl groups using succinate ester linkages. However, switching the linker moiety based on succinic acid to a linkage based on cqc(-dioxy-p-xylyl (DOX) allowed stable O-glycosidic attachment of MeO-PEG through the anomeric carbon as well as by ether linkages to other sugar positions [25°]. Hydrogenation liberated the bound oligosaccharide with a free hydroxyl group, or under milder conditions, a benzyl-like protecting group remained at the point of previous polymer attachment. T h e synthesis of D-mannopentaose, a structural moiety of cell surface D-mannans of pathogenic yeast and a target of therapeutic research, was achieved through stereocontrolled glycosylation reactions in a liquid-phase method incorporating M e O - P E G - D O X . Further work that optimizes glycosylation reactions in liquid-phase oligosaccharidc synthesis has been reported [23]. Based on the mechanism of glycosylation, several reaction conditions were varied to achieve successful attachment of 2-acetamidoglycopyranosyl derivatives to M e O - P E G - D O X . Side reactions, including degradation of the linker-support were eliminated through reaction optimization, and conditions were found to yield pure polymer-bound glycoside upon precipitation of the support using tert-butyl methyl ether. Changing the synthetic scheme to incorporate other glycosylating agents required additional modifications [26]. To minimize side reactions while improving reaction rate and yield in liquid-phase oligosaccharide synthesis, dibutvlboron triflate was found to be an efficient promoter

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of glycosylation with glycosyl trichloroacetimidates. Initial coupling of a saccharide to M e O - P E G - D O X proceeded with a 95% yield, and subsequent glycosylation with a disaccharide trichloroacetimidate in the presence of dibutylboron triftate gave the desired product in 85% yield. Two new examples of enzyme-mediated, liquid-phase oligosaccharide synthesis have been reported [27,28]. By reacting regioselectively and stereoselectively, enzymes provide an alternative method to achieve anomeric control during oligosaccharide synthesis without the need for protecting groups. Copolymerization of acrylamide and a sugar residue (n-acetyl-D-glucosamine, or GIcNAc) containing a terminal olefin connected by a phenylalanine-containing spacer unit (1, Fig. 1) provided a soluble support with GIcNAc pendant groups [27]. This glycopolymer served as a substrate for galactosyl- and sialyhransferases, and upon enzymatic cleavage from the polymer using ~-chymotrypsin, a trisaccharide was obtained in a good yield of 72%.

Figure 1 OH HO

O

HN~NH

o

°''1 ..... O 1

,oo--~o

r'~

OAe 2

@ 1997 Current Opinion in Chemical Biology

Copolymerization structure of 1 or 2 with acrylamide produces soluble gFycopolymers for enzyme-mediated oligosaccharide synthesis.

Water-soluble polymers were synthesized with improved loading capacities for use in enzymatic glycosylation reactions [28]. Copolymerization of acrylamide with an acryloyl saccharide derivative (2, Fig. 1) produced polymers containing up to 0.28 mmol/g glucose attached to acrylamide through a nitrobenzyl linker. Synthesis of a polymer-bound disaccharide was mediated by galactosyhransferase and resulted in an 86% yield, after which the lactose product was cleaved from the support by photolysis (yield 84%).

Synthesis of liquid crystalline c o m p o u n d s For potential material science applications such as displays in optoelectronics, liquid crystalline oligomeric compounds derived from terephthalic acid and 6t-hydroxv-eohydroxy-(oxy- 1,4-phenyloxy- 1,10-decamethyleneoxy-l,4phenylene) (Fig. 2) were synthesized on MeO-PEG [29]. Solution-based methods only succeeded in the stepwise synthesis of short oligomers (n=2) because of low product solubility in all solvents; however, the solubilizing power of MeO-PEG enabled synthesis of

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Combinatorialchemistry

larger oligomers. Furthermore, a sophisticated coupling scheme incorporating multiple PEG-protecting groups achieved highly pure final products even without reactions going to completion. T h e solubilizing power of MeO-PEG also allowed product identity and purity to be verified by solution-based analytical methods, before hydrolytic cleavage was carried out to isolate the insoluble oligomeric product.

Figure 2

a combinatorial library of neomycin B mimetics. After functionalizing M e O - P E G with an amino group through attachment of glycine, the library was rapidly prepared in a synthetic scheme that included an Ugi-type reaction to combine four components in a single step. This liquid-phase method was found to be superior to both classical or solid-phase methods. Library members were screened for binding to the RRE of HIV mRNA, and of the 52 compounds tested, nine members exhibited binding activity as good or better than neomycin B.

Figure 3 0

0/(cH2h°-O

,NH2 0

Lo

J.

© 1997 Current Opinion in Chemical Biology

Oligomeric compounds with liquid-crystalline properties produced by liquid-phase reaction of terephthalic acid and ~.-hydroxy-m-hydroxy- (oxy- 1,4-phenyloxy- 1,10-decamethyleneoxy- 1,4phenylene) on MeO-PEG.

Liquid-phase c o m b i n a t o r i a l synthesis (LPCS) of small m o l e c u l e libraries Nonoligomeric compounds may also be prepared by LPCS. This was demonstrated by the construction of a library of sulfonamides by parallel synthesis [14"]. In the past, sulfonamides have provided an inexpensive treatment for bacterial infections; however, their clinical usage has been limited by bacterial resistance, a narrow antibacterial spectrum, and side effects. In fact, the observed side effects have led to studies that suggest sulfonamides may be developed into antitumor agents, endothelin antagonists, and antiarrhythmic agents [30-32]. The screening of combinatorial libraries of this class of compounds may therefore lead to the discovery of new pharmaceutical agents. The main reaction step in this library synthesis consisted of condensing a MeO-PEG-arylsulfonyl chloride derivative with amines spanning a broad range of pKa values. Amines of various reactivities were successfully incorporated, ensuring that future syntheses can include numerous amines to provide a highly diverse library of sulfonamides. Cleavage of the urethane linkage to MeO-PEG produced the final library in analytically pure form in overall yields of 95-97%. A library of neomycin B mimetics was rapidly prepared on MeO-PEG [33°]. Neomycin B (Fig. 3) is an aminoglycoside antibiotic that has been shown to compete with the viral protein Rev for the binding of an RNA sequence (the Rev response element, RRE) of HIV [34,35]. However, the use of neomycin B as an inhibitory drug has been hampered by its toxicity, relative instability, and propensity toward in vivo enzymatic modification [33°]. Therefore, compounds with improved pharmacokinetic properties were sought through the synthesis and screening of

H O"

" ' ~ N H2

H

o"

© 1997 Current Opinion in Chemical Biology

To discover new agents active against HIV, liquid-phase methods were used to synthesize a library of compounds that mimic neomycin B, shown above.

Soluble polymer-supported reactions T h e macromolecular characteristics of liquid-phase synthesis not only facilitate product purification: in a novel application, a polymer support has enabled a temperature controlled switch for catalytic hydrogenation to be developed [36]. In this method, polymer-bound compounds may react with a heterogeneous catalyst at room temperature; however, above a certain temperature, the polymer precipitates from solution and prevents further hydrogenation. Thus, carbobenzyloxy-protected glycine attached through the phenolic hydroxyl group of the copolymer made from N-(p-hydroxyphenyl)acrylamide and N-isopropylacrylamide (1:20 ratio) was inert to hydrogenation above 38°C but was cleaved readily at 10°C. By incorporating different monomers in copolymerizations with N-isopropylacrylamide, various polymers were prepared that differed in the temperature at which phase separation occurred [36]. Such polymers could be useful for controlling reactivity during organic synthesis. Choice of solvent and polymer chain length have also been found to influence reactivity during catalytic hydrogenation of compounds bound to PEG [37]. With respect to the reduction of nitrophenyl groups linked to polymers,

SynthesisonsolublepolymersGraved and Janda

as the length of the polymer increased, the reaction rate decreased correspondingly. This decrease in reaction rate was suggested to arise by unfavorable steric interactions between the macromolecular support and heterogeneous catalyst. Changes in solvent also affected reactivity of PEG-supported compounds, as hydrogenation activity in ethanol was observed to increase with the addition of water. This may be caused by improved solvation of PEG as water is a better solvent than ethanol for this polymer. Linkers have also been developed that form an aliphatic C - H bond to target molecules after cleavage from M e O - P E G [38--40]. These 'traceless' linkers provide a method for increasing diversity of small molecule libraries by providing an alternative functionality to the heteroatom-hydrogen bond (such as an alcohol, amine, carboxylic acid, and amide) that commonly remains on the target molecule at the original site of polymer attachment. MeO-PEG was functionalized with a thiol that acted as a general carrier for various alkyl groups [38,39]. After the attached molecule underwent various synthetic transformations, cleavage from the polymeric linker by hydrogenolysis using a Raney nickel catalyst liberated the product containing a new C - H bond. After ether-induced precipitation of the polymer support followed by filtration, products were recovered in spectroscopically pure form by concentration of the filtrate. A complementary method permitted the synthesis of compounds that contain reduction-sensitive functional groups such as alkenes, alkynes, and epoxides [40]. In this modified procedure, the thioether linkage was selectively oxidized to the sulfone using KHSO5 (oxone) and subsequently reductively cleaved with sodium amalgam. This mild reaction scheme does not affect most reducible functional groups and provides for a variety of alkyl molecular scaffolds to be incorporated onto a polymeric matrix, resulted in increased diversity of small molecule combinatorial libraries.

Soluble polymer-supported reagents and catalysts Manganese phthalocyanine complexes bound to polyacrylamide can act as catalysts for the oxygenation of cyclohexene in the presence of base and oxidants [41]. These polymer-bound catalysts represent model systems of natural metalloenzymes and may be developed into useful reagents for organic synthesis. The polymeric catalyst was synthesized by copolymerization of acrylamide with a phthalocyanine vinyl monomer, followed by metal complexation [42]. Catalytic activity of these water-soluble polymers was greater in the presence of sodium hypochlorite than in hydrogen peroxide, with the best set of reaction conditions based on cyclohexene producing 1,2-cyclohexanediol in 79% yield and lesser amounts of other products. A polymer-bound palladium catalyst was highly active in the hydrodebromination of organic bromides [43].

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Catalyst preparation consisted of stirring commercially available poly(N-vinyl-2-pyrrolidinone) with PdCI z in acidified ethanol. Alternatively, RuCI 3 was subsequently added to obtain a bimetallic palladium-ruthenium catalyst. These polymeric catalysts successfully transformed aromatic bromides to their corresponding hydrodebrominated products. The bimetallic catalyst demonstrated higher activity for conversion of aliphatic bromides. Ligands bound to M e O - P E G were used to achieve catalytic asymmetric dihydroxylation of olefins and were later recovered for reuse [44]. T h e cinchona alkaloid ligand normally employed in this powerful and important method of stereoselective synthesis is expensive, and previous attempts to recover and reuse the ligand by linking it to insoluble polymer supports led to variable yields, increased reaction times, and lower enantioselectivity. However, since LPCS provides both homogeneous reactions and polymer-aided purification, it was reasoned that ligands bound to MeO-PEG (Fig. 4) would allow reagent recovery without adversely affecting the catalytic reaction. In fact, during asymmetric dihydroxylation of various olefins, ligands bound to MeO-PEG were as effective as free ligands in solution. Furthermore, MeO-PEG-bound ligands were recovered nearly quantitatively and reused several times without affecting product yields or enantioselectivity.

Figure4

0

0 ~ O M e

1997 Current Opinion in Chemical Biology

MeO-PEG is attached to a cinchona alkaloid used for the catalytic asymmetric dihydroxylation of olefins.

Linkage of triarylphosphines to PEG produced a polymersupported reagent useful in the Staudinger reaction and Mitsunobu ether synthesis [45]. During the reduction of azides in the Staudinger reaction, the PEG-based reagent exhibited faster reaction rates than an insoluble polystyrene-based reagent. Attachment of PPh 3 to PEG also allowed the production of aryl alkyl ethers using the Mitsunobu ether synthesis. In fact, this method permits the isolation of pure ethers without the use of chromatography. Furthermore, the liquid-phase reagent yielded products from starting materials that were unreactive toward the solid-phase reagent.

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Combinatorial chemistry

T h e development of a PEG-linked version of the Burgess reagent led to increased yields in the synthesis of labile oxazolines with improved case of reagent handling [46]. The Burgess reagent, methyl N-(triethylammonium sulfonyl)carbamate, permits the synthesis of five-membered heterocycles in high stereochemical purity. However, this reagent is oxidation and moisture sensitive and has a limited shelf-life even when stored at low temperatures. Although attachment of the Burgess reagent to modified Merrifield resin was unsuccessful, the polymeric reagent formed with MeO-PEG allowed the synthesis of oxazolines and thiazolines cleanly and in high yields even after extended storage at or below room temperature.

Conclusions Soluble polymer supports provide homogeneous reaction conditions and facilitate product purification as a result of our ability to selectively precipitate and filter the polymer-bound product. Using linear, non-cross-linked polymers in methods of liquid-phase synthesis in essence avoids the difficulties of solution- and solid-phase synthesis while preserving their positive aspects. Disadvantages of liquid-phase synthesis relative to solid-phase methods include lower loading capacities (fewer sites of attachment per gram of polymer) and an extra precipitation step prior to filtration of the polymer support. The insolubility of PEG in T H F below room temperature limits its utility for low temperature reactions; however, other soluble polymers may be suitable for these applications. In fact, the solubility properties of PEG, polyacrylamide, poly(N-isopropylacrylamide), and poly(N-vinyl-2-pyrrolidinone)have allowed the development of improved reaction methods and facilitated the synthesis of numerous molecules including azatides, oligonucleotides, and oligosaccharides. Although the choice of polymer used in liquid-phase methods may be tailored for specific applications, such as temperature-controlled hydrogenation using poly(N-isopropylacrylamide) copolymers, PEG is used most often because of its high solubilizing power and wide solubilit'y properties. Its usage has resulted in the method of LPCS wherein the integration of liquid-phase synthesis with combinatorial chemistry has produced small molecule libraries of peptides, sulfonamides, and neomycin B mimics. By employing the soluble polymer support MeOPEG, newly developed polymeric linkers allow for facile attachment and cleavage of a small molecule/molecular scaffold containing aliphatic C - H bonds. Furthermore, the modification of existing reagents with PEG has provided improved methods for synthesis of target molecules. These achievements verify the utility of liquid-phase synthesis and provide incentive for further research.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •* 1.

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2.

Mutter M, Bayer E: The liquid-phase method for peptide synthesis. In The Peptides, vol 2. Edited by Gross E, Meienhofer J. New York: Academic Press; 1979:285-332.

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4.

Pillai VNR, Mutter M: New perspectives in polymer-supported peptide synthesis. In Topics in Current Chemistry, vo1106. Edited by Boschke FL. New York: Springer-Verlag; 1986:119-175.

5. Geckeler KE: Soluble polymer supports for liquid-phase • synthesis. Adv Polym Sci 1995, 121:31-79. This recent review discusses the general features of synthesis on soluble polymers and evaluates their use in peptide and oligonucleotide synthesis. Gravert D, Janda K: Organic synthesis on soluble polymer supports: liquid-phase methodologies. Chem Rev 1997, in press. This review provides a comprehensive survey of peptide, oligonucleotide, oligosaccharide, and small molecule syntheses on soluble polymer supports. 6. ••

7.

Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 1963, 85:2149-2154.

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Han H, Wolfe MM, Brenner S, Janda KD: Liquid-phase combinatorial synthesis. Proc Nat/Acad Sci USA 1995, 92:6419-6423. Combinatorial chemistry on soluble polymers is demonstrated through the construction of a library of sulfonamides and the synthesis and screening of a peptide library. 14. •-

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Erb E, Janda KD, Brenner S: Recursive deconvolution of combinatorial chemical libraries. Proc Nat/Acad Sci USA 1994, 91:11422-11426.

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1Z

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Colonna FP, Scremin CL, Bonora GM: Large scale H.E.L.P. synthesis of oligodeoxynucleotides by the

Acknowledgements This work was supported in parr bs' The Scripps Research Institute, The Skaggs Institute for Chemical Biol~g~, the R W Johnson Pharnlaccutical Research Institute, and the Alfred P S[oan Foundation,

of special interest of outstanding interest

Synthesis on soluble polymers Gravert and Janda

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hydroxybenzotriazole phosphotriester approach. Tetrahedron Lett 1991, 32:3251-3254. Bonora GM, Biancotto G, Maffini M, Scremin CL: Large scale, liquid-phase synthesis of oligonucleotides by the phosphoramidite approach. Nucleic Acids Res 1993, 21:1213-1217. 21. Bonora GM: Polyethylene glycol. A high-efficiency liquid-phase • (HELP) for the large-scale synthesis of the oligonucleotides. App/ Biochern Biotechno/1995, 54:3-17. This paper describes the various HELP methods of oligonucleotide synthesis and includes a discussion of advantages and disadvantages compared to solid-phase methods.

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25. •

Douglas SP, Whiffield DM, Krepinsky JJ: Polymer-supported solution synthesis of oligosaccharides using a novel versatile linker for the synthesis of D-mannopentaose, a structural unit of D-mannans of pathogenic yeasts. J Am Chem Soc 1995, 117:2116-2117. This report demonstrates the versatility of the DOX linker in oligosaccharide synthesis and provides a method (in supplementary material) that may be applied to the synthesis of other carbohydrates. 26.

Wang Z-G, Douglas SP, Krepinsky JJ: Polymer-supported syntheses of oligosaccharides: using dibutylboron triflate to promote glycosylations with glycosyl trichloroacetimidates. Tetrahedron Lett 1996, 37:6985-6988.

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response elemenL J Am Chem Soc 1995, 118:10150-10155. This report demonstrates the advantages of liquid-phase synthesis over solution and solid-phase methods by synthesizing and screening a smallmolecule library for potential anti-HIV agents. 34.

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