Solid-Phase Synthesis and Combinatorial Technologies

1.1 SOLID SUPPORTS

3

bead (typically in the range of hundreds of picomoles) but small enough to be resistant to physical shock during handling. Their loading, defined as the number of sites per resin gram, is typically in the range of 1 mmol/g. They are cheap, when compared to other supports, and are commercially available with many different functional groups for a wide variety of loading chemistries. Examples are the chloromethylated- (Merrifield), aminomethylated-, carboxy, hydroxymethyl, formyl, and bromo resins (Fig. 1.2). All of these functionalized supports can be prepared from underivatized PS resin by simple chemical transformations.

1.1.2 Hybrid Hydrophilic Polystyrene Resins

A major drawback of the hydrophobic PS resins is their poor swelling in polar protic solvents, as mentioned previously. The grafting of hydrophilic monofunctional or bifunctional polyethylene glycol (PEG) chains to the PS resin to produce a hybrid support (10, 11) provides a solution to this problem that allows the use of hydrophilic solvents with these resins. The swelling properties of hydrophobic PS resins, monofunctional PEG-grafted (Tentagel, TG), and bifunctional PEG-grafted (Argogel, AG) PS resins are compared in Table 1.1. The structures of the two hybrid PEG–PS resins are shown schematically in Fig. 1.3.

These newer resins have had a great impact on SPS. The higher degree of flexibility of the terminal PEG chains produces a real “solutionlike” microenvironment and allows on-bead reaction monitoring and structure determination by gel-phase 13C nuclear magnetic resonance (NMR) or magic angle spinning (MAS) 1H-NMR spectroscopy. Many examples of the MAS–NMR spectra of compounds bound to a hydrophobic PS or to a hybrid PEG–PS resin clearly show the better signal resolution obtained with PEG–PS. The use of NMR in SPS will be described in more detail in Sections 1.3.4 and 1.4.6.

The grafting of hydrophilic groups onto PEG–PS resins decreases their loading, which typically drops to around 0.2–0.3 mmol/g for TG or 0.4–0.6 mmol/g for high-loading TG or AG. The PEG–PS-based resins have several disadvantages, and although they are commercially available with various grafted functionalities, they are significantly more expensive than hydrophobic PS supports. The introduction of PEG chains on the solid support sometimes has negative effects on the quality of the

COOH	CHO
Cl

NH2	Br
	OH

Figure 1.2 Selection of commerically available functional groups grafted onto PS resins.

4SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

TABLE 1.1 Swelling of PS-Based Resins in Common SP Solvents a

Resins/Solvents	THFb	DMF	DCM	MeOH	Water
Hydrophobic PS	8.8	5.6	8.3	1.6	—
TG–PS	5.8	5.6	5.1	4.2	4.2
AG–PS	5.7	5.8	7.2	4.1	3.2

aNote: THF = tetrahydrofuran; DMF = dimethyl formamide; DCM = dichloromethane. bResin swelling measured as volume increase in respect to the dry bead.

chemistry, which may be due to the leakage of PEG chains due to the use of aggressive reagents such as strong electrophiles or to the use of Lewis acids, which can form complexes with the PEG chains.

The general tendency is to use PEG–PS resins for detailed SP studies, where reaction monitoring by NMR is necessary or when polar protic solvents are required. When large quantities of supports must be used and the SPS strategy has been thoroughly validated, hydrophobic PS resins are normally used.

Other more hydrophilic, non-PS-based solid supports were reported with suitable properties for specific SP protocols. Radical polymerization of acrylamide-substituted PEG chains yields the so-called PEGA support (12), which is ideal for aqueous solvents and allows the inner penetration of macromolecular reagents (13; see also Section 7.2.3) but has a limited applicability for SP organic synthesis due to the reactivity of its numerous amide bonds. Polytetrahydrofuran (PTHF)-cross-linked PS resin is obtained from suspension copolymerization (14) of PTHF-linked styrenes with styrene and 4-vinylbenzyl chloride; its swelling properties and stability to commonly encountered organic reaction conditions is extremely encouraging, as confirmed by the compatibility with harsh reagents such as n-Buli (15). Newer, more stable and promising non-PS-based solid supports were recently reported and validated in demanding organic reaction conditions (16–19); they will soon represent further alternative support options for the bench chemist involved in SPS.

C C H

H2

O

X

X

O

O

CH3

O

O

X

O

n

n

n

Tentagel

Argogel

H

n = 70

Load)

n = 30-40

C

n = 30 (High

x = grafted group

CH2

x = grafted group

m

m

Figure 1.3 Structure of monofunctional hybrid PEG–PS resins (Tentagel, left) and of bifunctional hybrid PEG–PS resins (Argogel, right).

1.1 SOLID SUPPORTS

5

1.1.3 Macroporous Nonswelling Resins

A different class of supports contains macroscopic pores embedded in an extremely rigid structure that does not allow any swelling of the matrix. The SPS takes place on a gellike support that is polymerized into the permanent pores. These macroporous nonswelling resins are typically used for continuous-flow oligonucleotide and peptide synthesis (20, 21). The most popular are POLYHYPE resins, made of polyamide- (PA-) containing 10–50% cross-linked PS (22); PA-containing Kieselguhr, which is made from a silica-based support with a porous structure (23); and fully inorganic controlledpore glass (CPG) supports, made of highly porous pure silica (24), which are very common in oligonucleotide SPS and will be detailed in Section 2.2. A representation of a macroporous Kieselguhr support is shown in Fig. 1.4.

A recent addition to the family of macroporous supports was designed for the SPS of organic compounds (25) and has been used as an alternative to gelatinous supports. This resin, called AP–PS, consists of a highly cross-linked macroporous PS framework and has a loading of around 0.6–1 mmol/g. It does not swell appreciably in a wide range of solvents, allowing many different experimental conditions to be used. It is now commercially available with a number of different grafted functionalities, and it is in the same price range as the PEG–PS resins.

Two characteristics of macroporous supports are worth mentioning. First, the transfer of specific reaction conditions from solution to the macroporous support should be easier because the influence of swelling in different solvents and diffusion rates, typical for low-cross-linked PS resins, is not relevant. Second, this support can be washed more easily than the classical PS resins. A study of the retention of biphenyl in hydrophobic PS and AP–PS resins treated with solutions of biphenyl in methylene chloride (26) shows how after two identical wash cycles AP–PS retains 0.01% of biphenyl while hydrophobic PS retains 2.63% of the same impurity. This is due to the

Rigid skeleton

Permanent pores

Polyacrylamide gel

Figure 1.4 Schematic representation of a macroporous SP support.

6SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

easier accessibility for solvents of non-resin-bound impurities on macroporous resins. Disadvantages of macroporous supports are their extreme rigidity, which makes them more suitable to continuous-flow synthesis, even though they are claimed to exhibit a similar stability to physical shock as gelatinous resins (26), and the impossibility of performing on-bead NMR spectra on the rigid solid-like support.

1.1.4 Miscellaneous Solid Supports

The commercial availability of PS-based supports in nonbead format such as pins (27, 28), crowns (29, 30), microtubes (31), and recently discs (32) has also had a notable impact on the field of SPS. These devices allow the attachment of significantly higher amounts of compound onto a single support unit compared to resin beads. They are similar in nature to the PS resin supports, but their different morphology requires different handling procedures than resin beads; we will discuss some of their specific properties in the following chapters as related to specific examples.

While gellike and macroporous resins cover the vast majority of SPS, the use of other supports has also been explored. Cellulose (33, 34) in the form of paper sheets has been employed for multiple simultaneous SPS of peptides with a relatively low loading of 0.5–0.6 mol/cm2. Cotton (35) has been used for the same application with a loading of around 0.1 mmol/g. Glass (36) was among the first supports used for the synthesis of large numbers of peptides, due to its chemical inertness and solidity. Various polymeric membranes (37, 38) were also used to prepare peptides on SP. Several of these supports will be mentioned also as related to combinatorial library synthesis (see Section 6.4.1 and 6.4.2).

The class of so-called soluble supports, including PEGs, non-cross-linked PS, and the recently introduced high-loading dendrimers, will be covered in Section 8.5.

1.1.5 SPS: Reaction Kinetics and Work-Up Procedures

The use of a solid support for carrying out organic chemistry has a profound influence on some of the reaction parameters (9, 35); above all, there is a strong effect on the rate of reaction. When a reaction takes place in a homogeneous solution, the reactants can freely interact and the reaction rates depend on classical parameters (e.g., concentration and temperature). When a resin-bound reagent is involved, the reaction takes place in a heterogeneous medium and the reaction kinetics are also dependent upon the rate of diffusion of the reagent in solution into and out of the resin beads. Reaction rates are generally slower in SP than in solution-phase chemistry, and reagents supported on gelatinous resins exhibit different reactivities depending on the swelling properties of the resin in the solvent used for the reaction. Reaction rates are also highly influenced by the nature of the support (40). PEG–PS and hydrophobic PS resins show different reaction kinetics in different reactions, as expected, but it is not possible to predict their behavior due to the many factors influencing the reaction kinetics in each experimental condition (41).

1.1 SOLID SUPPORTS

7

Macroporous resins are not influenced by swelling, but their reactivity is comparable, and sometimes lower, than that of gelatinous resins.

Site–site interactions are an important parameter in SP reactions. When interand intramolecular reactions are possible in solution, their relative occurrence will depend on the number of encounters/collisions between two different molecules (intermolecular) and between two groups in the same molecule (intramolecular); the complete freedom of movement for each molecule in solution will not prevent a priori any intermolecular coupling. When the intramolecular process is disfavored (e.g. with macrocyclization of peptides), the undesired linear polypeptide will always be present in the crude reaction products even using the most careful reaction conditions (Fig. 1.5, top). The support significantly constrains the freedom of each supported molecule, thus favoring intraversus intermolecular reactions; the above-mentioned macrocyclization of peptides usually produces pure cyclic peptides in high yields (Fig. 1.5, bottom). It must be remembered, though, that the good solvation of beads brings them toward a solutionlike environment, thus lessening the constraints for each loading site to cross-react with another site. Site isolation is generally observed in SPS, and a high cross-linking and a high backbone rigidity for the support will favor it; a low level of cross-linking and a highly flexible support, as the PEG–PS resins, may rather cause significant site–site interactions. Examples of both negligible and significant site–site interactions during SP reactions have been reported; several recent, excellent reviews present in details the current knowledge regarding this issue (9, 42, 43). The extreme flexibility of two PS-based supports bearing respectively maleimide and anthracene has recently allowed an inter-bead Diels-Alder reaction to give stable, covalently linked bead aggregates (42); the potential of such a solid–solid interface chemistry needs to be fully assessed.

Another major difference is related to the work-up procedure for the reaction. When a reaction is carried out on SP, the reaction product remains attached to resin while all of the excess reagents, the catalysts, and the impurities remain in solution. A typical work-up procedure involves simple filtration of the resin followed by repeated washings with fresh solvents in which the resin has a good swelling and the reagents/impurities are soluble. A good degree of swelling helps the solvent to access the adsorbed impurities, which must be soluble in the same solvent to allow them to be removed from the beads. It is quite common to use a number of different solvents sequentially in the washing cycle in order to remove soluble reagents/impurities with different physicochemical properties. This work-up procedure is amenable to automation and, as we will see in the following chapters, is one of the features that make SPS so appealing for combinatorial technologies.

The tedious purification of intermediates in classical solution synthesis produces pure compounds, which are then carried forward to the subsequent steps of the synthetic scheme. When a side product of the SP reaction remains attached to the resin, it becomes impossible to separate it from the desired intermediate/target molecule, thus irreversibly affecting the quality of the synthesis and the purity of the final product. This and other factors, which will be discussed in the following chapters, are considered during the so-called chemistry assessment phase of the synthesis in which the

8SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

chemistry is transferred from solution to the solid phase and the reactions are optimized. In general, significant differences between a reaction in solution and the same reaction on a solid support are the rule rather than the exception. Several reviews related to the use of solids supports in SPS and/or in combinatorial chemistry have recently appeared (44–51); their content largely expands what has been covered in this Section and may be useful for more experienced and interested readers.

intramolecular

+

H2N

H2N

H2N

H2N

COOH H2N	COOH
	CONH
intermolecular	+

CONH

COOH

CONH

SOLUTION

CONH

COOH

intramolecular

COOH

CONH

SOLID-PHASE

Figure 1.5 Site–site interactions in solution phase and on SP: intraversus intermolecular reactions.

1.2 LINKERS 9

1.2 LINKERS

1.2.1 Properties of a Solid-Phase Linker

Successful SPS produces a final resin-bound target molecule that is released into solution by breaking a bond between the resin and a functional group in the final compound. Two examples are shown in Fig. 1.6. On the left, the basic hydrolysis of an ester bond releases a carboxylic acid into solution and simultaneously re-forms the original hydroxy PS resin. On the right, the acidic hydrolysis of an acetal function provides the starting aldehyde resin and a diol compound.

Often, the intermediates to be attached to the resin do not possess a suitable functional group. Moreover, the bond linking the solid support and the compound must be stable to all of the reaction conditions to be employed during the preparation of the final compounds. Even marginal sensitivity to one of the reagents used could result in the release of intermediates into solution and create free sites on the beads during the synthesis. This would decrease the amount of final compound produced and could also lead to the formation of resin-bound side products. Finally, the bond linking the substrate to the resin must be sensitive to a cleavage reaction condition that allows the release of the final compound in solution without degradation.

			O	CHO		OH
OH
	+	R	OH	+	HO	R
Esterification				Acetalization
O				O
O	R			C	R
				H O
SPS				SPS
O				O
O	Rdec			C	Rdec
				H O
Hydrolysis				Hydrolysis
			O	CHO		OH
OH
	+	HO	Rdec	+	HO	Rdec

Figure 1.6 SP attachment and cleavage of an acid onto a hydroxymethyl support (left) and of a diol onto an aldehydic support (right).

10 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

A direct bond between the precursor to the final compound and a commercially available PS resin hardly satisfies all of these requirements but the problem can be circumvented by the use of a so-called linker, which is a chemical structure inserted between the solid support and the compound to be prepared. The linker is stable to all of the reaction conditions used during the SPS scheme but is labile under well-defined conditions. The proper selection of the linker strongly influences both the quality and the success of the SPS strategy.

The use of SPS in combinatorial chemistry has given rise to the need to transpose many different chemistries onto SP and, as a result, has driven many efforts toward the design and preparation of new SP linkers. The ideal linker must be easy to prepare and be stable to the reaction conditions used during elaboration, but at the same time, it should be highly sensitive to one, or at most a small number, of specific cleavage reagents/conditions. It should not release troublesome by-products during the final cleavage and, ideally, should release different products into solution when cleaved under different conditions. It should also allow the selection of suitable protecting groups, either commercially available for a specific building block or easily prepared, to protect reactive functionalities during the SPS and to deprotect them selectively at different stages of the SPS without affecting the linker stability.

Many families of SP linkers are currently used in SPS and can be classified according to conditions used for their cleavage. The most commonly used are commercially available already anchored onto PS resins. We will describe the most frequently encountered families of linkers such as acidand base-labile linkers, photolabile linkers, safety-catch linkers, and traceless linkers. Several linkers could be attributed to more than one category, and the classification used will be motivated in the text. The so-called cyclative cleavage, where the last SPS step simultaneously cyclizes and cleaves the final compound from the solid support, will also be covered. An extensive bibliography will be provided, especially regarding recent examples of linkers with appealing features to recover pure compounds in high yields at the end of a SP synthesis; the reader should also refer to recent, exhaustive reviews related to SP linkers (52–56).

1.2.2 Acid-Labile Linkers

This is the most widely used class of SP linkers. Historically, the SPS of peptides (see Section 2.1) was developed using building blocks protected with acid-labile groups, thus allowing a convenient simultaneous cleavage and deprotection in the final step of the synthesis. Four commercially available acid-labile linkers are depicted in Fig. 1.7 in resinand compound-bound forms. The preferred cleavage conditions for each linker are also provided.

The Wang linker 1.1 (57) is a very popular choice that is based on a p-alkoxybenzyl alcohol moiety. It is typically cleaved by trifluoroacetic acid (TFA)–DCM 1/1 in 30 min at room temperature (rt) to produce carboxylic acids or alcohols. Two slight modifications to this linker have produced the super-acid-sensitive resin (SASRIN) 1.2 (58), in which an o-methoxy group increases the acid sensitivity allowing cleavage with 1% TFA–DCM, and the hypersensitive acid-labile (HAL) linker 1.3 (59), where a second o-methoxy group further increases the acid sensitivity allowing cleavage with

					1.2	LINKERS 11
		R	COOH	CORdec		HOOC
			SPS	O	TFA/DCM	Rdec
	O	or		or	1/1, 30', rt	or
			OH	Rdec		HO
1.1
		OH	R	O		Rdec
		OMe	COOH	CORdec		HOOC
		R	SPS	O	TFA 1%	Rdec
	O	or		or	DCM, rt	or
			OH	Rdec		HO
1.2
		OH	R	O		Rdec

		OMe
	O
N	O	as for
	( )	XH 1.1,2
H	4
		OMe

1.3 X=O 1.5 X=NH

	OMe
		as for
O	OMe	1.1,2
	XH

1.4 X=O 1.6 X=NH

			Rdec	TFA 0.1%	HO
	O				Rdec
					or
	or
				DCM, 5', rt (1.3)
			CORdec		HXOC
	X				Rdec
				TFA/PhOH
					or
	or
				95/5, 2 hrs,
					SO2NH2 Rdec
	N		SO2Rdec rt (1.5)
	H
		Rdec		TFA 0.2%	HO
	O				Rdec
					or
	or		DCM, 3', rt (1.4)
					HXOC
	CORdec
					Rdec
	X			TFA/DCM
					or
	or
			1/1, 15', rt (1.6)
					SO2NH2 Rdec
	SO2Rdec
	N

H

Figure 1.7 Acid-labile, commerically available SP linkers 1.1–1.6.

either 0.1% TFA–DCM in 5 min at rt, or 10% AcOH/DCM. Another popular choice is the Rink ester linker 1.4 (60), which contains a bis-benzylic hydroxyl function and can be cleaved with 10% AcOH–DCM for 1.5 h at rt, or 0.2% TFA–DCM in 3 min at rt. Both the HAL and the Rink linker exist in the corresponding NH2 versions, called PAL 1.5 [5-(4-(g-FMOC) aminomethyl-3,5-dimethoxy phenoxy) valeric acid] (61) and Rink amide 1.6 (62), respectively, which allow the formation and final release of amides or sulfonamides in acidic conditions. All of these linkers are reasonably stable to nonacidic conditions.

Seven noncommercial acid-labile linkers have been reported recently in the literature and are shown in Figs. 1.8. (1.7–1.10) and 1.9 (1.11–1.13). The THP (tetrahydropyran) linker 1.7 (63), which is easily grafted onto Merrifield resin, has been used to support primary alcohols, secondary alcohols, hydroxylamines, and carboxylic acids. It is stable to strong nucleophiles and basic conditions and can be cleaved by

12 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

O	OH	SPS			O	O		TFA/H2O
	R					Rdec			HO
O
								95/5, rt	Rdec
1.7
	NH	2	SO2Cl			R		TFA/H2O	R	1
	R		R			1
	1		2			N	R		HN	R
								2 95/5, rt			2
O	X						SO2			SO2
1.8 X=Br, I
			COOH			H		TFA/DCM		H	Rdec
	-Fmoc R			SPS					HO	N
O						N	Rdec
						O		5/95, rt		O
N	Fmoc
						O
H
1.9
							AcOH/THF/H2O
Et	OH						6/6/1, 4-8 hrs, 50°C
Si	R1		SPS		Si	Rdec		or	HO
						O				Rdec
Cl								HF.Py, THF
Et	O
	or							24 hrs, rt

1.10R2 R3

Figure 1.8 Acid-labile SP linkers 1.7–1.10.

95% TFA–water at rt or by PPTS (pyridinium p-toluensulfonate) in DCE (dichlo- roethane)–n-BuOH 1/1 at 60 °C for 16 h to release hydroxyl-containing compounds.

The halide linkers 1.8 (64), which can be prepared from Wang resin, have been used to support amines and to release sulfonamides using 95% TFA–DCM (R = H) or 5% TFA–DCM (R = OMe).

The N-Fmoc-amino-oxy-2-chlorotrityl linker 1.9 (65) is derived from the commercially available, extremely popular 2-chlorotrityl chloride PS resin and has been used to prepare functionalized hydroxamic acids or peptidyl hydroxamic acids. The final compounds can be removed from the resin using 5% TFA–DCM.

The silicon-based linker 1.10 (66), which can be readily prepared from commercially available PS–diethylsilane resin, has been successfully used to perform multistep SPS to give alcohols that could be cleaved with AcOH–THF–H2O in 4–8 h at 50 °C (48) or with HF.Py (hydrofluoric acid/pyridine complex) in THF at rt (67).

The indole linker 1.11 (68), easily prepared from aminomethyl PS resin and N-carboxyalkylated indole-3-carboxaldehyde, was used to support amines and to transform them on SP, obtaining, by release with TFA–DCM 1/1 in 30 min, a variety of compounds, including amides, sulfonamides, guanidines, ureas, and carbamates.