Solid-Phase Synthesis and Combinatorial Technologies

						1.2	LINKERS			23
	O
O	N	SPS	N	TMSI, dioxane, H				N
1.43	HN		N	MeCN, 72 hrs, 75°C			N			R2
		Cl	R2
	O		R1					R1
						Cu(OAc)2,
						Py/MeOH
H	O	H	O	H		rt, 2 hrs
			SPS
N	N	N		N		or			Ardec
		Ar	N	Ardec				H
O	H		H		NBS, Py,
	1.44
					DCM, 45', rt
					then MeOH
			Ph
SeCN Ph		COOH	O	m-CPBA		Ph		O	O
				O
1.45	CuCl2, PhCH3, 80°C		Se	DCM, rt
			Me	OTf					Me	OTf
			H				H
	SePh	HO				O
N	Si						H
			H
								Si
	O					O
O
	1.46	N-iodosuccinimide, TfOH,
		DCM/dioxane, 1 hr, rt
								SPS
		Me Rdec					Me Rdec
	H			TBAF 0.1N in
				tetramethyl urea

1 hr, 100°C

HO

H

Figure 1.16 Traceless SP linkers 1.43–1.46.

The oxidation-labile TL 1.44 (102) was readily prepared from aminomethyl PS resin and used to decorate the aryl moiety with C–C coupling reactions (e.g. Heck, Stille, Suzuki, and Sonogashira protocols). Oxidation of the hydrazide moiety to acyl diazene [copper acetate or N-bromosuccinimide (NBS)] and nucleophilic cleavage (methanol or n-propylamine) produced the desired aryl products.

24 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

The selenium linker 1.45 (103), obtained from Merrifield resin and potassium selenocyanate, was treated according to oxyselenylation conditions to give a supported selenolactone. Oxidative deselenylation (m-CPBA, DCM, rt) produced the unsaturated lactone in good yield and purity. An expansion of this chemistry, including several SP transformations prior to the cleavage, is mentioned in the original paper (103).

The selenium-silicon linker 1.46 (104), obtained from hydroxy PS resin and methyl glyoxylate through a simple five-step scheme, was coupled with a steroid scaffold to give an acetal then transformed on SP and finally cleaved with TBAF/tetramethyl urea, 1 h, 100 °C to give decorated alcohols via unstable hemiacetals with good yields and purities.

1.2.7. Cyclative Cleavage

A promising SP method of cleavage that is becoming more common is the so-called cyclative cleavage (CC). SPS produces an advanced open intermediate that undergoes cyclization with concomitant release of the final cyclized product. Only the desired product is released in the CC step, while any side product or remaining intermediate cannot cyclize and thus remains bound to the solid phase; this significantly eases the purification–work-up procedures to obtain the pure recovered compound from SPS. Several examples of CC are shown in Fig. 1.17 (1.47–1.50) and 1.18 (1.51–1.54).

In SP preparation of oxazolidinones, the carbamate SP linker 1.47 attached to a hydroxymethyl SP resin (105) was alkylated with a tosyl-epoxide and cyclized/cleaved at the end of the synthesis by aminolysis of the epoxide with spontaneous cyclization of the amino-alcohol to release the pure oxazolidinone into solution.

The allyl alcohol linker 1.48 (106), bound to an aldehyde PS resin, has been elaborated to the pentenoic acid derivative shown. This was cyclized with cleavage using ring-closing metathesis (RCM) (Ru catalyst in DCE at 80 °C for 16 h) to give pure Freidinger lactam in solution.

The commercially available, supported amino acid 1.49 was elaborated to complex linear intermediates that can easily be cyclatively cleaved to produce tetramic acids; the cleavage conditions included aqueous NaOEt (107), tetrabutyl ammonium hydroxide (108), and methanolic KOH (109). The procedures afforded the desired heterocycles in good yields and purities.

The TMS-exomethylene linker 1.50 (110), obtained from carboxyethyl PS resin and a suitable TMS alcohol, was reacted with an N-acylimine as in the imino-Sakurai protocol and then the SP transformations were performed; the intermediates were cyclatively cleaved with Pd(acac)2 and dppe in refluxing THF to give highly functionalized pyrrolidines.

Commercially available (4-(4-formyl-3-methoxyphenoxy)butyryl PS resin was loaded with primary amines to give the resin-bound secondary amines then transformed to the highly functionalized resin-bound secondary amines then transformed to the highly functionalized resin-bound anilines 1.51 (111) that were cyclatively cleaved with AcOH overnight at 80 °C to give tri-decorated imidazoles with good yields and purities. A similar approach provided the highly functionalized quinoxali-

1.2 LINKERS 25

O

R1

O N H

1.47

SPS

OH

1.48

NH2 SPS

O

R1

O

1.49

O

O

1.50

O		O		R1	H	O
	OTs O		N	R2	N	N		R1
					R3 O
				LiClO4, THF,
		O		16 hrs, rt
					N	R
					R3	2
		R3
			O
				(Cy3P)2Cl2Ru=CHPh				R
		N	R1					3
			R2	DCE, 16 hrs, 80°C				O
						R2	R1
				0.1M NaOEt
		O		24 hrs, 85°C				R2
					or
		R3
			N	R Bu4NOH, THF		R1	N	O
				2
		O	R1	6 hrs, rt, then
		O		Amberlyst A-15		O		R3
					or
				KOH, MeOH
					30', rt
		N
		Ar	boc			Ar
	Si
		BF3.Et2O, 3 hrs, rt
					HN	OtBu

O

SPS

		Pd(acac)2, dppe	Ardec
	N	Ardec
		3hrs, rt	NHdec
	dec

Figure 1.17 Examples of CC on SP: 1.47–1.50.

nones 1.52 (112), which were cyclized/cleaved with TFA/DCM/Et3SiH 10/30/1, 1 h, rt followed by heating at 50 °C in HCl, 4N, dioxane/MEOH 2/1, 3 h.

Even large linear precursors can be effectively cleaved and cyclized, releasing only the desired macrocycles; two examples (1.53 and 1.54) reported by Nicolaou et al. (113, 114) are shown to highlight the complexity of SP products that can be obtained and the usefulness of CC as a release protocol even for polyfunctional sensitive molecules.

A large number of additional examples of CC approaches to a wide panel of isolated and condensed ring systems can be accessed through a recent review (115).

26 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

	O	R3
	O2N
CHO	R2 SPS HN	AcOH	N
	N	R2 ovn., 80°C R2	R3
	N		N
	R1	1.51	R1
	R1

O2N	NO2	O	R3					H
			H	1) TFA/DCM/Et3SiH			O
								N	N
	SPS	HN	N	O	10/30/1, 1 hr, rt
N	F				2) 4N HCl,				R3
R				R			R2	N	N
		N	N		dioxane/MeOH				R1
1		R1	H	2	2/1, 3 hrs, 50°C			H
			1.52
	Bu Bu		O						O
	Sn		O	Pd(PPh3)4
									O
	1.53	I		toluene,
				48 hrs, 100°C
			O O					O
									O
	O	O					O
	P		K2CO3, 18-crown-6
	O								n = 7 to 9
	OMe					(n)
		O
O	(n)		toluene, 12 hrs, 65°C
							O
		O
1.54						O

Figure 1.18 Examples of CC on SP: 1.51–1.54.

1.3 REACTION MONITORING IN SOLID-PHASE SYNTHESIS

1.3.1 General Considerations

A key component of a successful organic synthesis is the constant monitoring of the reaction, which allows optimization of the yield of the target molecule while minimizing the products of side reactions. This monitoring is readily performed by either chromatographic analysis [thin-layer (TLC), high-performance liquid chromatography (HPLC), or gas (GC)] or spectroscopy. The use of excess reagents in SPS (typically 3 to 5 equivalents) precludes the monitoring of reactions by following the disappearance of reagents in solution and therefore alternative methods, specifically designed

1.3 REACTION MONITORING IN SOLID-PHASE SYNTHESIS

27

for studying reactions and determination of the structure in the solid phase, have been developed to guide the efforts of the SP chemist (116, 117).

Destructive methods, where the analytical sample is consumed by the analysis, and nondestructive methods will be presented and their qualitative or quantitative nature will be discussed. They will be divided into off-bead methods, where the resin-bound reaction product(s) are cleaved from the support with subsequent analysis of the cleavage solution, and on-bead methods, where single or multiple beads are analyzed directly.

1.3.2 Off-Bead Methods

The cleavage of resin-bound materials and their full analytical characterization in solution are used as the most accurate way to monitor the outcome of a reaction carried out in the SP. The methods used are those of classical organic chemistry and will not be commented on further. The reaction products can be weighed and an accurate structure determination can be obtained. There are, however, some limitations to the usefulness of off-bead methods for reaction monitoring in SPS.

First, the resin beads cleaved after each step of a multistep SPS are lost and the gravimetric yield determination requires a significant amount of compound. This may lead to a notable waste of precious materials and to a significant reduction of the target compound prepared.

The cleavage reaction may take hours, which prevents rapid monitoring of the reaction or necessitates complicated sets of parallel experiments that are quenched at different times. The reagents used for the cleavage may pollute the cleavage solution, thus requiring some purification steps prior to the analytical determination. Some reactive resin-bound intermediates may be sensitive to the cleavage conditions, thus leading to a misinterpretation of the reaction outcome.

The use of fast, reliable, sensitive on-bead methods circumvents the drawbacks to off-bead analysis outlined above. The modification of common analytical techniques has provided the SP chemist with valuable and often preferred alternatives to off-bead methods for SPS reaction monitoring.

1.3.3 On-Bead Methods: Colorimetric/Fluorescence Detection

Colored reagents to follow the appearance or the disappearance of a functional group have been widely used to monitor reactions in classical organic chemistry, particularly in TLC analysis. This technique has been successfully adapted to SPS; for example, ninhydrin (118), bromophenol blue (119), nitrophenyl isothiocyanate-O-trityl (120), picric acid (121), and malachite green isothiocyanate (122) have all been used to show the presence or the absence of free resin-bound amines. The presence of free resinbound thiol groups can also be detected (123).

In the commonly used Kaiser test, a few milligrams of resin beads is withdrawn from the reaction, thoroughly washed with a range of solvents, and treated with stock ethanolic solutions of ninhydrin and phenol followed by a solution of KCN in pyridine at 100 °C for 10 min. If the beads turn deep blue in color, there are free primary amine

28 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

groups on the support, while if the beads maintain their original color, no free primary amines are present. The method is strictly qualitative because beads containing between 10 and 100% of free amine groups give the same result. The quantitation of derivatized sites may be performed (124), but the procedure is complex and the risk of obtaining irreproducible results is high. The sensitivity of the test is at best around 4–5%, that is, less than 4–5% of sites bearing free amines can be overlooked using colorimetric detection. Nevertheless, the extreme rapidity of colorimetric methods and their simplicity make them useful for monitoring SPS, giving the best compromise for a qualitative evaluation of the reaction course.

Two recently reported colorimetric methods are particularly promising. The first is specific for resin-bound amines, including prolines and sterically hindered primary amines (125); the test relies on an accessible derivative of commercial Disperse Red 1 (three synthetic steps), is extremely sensitive (up to 2% of free amines on bead are spotted by a deep red color) and fast (10 min heating at 70 °C). The second is specific for hydroxyl groups via their tosylation, but also for any group which an undergo nucleophilic substitution (126); the test relies on a nucleophilic displacement with p-nitrobenzylpyridine and formation of an internal salt after treatment with piperidine, is extremely sensitive (up to 2% of free hydroxyls on bead are spotted by a pink/violet color) and fast (1 min heating with a heat gun).

The use of such methods for the detection and monitoring of other resin-bound functional groups will become common as more and more staining protocols will be transferred from solution synthesis to SPS. Adaptation of these reagents to the presence of the solid support, for example, by choosing a solvent where the beads swell properly, may sometimes be necessary.

1.3.4 On-Bead Methods: Nuclear Magnetic Resonance Spectroscopy

The use of NMR techniques to monitor reactions in solution has never been very popular because of the time required to prepare an aliquot of the solution and to record a meaningful spectrum. Faster, even if less information-rich, methods have been traditionally used.

The use of NMR in SPS is further complicated by two factors. First, the spectra of solid samples show broad NMR lines due to restricted molecular motion. This may be partially alleviated by a good swelling of the beads in a suitable deuterated solvent. Second, the heterogeneity of the resin slurry produces microenvironments with different magnetic susceptibilities that cannot be shimmed in the same way as homogeneous samples where the magnetic susceptibility is uniform. Again, this leads to broadening of the NMR signals. Nevertheless, solvent in which the resin swells properly allows the recording of an SP NMR spectrum using the so-called gel-phase NMR technique (127).

This method has found applications in SPS, particularly for those nuclei that are absent in the matrix and whose appearance can be easily monitored even with broad NMR signals; examples have been reported for 19F-NMR (128–130), 31P-NMR (131), and isotopically enriched 15N-NMR (132). While examples of 2H and 13C gel-phase

1.3 REACTION MONITORING IN SOLID-PHASE SYNTHESIS

29

NMR spectra exist, the technique is limited by line broadening, the long acquisition times, and the low abundance of 13C and 2H atoms in the matrix.

The use of a 13C-enriched building block anchored to a resin makes the gel-phase spectrum selective for the appearance of the new 13C signal, and the enrichment allows much shorter acquisition times (133, 134); a “real-time” kinetic was reported for the alkylation of amines with 13C-enriched bromoacetic acid (135). An example from our laboratories (136) shows the formation of a cyanohydrin on SP is monitored by 13C-enriched gel-phase NMR using 13C-benzaldehyde. The appearance of the cyanohydrin signal (63.2 ppm) and its increase at different reaction times is easily monitored by comparison with the constant signals of the solvent (deuterated benzene, 133–126 ppm, Fig. 1.19, spectra A–D). A major drawback of this technique is the cost and the limited availability of 13C-enriched building blocks, which currently severely limits its application.

While specific applications of gel-phase NMR have been useful for SPS reaction monitoring, the great potential of SPS NMR is in the determination of structure and the measurement of purity and yields, especially through the use of magic angle spinning–high-resolution (MAS–HR) NMR techniques. This important topic will be addressed in Section 1.4.6.

1.3.5 On-Bead Methods: Mass Spectrometry

The use of mass spectrometry (MS) techniques to monitor SP reactions has recently become possible through the use of matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) spectrometry (137) after in situ cleavage of a small number of resin beads (138–140). Although the compound is cleaved from the resin, the cleavage happens directly onto the center of the MALDI target and the method can be considered on-bead.

In a specific example (141), a small aliquot (10–100 beads) from an attempted Pd(0)-mediated deprotection of an allyl ester on Rink resin was placed on the MALDI–TOF sample plate and exposed to TFA vapor at rt for 20 min. The TFA was removed, the matrix and an internal standard were added, and the sample was analyzed. The incomplete removal of the allyl group under classical conditions was observed, and only repeated deprotection protocols with Pd(0) afforded the pure deprotected carboxylic acid.

The suitability of this technique for acid-labile (142) and photocleavable linkers (143) has been demonstrated. Other cleavage conditions that do not produce residues (e.g., cleavage with gaseous ammonia) could also be used in theory. TOF–secondary ion mass spectrometry (TOF–SIMS) (144) has also been validated to monitor SP peptide synthesis (145) and could in future increase the versatility of MS monitoring of SP reactions.

The requirement of an expensive MALDI MS spectrometer and the limited usefulness for compounds with molecular weight (MW) < 600 (presence of intense matrix signals) are serious limitations to the application of this method for the monitoring of SPS reactions. However, the extreme sensitivity (a single resin bead is usually enough)

30 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

Figure 1.19 Gel-phase 13C-NMR reaction monitoring of a cyanohydrin formation (63.2 ppm). Spectrum A: 10 min. Spectrum B: 1 h. Spectrum C: 2.30 h. Spectrum D: 4 h.

1.3 REACTION MONITORING IN SOLID-PHASE SYNTHESIS

31

and reliability of the method should help to increase its popularity in the near future, at least for the analysis of ionizable compounds.

A new reliable, sensitive, and fast MS monitoring approach, based on previous MS quantitation/encoding studies (146, 147), has been recently reported (148). The use of a carbamate-based photolabile linker 1.55 containing two cleavage sites developed in-house allowed the release of the target molecule via acid cleavage (site 1, Fig. 1.20); the so-called analytical construct also allowed the photolytic release for analytical determinations of a larger fragment containing the target molecule, the acid linker, and a free amine (site 2, Fig. 1.20). The amine acted as a mass sensitizer, increased the MS signal of the reaction product, and via an isotopic labeling protocol (146) allowed to discard the MS signals of unbound materials; a single bead was largely enough to determine the quality of the reaction and to spot elusive side products (147, 148). Additional analytical constructs were reported by the same researchers (149); the quantitation of mass-based analytical constructs is currently under study by several groups and holds the promise to accelerate and make more reliable the on-bead reaction monitoring in SPS.

	NO2	O	O
		O N	O
H			N
		H	H
N	O
O	OMe	site 2		O	O
			site 1
		photocleavable
	1.55		acid-cleavable
				HN
				H
				N	O
			O
		MS
		sensitizer
			O
HO	O		O
		H2N	N
			H
HN
H			photocleavage	O	O
N	O
			product
O			H	HN
acid cleavage
			N	O
product
			O

Figure 1.20 Structure of the carbamate-based dual linker 1.55 (analytical construct).

32 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

1.3.6 On-Bead Methods: Infrared Spectroscopy

The use of infrared (IR) spectroscopy as a reaction monitoring technique for SPS has become more frequent over the last few years with the introduction of technologies specifically aimed toward SP reactions. Even so, a number of reports describing the use of classical IR by thorough mixing of a few milligrams of grounded resin beads in KBr pellets have appeared (150, 151).

Single-bead Fourier transform IR (FTIR) spectroscopy is well established as a sensitive and reliable method to monitor SPS (152). Significant improvements have been obtained by flattening the bead in a manual pellet maker, thus reducing and making more homogeneous the pathlength of the incident beam compared to a spherical resin bead (153). For example, the transformation of a resin-bound aldehyde into a dansylhydrazone on hydrophobic PS and on Tentagel resin was monitored using this technique (154). The increase of the N(Me)2 and the N–H hydrazone stretches at 2790 and 3218 cm–1, respectively, and the decreases of the C–H stretch at 2728 cm–1 and of the carbonyl band at 1700 or 1723 cm–1 were easily monitored over time. The beads were pretreated simply by washing with DMF, THF, and DCM, followed by vacuum drying for 15 min. Other examples applied to different chemical transformations have also been reported (40, 155, 156); FTIR spectroscopy has also been used to evaluate the partitioning of reagents into PS beads and the influence of diffusion on SP reaction kinetics (157) or to evaluate the mechanism and kinetics of SP reactions (158).

Some related but more specialized SPS IR techniques have also been described. Attenuated total reflection (ATR) microspectroscopy is extremely sensitive (femtomolar quantities are routinely measured), detecting only surface-bound materials with an average penetration of about 2 µm (159), and has been used to monitor the SP synthesis of a polysubstituted piperazine (160) and of β-amino substituted piperidinols (161). Photoacoustic FTIR (PA-FTIR) provides spectra where only the absorption component of the IR radiation is measured, thus canceling interference due to light scattering and nonhomogeneity of the resin (162). Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) combines an extremely short analysis time of around 30 sec with negligible sample preparation (163, 164). Even more promising appears near-infrared (NIR) spectroscopy, which is also nondestructive and does not require any sample preparation; both a classical example where a single reaction was monitored (165) and the multiple monitoring and detection of NIR images (166) for several SP samples were reported, showing a great potential and flexibility for such a technique in SPS.

Recently several reports highlighting the usefulness of IR and Raman spectroscopy for SP synthesis, especially during the SP chemical assessment, were published; a multistep SP scheme was monitored successfully (167) and a new encoding technique (see Section 7.4) was reported (168). Both IR (169) and Raman (170) spectroscopy applications for SP synthesis were recently reviewed.

In summary, the use of IR either as a simple (KBr pellets) or sophisticated (single-bead techniques) reaction monitoring system for SPS has become very important. The technology behind the methods outlined above is constantly evolving, and