Solid-Phase Synthesis and Combinatorial Technologies

1.4 PURITY AND YIELD DETERMINATION IN SOLID-PHASE SYNTHESIS

33

these techniques will eventually form a complementary network for monitoring reactions in SP chemistry. An interesting comparison (171) between single-bead IR and MAS NMR techniques as tools to assess a novel SP protocol has recently highlighted the complementarity of the two, which should as much as possible be used in conjunction to maximize the quality and the reliability of the analytical monitoring studies.

The further development of these methods will allow chemists to select an appropriate method on the basis of the specific SP synthetic problem in hand and the instrumentation available in their laboratory. Eventually, analytical monitoring will become a routine element of SPS, as it is in classical solution-phase organic chemistry, and one of the major bottlenecks for the further development of SPS will disappear.

1.4 PURITY AND YIELD DETERMINATION IN SOLID-PHASE SYNTHESIS

1.4.1 General Considerations

The determination of the yield of a reaction carried out in the SP and the structure and purity of the product is an essential component of the process of SPS. The same analytical methods that we examined in the previous section for reaction monitoring can be used, but their usefulness for qualitative analysis may vary, as we will describe in this section.

The problems associated with high-throughput analysis of SP reaction products, automation of purification techniques, and automation of methods for structure determination in SPS are typically encountered in combinatorial chemistry and will be dealt with more thoroughly in Chapters 6–8.

1.4.2 Off-Bead Methods

The cleavage and characterization of intermediates or final compounds, as for off-bead reaction monitoring (Section 1.3.2), are the most reliable methods to quantify the outcome of an SPS. The final target molecule on SP has to be cleaved into solution so that it can be characterized by classical off-bead methods. The synthetic intermediates obtained after each SPS step can also be characterized off-bead when the cleavage conditions do not affect their structure and when the amount of resin beads lost during the characterization process is negligible.

Examples of off-bead yield estimation that do not require cleavage include reactions where new species are stoichiometrically formed in solution, and their quantification provides an indirect, but accurate, yield estimation of the SPS step. The classical Fmoc deprotection of amines in peptide synthesis is a widely used example. In a typical experimental procedure, the beads are treated with a 20% DMF solution of piperidine at rt for 20 min and the solution is recovered together with the resin washings. The solution is brought to a constant volume (typically 10 mL) by addition of DMF, and the quantitation is carried out by reading the UV absorbance of the piperidine–

34 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

dibenzylfulvene adduct in solution at 301 nm against a blank solution of piperidine in DMF. The loading can be determined from the equation

L = AV / 7.8W

where L is the Fmoc loading (mmol/g), A is the absorbance value at 301 nm, V is the solution volume in liters, 7.8 is a constant, and W is the weight of the resin in milligrams. The Fmoc loading compared with the known initial resin loading gives the yield of the deprotection. (Note: The UV readings are generally performed in triplicate and an average reading value is used in the equation.)

The outcome of an SPS can be monitored indirectly by reacting the functional group formed with an excess of a fluorescent dye and then monitoring the fluorescence decrease in the supernatant. A carefully planned and executed example (154) used dansylhydrazine to quantify the amount of carbonyl compounds on SP by forming the dansylhydrazone (DMF, at rt for 30 min). A panel of different commercially available (PS–CHO and TentaGel–CHO) and derivatized resins containing ketones or aldehydes were tested for their loading, and the results were checked with single-bead FTIR (see Section 1.3.6). The method requires the destruction of a few milligrams of resin but does not require cleavage and provides reliable results that are not affected by the presence of other functional groups on the support or by the nature of the solid support itself. The disappearance of dansylhydrazine from the supernatant is measured by subtraction of the fluorescence value at the end of the reaction (excess of reagent in solution) from the value at t = 0. This indirect measure of the loading sites of the support allows also a fast and reliable reaction monitoring. The same group reported recently sensible and reliable methods to quantify resin-bound hydroxylic groups via reaction with 9-anthroylnitrile and carboxylic groups via reaction with 1-pirenyldiazomethane (156); both methods could also be used qualitatively in an easy colorimetric test. The use of fluorescent dyes with other chemical functionalities should extend the usefulness of this method in the near future to the quantification of SPS reaction products for different resin-bound chemical groups.

There is a strong need for accurate quantitative and information-rich analytical methods for the on-bead estimation of purity and yield. In the transfer of the chemistry from solution to the SP, it is crucial to determine the yields of all the SPS steps and to monitor the yield and variations in the purity of products obtained by modifying selected reaction parameters. This can be done better, and with reduced amounts of materials, using nondestructive on-bead methods, which provide results that are not biased by the method of cleavage, thus showing if specific cleavage conditions are affecting a target molecule by allowing a comparison of the data obtained through on-bead and off-bead analyses.

1.4.3 On-Bead Methods: Colorimetric/Fluorescence Detection

These methods are typically qualitative rather than quantitative (see Section 1.3.3), and therefore their use for the determination of purity and yield in SPS has not been

1.4 PURITY AND YIELD DETERMINATION IN SOLID-PHASE SYNTHESIS

35

reported. The indirect use of fluorescent dyes for the estimation of yield using off-bead methods has already been discussed in the previous section.

1.4.4 On-Bead Methods: Infrared Spectroscopy

Infrared spectroscopy may be considered to be one of the analytical techniques best suited to the rapid monitoring of the progress of chemical reactions in the SP, as has been discussed in Section 1.3.6. There are some intrinsic limitations to the use of this technique in the determination of purity and yields. The difficulty in quantifying reaction yields by following the simple appearance or disappearance of IR bands is worsened by the broader bands often obtained on SP, especially when using KBr pellets. Small quantities of side products cannot be detected easily due to the reduced intensity (or absence) of IR-specific bands. While some attempts have provided quite accurate estimations of the yield in a few cases (40, 154–156), other analytical techniques appear to be more suited for the quantitative determination of yield and purity of SP reactions.

1.4.5 On-Bead Methods: Mass Spectrometry

The use of MALDI–TOF (see Section 1.3.5) in SPS purity and yield estimation takes advantage of the extreme sensitivity and reliability of the technique, which has other important advantages.

The extremely mild ionization process (137) produces no fragmentation ions, thus allowing the identification of each observed peak as corresponding to a specific reaction product. This is important for monitoring the complete disappearance of the starting material and the appearance of the expected reaction product but, most importantly, for detecting small amounts of side products through their molecular ions. Even impure samples, or mixtures, can be analyzed without affecting the reliability, rapidity, and sensitivity of the technique, which is typically in the femtomolar range. The usual drawbacks, such as the need for a cleavage step prior to analysis of the sample, do not significantly affect the usefulness of MALDI–TOF when used for a rapid estimation of the purity of the reaction products and for the monitoring conditions during the assessment of the chemistry.

The MALDI–TOF monitored SP preparation of lysobactin, a natural cyclopeptide antibiotic, on PS resin bearing the Rink amine linker 1.6 has been reported (141). The SPS scheme is shown in Fig. 1.21. The classical peptide coupling steps, the allyl deprotection (see Section 1.3.5), and the macrocyclization step were all monitored by MALDI–TOF, and the purity of the products was also determined using this technique. The presence of small impurities in compounds 1.56–1.61 was easily detected, and the reaction conditions for the key deprotection of 1.59 and cyclization of 1.60 were rapidly optimized. A total yield of 15% was obtained after HPLC purification of released 1.62 (lysobactin).

36

SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

a

b,c

d

RINK

RINK

RINK

1.57

NH2

1.56

NH

NH

1.6

Asp

Fmoc

Asp

Fmoc

AllylO

(OtBu)Ser

OAllyl

e

b

f

RINK

RINK

RINK

NH

NH

NH

Asp

Gly

Asp

Gly

Thr(OtBu)

Asp

Gly

(OtBu)Ser

Thr(OtBu)

(OtBu)Ser

(OtBu)Ser

Thr(OtBu)

Ile

Ile

Ile

OAllyl

OAllyl

OH

1.58

(Mtr)-D-Arg

Leu

(Mtr)-D-Arg

Leu

(Mtr)-D-Arg

Leu

Thr(OtBu)

b-OHPhe

Thr(OtBu)

Thr(OtBu)

Fmoc

Leu

b-OHPhe

D-Leu

Leu

1.59

D-Leu

1.60

Boc

Boc

Asp

COO

g

Gly

Ser

b-Phe

RINK

Thr

D-Arg

Thr

NH

Gly

Asp

COO

Ile

Leu

Leu

Ser b-Phe

Thr

D-Arg Thr

1.62

Leu

NH2

Ile

Leu

Leu

1.61

Leu

Lysobactin

NH

fully protected	Boc

a: DIC, HOBt, Fmoc-Asp(OH)-OAllyl; b: Pd(0); c: PyBOP, Ser(OtBu)-OAllyl; d: repeated DIC/HOBt amino acid couplings and Fmoc deprotections; e: repeated EEDQ amino acid couplings and Fmoc deprotections; f: DIC, HOBt, DMAP; g: TFA/TIS/H2O 95/2.5/2.5.

Figure 1.21 SPS of 1.62 (lysobaotin).

1.4.6 On-Bead Methods: Nuclear Magnetic Resonance Spectroscopy

The use of gel-phase NMR for monitoring SPS reactions has been described previously. The application of this method to the determination of the purity and yield of a product is not recommended for the reasons already discussed in Section 1.3.4. Another SPS NMR technique, MAS–HR NMR, is more suited to this purpose.

MAS NMR comes from an old observation (172) that spinning a heterogeneous NMR sample at an angle of 54.736° reduces the line broadening of solid/swollen

1.4 PURITY AND YIELD DETERMINATION IN SOLID-PHASE SYNTHESIS

37

polymer samples. Recent realization and use (173) of a HR NMR probe of reduced volume (NanoProbe) that holds all of the sample in the active region of the receiver coil allows the acquisition of SP MAS–HR 1H-NMR spectra with line widths as small as 1 Hz using a 500-MHz spectrometer (174). A few milligrams of resin beads is typically transferred into a Nano.NMR Probe cell, and 40 L of deuterated solvent is added. Further technical details on MAS–HR NMR can be found in a recent review (116).

A fundamental study (175) has shown that the quality of a MAS–HR spectrum is influenced primarily by the nature of the polymer matrix. Hybrid hydrophilic PS resins generally give well-resolved spectra, and the use of techniques such as presaturation of the PEG signals allows the recording of spectra of comparable quality to their solution state counterparts. The high mobility of the long and flexible tethered PEG chains is responsible for the high quality and the narrow signal widths. The use of solvents where both the matrix and the resin-bound compound have a good swelling/solubility further improves the quality of the spectra. Hydrophobic PS resins with short tethers such as the Wang linker generally produce poor-quality spectra, which may be improved by the use of appropriate solvents to swell the resin. Nontethered PS resins give poor-quality spectra under any experimental conditions, and the technique cannot be used to quantify the outcome of SPS on these resins.

Examples of MAS–HR applications to SPS reports can be accessed through recent reviews (116, 176, 177); among the most recent, significant papers, Riedl et al. (178) have brilliantly verified the feasibility and determined the scope of the asymmetric dihydroxylation of resin-bound alkenes, both in terms of reaction yields and of enantiomeric excess.

An emerging trend in the field of SPS is to use high-cost hybrid PS resins for the assessment of the chemistry and to reveal potential problems associated with a given synthetic strategy in the SP. Once this information is acquired, further preparation of the same target molecule on SP and possible exploitation of SPS to make combinatorial libraries can be carried out on cheaper hydrophobic PS resins, for which recent technological improvements (179) are significantly increasing the quality of MAS– NMR spectra. An example of the spectrum on TG–PS recorded in our laboratories (180) is shown in Fig. 1.22, pointing out both the high resolution and the ease of attribution for signals of complex molecules.

Despite the high cost of the equipment required and the time taken for sample preparation and spectra acquisition, MAS–HR NMR provides invaluable structural information about the species present in a reaction. Only a few milligrams of resin beads are required and they can be recovered as the technique is nondestructive. The complementarity of the technique with other analytical methods is clear: MALDI– TOF cannot discriminate among compounds with the same MW and depends on the ionization properties of the resin-bound compound, while FTIR depends on the presence of selected functional groups in the molecule. MAS–HR NMR can be used independently from the nature of the performed reaction and the functional groups formed or lost during the SPS step. Additionally, two-dimensional MAS techniques such as 2D-COSY (correlated spectroscopy) and TOCSY (total correlated spectroscopy) (171) or 2D-SECSY (spin echo correlation spectroscopy) (181) can provide more detailed information that may be useful in specific cases.

38 SOLID-PHASE SYNTHESIS: BASIC PRINCIPLES

Figure 1.22 MAS–HR 1H-NMR spectrum of N-Fmoc-6-amino hexanoic acid linked via amino photolinker to Argogel AG–PS resin.

It seems likely that the use of MAS NMR–related analytical techniques for quantitative SPS analysis will become more widespread in the future. Recent experiments have demonstrated the technical feasibility of improving the sensitivity (182) using also macrobeads (183); the integration of this with other techniques such as MALDI–TOF and the complete off-bead characterization intermediates and final products will provide fast, reliable, and complete information about the structure, purity, and yield of any product obtained through SPS. This will have a major impact on the assessment phase of the chemistry for SPS, which will, in turn, have a profound influence on combinatorial chemistry. These and related issues will be analyzed in depth in Chapters 6–8.

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