Monitoring solid phase synthesis by infrared spectroscopic techniques

Monitoring solid phase synthesis by infrared spectroscopic techniques

Walter Hubera,*, AndreÂ Bubendorf a, Alfred Griedera, Daniel Obrechtb

aF. Hoffmann-La Roche, PRPI 65/201, Postfach, CH-4070, Basel, SwitzerlandbPolyphor Ltd. Winterthurerstrasse 190, CH-8057, ZuÈrich, Switzerland

Received 10 August 1998; received in revised form 21 September 1998; accepted 22 October 1998

Abstract

The paper describes the use of attenuated total re¯ection fourier transform-infrared (ATR FT-IR) microspectroscopic

techniques for the veri®cation of the structure of chemical compounds synthesised on polystyrene beads in combinatorial

chemistry. A six step reaction sequence is characterised completely by infrared (IR) spectroscopic investigations of single

beads. Incomplete reactions or the occurrence of side products are clearly indicated. Quantitative information can be extracted

with high precision using absorption bands of the polymer matrix as internal standard. Compared to other IR techniques, the

ATR micro IR technique shows clear advantages with respect to sensitivity and resolution. The technique has the potential to

be used for the characterisation of single beads in split and combined synthesis techniques. # 1999 Elsevier Science B.V. All

rights reserved.

Keywords: Infrared; Microscopy; Combinatorial chemistry; Solid phase synthesis

1. Introduction

Since its introduction by Merri®eld in 1963, the

stepwise synthesis of peptides on solid supports has

become a powerful tool in the preparation of poly-

peptides. Recently, it has been demonstrated that solid

phase synthesis technique meets the demands of

combinatorial chemistry, a technology that has now

attracted high attention as a tool for the preparation of

large libraries of substances needed in pharmaceutical

drug screening [1±4]. A chemist starting to use this

technique is faced with the problem of developing

organic reactions at the surface of the solid support.

During this development phase, aiming at the optimi-

sation of the reaction for high yield, the chemist is

highly dependent upon analytical information about

product formation at the surface. This analytical

information is in addition also needed for quality

assurance during the production of compounds. In

general, the analytical problems arising are solved

by cleaving the products from the beads and charac-

terising them by the commonly used spectroscopic

techniques such as nuclear magnetic resonance

(NMR), infrared (IR) or mass spectroscopy (MS)

[5,6]. For the development of multistep syntheses this

methodology becomes time and material consuming.

Spectroscopic techniques suitable to detect such syn-

thetic products attached to the surfaces would be a

helpful analytical tool. Recently, special NMR tech-

niques have been introduced that enable direct on-

bead structure determination [7].

Analytica Chimica Acta 393 (1999) 213±221

*Corresponding author. Tel.: +41-61-688-76-17; fax: +41-61-

688-7408; e-mail: [email protected]

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 7 7 0 - 3

The usefulness of IR spectroscopy has already been

demonstrated in this area. One of the technique's

requires making KBr pellets from the beads [8], the

other makes use of an IR microscope in the transmis-

sion mode [9]. Fourier transform infrared (FT-IR)

internal re¯ection spectroscopy was applied to large

synthesis supports known as `Mega Crowns' [10].

These results indicated that the spectra monitored

by the internal re¯ection mode are less distorted by

the absorption band of the polymer support. The

present report demonstrates the use of attenuated total

re¯ection (ATR) FT-IR micro-spectroscopy for struc-

ture con®rmation of compounds synthesised on small

polymer beads. The beads are the so-called high

loaded Merri®eld resins frequently used for the pro-

duction of compounds for substance libraries by a

combinatorial approach. Taking a six step reaction

sequence as an example (Scheme 1) the sensitivity and

the diagnostic potential are discussed.

2. Materials and methods

Commercially available high loaded Merri®eld

resin (3.4 mmol gÿ1) was used as the solid support

in the synthesis. The diameter of the beads varied

between 50 and 200 mm. The reaction sequence car-

ried out on the beads and characterised by IR spectra is

depicted in Scheme 1. Experimental details of the

reaction conditions are given elsewhere [11,12].

Scheme 1.

214 W. Huber et al. / Analytica Chimica Acta 393 (1999) 213±221

The IR micro-spectroscopic measurements were

carried out on a Nicolet Magna 860 instrument

equipped with a Nicplan IR microscope. A ZnSe

ATR objective was used to monitor the ATR spectra

from single beads. Thereby, the bead was aligned with

the optical axis of the microscope and then pressed

against the objective. The pressure was optimised via

visible inspection of the single beam IR spectrum. For

the microscopic measurement on single beads in the

transmission mode the beads were held within a

diamond cell. KBr pellets were prepared by mixing

5 mg of bead material with 300 mg of KBr. The IR

measurements on these pellets were carried out on a

Nicolet 20SXB FT-IR instrument. About 500 scans

with a resolution of 4 cmÿ1 were accumulated for each

spectrum.

3. Results and discussion

3.1. Structure verification by the assignment of

absorption bands to group frequencies

Fig. 1 depicts a series of IR spectra taken with the

IR microscope (ATR mode) from polystyrene beads

taken from batches of beads subjected to the reaction

sequence given in Scheme 1. Each spectrum was taken

from a single bead. The signal to noise ratio of the

spectra is greater than 800. This series of spectra fully

characterises the intermediates synthesised on the

surface of the solid support by the preceding reaction

steps. For the interpretation of the spectra and the

elucidation of the structure of the intermediates one

can refer to tabulated group frequencies [13].

Fig. 1. IR spectra monitored on single beads subjected to the reaction sequence depicted in Scheme 1. The frequency of the most prominent

beaks characterising the synthesis product of a reaction step is given.

W. Huber et al. / Analytica Chimica Acta 393 (1999) 213±221 215

The spectrum in (Fig. 1(A)) shows the typical

spectral features of polystyrene. The C±C vibrations

of the aromatic rings are between 1600±1440 cmÿ1,

the respective overtones and combination modes of

the aromatic rings between 2000 cmÿ1 and 1700 cmÿ1

and the signals around 750 cmÿ1 for the C±H out-of-

plane vibrations of the aromatic rings. The formation

of the thiouronium salt in the ®rst reaction step

changes these spectral features completely

(Fig. 1(B)). The spectrum is no longer dominated

by the absorption bands of polystyrene. It is dominated

by absorption bands which can be assigned unambigu-

ously to vibrations of the thiouronium salt, the strong

absorption peak at 1645 cmÿ1 to the C=N bond and

the broad band at 3200 cmÿ1 to the N±H� stretching

mode. The small relative amplitudes of the polystyr-

ene absorption bands in this spectrum as well as in

the subsequent spectra are quite remarkable and can

be attributed to the surface selectivity of the ATR

measuring mode as well as to the high loading of the

beads.

The formation of the substituted pyrimidine ester

ring in the second step can be fully proven by the

respective IR spectrum (Fig. 1(C)). The absorption

band centred at 1729 cmÿ1, which appears as a doub-

let with maxima at 1718 cmÿ1 and 1741 cmÿ1, is due

to the C=O stretching vibration of the ester group and

the band at 1250 cmÿ1 is due to the C±O stretching

mode. The bands at 1565 cmÿ1 and 1520 cmÿ1 have to

be assigned to the C±C vibrations of the aromatic and

heteroaromatic ring systems. The sharp and intense

absorption band at 1366 cmÿ1 can be assigned to the

tert-butyl group of the ester. Complete conversion of

the uronium salt to the respective pyrimidine ester

derivative is indicated by the disappearance of the

signals assigned to the salt, especially the strong band

at 1645 cmÿ1. The cleavage of the ester group to the

respective carboxylic acid does not lead to signi®cant

changes in the spectrum (Fig. 1(D)) due to the simi-

larity of ester and carboxylic acid vibrations. It is,

therefore, dif®cult to decide by comparison of the two

respective spectra whether the conversion has

occurred. It is helpful in this respect to convert the

carboxylic acid into a carboxylate ion by treating the

bead with dilute NaOH. The spectrum of the carbox-

ylate derivative formed by this treatment is depicted in

Fig. 2 together with the spectrum of the carboxylic

acid derivative. It shows the spectral features of the

carboxylate group (absorption bands at 1616 cmÿ1 for

the asymmetric CO2 stretching and at 1408 cmÿ1 for

the symmetric CO2 stretching) and the absence of the

spectral features of the ester group (e.g., doublet with

maxima at 1718 and 1741 cmÿ1). The conversion of

the carboxylic acid group to an amide I group is

indicated in the respective spectrum (Fig. 1(E)) by

the appearance of an absorption band at 3383 cmÿ1

assigned to the N±H stretching vibration and at

1678 cmÿ1 assigned to the C=O stretching mode of

the amide group. The amide II mode appears as an

intense absorption signal at 1511 cmÿ1. The ®nal

oxidation of the sulfur atom is indicated by the two

bands appearing at 1325 cmÿ1 and 1125 cmÿ1

(Fig. 1(F)). Most of the remaining spectral features

of the amide remain nearly unchanged by this con-

version except the absorption band at 1250 cmÿ1,

which disappears. This band can be assigned to the

wagging mode of the CH2 group close to the sulphur

atom which is present only if the sulphur is in a non-

Fig. 2. Conversion of the carboxylic acid into the carboxylate salt

to show the absence of the ester by IR spectroscopic means.


oxidised state. This vibration band could thus be used

to check the completeness of conversion.

3.2. Incomplete conversion and formation of side

products

The above spectral sequence has been taken after an

intense optimisation of the chemistry involved in order

to avoid incomplete turnover or the formation of side

products. IR spectroscopy was heavily used in these

optimisation steps and the following example should

demonstrate the feasibility of such a spectroscopically

assisted optimisation of the reaction sequence. The

reaction step examined concerns the formation of the

pyrimidine ring starting from the thiouronium salt

(step 2 in reaction Scheme 1). The respective reaction

in homogeneous solution is in general fast and com-

plete within 2 h. The attachment of the thiouronium

salt to the solid phase decelerates the reaction dras-

tically, although the reactant is present in excess. The

spectrum depicted in (Fig. 3(A)) was taken from

beads treated for 6 h. It clearly shows the typical

absorption bands of the desired ester derivative (com-

pare the spectrum with the spectrum in (Fig. 3(B))

indicating that the correct product is formed in this

reaction. However, even after this prolonged reaction

time the absorption band of the thiouronium salt at

1645 cmÿ1 is still visible indicating that the reaction

time has to be prolonged further for complete turnover

of the thiouronium salt. (Fig. 3(C)) shows the spec-

trum of a byproduct that is formed during the synthesis

of a structurally slightly modi®ed pyrimidine deriva-

tive, i.e., the phenyl ring is substituted by a alkyl

residue. From the spectrum of the reaction product it is

straightforward to conclude that the aromatic pyrimi-

dine ring is not formed in this reaction. The most

prominent indication is the frequency of the absorp-

tion band of the C=O vibration of the ester, which is

located above 1715 cmÿ1 for all pyrimidine deriva-

tives so far synthesised. For the derivative isolated in

this reaction the C=O absorption of the ester is located

at 1680 cmÿ1. Structural analysis on an isolated pro-

duct supported by MS and NMR spectroscopy led to

the conclusion that the unknown side product has the

structure given in (Fig. 3(C)). Elimination of H2O and

aromatisation of this ring system can be induced by

treating the beads with bases.

3.3. Quantitative analyses

In the case of incomplete turnover it is desirable to

have an estimate of product formation or educt turn-

over. Infrared spectroscopy can be used for semi-

quantitative analysis because signal intensity is,

among other parameters, also dependent on the con-

centration of the substance. It is questionable in the

present case whether such a semi-quantitative analysis

will work on single beads. The main problem coupled

with these question concerns the bead-to-bead repro-

ducibility of the absorption band amplitude or area.

For proof of concept, 40 spectra were recorded with

the ATR-IR microscope from different beads of the

same synthetic batch. Fig. 4 depicts a selection of this

series that includes the extremes. Table 1 contains the

Table 1

Peak variation of experimental absorption peaks and standard peaks of the supporting polystyrene material and the variation of these peak

areas after normalization

Peak areas of experimental peaksa Normalized peak areas

Peak position 1741 � 1718b 1449c 1741 � 1718/1449d

Mean 6.48 1.88 3.44

Maximum 9.62 2.74 3.89

Minimum 2.88 0.88 2.92

S.D. 1.7 0.45 0.26

S.E. 0.38 0.1 0.05

Note: The spectra and the spectrum deconvolution shown in Fig. 4 have been used.

a) Peak area determined via deconvolution.

b) Sum of the two peak areas forming the doublet of the ester CO-band centered at 1729 cmÿ1.

c) Peak area of the polystyrene band at this position (internal reference peak).

d) Sum of the to peak areas forming the doublet of the ester CO-band centered at 1729 cmÿ1 divided by the peak area of the polystyrene band

at 1449 cmÿ1.


mean peak areas of the most characteristic peaks of the

ester derivative (doublet around 1729 cmÿ1) and the

polystyrene (peak at 1449 cmÿ1 ) investigated in this

case.

It is obvious from the representation of these spectra

that the absorption band intensities differ drastically

from bead to bead. At ®rst glance, a determination of

product concentration seems to be impossible. There

could be two reasons for this intensity variation: (i) the

variation occurs because of a variation in the loading

from bead to bead or (ii) the variation is due to the

spectroscopic parameters that are mainly responsible

for the spectral intensity, such as contact area between

bead and ATR crystal or the tightness of the contact. In

both cases, the variation could be eliminated by taking

spectra from a large ensemble of beads and using an

Fig. 3. Incomplete turnover (A) and formation of side product (C) monitored by IR spectroscopy. The spectrum of the desired product is given

for comparison (B).


averaged spectrum for the determination of the

amount of product formed. This would be a very time

consuming procedure and would not be compatible

with single bead analysis. However, if the ®rst point is

mainly responsible then this would be the only way

and it would be questionable if this technique is really

adequate to solve quantitative problems. If the second

reason is mainly responsible then one could try to

eliminate this variation by using internal standard

signals such as the polystyrene absorption bands.

Since the polystyrene concentration can be taken as

a constant for all beads, variation in their peak inten-

sity can be assigned to variation in the measuring

parameters such as contact area between the ATR-IR

crystal and the bead. In order to examine this hypoth-

esis, the spectra depicted in Fig. 4 (bottom) were

subjected to a deconvolution into the underlying

Gaussian or Lorentzian curves. It has to be emphasised

that the parameter set for deconvolution (peak posi-

tion, peak width, and peak shape) remained constant

within certain limits for the deconvolution of all

spectra. The parameter set was worked out on one

spectrum representing an average spectrum. The stan-

dard deconvolution is depicted in Fig. 4 (top). The

deconvolution enables the proper separation of pro-

duct and support absorption bands and the determina-

tion of their peak areas by integration. The peaks

chosen in this example are highlighted in Fig. 4:

the doublet of the C=O stretching as a speci®c product

absorption band and the peak at 1449 cmÿ1 as the

support absorption band. The respective peak areas are

given in Table 1 for the doublet of the C=O peak of the

ester centred at 1729 cmÿ1 and for a polystyrene peak

at 1449 cmÿ1. The areas of the C=O bands vary by a

factor of three and the standard error of these non-

normalised peak areas is 38%. In contrast, the standard

error drops to 5% upon peak normalisation using the

area of the polystyrene peak in the respective spectrum

as an internal standard (Table 1). The use of spectra

monitored by the ATR-IR spectroscopic method for

semi-quantitative analyses is feasible within a 5%

standard deviation.

3.4. Comparison of the ATR-IR micro-spectroscopic

technique to other IR-spectroscopic techniques

There are other IR techniques in use for an in situ

spectroscopic characterisation of products prepared on

polymer beads. The ATR-IR micro-spectroscopic

technique offers clear advantages with respect to

sample handling and the quality of spectra.

Fig. 5 presents IR-spectra taken from the beads

using the two technologies presented in the literature

The depicted spectra are all original spectra, and have

not been subjected to any corrections (baseline correc-

tions, smoothing etc.). The differences became clearly

obvious from this comparison. In the transmission

spectrum taken from a KBr pellet (Fig. 5(C)) the

signals of the supporting material are of similar

intensity as the signals of the material synthesised

on the solid support. The overlap of these signals with

the signals of the supporting material leads to an

apparent line broadening. Additional line broadening

also occurs due to light scattering effects at the beads

Fig. 4. Overlap of the spectra of different beads of the same

synthetic batch (bottom) and deconvolution of an average spectrum

into the underlying absorption peaks (top). Typical peaks of the

support material (1449 cmÿ1) used as standard and of the synthesis

product (1741 cmÿ1 and 1718 cmÿ1) are marked.


enclosed in the KBr matrix. These two line-broad-

ening effects decrease the apparent line separation and

makes the interpretation of the spectra and hence the

structure elucidation more dif®cult. The ®gure also

depicts two spectra of a single bead with a diameter

�100 m measured in the transmission mode

(Fig. 5(A) and Fig. 5(B)). The bead investigated is

nearly non-transparent to IR radiation in the spectral

region of interest (1700±600 cmÿ1). Flattening of the

bead by applying high pressure increases the transpar-

ency and the absorption bands of the product are then

visible. The microscope transmission mode is

obviously only suitable for beads with a diameter

�100 mm. With respect to the apparent line width

and the apparent resolution, the observed spectrum

(Fig. 5(B)) is comparable to the spectrum obtained

from measurements on KBr and is therefore less

suitable for interpretation than the spectrum taken

by the ATR-IR technique. Moreover, sample handling

and microscope alignment is much more dif®cult and

time consuming in the transmission than the ATR-IR-

microscopic measurement. The applicability of the

technique and the high spectral quality is demon-

strated for so-called high loaded beads. These beads

are generally used for the production of compounds

for substance libraries. The high signal-to-noise ratio

of >800 indicates that beads with a lower loading can

also be characterised. In fact, we have applied the

technology (results not shown) for beads with a load-

ing of 1 mmol gÿ1. Beads with lower loading are

generally not used for the production of compounds

for substance libraries. They are mainly used in split

and mix techniques, where screening assays are car-

ried out with the compounds still bound to the solid

support.

4. Conclusions

The present paper introduces a novel IR spectro-

scopic technique for the in situ characterisation of

solid phase-bound reaction products in combinatorial

chemistry. The most prominent advantages of the

technique are the speed of the analysis (no sample

preparation) and the quality of the spectra obtained.

The feasibility of giving semi-quantitative results

has been shown in this paper. Such an opportunity is

particularly helpful in combinatorial chemistry for the

quantitative examination of identical beads from sub-

sequent productions or for the quantitative examina-

tion with respect to turnover of beads with structurally

related but diverse synthesis products often generated

for substance libraries.

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Fig. 5. Comparison of spectra taken by the micro transmission

technique (A and B) and the KBr technique (C) with spectrum

taken by the ATR-IR micro-spectroscopic technique (D).


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Monitoring solid phase synthesis by infrared spectroscopic techniques