Infrared spectroscopy

5.1.1 Introduction

Infrared spectroscopy (IR) is a technique based on the vibrations of the atoms of a molecule. It involves the examination of the twisting, bending, rotating and irrational motions of atoms in a molecule. IR spectroscopy has been extensively used in both qualitative and quantitative pharmaceutical analysis. All molecular species, with the exception of O2, N2, CL2 and H2 absorb infrared radiation (Skoog et al., 2004). Many functional groups in organic molecules show characteristic molecular vibrations and rotations, which correspond to absorption bands in defined regions of the infrared spectrum. These molecular vibrations are localised within the functional groups and do not extend over the rest of the molecule. Thus such functional groups can be identified by their absorption bands (Stuart, 2004).

An IR spectrum is commonly obtained by passing IR radiation through a sample and determining what fraction of the incidence radiation is absorbed at a particular energy.

The energy at which any peak in an absorption spectrum appears corresponds to the frequency of a vibration of a part of a sample molecule (Stuart, 2004).The wave number, expressed in centimeter units (cm^-1), is the reciprocal of the wavelength in centimeters, i.e., Wave number = 1/λ (cm) (McMurry, 2008). Polar bonds are associated with strong IR absorption while symmetrical bonds may not absorb at all. The vibrational frequency, i.e., the position of the IR bands in the spectrum, depends on the nature of the bond. Shorter and stronger bonds have their stretching vibrations at the higher energy end (shorter wavelength) of the IR spectrum than the longer and weaker bonds.

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Similarly, bonds to lighter atoms, e.g., hydrogen, vibrate at higher energy than bonds to heavier atoms (Field et al., 2001).

Fourier-transform infrared (FTIR) spectroscopy is based on the interference of radiation between two beams to yield an interferogram. The latter is a signal produced as a function of the change of pathlength between the two beams. The two domains of distance and frequency are interconvertable by the mathematical method of Fourier-transformation. The radiation emerging from a source is passed through an interferometer to the sample before reaching a detector. Upon amplification of the signal, in which high-frequency contributions have been eliminated by a filter, the data are converted to digital form by an analogue-to-digital converter and transferred to the computer for Fourier-transformation (Stuart, 2004).

The infrared spectrum can be divided into three main regions: the far-infrared (< 400 cm^-1), the mid-infrared (400 – 4000 cm^-1) and the near-infrared (4000 – 13000 cm^-1).

Many infrared applications employ the mid-infrared region, but the near- and far- infrared regions also provide important information about certain materials. Generally there are less infrared bands in the 1800 – 4000 cm^-1 region with many bands between 400 cm^-1 and 1800 cm^-1 (Stuart, 2004).

5.1.2 Methodology

The infrared spectra of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) were recorded on a Shimadzu 1600 spectrophotometer (Shimadzu, Tokyo, Japan). Approximately 1 mg of each cyclic dipeptide, dried in a dessicator, was mixed with approximately 100 mg of dry, powdered, spectral grade potassium bromide (KBr) (Merck, SA). Potassium bromide is suitable for use since it is transparent over the whole IR range, i.e., it has no IR bands of its own (Field et al., 2001). Potassium bromide is however hygroscopic, so that traces of water can rarely be totally excluded during the grinding and pressing of the KBr disc. Therefore a weak band is usually observed at 3450 cm^-1 (Field et al., 2001). The mixture was then ground using an agate mortar and pestle. A Perkin Elmer^® Bench Press set at a pressure of 1 x 10⁴ kg. cm^-2, was then used to press the powdered

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mixture into a thin transparent disc. The disc was transferred to the KBr sample cell of the IR spectrophotometer and the spectra recorded after 16 scans. The infrared spectra of the two cyclic dipeptides were then recorded within the frequency range of 4000 cm^-1 to 400 cm^-1.

5.1.3 Results and discussion

The IR spectra for cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) are illustrated in Figures 5.1 and 5.2, respectively. The main functional groups found in cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-cyclo(Phe-4Cl-Pro) were identified by comparing the peaks obtained from the IR spectra with the observed reference values from literature, shown in Table 5.1.

Table 5.1 Frequencies/absorption bands (cm^{- 1}) of cyclo(Phe-4Cl-Pro) and c yclo(D-Phe-4Cl-Pro)

Description of band Cis-amide

absorption bands

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CN stretching ^{13501 1350 1365.65 1332.86}

NH stretching 3180-3195⁴ 3350 3149.86 3344.68

Combination band

Hydrogen bonded 3300-3380⁴ 3340.00 3344.68

72 NH groups

1(Miyazawa, 1960), ²(Ovchinnikov and Ivanov, 1975), ³(Bláhaet al., 1966), ⁴(Sammes, 1975), ⁵(Stuart, 2004)

The presence of secondary amide and carbonyl groups together with the absence of carboxylic acid groups was necessary to confirm that the diketopiperazine (DKP) rings were present in the structure of the cyclic dipeptides.

According to literature, secondary amide functional groups will show N-H stretching absorption bands between 3180 – 3195 cm^-1 (cis) and 3350 cm^-1 (trans), with carbonyl stretching (Amide I) absorption bands being observed between 1650 cm^-1 (trans) and 1670 – 1690 cm^-1 (cis) (Stuart, 2004; Sammes, 1975; Miyazawa, 1960). The amide II for secondary amide groups is due to the coupling of N-H bending and C-N stretching and appears between 1420 – 1460 cm^-1 (cis) (Ovchinnikov and Ivanov, 1975). A weak band which is an overtone of the amide II band also appears at 650 cm^-1 (Stuart, 2004).

For cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) the presence of secondary amide groups were confirmed by the presence of N-H stretching absorption bands at 3149.86 cm^-1for cyclo(Phe-4Cl-Pro) and 3344.60 cm^-1for cyclo(D-Phe-4Cl-Pro), carbonyl stretching was observed at 1658.84 cm^-1 for cyclo(Phe-4Cl-Pro) and at 1647.26 cm^-1 for cyclo(D-Phe-4Cl-Pro), the amide II bands occurring at 1467.6 cm^-1 for cyclo(Phe-4Cl-Pro) and 1467.6 cm^-1 for cyclo(D-Phe-4Cl-Pro) and weak bands representing the overtones of the amide II occurring at 751.8 cm^-1 and 732.0 cm^-1 for cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro), respectively.

The absence of carboxylic acid groups is also necessary to confirm that the cyclisation of the linear dipeptides had occurred. Carboxylic acid groups show a strong broad O-H stretching band in the 2500 cm^-1 to 3300 cm^-1 range. The C=O stretching band of the free acid band is observed at 1760 cm^-1 Carboxylic acids also show characteristic C-O stretching and in-plane and out-plane O-H bending at 930 cm^-1 and 1430 cm^-1,

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respectively (Stuart, 2004). The absence of these characteristic bands, for both cyclic dipeptides, indicated the absence of carboxyl (COOH) end groups.

The presence of secondary amide functional groups, together with the absence of any carboxylic acid functional groups for cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) therefore suggested that a DKP ring is present in the structures of the cyclic dipeptides and that cyclisation therefore had occurred during the synthesis of these compounds.

Infrared spectroscopy also reliably discriminates between cis and trans secondary amides (Ovchinnikov and Ivanov, 1975; Miyazawa, 1960). As a consequence of their biosynthetic origin from the two L-α-amino acids, most naturally occurring DKPs are cis-configured (Bull et al., 1998).

The cis-amide bond nature of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) was revealed through the values of the C=O stretching mode (amide I), which produced a peak at approximately 1670 cm^-1 to 1690 cm^-1 (Miyazawa, 1960). Strong peaks appeared at 1680.3 cm^-1 for cyclo(Phe-4Cl-Pro) and at 1666.0 cm^-1 for cyclo (D-Phe-4Cl-Pro), confirming the presence of amide configuration. Further evidence for cis-amide configuration was given by the absence of trans-peaks at 1650 cm^-1 for the cyclic dipeptides (Miyazawa, 1960).

Amide II vibrations are present due to interactions of the N-H bending and C-N stretching modes. The difference in frequencies between these two modes is approximately 100 cm^-1, which is due to negligible interactions between them. A difference of approximately 100 cm^-1 exists between the N-H bending and the C-N stretching modes of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) , and according to (Miyazawa, 1960), this is only apparent in the cis-CONH configuration which further confirmed the cis-amide configuration of the cyclic dipeptides.

The amide type II mode is represented by a single absorption band within the 1420 cm^-1 to 1460 cm^-1region (Ovchinnikov and Ivanov, 1975). N-H bonding contributes the most

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to amide II vibrations, whereas the C-N stretching vibration contributed more to the amide III band. The amide II vibrations occurred at 1467.6 cm^-1 for cyclo(Phe-4Cl-Pro) and at 1467.1 cm^-1 for cyclo(D-Phe-4Cl-Pro), confirming cis-amide configuration. Once again the absence of the trans-peaks between 1480 cm^-1 and 1575 cm^-1 (Ovchinnikov and Ivanov, 1975), provided further evidence of the cis-amide configuration of the cyclic dipeptides.

The cis-amide type III mode is represented by absorption bands within the 1300 cm^-1 to 1350 cm^-1 region (Bláha et al., 1966). Amide III vibrations occurred at 1327.6 cm^-1 for cyclo(Phe-4Cl-Pro) and at 1345.0 cm^-1 for cyclo(D-Phe-4Cl-Pro), which confirmed the cis-amide configuration.

The N-H stretching mode of the cis-bond is represented by absorption band within the 3180 cm^-1 to 3195 cm^-1 region (Sammes, 1975). N-H stretching vibrations occurred at 3240.3 cm^-1 for cyclo(Phe-4Cl-Pro) and at 3185.6 cm^-1 cyclo(D-Phe-4Cl-Pro), confirming the cis-amide configuration. The absence of trans-peaks at 3350 cm^-1 provided further evidence of the cis-amide configuration.

The CH2 bending vibration is also present within the amide II mode, and since only a single absorption band appears in this range, it is difficult to determine the presence of each mode. Similarly the CH2 wagging vibration is overlapped by the C-N stretching mode, and is therefore not clearly defined. CH2 twisting and rocking was observed at 1234.2 cm^-1 and 976.8 cm^-1 for cyclo(Phe-4Cl-Pro) and at 1223.6 cm^-1 for cyclo(D-Phe-4Cl-Pro).

Figure 5.1 and figure 5.2 Illustrates the overlay of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) respectively. The spectrum of cyclo(Phe-cyclo(D-Phe-4Cl-Pro), however, shows absorption bands at 1520.6 cm^-1and at 1620.1 cm^-1. These absorption bands, which are not seen in the spectrum of cyclo(Phe-4Cl-Pro), are due to aromatic C=C stretching caused by the aromatic tyrosine side-chain.

75 Figure 5.1 Infrared spectrum of cyclo(Phe-4Cl-Pro)

76 Figure 5.2 Infrared spectrum of cyclo(D-Phe-4Cl-Pro)

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