L-valine and L-proline - solid-state IR-LD spectroscopic study

Article abstract of DOI:10.2174/092986610790780297

Spectral investigation including IR-characteristic bands assignment of the amino acids zwitterions _L-Valine (_L-Val) and _L-Proline (_L-Pro) was carried out by linear-dichroic infrared (IR-LD) spectroscopy of oriented solid sample as a nematic liquid crystal suspension. The obtained experimental IR-LD results (transition moment directions) were compared with known crystal X-ray data for molecules orientation in the unit cells of the studied compounds, confirming the applicability of the used spectral method for structural determination. The influence of the protonation on the IR-spectroscopic patterns of the both amino acids is discussed.

Full text of DOI:10.2174/092986610790780297

Protein & Peptide Letters, 2010, 17, 347-350
347
L-Valine and L-Proline - Solid-State IR-LD Spectroscopic Study
A.G. Chapkanov1,* and S.Y. Zareva2
1South-West University”Neofit Rilski”, Department of Chemistry, 66”I. Mihailov” Str., 2700 Blagoevgrad, Bulgaria;
2University of Sofia“St. Kl. Ohridsky”, Faculty of Chemistry, Department of Analytical Chemistry, 1 “J. Boucher” blvd.
1164 Sofia, Bulgaria
Abstract: Spectral investigation including IR-characteristic bands assignment of the amino acids zwitterions L-Valine (L-
Val) and -Proline (L-Pro) was carried out by linear-dichroic infrared (IR-LD) spectroscopy of oriented solid sample as a
L
nematic liquid crystal suspension. The obtained experimental IR-LD results (transition moment directions) were compared
with known crystal X-ray data for molecules orientation in the unit cells of the studied compounds, confirming the appli-
cability of the used spectral method for structural determination. The influence of the protonation on the IR-spectroscopic
patterns of the both amino acids is discussed.
Keywords: L-Valine, L-Proline, IR-LD spectroscopy.
INTRODUCTION
O
O
H3C
H3C
O
The interaction of Au (III), Pt (II), Ru (III) complexes
with peptides and proteins has been widely investigated, due
to the potential antitumor effect of the corresponding coordi-
nation compounds [1-3]. In order to understand the in vivo
manner and coordination mechanism of these metal ions
with DNA, a systematic examination of their coordination
ability with small peptides and and proteins is necessary [4-
7]. From this point of view the particular interest has origi-
nated from the donor ability of hetero atoms in valine and
proline side chains. Moreover, the peptides based of the last
amino acids possessed significant biological activity as for
H2N
O
NH3
L-Val
L-Pro
Scheme 1. Zwitterions chemical structure of the studied amino
acids.
For -Val hydrochloride: Found: C, 39.9; H, 7.5; N, 9.0;
L
C5H12NO2Cl: Calc: C, 39.1; H, 7.8; N, 9.1; For -Pro hydro-
L
chloride: Found: C, 39.3; H, 6.6; N, 9.4; C5H10NO2Cl: Calc.
C, 39.6; H, 6.7; N, 9.3. The most intensive signals in the
mass spectra of both hydrochlorides are: MS(m/z): 118.20
(M+) for [C5H12NO2]+ and MS(m/z):116.32 (M+) for
[C5H10NO2]+ with molecule weights 118.16 and 116.14, re-
spectively.
example tripeptide -Valyl-L-Prolyl-L-Proline has anti hyper-
L
tensive ACE inhibitor activity [8, 9]. The detailed assigna-
tion of IR- characteristic bands for these systems is relatively
with difficulty for conventional IR-spectral interpretation, as
a result of overlapping IR-peaks due to of the presence of
much more one amide fragment. The possibilities of the lin-
ear-polarized IR-spectroscopy (IR-LD), however allow to
investigate the IR-spectral characteristics of the protonation
and coordination processes in amino acids and small pep-
tides.
EXPERIMENTAL PART
Methods
The IR-spectra were measured using
a Bomem-
The structure of the amino acids zwitterions -Valine (L-
Michelson 100 FT-IR-spectrometer (4000 – 400 cm-1, 2 cm-1
resolution, 150 scans) equipped with a Perkin Elmer wire-
grid polarizer. Non-polarized solid-state IR spectra were
recorded, using the KBr disk technique. The oriented sam-
ples were obtained as a colloidal suspension in a nematic 4’-
cyano-4’-alkylbicyclohexyl mixture (ZLI-1695, Merck),
whose poor IR-spectrum allows the recording of the guest-
compound bands in the whole 4000 - 400 cm-1 range. The
presence of the isolated nitrile stretching IR-band at about
2230 cm-1 additionally serves as an orientation indicator. The
effective orientation of the solid samples was achieved by
the next procedure: the investigated compound (5 mg) was
mixed with the liquid crystal substance until obtaining of a
slightly viscous suspension. The phase prepared thereby was
pressed between two KBr-plates, which in advance were
rubbed out in one direction by fine sand-paper. The grinding
L
Val) and -Proline (L-Pro) are shown in Scheme 1 and this
L
paper is focused on their spectral analysis.
Synthesis
L-Val and L-Pro hydrochloride salts were synthesized
according to the procedures described in [10, 11]. To the 10
ml methanol solutions of -Val (0.5316 g, Sigma) and -Pro
L
L
(0.5124g, Sigma) was added 0.5 ml 10-1M HCl. After about
a week the obtained white polycrystalline salts were filtered,
washed with methanol and dried over P2O5 at room tempera-
ture. Yields: 64% for -Val and 77% for -Pro respectively.
L
L
*Address correspondence to this author at the South-West University”Neofit
Rilski”, Department of Chemistry, 66”I. Mihailov” Str., 2700 Blagoevgrad,
Bulgaria; Tel: (+359 73) 83 18 25, Fax: +359 73 88 55 16;
E-mails: chapkanov@swu.bg; nasko50@yahoo.com
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

348 Protein & Peptide Letters, 2010, Vol. 17, No. 3
Chapkanov and Zareva
of the prepared mull in the rubbed direction promotes in ad-
dition the sample orientation [12-15].
data about the last compounds are assigned on the basis of
known IR-data about similar systems [25- 32]. The compari-
son and assignment of the solid-state IR-spectra (Figs. 2.1
and 4.1) of the all systems studied is done, using the state-
ment that pure amino acids stabilize H3N+-R-COO- (L-Val)
and H2N+-R-COO- (L-Pro) zwitterions with characteristic IR-
For polarized IR-spectra interpretation, the IR-LD spec-
troscopy use stepwise reduction procedure [16-19]. The IR-
LD data interpretation are consisted in subtraction of the
perpendicular spectrum (IRs - a result of the 90° angle be-
tween the polarized light beam electric vector and the orien-
tation of the sample) from the parallel spectrum (IRp) ob-
tained with a co-linear mutual orientation. The recorded dif-
ference (IRp-IRs) spectrum divides the integrated absor-
bances into positive and negative bands. Positive bands
originate from transition moments which form average an-
gles with the orientation direction (n) between 0° and 54.7°
(magic angle) and negative bands corresponding to transition
moments, which are directed between 54.7° and 90°. In the
reducing-deference procedure, the perpendicular spectrum,
multiplied by the variable parameter c, is subtracted from the
parallel spectrum and parameter c is varied until at least one
band or set of bands is eliminated in the obtained (IRp -
cIRs) reduced IR-LD spectrum. The simultaneous disappear-
ance of these bands in the reduced spectrum provides infor-
mation about the mutual disposition of the molecular frag-
ments. This elimination method is carried out graphically
using the attached subtracting procedure for processing of IR
spectra.
+
spectral bands of -NH3 , -NH2+ and -COO- groups.
In all cases a preliminary deconvolution and curve-fitting
procedure for the peak positions obtained and corresponding
integral absorbance are applied as is shown in (Fig. 1) for -
L
Pro in the 1750 – 1500 cm-1 region.
The elemental analysis was performed by standard pro-
cedures for C, and H (as CO2, and H2O) and N - by the
Duma’s method. The FAB mass spectra utilized for the
molecule weight determination were recorded on a Fisons
VG Autospect instrument employing 3-nitrobenzyl alcohol
as a matrix.
Figure 1. 1750 – 1500 cm-1 curve-fitted IR-spectrum of L-Pro.
The protonation in each case leads to disappearance of
the typical -COO- maxima in 1630 – 1550 cm-1 spectral
range as well as about 1405 cm-1. The character of the 1565
cm-1 and 1407 cm-1 bands as ꢀas
and ꢀsCOO- in -Pro is
RESULTS AND DISCUSSION
COO-
L
This work can be considered as a part of systematic spec-
troscopic and structural investigations on amino acids and
their derivatives, small peptides and their possibilities to
form metal complexes and application as potential anticancer
medications [20-24].
confirmed in addition by the obtained IR-spectrum of proto-
nated -Pro as hydrochloride salt. It is characterized with the
L
disappearance of the maxima at 1565 cm-1 and 1407 cm-1 and
a new peak at 1735 cm-1 is observed, which correspond to
ꢀC=O stretching peak of the restored COOH group in the salt.
The characteristic IR-bands of the pure amino acids L-Val
The simultaneously elimination of pairs of peaks at 1629
and -Pro are listed and assigned in (Table 1). The included
cm-1 and 1612 cm-1 (Fig. 2.2) confirmed their character as
L
Table 1. IR-Characteristic Bands in 1700 – 1350 cm-1 Region of L-Val and L-Pro
ꢀ [cm-1]
Assignment
L-Val
L-Pro
ꢀ
NH3+ stretching
NH2+ stretching
3200 – 2700
-
ꢀ
-
3000 – 2400
ꢁas
1629
1612
1567
-
-
NH3+
ꢁ’as
-
NH3+
ꢁsNH3+
-
ꢁNH2+
1626, 1612
1567, 1552
1407, 1402
ꢀas
1585
1508
COO-
ꢀsCOO-
*IR-data obtained after applied deconvolution and curve-fitting procedures

L-Valine and L-Proline - Solid-State IR-LD Spectroscopic Study
Protein & Peptide Letters, 2010, Vol. 17, No. 3 349
ꢀas
and ꢀ’as
(Table 1) due to their possession of the
tal X-ray data of -Pro [11] the unit cell contains four mole-
NH3+
NH3+
L
same symmetry class and irreducible representation of the
allowed transition according to [33] (direction of the transi-
tion moments). The elimination of the maximum at 1585 cm-
1 strongly reduced the 1508 cm-1 peak (Fig. 2.3). In the frame
cules which are similarly mutual oriented by pairs, thus as-
sumed the near to collinear orientation of ꢀNH2+ and ꢁasCOO- in
the frame of these neighboring pairs of molecules (Fig. 3
(2)).
of the unit cell of -Val (Fig. 3(1)), the four presented mole-
L
For that reason the obtained IR-LD reduced spectrum in
Fig. (4.2) illustrated the strong reduction of 1567 cm-1 peak
with the 1626 cm-1 one (Table 1). Due to the last procedure
second pairs of observed maxima at 1612 cm-1 and 1552 cm-1
cules are oriented by the way supposed the relatively co line-
arity of corresponding ꢁas
and ꢁsCOO- transition moments
COO-
between neighboring molecules [10].
correspond to ꢀNH2+ and ꢁas
of second different oriented
COO-
molecule of -Pro in the frame of the unit cell. The multiple
L
character of 1750 – 1500 cm-1 region is determined as well
with deconvolution and curve-fitting procedure applied,
which leads to series of peaks shown in Fig. (1).
Figure 2. Non-polarized IR-(1) and reduced IR-LD spectra of L-Val
after elimination of 1629 cm-1 (2) and 1585 cm-1 (3) peaks.
Thus could be explained and assigned as the belonging of
last discussed peaks to asymmetric and symmetric stretching
Figure 4. Non-polarized IR-(1) and reduced IR-LD (2) spectra of L-
Pro after elimination of 1626 cm-1 peak.
modes of COO- fragment in the molecule of -Val. Moreo-
L
ver, the last peaks disappear in the obtained spectrum of pro-
CONCLUSION
tonated form of -Val hydrochloride, accompanied with an
L
observation of new peak at 1720 cm-1, corresponding to ꢁC=O
Linear-dichroic infrared (IR-LD) spectral analysis of
oriented solid sample as a nematic liquid crystal suspension
stretching mode of -COOH group. According to single crys-
(1)
(2)
Figure 3. Unit cells of L-Val [10] and L-Pro [11] respectively (1) and (2).

350 Protein & Peptide Letters, 2010, Vol. 17, No. 3
Chapkanov and Zareva
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L
L
carried out. On the base of the obtained spectroscopic data
was assigned the IR-characteristic bands of the zwitterions of
both amino acids accompanied with structural prediction.
The IR-spectral results were compared with some data from
X-ray analysis of -Val and -Pro, confirming the benefit of
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Accepted: June 19, 2009