Journal of Molecular Structure 826 (2007) 120125

www.elsevier.com/locate/molstruc

Molecular mechanism of high pressure action on lupanine

Magorzata Giel-Pietraszuk a, Zoa Gdaniec a, Tadeusz Brukwicki b, Jan Barciszewski
a

a,*

Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12, 61-704 Poznan, Poland
b
Faculty of Chemistry of the Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
Received 28 February 2006; received in revised form 20 April 2006; accepted 23 April 2006
Available online 30 May 2006

Abstract
High hydrostatic pressure (HHP) is an emerging tool for studying conformational changes in lipids, proteins and nucleic acids.
Although many thermodynamic parameters describing those processes have been determined, a molecular mechanism of HHP action
is poorly recognized. To get insight into that, we have studied quinolizidine alkaloid, lupanine. It consists of two quinolizidine moieties,
one of which contains lactam group. Using Fourier-transform infrared (FT-IR) spectroscopy, we have showed that at 6 kbar, the intensity of amide band at 1589 cm 1 decreased and a new band at 1556 cm 1 appeared. These changes are due to the hydrolysis of lupanine
to lupanic acid. That reaction was conrmed with 13C NMR spectra of lupanine exposed to HHP. The NMR signals at 176.6 and
184.3 ppm were assigned to lactam group of lupanine and carboxylic group of lupanic acid, respectively. The ring opening reaction
of lupanine under HHP is reversible at ambient pressure, as evidenced by CD measurements. A slightly acidic condition induced by
HHP causes protonation of lactam group and carbocation is formed, while on the other hand, water molecule as nucleophile attacks
electrophilic carbon of lactam and electrons move towards oxonium ion. Finally, CAN bond breaks down and carboxyl group is formed.
 2006 Elsevier B.V. All rights reserved.
Keywords: High hydrostatic pressure; Lupanine; Lupanic acid

1. Introduction
High hydrostatic pressure (HHP) has recently gained
renewed interest in bioscience and biotechnology [1]. It is
a unique tool to obtain unperturbed thermodynamic and
kinetic information on denaturation and dissociation equilibria of proteins, nucleic acids and lipids [24]. In contrast
to chemical denaturants, HHP aects structure of biomolecules by altering intra- and inter-molecular interactions
also with solvent [4]. Oligomeric proteins dissociate at pressure above 1 kbar. Thus, it implies a smaller molecular volume for monomers due to electrostriction (reduction in

Abbreviations: HHP, high hydrostatic pressure; FTIR, Fourier-transform infrared spectroscopy; CD, circular dichroism; NMR, nuclear
magnetic resonance.
*
Corresponding author. Tel.: +48 61 8528503x132; fax: +48 61
8528532.
E-mail address: Jan.Barciszewski@ibch.poznan.pl (J. Barciszewski).
0022-2860/$ - see front matter  2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2006.04.038

volume on hydrophobic hydration), or by imperfect van

der Waals interactions [5,6].
It was shown that conformation of DNA and RNA
changes under HHP [711]. In addition to conformational
changes of biomolecules, HHP induces DielsAlder condensation (CAC bond formation) of 6-furfuryladenine
with maleic anhydride and dimethyl acetylenedicarboxylate
catalyzed by tRNA [12]. Interestingly, tRNA can be nonenzymatically aminoacylated at HHP [13,14]. It was
observed that HHP accelerated degradation of aspartame
in milk and patulin in apple juice [16,17]. The reaction, carried out in water, showed fewer degradation products
probably due to slightly acidic pH [16,17]. To get a clear
insight into a mechanism of reaction induced under
HHP, we analyzed structural changes of quinolizidine
alkaloid, lupanine. It is composed of two quinolizidine
moieties, one of them containing lactam group. The conformation and molecular properties of lupanine (Mw
248 Da) were studied in detail, and lots of structural data

M. Giel-Pietraszuk et al. / Journal of Molecular Structure 826 (2007) 120125

121

3. Results
3.1. FTIR spectra of lupanine at ambient and high
hydrostatic pressure

Fig. 1. An equilibrium between lupanine in: trans (boatchair conformation of ring C and D) and cis conformation (chairchair). The formula
shows four rings (AD) and atom numbering.

were accumulated [1823]. In solution, lupanine occurs in

conformational equilibrium with the dominance of the
boat form of ring C (9:1), (Fig. 1). Stereochemistry of lupanine was correlated with IR absorption bands in the region
28402700 cm 1, which consists of two main bands at 2808
and 2761 cm 1, assigned to CAH vibrations of methylene
groups at C15 and C17. The characteristic trans band
is present only in the spectra of lupanine in boatchair conformation of rings CAD [18,19].
In this paper we showed that HHP induces lupanic
acid formation by hydrolysis of lactam bond of
lupanine. That result was conrmed by measuring FTIR
and 13C NMR spectra of pressured lupanine. These
data shed light on the mechanism of induction of chemical reactions by HHP.

The IR spectra of lupanine and lupanic acid measured at

ambient pressure are shown in Fig. 2a and b. The comparison of FTIR of lupanine at ambient pressure and 13 kbar
showed essential dierences in the range of 1700
1500 cm 1. A decrease of band intensity at 1589 cm 1 is
accompanied by an increase of band at 1556 cm 1 as pressure increases from 1 bar up to 13 kbar (Fig. 2c). To assess
the yield of the reaction, we compared the area under these
bands, which showed that the total intensity is preserved
(area under the band II amounts to 94.4% of the band I).
A plot of intensity of those bands versus pressure (Fig. 3)
showed that the highest changes in lupanine conformation
take place at 6.35 kbar. The plots of wavelength maxima of
the bands at 2883, 1585, 1451, 1420 and 1029 cm 1 versus
HHP recorded at increasing and decreasing pressure
(Fig. 4) show that the pressure-induced conformational
changes are reversible.
3.2. CD spectra
The CD spectra of lupanine in water subjected to HHP
recorded after 15 min, 21, 42 and 67 h of pressure release
conrm that HHP-induced changes in its structure are
reversible and the equilibrium between lupanine and lupanic acid is established (Fig. 5).

2. Experimental
3.3. NMR analysis
()Lupanine was isolated from lupin seeds with the
method described in Ref. 24. Lupanic acid was prepared from lupanine (0.5 g), dissolved in 10% HCl
and heated at 95 C for 2 h. The reaction product
was extracted with n-butanol [25]. The NMR spectra
of lupanine and lupanic acid were measured in D2O
(10 mg/ml). For the HHP experiment, lupanine was
exposed to 10 kbar pressure for 24 h at 25 C using
high-pressure apparatus U101 (Unipress, Warsaw).
After pressure release, the 13C NMR spectra were
recorded with Varian Unity 300 spectrophotometer.
Similar to the 13C NMR, the CD spectra were measured after releasing of pressure at dierent time on
Jasco J-20 spectropolarimeter.
For the Fourier-transform infrared spectroscopy
(FTIR) measurements, lupanine was dissolved in D2O
or H2O (100 mg/ml) and placed in a diamond anvil
cell. The initial gasket thickness was 0.075 mm. The
measurements of the spectra were carried out at room
temperature starting from ambient pressure up to
13 kbar. After increasing (or decreasing the pressure)
of 1 kbar, the sample was left for equilibration for
1 h. The infrared spectra were recorded with Bruker
FT-IR spectrometer. The IR spectrum of lupanic acid
was recorded in nujol.

The 13C NMR spectrum recorded for lupanine showed a

signal at 176.6 ppm, but the spectrum for lupanic acid at
184.3 ppm. The spectrum of lupanine treated with HHP
showed two signals at 176.6 and 184.0 ppm at ratio 2:1
(Fig. 6).
4. Discussion
Despite broad application of HHP in chemistry and biotechnology, we are still far back from understanding its
action at molecular level [17]. High pressure of 6 kbar
induces B-Z DNA transition [8]. RNA as a more stable
molecule changes its conformation at 6 kbar in the presence of high salt concentration [9,10]. To explain that phenomenon, it has been proposed that the structural
transitions of nucleic acids are driven by water structure
aected by HHP. At ambient pressure water exists mainly
in tetrameric form, while under high pressure converts into
octameric, which has been well described theoretically and
proved experimentally [2630].
Mechanism of HHP action is not known despite the fact
that this technique has been used in chemical and biochemical technology for a long time. Wealth of data show that in
the case of reaction carried out in water, a key role plays

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M. Giel-Pietraszuk et al. / Journal of Molecular Structure 826 (2007) 120125

1132.7

1095.1

1281.2
1251.3

1362.2
1339.1

1422

1485.7

1451.0

Absorbance (a.u.)

4
N
C

1588.9

1500

1000

Wavelength (cm-1)

Absorbance (a.u.)

80
Fig. 3. Pressure eects on the band intensity ratio 1589/1556 cm 1. Inset:
FTIR spectra of lupanine in region for diagnostic band of lactam bond.

60

40

20

sym
asym
COO-

1600

1400
Wavelength

1200

1000

(cm-1)

2.50
2.25

Absorbance (a.u.)

2.00
1.75

Intensity at 1589 cm-1

1.50

4
3
2
1
0
0 2 4 6 8 10 12 14
Pressure (kbar)

1.25

II
1.00
0.75
0.50
0.25
0.00
1700 1650 1600 1550 1500 1450 1400 1350 1300 1250 1200

Wavenumber (cm-1)
Fig. 2. IR spectra of (a) lupanine in D2O measured at normal pressure, (b)
lupanic acid in nujol. (c) The FT IR spectra of lupanine (100 mg/ml D2O)
in the region 17001200 cm 1. Measuring was done in diamond anvil cell
at 1 bar (I) and 14 kbar (II). Inset: pressure eect on the intensity of the
band at 1589 cm 1.

the electrostriction leading to a volume reduction and lowering of pH [15]. To verify that idea, we used lupanine
(Fig. 1). That molecule was selected on the basis of its good
solubility in water and presence of lactam group, which is
decomposed at acidic conditions. A general mechanism

of lactam hydrolysis in acidic condition is well known.

Thus having same observation on small molecule, we can
conclude about the inuence of HHP on other molecules
including biological compounds.
The analysis of FTIR spectra of lupanine at HHP
showed that the most signicant dierences occurred in
the range 17001500 cm 1 (Fig. 2). The band at
1589 cm 1 was assigned to lactam group and originated
from its stretching vibrations [18]. The spectra of lupanine
recorded at ambient pressure and 13 kbar showed a clear
dierence of the intensity of that diagnostic band. It
decreases as pressure increases from 1 to 13 kbar and, at
the same time, a new band at 1556 cm 1 becomes visible
(Figs. 2c and 3). The shift of the band at 1589 cm 1
towards lower frequency was attributed to hydrolysis of
lactam bond and lupanic acid formation [25,31]. The comparison of position of this band at 1589 cm 1 recorded during slow releasing of pressure showed that hydrolysis of
lactam bond is reversible (Fig. 4a). It was also conrmed
by measurement of the Cotton eect at 220 nm characteristic for lupanine. The solution of lupanine after pressuring
was left at ambient pressure for several hours, the CD spectra show that the Cotton eect rises up along with establishing of lupanic acidlupanine equilibrium (Fig. 5).
After 67 h the equilibrium was nally achieved in ratio
around 15:85, respectively [18,32]. The analysis of bands
around 1451 cm 1, which can be attributed to NAH bending, showed similar behaviour which suggests that the ring
opening reaction at HHP is reversible. The band at
1029 cm 1 consists of overlapping signals originating from
CAN and CAC ring stretching vibration which makes the
eect of hydrolysis less spectacular comparing to the above
results (Fig. 4b and c). The band at 1420 cm 1 depicted to
bending vibration of methyl groups did not show any
hysteresis, which shows that conformation of these groups
is fully reversible upon decreasing of pressure (Fig. 4d).
The presence of a band at 2883 cm 1 indicated that the

M. Giel-Pietraszuk et al. / Journal of Molecular Structure 826 (2007) 120125

1589

1453

1032

1588

1452

1031

Wavelength (cm-1)

1587

123

1030

1451
1586

1029
1450

1585
1584

1028
1027

1449
0

8 10 12 14

8 10 12 14

8 10 12 14

Pressure (kbar)

Wavelength (cm-1)

d
1422

2888

1421

2887
2886

1420

2885
2884

1419

2883

1418

2882

1417

2881
0

8 10 12 14

8 10 12 14

Pressure (kbar)
Fig. 4. Pressure eects on the wavelength maxima of the bands of lupanine (100 mg/ml of H2O) up to 14 kbar. (d) increasing pressure; (s) decreasing
pressure.

176.6
N

3
2

175

[ppm]

175

[ppm]

(M-1cm-1)

1
H 2N +
O

O-

184.3

c
176.6
184.0

190

220
Wavelength

250
(cm-1)

Fig. 5. The circular dichroism spectra of lupanine in water. The spectrum

was measured in 0.5 cm cell at concentration of 1.55 10 4 M: (1) 15 min;
(2) 21 h; (3) 42 h; (4) 67 h.

175

[ppm]

Fig. 6. The 13C NMR spectra of (a) lupanine at ambient pressure, (b)
lupanic acid, and (c) lupanine after exposition at 10 kbar.

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M. Giel-Pietraszuk et al. / Journal of Molecular Structure 826 (2007) 120125

Fig. 7. The mechanism of lupanine hydrolysis at high hydrostatic pressure.

boatchair conformation of the rings CAD remained

unchanged during the experiment.
The 13C NMR spectra recorded for lupanine and
lupanic acid showed bands at 176.6, 184.3 ppm, which
were attributed to carbonyl group of lactam and carboxyl moiety, respectively [33,34]. The spectrum of lupanine
at HHP showed two signals at 176.6 and 184.0 ppm at
ratio 2:1, indicating hydrolysis of the lactam bond
(Fig. 6). A similar shift was observed for hydrolysis of
b-lactam group of nitrocen catalyzed by metallo-b-lactamase [31]. The spectroscopic data of nitrocen showed
b-lactam resonance at 165.3 ppm, which disappeared
upon hydrolysis and was replaced by a new band at
171.5 ppm. The NMR data were supported by an IR
experiment, in which stretching frequency of lactam
carbonyl group at 1781 cm 1 was replaced by a new
band at 1673 cm 1 [31].
It is known that HHP induces self-ionisation of water
[15]. At 1 kbar, pH of water decreases to 6.23 [35]. We considered dissociation of water molecules induced by HHP as
the rst step in a mechanism of lactam hydrolysis (Fig. 7).
Following that, proton originating from high pressure
action on water molecule binds to oxygen atom of lactam
forming carbocation. The water functioning as the nucleophile attacks the electrophilic carbon of the C@O group. It
forces the electrons to move towards the oxonium ion and
proton to lactam nitrogen, which, in turn, is followed by
breaking of CAN bond and formation of carboxyl group.
In summary, that mechanism has been the rst one to
describe precisely a chemical reaction at high pressure. It
combines the properties of lactam group as well as the
eects of pressure on water.
Acknowledgements
This work is dedicated in remembrance of our colleague
Dr. Anna Perkowska who passed away. She was an expert
in structure and function analysis of quinolizidine
alkaloids. We thank Geert Vermeulen and Karel Heremans
of Catholic University of Leuven for performing FTIR.

Also we would like to thank the Reviewers for their very

helpful comments.
References
[1] C. Balny, P. Masson, K. Heremans, Biochim. Biophys. Acta 1595
(2002) 3.
[2] J.L. Silva, D. Foguel, A. Royer, Trends Biochem. Sci. 26 (2001) 612.
[3] R.B. Macgregor Jr., Biopolymers 48 (1998) 253.
[4] S. Marchal, J. Torrent, P. Masson, J.M. Kornblatt, P. Tortora, P.
Fusi, R. Lange, C. Balny, Braz. J. Med. Res. 38 (2005) 1175.
[5] V.V. Mozhaev, K. Heremans, J. Frank, P. Masson, C. Balny,
Proteins 24 (1996) 81.
[6] V.V. Mozhaev, K. Heremans, J. Frank, P. Masson, C. Balny, Trends
Biotechnol. 12 (1994) 493.
[7] G. Weber, H.G. Drickamer, Q. Rev. Biophys. 16 (1983) 89.
[8] A. Krzy_zaniak, P. Saanski, J. Jurczak, J. Barciszewski, FEBS Lett.
279 (1991) 1.
[9] A. Krzy_zaniak, J.P. Furste, R. Bald, P. Saanski, J. Jurczak, V.A.
Erdmann, J. Barciszewski, Int. J. Biol. Macromol. 16 (1994) 159.
[10] A. Krzy_zaniak, J.P. Furste, V.A. Erdmann, P. Saanski, J. Jurczak, J.
Barciszewski, Biochimie 78 (1996) 862.
[11] J. Barciszewski, J. Jurczak, S. Porowski, T. Specht, V.A. Erdmann,
Eur. J. Biochem. 260 (1999) 293.
[12] M. Mielcarek, M.Z. Barciszewska, P. Salanski, M. Stobiecki, J.
Jurczak, J. Barciszewski, Biochem. Biophys. Res. Commun. 294
(2002) 145.
[13] A. Krzy_zaniak, P. Saanski, J. Jurczak, J. Barciszewski, Int. J. Biol.
Macromol. 16 (1994) 153.
[14] A. Krzy_zaniak, P. Saanski, T. Twardowski, J. Jurczak, J. Barciszewski, Biochem. Mol. Biol. Int. 45 (1998) 489.
[15] B. Tauscher, Z. Lebens. Unters. Forsch. 200 (1995) 3.
[16] P. Butz, A. Fernandez, H. Fister, B. Tauscher, J. Agric. Food Chem.
45 (1997) 302.
[17] D. Bruna, M. Voldich, M. Marek, J. Kamarad, in: K. Heremans
(Ed.), High Pressure in Biosciences and Biotechnology, Leuven
University Press, Leuven, 1997, p. 335.
[18] M. Wiewiorowski, O.E. Edwards, M.D. Bratek-Wiewiorowska,
Canad. J. Chem. 45 (1967) 1447.
[19] J. Skolik, P.J. Krueger, M. Wiewiorowski, Tetrahedron 24 (1968)
5439.
[20] N.J. Leonard, in: R.H.F. Manske (Ed.), The Alkaloids, vol. 7,
Academic Press, New York, London, 1960, p. 253.
[21] H.G. Boit, Ergebnise der Alkaloidchemie bis 1960, Academic Verlag,
Berlin, 1961.
[22] P.H. Doucerain, A. Chiaroni, C. Riche, Acta Cryst. B32 (1976) 3213.
[23] W. Wysocka, T. Brukwicki, J. Mol. Struct. 385 (1996) 23.

M. Giel-Pietraszuk et al. / Journal of Molecular Structure 826 (2007) 120125

[24] W. Wysocka, A. Przyby, Sci. Legumes 1 (1994) 37.
[25] J. Suszko, M. Wiewiorowski, W. Meissner, Bull. Acad. Polom. Sci.,
Ser. sci. chi,. geol. et geogr. 7 (1959) 87.
[26] S.W. Benson, E.D. Siebert, J. Am. Chem. Soc. 114 (1992) 4269.
[27] K.S. Kim, M. Dupuis, G.C. Lie, E. Clementi, Chem. Phys. Lett. 131
(1986) 451.
[28] C.J. Gruenloh, J.R. Carney, C.A. Arrington, T.S. Zwier, S.Y.
Frederics, K.D. Jordan, Science 276 (1997) 1678.
[29] W.B. Blanton, S.W. Gordon-Wylie, G.R. Clark, K.D. Jordan, J.T.
Wood, U. Geiser, J. Collins, J. Am. Chem. Soc. 121 (1999) 3551.

125

[30] D.E. Khoshtariya, A. Zahl, T.D. Dolidze, A. Naubrand, R. van

Eldik, Chem. Phys. Chem. 5 (2004) 1398.
[31] N.V. Kaminskaia, B. Spingler, S.J. Lippard, J. Am. Chem. Soc. 123
(2001) 6555.
[32] W. Klyne, P.M. Scopes, R.N. Thomas, J. Skolik, J. Gawrosnki, M.
Wiewiorowski, J. Chem. Soc. Perkin I (1974) 25652570.
[33] V. Galasso, F. Asaro, F. Berti, I. Habus, B. Kovac, C. De Risi, Chem.
Phys. 301 (2004) 33.
[34] R. Kolanos, W. Wysocka, T. Brukwicki, Tetrahedron 59 (2003) 5531.
[35] R.E. Marquis, Adv. Microbiol. Physiol. 14 (1976) 159.