Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene, homooxacalix[3]arene and homoazacalix[3]arene as extractant agents for amino acids from the aqueous extract

Article abstract of DOI:10.1007/s00726-019-02809-z

The task of this work was to investigate the extraction capacity of various calixarenes for free and esterified amino acids from aqueous acid phases. Furthermore, this method was applied to aqueous extracts of Helleborus purpurascens. Generally, it is known that calixarenes can be used as extractants for ammonium compounds due to π-cation and lone pair cation interactions. As first, tert-Butyl-calix[6]arene and derivatives thereof were used. They had already proven their worth in previous investigations. In addition, tert-Butyl-hexahomooxa-calix[3]arene was used also, which can also enter into lone pair cation interactions. In addition to these well-known calixarenes, new calixarenes were produced and tested. Based on the tert-Butyl-hexahomooxa-calix[3]arene, a phosphor(III)bridged derivative was prepared, combining the three aromatic hydroxyl groups to a phosphite. As a seldom-described class of calixarenes, tert-Butyl-hexahomoaza-calix[3]arene derivatives were used. The nitrogen analogues of tert-Butyl-hexahomooxa-calix[3]arene could be produced as N-benzyl derivatives. The structure of the esterified carboxymethylated derivative of N,N′,N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene could be verified by X-ray structure analysis. It crystallized as a partial cone. The extraction capacity of the described calixarenes was investigated for amino acids from aqueous acidic solutions into an organic phase. For the testing were chosen asparagine, aspartic acid, tyrosine, tryptophane, phenylalanine and pipecolinic acid and their methyl esters. The amino acids and their methyl esters were dissolved in water at different pH values. The calixarenes were dissolved in dichloromethane (DCM) or chloroform. After this preparation, the aqueous acidic amino acid solutions were mixed with the solutions and shaken intensively. In addition, blank values were determined by extracting the aqueous stock solutions of the amino acids and their methyl esters with pure solvents. To determine the extraction rate, the phases were separated and each analysed using GC-FID, partially GC–MS(EI). The evaluation is performed in two ways. On the one hand the depletion in the aqueous phase and on the other hand the content in the organic phase was determined.

Full text of DOI:10.1007/s00726-019-02809-z

Amino Acids
https://doi.org/10.1007/s00726-019-02809-z
ORIGINAL ARTICLE
Studies on the constituents of Helleborus purpurascens: use
of derivatives from calix[6]arene, homooxacalix[3]arene
and homoazacalix[3]arene as extractant agents for amino acids
from the aqueous extract
M. Heiko Franz1,2 · Mirela Iorga1 · Catalin V. Maftei1,3 · Elena Maftei1,3 · Ion Neda1,3
Received: 29 October 2019 / Accepted: 27 November 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
The task of this work was to investigate the extraction capacity of various calixarenes for free and esterifed amino acids from
aqueous acid phases. Furthermore, this method was applied to aqueous extracts of Helleborus purpurascens. Generally, it
is known that calixarenes can be used as extractants for ammonium compounds due to π-cation and lone pair cation interac-
tions. As frst, tert-Butyl-calix[6]arene and derivatives thereof were used. They had already proven their worth in previous
investigations. In addition, tert-Butyl-hexahomooxa-calix[3]arene was used also, which can also enter into lone pair cation
interactions. In addition to these well-known calixarenes, new calixarenes were produced and tested. Based on the tert-Butyl-
hexahomooxa-calix[3]arene, a phosphor(III)bridged derivative was prepared, combining the three aromatic hydroxyl groups
to a phosphite. As a seldom-described class of calixarenes, tert-Butyl-hexahomoaza-calix[3]arene derivatives were used. The
nitrogen analogues of tert-Butyl-hexahomooxa-calix[3]arene could be produced as N-benzyl derivatives. The structure of
the esterifed carboxymethylated derivative of N,N′,N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene could be verifed by
X-ray structure analysis. It crystallized as a partial cone. The extraction capacity of the described calixarenes was investigated
for amino acids from aqueous acidic solutions into an organic phase. For the testing were chosen asparagine, aspartic acid,
tyrosine, tryptophane, phenylalanine and pipecolinic acid and their methyl esters. The amino acids and their methyl esters
were dissolved in water at diferent pH values. The calixarenes were dissolved in dichloromethane (DCM) or chloroform.
After this preparation, the aqueous acidic amino acid solutions were mixed with the solutions and shaken intensively. In
addition, blank values were determined by extracting the aqueous stock solutions of the amino acids and their methyl esters
with pure solvents. To determine the extraction rate, the phases were separated and each analysed using GC-FID, partially
GC–MS(EI). The evaluation is performed in two ways. On the one hand the depletion in the aqueous phase and on the other
hand the content in the organic phase was determined.
Keywords Helleborus purpurascens · Amino acid · Extraction · Calixarene · Homoazacalix[3]arene · Homooxacalix[3]
arene
Introduction
Handling Editor: V. Soloshonok.
* Ion Neda
The multifunctional calixarenes have wide range of appli-
cation in chemistry (Durmaz et al. 2018; Morohashi and
Hattori 2018; Arnott 2018). A well-known property of them
is their ability to extract selectively diferent species from
various solutions, using their cavity (Oshima 2014; Mutihac
et al. 2011). This feature shall be used for the extraction of
amino acids from plant extracts. Recently, the constituents
of aqueous extracts from winter-blooming Helleborus pur-
purascens were presented (Franz et al. 2018). It is a wild
i.neda@tu-bs.de
1
Institutul National de Cercetare Dezvoltare pentru
Electrochimie si Materie Condensata, Str. Dr. A. Paunescu
Podeanu, Nr. 144, 300569 Timisoara, Romania
2
InnoChemTech GmbH, Hagenring 30, 38106 Brunswick,
Germany
3
Institut für Anorganische und Analytische Chemie,
Technische Universität Braunschweig, Hagenring 30,
38106 Brunswick, Germany
Vol.:(0123456789)
1 3

M. H. Franz et al.
plant native in eastern Europe mainly Romanian Carpathians
belonging to the family of Ranunculaceae whose extracts
are used in the Romanian folk medicine since ancient times
and are still used today. It was found that major contents are
asparagine and pipecolic acid. Although pipecolic acid is not
a part of the proteinogenic amino acid cocktail, it is known
in plant physiology and plays in the defence of plants an
important role and it regulates the inducible plant immunity
(Zeier 2013; Návarová et al. 2012). Also, in pharmaceuti-
cal active compounds like FK506 and VX710, it is a core
structure (Gopalakrishnan et al. 2012; Germann et al. 1997).
Already in the middle of the nineteenth century chemists
and pharmacists started to search for interesting biological
compounds. Hellebrin was identifed as main compound
(Schmutz 1947), belonging to the family of cardiac glyco-
sides. Muhr et al. (1995) determined the confguration of
the anomeric centres. Based on their knowledge, the group
of Franz Kerek developed a formulation of H. purpurascens
at the Institute of Chemistry Timisoara of the Romanian
Academy, which is commercialized by the company S.C.
Exhelios S.R.L. The Institute of Chemistry, Timisoara of the
Romanian Academy stayed a centre for research on H. pur-
purascens. Hydrogene-free macrocyclic carbon suboxides
(MCS) were found (Kerek 2000) which exhibit interesting
biological activity by acting as potent Na,K-ATPase inhibi-
tors (Kerek et al. 2008; Neacsu et al. 2010). We started our
work on the extraction of amino acids from H. purpurascens,
using unusually substituted calixarenes in the context of the
project “EXPLORATORY RESEARCH PROGRAM IDEI-
PCE-PROJECT NR. 341-/05.10.2011-Immunomodulatory
Fluoroglycopeptide Molecular Architectures and the PRO-
JECT NR. 18360301, Contract: 42N/2018”. Our interest
was focused on one direction on polar substances in general
(Neda et al. 1996, 1999, 2000) and especially those which
can be used as interesting substituents for 1,2,4-oxdiazoles
and N-heterocyclic carbenes (NHC) which were published
recently (Maftei et al. 2016a, Maftei et al. 2016b). This inter-
est should be combined with the competence of our group
in synthesis and use of all varieties of calixarenes (Voll-
brecht et al. 1997; Neda et al. 1998; Dieleman et al. 1999;
Kunze et al. 2001). Further inspiration we get by the work of
Macarie et al. (2017) and Kadyrov et al. (1996). Siedentop
et al. (1999) have investigated the reactions of tert-butylca-
lix[4]arenes with tert-butyl-bis[N-(N′,N′,N″,N″-tetramethyl)
guanidinyl] phosphine with formation of the frst complex
calix[4]arene-anion with the protonated phosphonium cat-
ion. The complexes are stabilized by π-cation interactions.
Goto and Furusaki observed that neither 4-tert-octyl-calix[6]
arene extracted anything nor unprotected amino acid could
be extracted in any way (Oshima et al. 2002). Obviously,
the Nernst’s distribution coefcient (Nernst 1891) is far on
the side of the water in this case. Therefore, the amino acids
remained practically in the aqueous phase. The Nernst’s
distribution coefcient changed when methyl esters of amino
acids and carboxylated calixarenes were used. Oshima et al.
found that methyl esters of tryptophan, phenylalanine and
tyrosine could be extracted when O-carboxymethylated
calixarenes were used. The best one was the calix[6]arene
1 (Fig. 1).
The group from Araki and Shinkai chose another type
of calixarene. They studied widely the interactions between
derivatives of 15,55,95-tri-tert-butyl-3,7,11-trioxa-1,5,9
(1,3)-tribenzenacyclododecaphane-12,52,92-triol (2) (tert-
butyl-hexahomo-trioxacalix[3]arene) and ammonium spe-
cies as neopentylammonium picrate or ethyl phenylalaninate
(Araki et al. 1993a, b). A special derivative was the triple
alkylated homooxacalix[3]arene 3. This compound has a
hindered rotation and forms a stable partial cone which has
a pseudo-C2-symmetry (Fig. 2). The alkyl group of the guest
is located in the cavity formed by the methoxy-substituted
phenyl-ring and one of the butoxy-substituted phenyl-rings
(Araki et al. 1996).
It can be concluded that diferent types of calixarenes can
extract ammonium compounds from aqueous acidic solu-
tion into an organic phase due to π-cation interactions. The
aim of this work was to investigate various calixarenes for
their ability to extract amino acids. The focus should be on
the amino acids found mainly in the aqueous extract of H.
purpurascens which are asparagine as the dominant com-
pound, pipecolic acid, aspartic acid, γ-aminobutyric acid
and glutamine. In addition to some of the calixarenes already
described, further unconventionally substituted derivatives
are to be used (Kawaguchi et al. 2000; Maftei et al. 2015;
Neda et al. 1993). Finally, this procedure should be applied
directly to an aqueous extract of H. purpurascens.
Experimental section
General aspects
The work was made within the project “EXPLORATORY
RESEARCH PROGRAM IDEI-PCE-PROJECT NR.
341-/05.10.2011-Immunomodulatory Fluoroglycopeptide
Molecular Architectures and the PROJECT NR. 18360301,
Contract: 42N/2018”. Dried rhizome and roots of H. pur-
purascens along with the hydroalcoholic extract thereof1
were obtained from the company S.C. Exhelios S.R.L.
(Timisoara, Romania). The plant material was collected in
1
The term “hydroalcoholic” is not precisely but commonly used.
Therefore, the term is also used by the authors for the extract which
was generated with a mixture of water and ethanol.
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Fig. 1 Structure of p-tert-octylcalix[6]arene hexacarboxylic acid derivative 1 (t-Oct[6]CH2COOH) and the used methylesters of tyrosine, try-
tophane and phenylalanine by Oshima et al. (2002)
Synthesis of calixarenes
15,55,95‑tri‑tert‑butyl‑3,7,11‑trioxa‑1,5,9(1,3)‑tri
benzenacyclododecaphane‑12,52,92‑triol (2)
(tert‑Butyl‑hexahomooxa‑calix[3]arene)
Fig. 2 Complex of 12,52-dibutoxy-15,55,95-tri-tert-butyl-92-methoxy
-3,7,11-trioxa-1,5,9(1,3)-tribenzenacyclododecaphane (3, O,O-Di-
n-butyl-O-methyl-tert-butyl-hexahomotrioxacalix[3]arene) and neo-
pentylammonium suggested by the group of Araki and Shinkai based
on the analysis of NMR experiment (Araki et al. 1996)
2.1 g of (5-(tert-butyl)-2-hydroxy-1,3-phenylene)dimethanol
(10 mmol) is dissolved in 10 mL Dimethoxyethane (DME).
A catalytic amount of methane sulfonic acid (65 μL, 1 mmol,
10 mol%) and anhydrous sodium sulphate (2.85 g, 20 mmol)
are suspended in 90 mL DME. The reaction mixture is
heated under refux for 12 h. During the frst hour is added
the previously prepared solution of (5-(tert-butyl)-2-hy-
droxy-1,3-phenylene)dimethanol. Then the DME is removed
the region Bistrita Nasaud, Romania. All used solvents and
reagents were of analytical grade. Solvents, reagents and
amino acids were purchased from Sigma Aldrich Chemie
GmbH (Munich, Germany).
1 3

M. H. Franz et al.
under reduced pressure and the residue is taken up in 100 mL
Dichloromethane (DCM) and 100 mL of water. The phases
are separated and the organic phase is washed with sodium
hydroxide solution (10%, 50 mL), water (50 mL) and brine
(50 mL). The organic is dried with sodium sulphate and
the solvent is removed under reduced pressure. The residue
is purifed by column chromatography (DCM:hexane//1:4).
It results in an of-white solid (1.14 g, 1.98 mmol, yield:
59%). 1H-NMR (200 MHz, CDCl3) δ 8.57 (s, 3H, –OH);
7.13 (s, 6H, CHAr); 4.72 (s, 12H, Ar–CH2–O); 1.24 (s, 27H,
–C(CH3)3); ESI–MS: m/z 599.3 [M+Na]+, 577.4 [M+H]+.
alternative name: 7,15,23-tri-tert-butyl-25,26,27-trihydroxy-
2,3,10,11,18,19-hexahomo-3,11,19-trioxacalix[3]arene.
714 mg of tris-O-Trimetylsily-tert-butyl-hexahomooxa-
calix[3]arene (0.9 mmol) is dissolved in Toluene (45 mL).
The reaction mixture is cooled to 0 °C. Then Chlorodifuo-
rophosphane (193 μmL, 282 mg, 2.7 mmol) is added. The
prepared reaction mixture is stirred for 3 h at room tem-
perature. Then the temperature is raised to 115 °C and the
solution is kept for 24 h under refux. After this the solvent
is removed under reduced pressure. It results in a beige
1
solid. H-NMR (200 MHz, CDCl3) δ 7.13 (s, 6H, CHAr);
4.52 (s, 12H, Ar–CH2–O); 1.29 (s, 27H, –C(CH3)3); Just
the cone conformer is detected; 31P-NMR (200 MHz,
CDCl3) δ 143.3; ESI–MS: m/z 627.3 [M + Na]+, 605.3
[M + H]+.
Method B: 275 μL of Tris(diethylamino)phosphine
(248 mg, 1 mmol) is dissolved in Toluene (125 mL).
Then, 577 mg of tert.-Butyl-hexahomooxa-calix[3]arene
(1 mmol), dissolved in Toluene (150 mL), and Imidazole
(3.5 mg, 0.05 mmol) is added to the reaction mixture. The
resulting solution is heated for 3 days at 75 °C. The devel-
oped Diethylamine is distilled out. After 3 days the reac-
tion mixture is cooled to room temperature and 100 mL
of water added. The phases are separated and the organic
phase is washed with saturated sodium hydrogen carbonate
solution (50 mL), water (50 mL) and brine (50 mL). The
organic phase is dried using sodium sulphate and the sol-
vent is removed under reduced pressure. It results in a solid
(596 mg). The residue is purifed by column chromatogra-
phy (TBME: Petrol ether//1:7). It results an of-white solid
3,15,26‑tri‑tert‑butyl‑5H,7H,11H,13H‑8,10‑(epiprop[1]en[1]
yl[3]ylidene)‑9,19‑epoxy‑1,17‑(methanooxy‑methano)
dibenzo[d,o][1,3,7,13]tetraoxa[2]phosphacyclohexadecine
(4) (phosphorous(III)‑tert‑Butyl‑hexahomooxa‑calix[3]
arene)
1
(374 mg, 0.62 mmol, yield: 62%). H-NMR (200 MHz,
CDCl3) d 7.13 (s, 6H, CHAr); 4.52 (s, 12H, Ar–CH2–O);
1.29 (s, 27H, –C(CH3)3); 31P-NMR (200 MHz, CDCl3)
d 143.3; ESI–MS: m/z 678.4 [M + HNEt2 + H+]; 627.3
[M + Na]+, 605.3 [M + H]+.
Method A: 577 mg of tert.-Butyl-hexahomooxa-calix[3]
arene (1 mmol) is dissolved in 58 mL Tetrahydrofuran
(THF). The mixture is cooled to 0 °C. Then Trimethylsi-
lylchloride (0.4 mL, 358 mg, 3.3 mmol) and Triethylamine
(0.7 mL, 506 mg, 5 mmol) are added to the reaction mix-
ture at the same time. The reaction mixture is stirred for
another 8 h; meanwhile the temperature of the reaction
mixture is allowed to warm to room temperature. Then
100 mL of DCM and 100 mL of water are added. The
phases are separated and the organic phase is washed with
sodium hydroxide solution (10%, 50 mL), water (50 mL)
and brine (50 mL). The organic phase is dried with sodium
sulphate and the solvent is removed under reduced pres-
sure. It results in an oil (790 mg, quantitative),which is
used without further purifcation. 1H-NMR (main con-
former, 200 MHz, CDCl3) δ 7.15 (s, 6H, CHAr); 4.63 (s,
12H, Ar–CH2–O); 1.26 (s, 27H, –C(CH3)3); 0.21 (s, 27H,
–Si(CH3)3); The ratio between cone conformer and the
partial cone conformer is 75 to 25. ESI–MS: m/z 815.5
[M + Na]+, 793.5 [M + H]+.
3,15,26‑tri‑tert‑butyl‑5H,7H,11H,13H‑8,10‑(epiprop[1]
en[1]yl[3]ylidene)‑9,19‑epoxy‑1,17‑(methanooxymethano)
dibenzo[d,o][1,3,7,13]tetraoxa[2]phosphacyclohexadecine
19‑sulfde (5) (Phosphorous(V)sulphide‑tert.‑Butyl‑hexaho‑
mooxa‑calix[3]arene)
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Sulphur (16 mg, 0.5 mmol) is suspended in DCM (5 mL).
Then, 151 mg of Phosphorous(III)-tert.-Butyl-hexaho-
mooxa-calix[3]arene (0.25 mmol), dissolved in DCM
(5 mL), is added to the reaction mixture. The resulting
solution is heated for 2 h at refux. The reaction mixture is
cooled to room temperature and 10 mL of water i added.
The phases are separated and the organic phase is washed
with saturated sodium hydrogen carbonate solution
(5 mL), water (5 mL) and brine (5 mL). The organic phase
is fltered through a cellite pad and dried over sodium sul-
phate. The DCM is removed under reduced pressure. It
results in a solid (132 mg). The residue is used without
3.60 (s, 6H, Ph–CH2–N); 1.27 (s, 27H, –C(CH3)3); 13C-NMR
(75 MHz, CDCl3) δ 154.76 (Cq, 3C); 140.50 (Cq, 6C); 136.94
(Cq, 3C); 129.78 (CH, 6C); 127.81 (CH, 6C); 126.73 (CH,
3C); 126.54 (CH, 6C); 123.06 (Cq, 3C); 57.65 (CH2, 6C);
56.84 (CH, 3C); 33.83 (Cq, 3C); 31.65 (CH3, 9C); ESI–MS:
m/z 866.6 [M+Na]+, 844.6 [M+H]+.
Triethyl 2,2′,2″-((3,7,11-tribenzyl-15,55,95-tri-tert-
butyl-3,7,11-triaza-1,5,9(1,3)-tribenzenacyclodode-
caphane-12,52,92-triyl)tris(oxy))triacetate (7) (N, N′,N″-
Tribenzyl-(tert-Butyl-hexahomoaza-calix[3]arene)-tri O-ethyl
ester)
1
further purifcation. H-NMR (200 MHz, CDCl3) d 7.14
(s, 6H, CHAr); 4.67 (s, 12H, Ar–CH2–O); 1.29 (s, 27H,
–C(CH3)3); 31P-NMR (200 MHz, CDCl3) d 61.9; ESI–MS:
m/z 658.3 [M + Na]+, 637.3 [M + H]+.
3,7,11‑tribenzyl‑15,55,95‑tri‑tert‑butyl‑3,7,11‑tri‑
aza‑1,5,9(1,3)‑tribenzenacyclododecaphane‑12,52,92‑triol
(6) (N, N′,N″‑Tribenzyl‑tert‑Butyl‑hexahomoaza‑calix[3]
arene)
4 g of N, N′, N″-Tribenzyl-tert-Butyl-hexahomoaza-
calix[3]arene (4.74 mmol) and 6.5 g of potassium carbon-
ate (47.4 mmol) are suspended in 75 mL Acetonitrile. Then
5.3 mL of ethyl bromoacetate (47.4 mmol, 7.92 g) is added.
The suspension is heated for 48 h under refux. Then, the
reaction mixture is fltered and the solvent is removed under
reduced pressure. The residue is purifed by column chroma-
tography (Diethylether:Hexane//1:1). It results in a yellowish
solid (2.64 g, 2.4 mmol, yield: 51%). 1H-NMR (300 MHz,
CDCl3) δ 7.67–6.94 (m, 21H, –Ph and CHAr); 5.05–3.24 (m,
30H, Ar–CH2–N, Ph–CH2–N, O–CH2–CO, O–CH2–CH3);
1.35–1.13 (m, 36H, O–CH2–CH3 and –C(CH3)3: shifting
of tert-butyl: 1.30 (partial cone, 1.27 (cone), 1.20 (partial
cone)); ESI–MS: m/z 1124.5 [M+Na]+, 1102.7 [M+H]+.
5 g of (5-(tert-butyl)-2-hydroxy-1,3-phenylene)dimethanol
(29.7 mmol) and 3.25 mL Benzylamine (3.18 g, 29.7 mmol)
are dissolved in 150 mL xylene. Anhydrous sodium sulphate
(50 g, 352 mmol) is added to the reaction mixture. The reac-
tion mixture is heated under refux for 3 days. Then the xylene
is removed under reduced pressure and the residue is taken up
in 200 mL Dichloromethane (DCM) and 100 mL of water.
The phases are separated and the organic phase is washed with
sodium hydroxide solution (10%, 100 mL), water (100 mL)
and brine (100 mL). The organic is dried with sodium sulphate
and the solvent is removed under reduced pressure. The residue
is purifed by recrystallisation (Acetone/Methanol) and column
chromatography (DCM:Methanol//10:1). It results in a yellow-
ish powdery solid (1.35 g, 1.6 mmol, yield: 16%). 1H-NMR
(300 MHz, CDCl3) δ 11.07 (bs, 3H, –OH); 7.34–7.04 (bs,
15H, –Ph); 6.96 (s, 6H, CHAr); 3.74 (bs, 12H, Ar–CH2–N);
Nα‑(tert‑butoxycarbonyl)‑l‑asparagine (8)
l-asparagine monohydrate (1.5 g, 10 mmol) and sodium
hydroxide (0.88 g, 22 mmol) were dissolved in a mixture
of water (25 mL) and 1,4-dioxane (15 mL). A solution of
di-butyloxydicarbonate was added to this mixture. (3.27 g,
15 mmol) in 1,4-dioxane (10 mL) dropwise while stirring
at 0 °C. The reaction solution is stirred for 16 h at room
temperature. After the end of the reaction, the solvent was
removed in vacuum. The residue was dissolved under cool-
ing (0 °C) in diethyl ether (50 mL) and washed with a 5%
solution of citric acid (50 mL). The organic solution is dried
with sodium sulphate and the solvent is removed under
reduced pressure. An oil is obtained which solidifes over
1 3

M. H. Franz et al.
1
time. The yield is m = 1.58 g (6.8 mmol; 68%). H NMR
X‑ray structure determinations
(DMSO-d6, 300 MHz): δ 7.06 (s, 1H; NH), 6.88 (s, 2H,
NH2), 4.22 (dd, 1H, CH), 2.48 (m, 1H, CH2), 2.09 (m, 1H,
CH2), 1.38 (s, 9H, C(CH3)3). Elemental analysis: Calculated
for C9H16N2O5: C, 46.54; H, 6.94; N, 12.07%. Found: C,
46.72; H, 7.02; N, 12.02%. ESI–MS: m/z 255.1 [M+Na]+,
233.1 [M+H]+.
Data are summarized in Table 1. Crystals were mounted
in inert oil on glass fbres and transferred to the cold gas
stream of the difractometer (Oxford Difraction Nova A
with mirror-focussed Cu Kα radiation or Xcalibur E with
monochromated Mo Kα radiation). Absorption corrections
were implemented on the basis of multi-scans. The struc-
tures were refned anisotropically on F2 using the program
SHELXL-97 (Sheldrick 2008). NH and OH hydrogens were
refned freely; other hydrogens were refned using rigid
methyl groups or a riding model starting from calculated
positions. Special features and exceptions: Crystallographic
data have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publications no.
CCDC-1960692 (7). Copies of the data can be obtained free
of charge from www.ccdc.cam.ac.uk/data_request/cif.
Methyl (tert‑butoxycarbonyl)‑L‑asparaginate (9)
N-(tert-butoxycarbonyl)-l-asparagine (1.16 g, 5 mmol) was
dissolved in anhydrous methanol (5 mL) and cooled to 0 °C.
An ethereal solution of diazomethane (c=~0.5 mol/L) was
added to this solution while stirring until a permanent yellow
colour was achieved. The mixture was further stirred in an
ice bath for 2 h. At the end of the reaction, the solvent was
removed under reduced pressure. The residue was dissolved
under cooling (0 °C) in diethyl ether (50 mL) and washed
once with a 5% solution of citric acid (50 mL) and once with
a diluted sodium hydroxide solution (50 mL, c=0.1 mol/L).
The organic solution was dried with sodium sulphate and
the solvent was removed under reduced pressure. An oil
is obtained. The yield is m = 0.96 g (3.9 mmol; 78%).1H
NMR (CDCl3, 300 MHz): δ 7.11 (s, 1H; NH), 6.91 (s, 2H,
NH2), 4.52 (dd, 1H, CH), 3.62 (s, 3H, CH3), 2.86 (m, 1H,
CH2), 2.38 (m, 1H, CH2), 1.40 (s, 9H, C(CH3)3). Elemental
analysis: Calculated for C10H18N2O5: C, 48.77; H, 7,37; N,
11,38%. Found: C, 49.12; H, 7.42; N, 11.27%. ESI–MS: m/z
269.1 [M+Na]+, 247.1 [M+H]+.
Extraction of amino acids form Helleborus
purpurascens
Defatted plant material
The rhizome and the roots were ground carefully until the
material was powdery. 100 g of this ground material was
stirred in dichloromethane (500 mL) for 18 h at 25 °C. Then
the solvent was decanted and the plant material was dried in
vacuum for 1 h. Afterwards, the plant material was stirred in
hexane (300 mL) for 3 h. After the removing of the hexane
the plant material was dried in vacuum until the weight was
constant. Finally, the plant material was frozen with liquid
nitrogen and dried in vacuum until it was weight constant.
This dried and defatted plant material was used further for
direct extractions. This protocol is used from the company
S.C. Exhelios S.R.L. (Timisoara, Romania) and was made
available to us.
Methyl L‑asparaginate (10)
Methyl N-(tert-butoxycarbonyl)-L-asparagate (0.86 g,
3.5 mmol) was dissolved in diethylether (7 mL) and
cooled to 0 °C. An ethereal solution of Hydrogenchloride
(c=1.0 mol/L) was added dropwise to this solution. While
stirring a gas evolution was observed and a white solid
appeared. The mixture was further stirred at room tempera-
ture for 1 hour. At the end of the reaction the suspension was
diluted with Diethylether (30 mL) and washed twice with a
diluted sodium hydroxide solution (50 mL, c =0.2 mol/L).
The organic solution was dried with sodium sulphate and
the solvent was removed under reduced pressure. An oil is
obtained which solidifes over time. The yield is m=0.48 g
(3.3 mmol; 94%).1H NMR (CDCl3, 300 MHz): δ 8.56 (s,
2H; NH2), 6.89 (s, 2H, NH2), 4.48 (dd, 1H, CH), 3.56 (s,
3H, CH3), 2.91 (m, 1H, CH2), 2.42 (m, 1H, CH2). Elemental
analysis: Calculated for C5H10N2O3: C, 41.09; H, 6.90; N,
19,17%. Found: C, 41.22; H, 6.99; N, 18.96%. ESI–MS: m/z
169.1 [M+Na]+, 147.1 [M+H]+.
Extracts
The extract was made directly from authentic defatted plant
material of H. purpurascens. Industrially, the rhizome and
roots are extracted in a ratio of one to fve with a mixture of
water and ethanol (40:60). As already mentioned S.C. Exhe-
lios S.R.L. provided the already prepared hydroalcoholic
extract which is more precisely a hydroethanolic one. This
extract was called Extract-HA (Extract-Hydro Alcoholic).
Additionally, a second extraction was made directly from the
defatted plant material. This extract was called Extract-W
(Extract-Water). Therefore, the material was ground until it
was fne powdery. Deionized water was used as extraction
solvent (Dunn and Ross 1938; Otsuka 2005; Nozaki and
Tanford 1971). For aqueous system was used two diferent
extraction conditions. One type of extraction was made with
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Table 1 Summary of X-ray
Empirical formula
C69H87N3O9
crystallographic data for 7
Formula weight
Temperature
1102.42
100(2) K
Wavelength
Crystal system
Space group
1.54184 Å
Triclinic
P-1
Unit cell dimensions
a=12.2369(7) Å
b=13.7679(8) Å
c=20.5750(9) Å
3130.0(3) Å3
α=81.469(4)°
β=76.288(4)°
γ=68.692(6)°
Volume
Z
2
Density (calculated)
Absorption coefcient
F(000)
Crystal size
Theta range for data collection
Index ranges
Refections collected
Independent refections
Completeness to theta=75.00°
Absorption correction
Max. and min. transmission
Refnement method
Data/restraints/parameters
Goodness-of-ft on F2
Final R indices [I>2sigma(I)]
R indices (all data)
Largest dif. peak and hole
1.170 Mg/m3
0.608 mm−1
1188
0.30×0.12×0.08 mm3
3.45°–75.87°
−15≤h≤15, −17≤k≤17, −25≤l≤24
71,709
12,972 [R(int)=0.0278]
99.9%
Semi-empirical from equivalents
1.00000 and 0.58241
Full-matrix least-squares on F2
12,972/21/758
1.031
R1=0.0409, wR2=0.1054
R1=0.0446, wR2=0.1086
0.423 and −0.283 e.Å−3
ultrasound treatment at 60 °C for 3 h (method A) and second
type of extraction was stirred just at room temperature for
24 h (Method B). For the hydroalcoholic system was used
a further variation. Here, the defatted material was heated
to 60 °C for 6 h.
samples were left to stand for 8 h so that the phases sepa-
rated completely. For the analysis the phases were separated
and analysed by GC-FID and partly by GC–MS (EI). The
evaluation is performed twice. On the one hand the depletion
in the aqueous phase and on the other hand the content in
the organic phase is determined. The determination of the
respective content was carried out by a single-point calibra-
tion. Blank values were determined by extracting the aque-
ous stock solutions, amino acids and their methyl derivatives
with pure solvents (DCM or chloroform). Furthermore, this
procedure was applied to aqueous extracts of H. purpuras-
cens (Extract-HA and Extract-W). In addition, the extracts
were further processed. The contained amino acids were
converted into the respective methyl esters by a sequence
of Boc protection, methylation with diazomethane and sub-
sequent deprotection. The conditions corresponded to the
protocol used for the synthesis of the methyl asparaginate.
Extraction of amino acids using calixarene derivatives
The extraction follows a protocol from Oshima et al. (2002).
As amino acids were chosen asparagine (Asn), aspartic
acid (Asp), tyrosine (Tyr), tryptophan (Trp), phenylalanine
(Phe) and pipecolinic acid (Pip) and their methyl esters. The
amino acids and their methyl esters were each dissolved in
water at diferent pH values, resulting in a concentration
of 1.0 mmol/L. The amino acids and their methyl esters
were dissolved in water at diferent pH values. The pH val-
ues were between 1.5 and 5.0 (1.5; 2.0; 2.5; 3.0; 4.0; 5.0).
The calixarenes were dissolved in dichloromethane (DCM)
and chloroform, respectively, resulting in a concentration
of 5.0 mmol/L. After this preparation, 2 mL of an aque-
ous acidic amino acid solution was intensively shaken
with 2 mL of a calixarene solution for 8 h. Afterwards, the
1 3

M. H. Franz et al.
were identifed by three methods. First, the derivatized sam-
ples were analysed by GC-FID on a 10-m column, belong-
ing to the Phenomenex kit. This short column is optimized
for amino acid separation and allows short run times. The
retention times were calibrated by a standard which con-
tains 18 proteinogenic amino acids (missing arginine and
cysteine), cysteine, 9 other amino acids and two dipeptides.
Second, the modifed extracts were analysed by a GC–MS
machine which runs in the SIM mode on the same column.
In this mode, we could identify compounds which have
documented SIM values. Therefore, we analysed enriched
samples on a GC–MS machine recording MS (EI) spectra
as a third identifcation method. Especially the last method
allowed us to identify compounds which have unknown
retention times and unknown SIM values. Furthermore, we
used for the third method a longer column (30 m) which
allows a better separation of the peaks. The recorded spectra
could be compared either with literature data bases or with
chromatograms of authentic material as a fnal prof.2
Gas chromatography
Preparation of the samples from pure amino acids,
O‑methylated derivatives and aqueous solutions
A mixture of petrol ether and chloroform in a rate of three
to one is prepared. Using this solvent mixture, a 10% solu-
tion of pyridine and a 10% solution of ethyl chloroformiate
are made. Having these solutions by hand, the samples are
made as following: The amino acid is dissolved in water or
an already prepared aqueous solution is used. The concentra-
tion should be like c=0.1 μmol/mL). A sample of the amino
acid solution is mixed with 0.5 mL of methanol and 1.5 mL
of the pyridine solution. The mixture is shaken carefully
for 15 s. Then, a portion (1 mL) of the ethyl chloroformiate
solution is added. The sample is shaken carefully for two
times (each 30 s) with a break of 1 min. After the phases are
separated the organic upper phase is analysed.
Analysis
Mass spectrometry
GC-FID: Analysis was performed either on a GC-2010
(Shimadzu, Kyoto, Japan) using the column forte BP5
(30 m × 2.5 mm × 2.5 μm) from SGE (Melbourne, Aus-
tralia) or on a GC-2014 (Shimadzu, Kyoto, Japan) using the
column Zebron ZB-AAA (10 m × 2.5 mm × 2.5 μm) from
Phenomenex (Torrance, CA, USA). A split injection port at
250 °C was used for sample introduction and the split ratio
was set to 15:1. Temperature program was on the GC-2010:
50 °C (2 min)-10 °C/min-30 °C (5 min) or on the GC-2014:
110 °C (0 min)-30 °C/min-320 °C (1 min). The helium car-
rier gas was set to 1.1 ml/min fow rate (constant fow mode).
GC–MS (EI): Gas chromatography: An Agilent 6890 gas
chromatograph was equipped with a 30-m analytical column
(Phenomenex ZB5-MS, 30 m×0.25 mm ID, tf=0.25 µm). A
split injection port at 270 °C was used for sample introduc-
tion and the split ratio was set to 5:1. Temperature program
100 °C (1 min)-10 °C/min-320 °C (3 min)). The helium
carrier gas was set to 1.0 ml/min fow rate (constant fow
mode). Mass Spectrometry: A JMS-T100GC (GCAccuTOF,
JEOL, Japan) time of fight mass spectrometer in electron
ionization (EI) mode at 70 eV and JEOL MassCenter™
workstation software was used. The source and transfer line
temperature were set at 200 °C and 290 °C, respectively. The
detector voltage was set at 2000 V. The acquisition range
was from m/z 41 to 600 with spectrum recoding interval of
0.4 s. The system was tuned with Perfuoro kerosene (PFK)
to achieve a resolution of 6000 (FWHM) at m/z 292.9824.
The mass spectrometry was recorded on a linear ion trap cou-
pled with orbitrap mass analyser from Thermo Fisher Scien-
tifc (Bremen, Germany, Model: LTQ-Orbitrap Velos). The
resolution was 100,000 FWHM at m/z=400 Da. The mass
spectra were acquired in the mass range m/z=130–2000 Da
(resulting in acquisition times of 1.6 s per cycle) Electro-
spray measurements were performed in direct infusion mode
using a custom-made microspray-device mounted on a Prox-
eon nanospray ion source. The microspray-device allows
for the sample infusion through a stainless-steel capillary
(90 μm I. D.). Accurate mass measurements in the orbitrap
were per formed using the lock mass option of the instru-
ment control software using the cation of tetradecyltrimeth-
ylammonium bromide (256.29988 amu) as internal mass
reference. The sample concentration was approx. 50 μg/mL
in methanol spiked with 0.1 mg/mL tetradecyltrimethylam-
monium bromide. The fow was approx. 1 μL/min and the
typical spray voltage (positive mode) was between 2.3 and
2.8 kV.
Results and discussion
Calixarenes can stabilize ammonium compounds due to
π-cation interactions. This allows them basically to extract
such ammonium species from an aqueous phase into an
Identifcation of the amino acids
2
A congruence of retention time and fragmentation pattern between
For the identifcation of the amino acids the extracts were
treated with the Phenomenex kit. The modifed amino acids
an unknown substance and an authentic material proves the identity
of an unknown substance.
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Fig. 3 Tert-Butyl-calix[6]arene 11 and derivatives thereof used for extraction od amino acids
organic phase. The task of this work was to investigate this
ability using the example of extraction of amino acids and
amino acid methyl esters in the presence of diferent calix-
arenes. The extraction was carried out under both neutral
and acidic conditions. The extractants used were more well-
known calixarenes, such as calix[6]arene, but also unusu-
ally substituted or specially constructed calixarenes, such as
homoazacalix[3]arene. Finally, this procedure was applied to
a processed aqueous extract from the rhizome and roots of
H. purpurascens. A sample of authentic material was given
by S.C. Exhelios S.R.L. (Timisoara, Romania). The plant
material was dried and defatted before the extraction process
can start.
In a modifcation of their work, tert-butyl calix[6]arene 11
was used instead of tert-octyl calix[6]arene. In addition to
the parent system 11, the hexacarboxylic acid 12 and its cor-
responding ester 13, and the hexa ethylene-diphenyl-phos-
phate 14 (Arnaud-Neu et al. 1999) were chosen as extractant
(Fig. 3). The last already fnds commercial use as a calcium
ionophore.
Homooxa‑calix[3]arene
In addition to the above-mentioned calix[6]arenes, derivat-
ized homooxa-calix[3]arenes should be used as extract-
ants. The most known representative of them is the tert.-
Butyl-hexahomooxa-calix[3]arene whose easy and straight
forward synthesis starts from the para-(tert-Butyl)-phenol
(Scheme 1). Because of the longer connections between the
aryl rings the shape is like a fat bowl.
Calixarenes
Tert‑Butyl‑calix[6]arene
An interesting derivative thereof is the phosphorous (III)-
bridged one in which a phosphite subunit is established.
Kawaguchi et al. (2000) investigated the complexation and
stabilization of neutral and reduced fullerenes by this unu-
sually derivatized hommooxacalix[3]arene. They observed
high complex formation constants. The peculiarity of this
A frst group of Calixarene based on the work of Oshima
et al. (2002). The best extraction efect was observed by the
authors when derivatives of tert-octyl-calix[6]arene were
used. In addition to the parent system, a hexacarboxylic acid
and its corresponding ethyl ester was investigated (Fig. 1).
1 3

M. H. Franz et al.
Scheme 1 Synthesis of tert.-Butyl-hexahomooxa-calix[3]arene 2
calixarenes is certainly the free electron pair of trivalent
phosphorus. The synthesis starts from the unprotected tert-
butyl hexahomooxa-calix[3]arene 2 (Scheme 2). In the frst
step, the hydroxy groups are silylated with trimethylsilyl-
chloride. This prevents the dynamic swinging through. The
result is a mixture of diastereomers with a cone/partial-cone
ratio of three to one. In the next step, the silylated diastere-
omers are reacted with dichlorofuorophosphane: initially,
the triply phosphorylated ((15,55,95-tri-tert-butyl-3,7,11-
trioxa-1,5,9(1,3)-tribenzenacyclododecaphane-12,52,92-
triyl)tris(oxy))tris(difuorophosphane) (18). By cleavage of
two Trifuorophospanemoleküle the bridged phosphite 19 is
formed (Scheme 2).
further described. Compound 6 is mentioned again in the
literature without describing spectroscopic data or synthe-
sis (Khan et al. 1993; Chirakul et al. 2000).
The synthesis of compound 6 based on (5-(tert-butyl)-
2-hydroxy-1,3-phenylene)dimethanol (16) and benzylamine
(22). Based on an idea by Takemura et al. (1992), the sub-
stances were heated for 3 days in xylene at 135 °C with
refux. The primary alcohol groups of triol 16 condense with
the amine function of aromatic amine 22 with elimination
of water. The resulting water is removed from the reaction
mixture by the vapours of the refuxing xylene and separated
at a Dean-Stark apparatus (Scheme 4).
The free hydroxy groups of tribenzyl-tert-Butyl-hexa-
homoaza-calix[3]arens 6 are the target of further modif-
cations of this molecule. The hydroxy groups are deproto-
nated by base and ethyl bromate acetic acid ester is reacted.
The saponifcation of the triethyl 2,2′,2″-((3,7,11-triben-
zyl-15,55,95-tri-tert-butyl-3,7,11-triaza-1,5,9(1,3)-
tribenzeneacyclododeca phane-12,52,92-triyl)tris(oxy))
triacetate (7) to the corresponding tricarboxylic acid and
the subsequent isolation failed.
The reaction sequence shown in Scheme 2 is subop-
timal in two aspects. On the one hand, it is a multi-step
synthesis and, on the other, toxic trifluorophosphane is
formed. In addition, you need three equivalents of phos-
phorus reagent. This moved us to work out an alternative.
Instead of the chlorodifuorophosphane, the tris (diethyl-
amino) phosphine was used as the source of phosphorus.
Catalysed by imidazole (5 mol%), the phosphorylated prod-
uct 4 is formed successfully. This improved protocol pro-
duces just diethylamine, which absorbs the proton of the
hydroxy groups. Furthermore, no toxic, volatile phosphorus
halides are formed. In a subsequent step, compound 4 was
converted with elemental sulfur into 3,15,26-tri-tert-butyl-
5H,7H,11H,13H-8,10-(epiprop[1]en[1]yl[3]ylidene)-9,19-
epoxy-1,17-(methanooxymethano)dibenzo[d,o] [1,3,7,13]
tetraoxa[2]phosphacyclohexadecine 19-sulfdes (20). The
transformation is quantitative (Scheme 3).
X‑ray
The structure of compound 7 could be confrmed by X-ray
analysis (Fig. 5). The compound 7 crystallizes in the tri-
clinic space group type P–1 with one molecule in the
asymmetric unit. The molecular structure of 7 (Scheme 4)
corroborates the proposed connectivity.
Methylation of amino acids
Homoaza‑calix[3]arene
The methyl esters of aspartic acid, tyrosine, trypto-
phan, phenylalanine and pipecolinic acid could easily be
obtained from the corresponding amino acid and methanol
in the presence of thionyl chloride. One gramme of the
respective amino acid was suspended in 20 mL metha-
nol and 4 mL thionyl chloride was added. After 24 h the
LM was removed and the respective ester was obtained in
sufcient purity. The synthesis of the asparagine methyl
ester is only partially successful with this method, since
As third category was chosen to use homoaza-calix[3]
arene (Fig. 4). This class of calix[3]arene ofers another
interesting possibility of variation by the third substitu-
ent. The methyl-substituted derivative 21 was introduced
into the literature by Takemura et al. (1992) and described
extensively spectroscopically. He also mentions the tert-
butyl derivative 6, the synthesis of which, however, is not
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Scheme 2 Synthesis of P(III)-oxo-bridged tert.-Butyl-hexahomooxa-calix[3]arene 4 in a two steps sequence
Scheme 3 Synthesis of sulphur-P(V)-oxo-bridged tert-Butyl-hexahomooxa-calix[3]arene 20 via compound 4 in a two step sequence
1 3

M. H. Franz et al.
Scheme 4 Synthesis of Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene 6 and its ester derivative 7
Fig. 4 Structures of the homoazacalix[3]arene type described by Takemura et al. (1992)
a large part hydrolyses into the aspartic acid. Therefore,
the asparagine was frst BOC-protected (Li and Sha 2008),
then methylated with diazomethane (Capasso et al. 1989)
and fnally deprotected again using an ethereal solution of
hydrogen chloride (Scheme 5).
Extraction capability of calixarenes against selected
amino acids—analysis by GC
The extractability of the above calixarenes against aspara-
gine (Asn), aspartic acid (Asp), tyrosine (Tyr), tryptophan
(Trp), phenylalanine (Phe) and pipecolinic acid (Pip) is
described.
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
tert-butyl-calix[6]arene hexaesters the extraction behaviour
largely corresponded to the extraction capacity of the calix-
arene-free solvents. This means that practically no extraction
(E) was observed up to a pH value of pH≤4 (E≤2%). At a
pH value of pH=5, the average extraction rate for the amino
acid methyl esters was E = 5%. For the free amino acids,
the extraction rate was still practically zero. The situation
for tert-butyl-calix[6]arene hexasic acid is somewhat more
diferentiated. With the free amino acids, the situation was
similar to the other two calixarenes. Practically no extraction
could be observed that was noticeably higher than the blank
values. With the methyl esters on the other hand a clear
extraction ability was to be observed (Table 2).
Table 2 summarizes the values from the extraction tests
of the amino acid methyl esters with tert-butyl-calix[6]arene
hexasic acid dissolved in chloroform. The values are the
mean values of the measured values determined by deple-
tion in the aqueous phase and by extraction in the organic
phase. This shows that L-H-Trp-OMe (entry 1) can best be
extracted. Even at a pH value of pH= 3, three-quarters of
the initial amount is extracted. At a pH value of pH = 4,
L-H-Phe-OMe (entry 2), L-H-Asn-OMe (entry 4) and L-H-
Pip-OMe (entry 6) have already extracted more than 50%.
The two more polar amino acids L-H-Tyr-OMe (entry 3)
and L-H-Asp-OMe (entry 5) have already been extracted
by almost 50% at a pH value of pH = 5. In all cases, the
extraction rate at a pH value of pH = 5 is significantly
higher than the extraction rate of pure chloroform without
Fig. 5 Molecular structure of the partial-cone isomer of Tribenzyl-
tert.-Butyl-hexahomooxa-calix[3]arene ester derivative 7. Hydrogen
atoms and a disordered tert-Butyl moiety are omitted for clarity; ther-
mal ellipsoids are drawn at the 50% probability level
Calix[6]arene
The strain system tert-butyl-calix[6]arene and the two
O-derivatives tert-butyl-calix[6]arene hexanoic acid and
tert-butyl-calix[6]arene hexanoate were used here as calix-
arenes. In the strain system tert-butyl-calix[6]arene and in
Scheme 5 Synthesis of methyl L-asparaginate (10)
Table 2 Extraction capacity of
pH-value
Extraction capacity at various pH values in percent
tert-butyl-calix[6]arene hexasic
acid 12 dissolved in chloroform
as a function of the pH value
1.5
2.0
2.5
3.0
4.0
5.0
1
2
3
4
5
6
H-Trp-OMe
H-Phe-OMe
H-Tyr-OMe
H-Asn-OMe
H-Asp-OMe
H-Pip-OMe
23
2
2
5
2
39
10
3
12
4
54
31
3
29
6
74
48
10
42
12
46
–
–
–
46
63
42
84
of the aqueous solution to be
extracted
68
25
55
21
65
3
15
32
1 3

M. H. Franz et al.
Table 3 Extraction capacity
of the phosphorus(III)-tert-
butyl-hexahomocalix[3]arene 4
dissolved in DCM as a function
of the pH value o the aqueous
solution to be extracted
pH-value
Extraction capacity at various pH values in percent
1.5
2.0
2.5
3.0
4.0
5.s0
1
2
3
4
5
6
H-Trp-OMe
H-Phe-OMe
H-Tyr-OMe
H-Asn-OMe
H-Asp-OMe
H-Pip-OMe
12
8
7
4
2
19
15
12
9
4
5
29
24
19
14
8
37
31
27
25
16
14
44
39
35
32
21
20
52
47
42
39
26
24
2
8
tert-butyl-calix[6]arene hexasic acid (L-H-Trp-OMe: 8%,
L-H-Phe-OMe: 5%, L-H-Tyr-OMe: 0%, L-H-Asn-OMe: 6%,
L-H-Asp-OMe: 2%, L-H-Pip-OMe: 7%). Extractions with
DCM were also performed. The results difer only minimally
in tendency and absolute values with those of chloroform
extractions. Overall, the results confrm the work of Goto
and Furusaki (Oshima et al. 2002). It is also gratifying to
note that a high extraction rate could also be observed for
asparagine methyl ester and pipecolic acid methyl ester.
(L-H-Trp-OMe: 7%, L-H-Phe-OMe: 5%, L-H-Tyr-OMe:
1%, L-H-Asn-OMe: 5%, L-H-Asp-OMe: 2%, L-H-Pip-OMe:
6%). It can be observed that the aromatic amino acid methyl
esters are better extracted at all pH values (e.g. pH = 5:
42–52%; entry 1–3) than the non-aromatic ones (e.g. pH=5:
24–39%; entry 4–6). Extractions with chloroform were also
performed. The results difer only minimally in tendency and
absolute values with those of the DCM extractions. Despite
the observed extraction rate, phosphor(III)-tert-butyl-hex-
ahomocalix[3]arene 4 is not optimal for the extraction of
methyl asparaginate ester and methyl pipecolinate.
Homooxacalix[3]arene and phosphorous derivative
The strain system tert-butyl-hexahomocalix[3]arene 2 and
the P-bridged phosphor(III)-tert-butyl-hexahomocalix[3]
arene 4 were used here as calixarenes. In the strain sys-
tem tert-butyl-hexahomocalix[3]arene 2, the extraction
behaviour largely corresponded to the extraction capacity
of the calixarene-free solvents. This means that practically
no extraction (E) was observed up to a pH value of pH≤4
(E≤2%). At a pH value of pH=5, the extraction rates for
the amino acid methyl esters were: L-H-Trp-OMe: 7%, L-H-
Phe-OMe: 4%, L-H-Tyr-OMe: 2%, L-H-Asn-OMe: 8%, L-H-
Asp-OMe: 4%, L-H-Pip-OMe: 9%. For the free amino acids,
the extraction rate was still practically zero. The situation
for phosphor(III)-tert-butyl-hexahomocalix[3]arene 4 is
somewhat more diferentiated. With regard to the free amino
acids, there was also practically no extraction to be observed,
which was noticeably higher than the blank values. With
regard to methyl esters, however, a clear extraction capacity
could be observed (Table 3).
Homoazacalix[3]arene
The formal strain system would be the O- and N-non-substi-
tuted tert-butyl-hexahomoaza-calix[3]arene. However, this is
not accessible as such. Therefore, the N, N′,N″-Tribenzyl-tert-
butyl-hexahomoaza-calix[3]arene and the N, N′,N″-Tribenzyl-
tert-butyl-hexahomoaza-calix[3]arene were used here as calix-
arene. The extraction behaviour largely corresponded to the
extraction capacity of both calixarenes and the calixarene-free
solvents. This means that practically no extraction (E) was
observed up to a pH value of pH≤4 (E≤2%). At a pH value
of pH=5, the extraction rates for the amino acid methyl esters
of both calixarenes were: L-H-Trp-OMe: 8%, L-H-Phe-OMe:
4%, L-H-Tyr-OMe: 3%, L-H-Asn-OMe: 7%, L-H-Asp-OMe:
5%, L-H-Pip-OMe: 6%. Interestingly, at a pH value of pH=5,
increased extraction rates could also be observed for free
amino acids: L-H-Trp-OH: 7%, L-H-Phe-OH: 5%, L-H-Tyr-
OH: 3%, L-H-Asn-OH: 5%, L-H-Asp-OH: 2%, L-H-Pip-OH:
8%. Overall it can be stated that the extraction capacity of the
two homoazacalix[3]arenes is insufcient with regard to free
or protected amino acids.
Table 3 summarizes the values from the extraction tests
of amino acid methyl esters with phosphorus(III)-tert-
butyl-hexahomocalix[3]arene 4 dissolved in DCM. The
values are the mean values of the measured values deter-
mined by depletion in the aqueous phase and by extraction
in the organic phase. This shows that L-H-Trp-OMe (entry
1) can best be extracted. At a pH value of pH=5, more than
ffty per cent of the initial amount was extracted. All others
are extracted at a pH of pH= 5 only between 25 and 50%
(entry 2–6). In all cases the extraction rate at a pH value
of pH=5 is clearly above the extraction rate of pure DCM
without phosphor(III)-tert-butyl-hexahomocalix[3]arene
Calixarenes’ ability to extract amino acids
from extracts of Helleborous purpurascens
The aqueous extract
Two extracts were made using diferent ways of substance
isolation. In one case, the defatted rhizome was stirred for
24 h at room temperature (Extract-W-rt) and in the other
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Table 4 GC–MS analysis of
the aqueous extract (Extract-
W-ultra)
Entry
Retention timea Retention timeb SIM valuesc (Da)
Amino acid
Intensity
(min)
(min)
1
2
3
3
4
5
6
7
8
9
10
1.74
2.07
2.21
2.59
2.63
2.72
2.82
2.82
3.40
3.77
4.48
15.30
16.72
17.14
17.79
17.99
18.07
18.25
18.77
20.53
21.74
22.48
130, 88
158, 116
158, 116, 72
160, 101
172, 144, 130
156, 114, 70
155, 69
170, 128, 84
216, 130, 88
230, 170, 142
187, 112, 84
Alanine
W
vW
M
vW
M
W
vS
S
Valine
Norvalined
Threonine
GABA
Proline
Asparagine
Pipecolic acid
Aspartic acid
Glutamic acid
Glutamine
S
W
M
aPhenomenex column
bNormal GC-FID column
cSingle ion monitoring
dInternal standard
Table 5 GC–MS analysis of the
Entry
Retention timea Retention timeb SIM valuesc (Da)
Amino acid
Intensity
extract-HA
(min)
(min)
1
2
3
4
5
6
7
8
9
1.74
2.21
2.63
2.63
2.72
2.82
2.82
3.40
3.77
7.76
9.12
130, 88
Alanine
W
M
Vw
M
M
S
S
M
W
158, 116, 72
203, 146, 101
172, 144, 130
156, 114, 70
155, 69
170, 128, 84
216, 130, 88
230, 170, 142
Norvalined
Serine
10.15
10.37
10.43
10.65
11.10
12.85
14.04
GABA
Proline
Asparagine
Pipecolic acid
Aspartic acid
Glutamic acid
aPhenomenex column
bNormal GC–MS column
cSingle ion monitoring
dInternal standard
case, the plant material was extracted at 60 °C under the
infuence of ultrasound (Extract-W-ultra). The result of
GC–MS (EI) analysis for Extract-W-ultra is given in Table 4.
The amino acids were identifed via their specifc SIM val-
ues. The dominating peaks represented asparagine, pipecolic
acid and aspartic acid.
The hydroalcoholic extract
The hydroalcoholic extract Extract-HA was also analysed.
Generally, the composition is similar to that one of the
Extract-W. The results of the GC-FID analysis are collected
in Table 5. All amino acids were characterized by their reten-
tions times, their SIM values and MS(EI) spectra. Entry 2
represents the internal standard. Entries 1, 3–6, 8 and 9 were
identifed by comparison with a standard mixture.
The composition of Extract-W-rt and Extract-W-ultra
is similar but the relative intensities of the detected amino
acids are diferent. Asparagine was in Extract-W-ultra the
dominating one while its peak has in Extract-W-rt just the
same intensity like the pipecolic acid. It could be concluded
that Asn is reduced while the Aspartic acid was enriched. A
conclusion could be that the amide function was saponifed.
Extraction of amino acid from a extract of Helleborous
purpuresens
Based on the previous results, tert-butyl-calix[6]arene hexa-
sic acid 12 was the most suitable to mediate the extraction of
1 3

M. H. Franz et al.
Table 6 Extraction capacity of
tert-butyl-calix[6]arene hexasic
acid 12 dissolved in chloroform
as a function of the pH value
of the prepared aqueous H.
purpurascens extracts (Extract-
W-ultra)
pH-value
Extraction capacity at various pH values in percent
Intensity
Relative
2.0
3.0
4.0
5.0
1
2
3
4
5
6
7
8
Alanine
GABA
Proline
Asparagine
Pipecoline
Aspartic acid
Glutamic acid
Glutamine
9
6
14
11
16
3
27
15
42
39
45
10
11
37
37
31
59
56
62
22
24
58
59
50
83
67
86
43
38
72
W
W
W
S
S
M
W
M
2
10
amino acids from an acidic environment. All three extracts
(Extract-W-rt, Extract-W-ultra and Extract-HA) were ana-
lysed. The hydroalcoholic extract (Extract-HA) was dried
frst and then resolved in water. For the extraction, the amino
acids, however, had to be present as methyl esters. Thus,
the following protocol for the extraction was carried out.
The amino acids were extracted at 60 °C for 24 h from the
degreased plant material. The extracted amino acids were
then enriched in an acidic ion exchanger and subsequently
converted into methyl ester. Since asparagine in particular
is not reliably transferred directly into the methyl ester, the
entire mixture was frst protected as tert-butyloxycarbamate
and then converted into the methyl ester with diazomethane.
Finally, the protective group was acidically split of under
mild conditions. The conditions of the protocol follow the
above described protocol for the asparagine (Scheme 5). The
extract thus prepared was divided into four portions and dis-
solved in water at diferent pH values (pH= 2.0; 3.0; 4.0;
5.0) in such a way that a concentration of approximately
camino acid = 1.0 mmol/L is adjusted. As this is a mixture,
an average molar mass of M= 130 g/mol was calculated.
5 mL of the amino acid methyl ester solutions prepared
in this way was shaken intensively with 5 mL of a tert-
butyl-calix[6]arene hexasic acid 12 solution in chloroform
(c=5.0 mmol/L) for 8 h. The samples were then left to stand
for 8 h so that the phases could separate completely. For
analysis, the separated phases were derivatized for GC anal-
ysis and analysed in the known manner using GC-FID and
GC–MS (EI). The result for the analysis of Extract-W-ultra
is summarized in Table 6. The other extracts (Extract-W-rt
and Extract-HA) gave similar results.
Conclusion
One focus of the work was on the synthesis of novel
calixarenes. The well-known tert-Butyl-hexahomooxa-
calix[3]arene 2 was obtained by an improved synthesis.
Based on this, the synthesis of a structurally innovative
phosphorus(III)-bridged tert-Butyl-hexahomooxa-calix[3]
arene phosphite 4 was described. This is achieved on the one
hand by a one-pot method with Tris(diethylamino)phosphine
and on the other hand by a two-stage sequence with the
intermediately isolated triple O-silylated tert-Butyl-hexaho-
mooxa-calix[3]arene 17. Furthermore, the presentation of
triple N-benzylated tert-Butyl-hexahomoaza-calix[3]arene
6 is described. Ethyl esterifed carboxymethylated deriva-
tive of N,N′,N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]
arene 7 was obtained by reaction with ethyl bromoacetate.
The structure of this compound could be confrmed by X-ray
structure analysis. With regard to the extraction capacity
of the calixarenes used, it must frst be noted that the free
amino acids could not be extracted in any combination.
The methyl-protected amino acids could be successfully
extracted with O-carboxymethylated-tert-butyl-calix[6]
arene hexasic acid 12 and phosphorus(III)-tert-Butyl-hex-
ahomooxa-calix[3]arene phosphite 4. In both cases methyl
tryptophanate was best extracted into the organic phases.
In the case of phosphorus(III)-tert-Butyl-hexahomooxa-
calix[3]arene phosphite 4 there was also a clear tendency
that the aromatic amino acids were extracted better than
the aliphatic ones. The transfer to the Helleborous extract
succeeded well. The amino acids known from the aqueous
extracts were found with a similar intensity. It is regretta-
ble that the derivatization is quite complex, because oth-
erwise asparagine is not found well again. The reason for
the good extraction capacity of phosphorus(III)-tert-Butyl-
hexahomooxa-calix[3]arene phosphite 4 in comparison
to tert-Butyl-hexahomooxa-calix[3]arene 2 could not be
conclusively clarified. One reason will certainly be the
free electron pair at the phosphorus. Another reason could
be that the structure is much more rigid. A task of further
work is the synthesis of carboxymethylated derivative of
The extractions showed several tendencies. The relative
intensity distribution corresponds to the original pattern.
Asparagine is still one of the dominant signals (entry 4).
The very thin signals for valine and threonine in the original
extract (Table 3) could not be clearly found again after the
long procedure. Otherwise, the extraction of the H. purpura-
scens extract worked well as expected.
1 3

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene,…
Arnott GE (2018) Inherently chiral calixarenes: synthesis and applica-
N,N′,N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene.
This is interesting because this compound is expected to
have a good extraction efect on free amino acids due to
its amphoteric character. In general, hexahomoaza-calix[3]
arenes will be an interesting synthesis target in any type of
decoration.
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Acknowledgements The authors want to thank Ing. Geta Serbanescu
and Ing. Draga Todorov (both S.C. Exhelios S.R.L.) for fruitful dis-
cussions and for the support of this work with rhizome and roots of H.
purpurascens and an already prepared hydroalcoholic extract thereof.
The authors want to thank also Dr. Macarie A. Corina and Dr. Ionel
Balcu, (both: Institutul National de Cercetare Dezvoltare pentru Elec-
trochimie si Materie Condensata, Timisoara, Romania) for their con-
tributions for this publication and also Dr. Matthias Freytag (Institut
für Anorganische und Analytische Chemie, Technische Universität
Braunschweig, Braunschweig, Germany); the authors want to thank
for the measuring of the x-ray structure. In addition, this work was
supported by the Romanian National Authority for Scientifc Research
through the exploratory research program: “DEI-PCE-PROJECT NR.
341-/05.10.2011-Immunomodulatory Fluoroglycopeptide Molecular
Architectures and PROJECT NR. 18360301, Contract: 42N/2018”.
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Author contributions IN, MI and MHF contributed to the study con-
ception and design. EM synthesised the Homoaza-calix[3]arene; IN
and CVM synthesised the phosphorous-bridged Homooxa-calix[3]
arene, MHF synthesised the esters of the amino acids; excepted the
extractions and performed the GC analysis. The frst draft of the manu-
script was written by MHF and all authors commented on previous
versions of the manuscript. All authors read and approved the fnal
manuscript.
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periluorinated α-diketones. Chem Ber 129:725–732
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ies of calixarene-[60]fullerene inclusion processes. J Incl Phenom
Mol Recognit 37:253–258
Compliance with ethical standards
Kerek F (2000) The structure of the digital is like and natriuretic fac-
tors identifed as macrocyclic derivatives of the inorganic carbon
suboxide. Hypertens Res 23:S33–S38
Conflict of interest The authors declare that they do not have any con-
fict of interest.
Kerek F, Szegli G, Cremer L, Lupu AR, Durbaca S, Calugaru A, Her-
old A, Radu DL (2008) The novel arthritis-drug substance MCS-
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Research involving human participants and/or animals This article
does not contain any studies with human participants or animals per-
formed by any of the authors.
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