Distant precursors of benzylisoquinoline alkaloids and their enzymatic formation.

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Section a
Physics, Physical Chemistry, Cosmic Physics
Section b
Inorganic and Organic Chemistry
Contents V
Contents of Number 4
Original Communications
Stereochemistry and Mechanism of Reactions Cata
lyzed by Tyrosine Phenol-Lyase from Escherichia
intermedia
M. M. PALCIC, S.-J. SHEN, E. SCHLEICHER, H.
KUMAGAI, S. SAWADA, H. YAMADA, and H. G.
FLOSS 307
Distant Precursors of Benzylisoquinoline Alkaloids
and Their Enzymatic Formation
M. RUEFFER and M. H. ZENK 319
Characterization of 2ß(R)-17-0-Acetylajmalan:
J
Acetylesterase — a Specific Enzyme Involved in
the Biosynthesis of the Rauwolfia Alkaloid Ajma-
line
L. POLZ, H. SCHÜBEL, and J. STÖCKIGT 333
Induction and Characterization of a NADPH-De-
pendent Flavone Synthase from Cell Cultures of
Soybean
G. KOCHS and H. GRISEBACH 343
Proposal for the Mechanism of Action of Urocanase.
Inference from the Inhibition by 2-Methyluroca-
nate
E. GERLINGER and J. RETEY 349
Re-Investigation of the Protein Structure of Co
enzyme B -Dependent Diol Dehydrase
12
K. TANIZAWA, N. NAKAJIMA, T. TORAYA, H. TANA-
KA, and K. SODA 353
VI Contents
Methanogenesis from Acetate by Methanosarcina Functional Group Recognition of Pheromone Mole
barkeri: Catalysis of Acetate Formation from cules by Sensory Cells of Antheraea polyphemus
Methyl Iodide, C0, and H by the Enzyme Sys and Antheraea pernyi (Lepidoptera: Saturniidae)
2 2
tem Involved H. J. BESTMANN, W. CAI-HONG, B. DÖHLA, LI-
K. LAUFER, B. EIKMANNS, U. FRIMMER, and R. K. KEDONG, and K. E. KAISSLING 435
THAUER 360
Building Blocks for Oligonucleotide Syntheses with
Divergent Evolution of 5S rRNA Genes in Methano-
Uniformly Fragmentable ß-Halogenated Protect
coccus
ing Groups (In German)
G. WICH, L. SIBOLD, and A. BÖCK 373
P. LEMMEN, R. KARL, I. UGI, N. BALGOBIN, and J.
Characterization of Some Claviceps Strains Derived CHATTOPADHYAYA 442
from Regenerated Protoplasts
Experiments on the Optical Resolution of Condur-
B. SCHUMANN, W. MAIER, and D. GRÖGER 381
amine Analogs by Enzymatic Transesterification
Phenylalanine and Tyrosine Biosynthesis in Spore- in Organic Solvents (In German)
forming Members of the Order Actinomycetales G. KRESZE and M. SABUNI 446
H. -K. HUND, B. KELLER, and F. LINGENS 387
Steric Course of the Rhodium-Catalyzed Decarbony-
lation of Chiral 4-Methyl-[l-3H,2-2H]pentanal
E. coli Maltodextrin Phosphorylase: Primary Struc 1
ture and Deletion Mapping of the C-Terminal Site H. OTSUKA and H. G. FLOSS 449
D. PALM, R. GOERL, G. WEIDINGER, R. ZEIER, B.
Synthesis of Immobilized Peptide Fragments on
FISCHER, and R. SCHINZEL 394
Polystyrene-Polyoxyethylene for Affinity Chro
Fermentation of D-Xylose to Ethanol by Bacillus matography (In German)
macerans E. BAYER, H. HELLSTERN, and H. ECKSTEIN 455
H.-J. SCHEPERS, ST. BRINGER-MEYER, and H. SAHM
401 Biopterin Synthesis in Mouse Spleen during Bone
Marrow Transplantation Correlates with Unim
Semicontinuous and Continuous Production of Citric paired Hemopoietic Engraftment
Acid with Immobilized Cells of Aspergillus niger I. ZIEGLER and ST. THIERFELDER 461
H. EIKMEIER and H. J. REHM 408
In vivo Screening of Glutathione Related Detoxifica
Microbial Hydroxylation of Cedrol and Cedrene tion Products in the Early State of Drug Devel
W.-R. ABRAHAM, P. WASHAUSEN, and K. opment
KIESLICH 414
A. PROX, J. SCHMID, J. NICKL, and G. ENGEL
HARDT 465
6-Methylpurine, 6-Methyl-9-ß-D-ribofuranosylpu-
rine, and 6-Hydroxymethyl-9-ß-D-ribofuranosyl- Synthesis and Complexing Features of an Artificial
purine as Antiviral Metabolites of Collybia Receptor for Biogenic Amines (In German)
maculata (Basidiomycetes) F. P. SCHMIDTCHEN 476
K. LEONHARDT, T. ANKE, E. HILLEN-MASKE, and
Metabolism of the Herbicide 2-(2,4-Dichloro-
W. STEGLICH 420
phenoxy)-propionic Acid (Dichlorprop) in Barley
Enzymatic Synthesis of Riboflavin and FMN Specifi (Hordeum vulgare)
cally Labeled with 13C in the Xylene Ring G. BÄRENWALD, B. SCHNEIDER, and H.-R.
H. SEDLMAIER, F. MÜLLER, P. J. KELLER, and A. SCHÜTTE 486
BACHER 425
Site Directed Antisera to the D-2 Polypeptide Sub-
A Vitamin D Steroid Hormone in the Calcinogenic unit of Photosystem II
3
Grass Trisetum flavescens R. GEIGER, R. J. BERZBORN, B. DEPKA, W. OETT-
W. A. RAMBECK, H. WEISER, and H. ZUCKER 430 MEIER, and A. TREBST 491
Distant Precursors of Benzylisoquinoline Alkaloids
and their Enzymatic Formation
Martina Rueffer and Meinhart H. Zenk
Lehrstuhl für Pharmazeutische Biologie, Universität München, Karlstraße 29,
D-8000 München 2, Bundesrepublik Deutschland
Z. Naturforsch. 42c, 319-332 (1987); received October 30, 1986
Dedicated to Professor Helmut Simon on the occasion of his 60th birthday
Berberis Species, Suspension Cultures, Cell-Free Systems, Benzylisoquinoline Alkaloids,
Enzymes of Tyrosine Metabolism
The incorporation rates of labelled tyrosine, DOPA, tyramine, and dopamine have been inves
tigated during the in vivo formation of the protoberberine alkaloid, jatrorrhizine, in callus cul
tures of Berberis canadensis. While tyrosine was equally well incorporated into both the iso-
quinoline (54%) and benzyl (46%) portions of the alkaloid, DOPA was almost exclusively (91%)
transformed into the isoquinoline moiety. However, tyramine (25%) and to a lesser extent,
dopamine (15%) were incorporated into the aldehyde-derived, benzylic half of the isoquinoline
molecule as well. In order to investigate further the precursory roles of these compounds, select
enzymes involved in tyrosine metabolism in alkaloid-producing cell cultures have been studied.
The occurrence of tyrosine decarboxylase, phenolase, transaminase, p-hydroxyphenylpyruvate
decarboxylase, amineoxidase and methionine adenosyl transferase was demonstrated in suspen
sion cells of Berberis. These enzymes were partially purified and a preliminary characterization
was performed. In the light of these and previous data, the differential metabolism of tyrosine and
DOPA in the early steps of isoquinoline alkaloid biosynthesis is discussed. Conclusive evidence as
to the biosynthetic origin of the phenylacetaldehydes which furnish the benzylic moiety of the
alkaloids is precluded by the presence of both amineoxidase and phenylpyruvate decarboxylase
activities in these cultures.
Introduction amino acid is metabolized solely via decarboxylation
to dopamine, which is in turn incorporated almost
In 1910 Winterstein and Trier [1] suggested that
exclusively into the upper isoquinoline portion of the
two molecules of 3,4-dihydroxyphenylalanine
benzylisoquinoline alkaloids [3]. While the forma
(DOPA) may be modified in the plant to yield
tion of the isoquinoline part of the molecules in ques
dopamine and 3,4-dihydroxyphenylacetaldehyde.
tion seems to be clear, there is considerable confu
These could subsequently condense to yield nor-
sion concerning the origin of the lower benzylic part.
laudanosoline, already considered as a potential pre
Reports [4] that DOPA is also incorporated via 3,4-
cursor for more complex isoquinoline alkaloids, L-
dihydroxyphenylpyruvic acid into the "lower" por
Tyrosine is an immediate precursor of L-DOPA and
tion and postulation of norlaudanosolinecarboxylic
numerous reports [see 2] have appeared demonstra
acid as an intermediate, which was apparently ex
ting the incorporation of this primary amino acid into
perimentally supported by three other research
benzylisoquinoline-derived alkaloids. Chemical de
groups [5—7], were refuted by in vivo [3] and in vitro
gradation of the benzylisoquinoline skeleton, which
[8, 9] experiments. Holland et al [3] investigated the
has been labelled by application of specifically label
incorporation of DL-[3-14C]DOPA into 6 different
led tyrosine, demonstrated in numerous cases that
isoquinoline alkaloids in 3 different plant species. In
two molecules of tyrosine form the isoquinoline
all cases incorporation was found predominantly
"upper" and benzylic "lower" portion of these com
(93-99%) in the isoquinoline half of the target al
pounds [2. 3]. In contrast, feeding experiments using
kaloid. We [8, 9], on the other hand, discovered a
specifically labelled DOPA demonstrated that this
specific enzyme which, in a stereospecific manner,
Abbreviations: DOPA, 3,4-Dihydroxyphenylalanine; condenses dopamine with 3,4-dihydroxy- and 4-hy-
SAM, S-Adenosylmethionine. droxyphenylacetaldehyde to yield (S)-norlaudanoso-
Reprint requests to Dr. M. Rueffer. line and norcoclaurine respectively, ruling out the
above postulated norlaudanosolinecarboxylic acid as
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
0341-0382/87/0400-0319 S 01.30/0 an obligatory intermediate.
320 M. Rueffer and M. H. Zenk • Distant Precursors of Benzylisoquinoline Alkaloids
This present study was therefore undertaken to Chemicals
further clarify the true nature of the distant precur
Tyrosine, tyramine, DOPA, and dopamine were
sors in the formation of the benzylisoquinoline sys
purchased from Fluka (Neu-Ulm),/?-hydroxyphenyl-
tem. This investigation was conducted in a dual man
pyruvate from Sigma (München). All biochemicals
ner, using callus and suspension cultures of Berberis,
were obtained from Boehringer (Mannheim). Pal-
which are known to produce large amounts of pro-
matine was a kind gift of Prof. N. Nagakura (Kobe).
toberberine alkaloids of the jatrorrhizine type [10].
The following chromatography gels were used:
Firstly, the incorporation of distant potential precur
Sephadex G-25 and QAE-Sephadex (Pharmacia,
sors into the protoberberine alkaloids was checked.
Uppsala), DEAE-Matrex™Cellufine (Amicon, Wit-
The advantage is that considerably higher rates of
ten), TSK-HW 55S (Merck, Darmstadt), Hydroxyl-
incorporation of precursors can be achieved with the
apatite (Bio-Rad, Richmond). TLC-Plates (Silica
callus system as compared to intact plants. Secondly,
60-Polygram sheets and Cellulose MN 300) were ob
the enzymes of the tyrosine metabolic pathway were
tained from Macherey and Nagel (Düren). Rotiszint
investigated in protoberberine producing cell suspen
22 (Roth, Karlsruhe) and Quickszint 2000 (Zinsser,
sion cultures to gain an insight into the formation of
Frankfurt) were used as scintillation cocktails. All
dopamine and the hydroxylated phenylacetalde-
other chemicals and solvents were purchased from
hydes, the known precursors of the benzylisoquino-
Merck (Darmstadt) or Roth (Karlsruhe).
lines. As already pointed out by Spenser [2], every
known isoquinoline alkaloid contains one or more
Radiochemicals
methyl or methylenedioxy groups. Both entities are
derived from the S-methyl group of methionine. The The following radiochemicals were purchased
surprisingly specific O- and N-methyltransferases in from Amersham-Buchler (Braunschweig): [U-14C]ty-
volved in the later steps of benzylisoquinoline mod rosine (58 uCi/umol), [14COOH]tyrosine (58 uCi/
ification have become increasingly known [11]. In an umol), [7-14C]tyramine (56 |iCi/urnol), [7-14C]dop-
attempt to study the distant precursors of the iso amine (54 uCi/urnol), [3-14C]DOPA (10.9 uCi/urnol),
quinoline alkaloids, we also included the enzyme re [2,3-3H]tyrosine (16 mCi/urnol), [l-14CH3]meth-
sponsible for the synthesis of S-adenosylmethionine ionine (50 jiCi/umol), [S-C3H3]methionine (70 mCi/
(SAM) from L-methionine and ATP. umol). [Ring-3H]tyramine (29.8 mCi/urnol) was
obtained from NEN (Dreieichenhain).
By using both of the approaches depicted above —
precursor feeding and enzyme studies — it was hoped [14COOH]-/?-Hydroxyphenylpyruvate was synthe
to gain an insight into the nature of the true precur sized by incubating [14COOH]tyrosine (5 uCi;
sors and the enzymes involved in the formation of 0.5 umol) in 0.1 M KP042" buffer, pH 7.2, contain
the building blocks of the benzylisoquinoline system. ing 1 mg catalase (beef liver, Boehringer, Mann
heim; 6000 units) and 30 u.g L-amino acid oxidase
(Crotalus durissus, Boehringer, 0.2 units) in a total
volume of 400 ul. After incubation for 4 h at 30 °C
Materials and Methods
the mixture was acidified with 0.2 N HCl and ex
Plant material tracted 5 times with 2 ml ethylacetate. The keto acid
was recovered from the organic phase and subse
Callus and suspension cells of Berberidaceae,
quently purified by TLC (Polygram) with the solvent
Papaveraceae, Menispermaceae and control species
system: benzene: dioxane : acetic acid = 90:25:4 (R:
have been maintained in this laboratory for the past {
0.4). The yield of purified p-hydroxyphenylpyruvic
12 years. The calli were grown on LS-medium [12]
acid under these conditions was 85%.
solidified with agar (1%). The suspended cells were
cultivated in 2.5 1 Fernbach flasks in a volume of
TLC-Systems
1000 ml LS-medium on a gyratory shaker (100 rpm)
in diffuse light (750 lux) at 24 °C and were subcul- The following solvent systems were used with Sili
tured at weekly intervals using about 10% inoculum ca 60-Polygram sheets: ethylacetate: methylethyl-
(v/v). The cells were harvested immediately after ketone: formic acid: water = 50:30:10:10 (jatror
reaching the stationary phase, frozen in liquid nitro rhizine, R: 0.80); chloroform: methanol: ammo
f
gen and stored at -20 °C. nia = 68:18:0.6 (palmatine, R: 0.52); chloroform:
(
M. Rueffer and M. H. Zenk • Distant Precursors of Benzylisoquinoline Alkaloids 321
methanol = 95:5 (corydaldine, Rf 0.73); benzol: (d). The aqueous phase was basified with 15%
acetic acid =8:2 (2,3-dimethoxyphthalic acid, Rf NaOH and extracted with chloroform. The organic
0.51); /i-butanol:acetic acid:water = 4:1:5 (upper layer was concentrated and chromatographed,
phase) (tyramine, Rf 0.43; dopamine, Rf 0.22); resulting in the upper isoquinoline portion of the
chloroform:ethylacetate = 8:2 (4-hydroxyphenyl- alkaloid, 6,7-dimethyl-l,2,3,4-tetrahydro-l-iso-
acetaldehyde, Rf 0.70; 3,4-dihydroxyphenylacetal- quinoline = corydaline (17%). MS: 207 (M+, 100),
dehyde, Rf 0.63). For separation on Cellulose MN 178, 150, 135; NMR (CDC1): 290 (m), 3.48 (m), 390
3
300 plates the solvent system: n-butanol:acetic acid: (s), 6.65 (s), 7.56 (s).
water = 4:1:5 (upper phase) (tyrosine, Rf 0.29; At each stage of purification the specific activity of
tyramine, Rf 0.53; DOPA, Rf 0.24; dopamine, Rf the products was determined.
0.38) was used.
Enzyme assays
Feeding procedure
1. Tyrosine decarboxylase
The labelled precursor (50 uJ, 2.5 uCi, 0.05 umol)
The assay system consisted of 62.5 nmol L-
was applied to a vigorously growing 14 day old callus
[,4COOH]tyrosine (2.5 x 104 cpm) and 50 nmol py-
culture of Berberis canadensis. The callus was al
ridoxalphosphate in 1.6 x 10~3 M KP02" buffer pH
lowed to metabolize for 5 days at 24 °C in the dark. 4
6.5, and enzyme in a total volume of 300 ul, incu
Thereafter the callus was harvested quantitatively
bated in an Eppendorf vial for 1 h at 30 °C. The
and the major alkaloid, jatrorrhizine, was isolated
Eppendorf vial was then placed into a scintillation
and subsequently analyzed.
vial containing 0.2 ml hyamine (Zinsser, Frankfurt)
and subsequently sealed with a rubber stopper. The
Degradation of jatrorrhizine
reaction was terminated by addition of 50 uJ of a
Callus tissue (approx. 0.3 g dwt) was extracted
0.2 N perchloric acid solution which was injected into
with MeOH (2x20 ml). The extract was concen
the Eppendorf vial. The evolving 14C0 was ab
trated and chromatographed on Polygram Silica 2
sorbed by the hyamine solution during a period of
plates (0.25 mm; Macherey and Nagel, Düren)
1 h and the Eppendorf vial was subsequently
using the solvent system ethylacetate: methylethyl-
discarded. Scintillator (Quickszint 2000; Zinsser,
ketone:formic acid:water = 50:30:10:10 (jatror
Frankfurt) was added and the sample was counted.
rhizine, Rf 0.8). The alkaloid was eluted and methyl
The recovery rate of this assay was tested with label
ated with dimethyl sulfate/acetonitrile. The resulting
led Na14C0 and averaged 90%. Protein contents
palmatine was appropriately diluted with carrier al 2 3
were determined by the method of Bradford [13]
kaloid (average: total of 30 umol) and chromato
using bovine serum albumin as standard.
graphed with chloroform: methanol: NHOH =
4
68:18:0.6 as solvent (palmatine, Rf 0.5). The eluted
2. Phenolase
palmatine was reduced with NaBH in MeOH, the
4
solvent evaporated and the residue taken up in H0 [Ring-3H]tyramine 10 nmol (2xl03 cpm), ascor-
2
and extracted with ethylacetate. The organic layer bate 50 umol, KP042~ buffer 50 umol and enzyme
(up to 1 mg protein) were incubated in a total vol
was concentrated in vacuo and taken up in 2 ml 10%
ume of 300 ul for 1 h at 30 °C and the reaction termi
HS0. The acidic solution was cooled in an ice water
2 4
nated by the addition of 300 ul charcoal suspension
bath and 5 mg KMn0 dissolved in 1 ml H0 were
4 2
(10 g/100 ml; dextran coated). The mixture was cen-
slowly added while stirring. After 2 h the mixture
trifuged for 5 min (Eppendorf system) and an aliquot
was acidified to pH 3 and extracted with ethylace
of 300 ul of the aqueous phase containing tritiated
tate. This procedure yielded the lower benzylic por
water was removed for liquid scintillation counting.
tion of the alkaloid as 2,3-dimethoxyphthalic acid
(10% yield). TLC of the product in benzene: glacial
acetic acid = 8:2 (Rf. 0.5) and treatment with 3. Transaminase
diazomethane yielded the methylated product. MS [2',3'-3H]Tyrosine or DOPA (synthesized from
(m/z) 254 (M+), 223 (100), 207, 191 and NMR tyrosine by phenolase) 250 nmol (104 cpm) were
(CDC1): 3.95 (s), 405 (s), 7.15-7.19 (d), 7.51-7.55 incubated in the presence of pyridoxalphosphate
3
322 M. Rueffer and M. H. Zenk • Distant Precursors of Benzylisoquinoline Alkaloids
750 nmol, a-ketoglutarate 750 nmol and enzyme (up (15 min). The supernatant was saturated to 70%
to 2 mg protein) in a total volume of 300 ul. The (NH)S0 and centrifuged again. The pellet was re-
42 4
mixture was incubated for 1 h at 30 °C, the reaction suspended in 10 mM KP02" buffer at pH 7.5 and
4
terminated by addition of charcoal, and monitored as separated from most of the phenols and alkaloids
above. by passage through a Sephadex G-25 column
(70 x 2.5 cm: 90 ml/h, elution buffer: 10 mM KP02~
4
4. p-Hydroxyphenylpyruvate decarboxylase pH 7.5). The protein containing eluates were com
bined (63 ml) and concentrated by pressure filtration
In 300 ul, the assay system contained [I4COOH]-
(YM10; Amicon, Witten) to 20 ml. The protein so
p-hydroxyphenylpyruvate 25 nmol (104 cpm), thia
lution was applied to a QAE-ion exchange column
mine pyrophosphate 50 nmol, MgCl 100 nmol,
2 (10x2.5cm; Pharmacia, Freiburg; 30 ml/h) and
phosphate buffer 66 (mmol (pH 7.0) and enzyme (up
eluted with a 0—1 M KCl gradient in suspension buf
to 1 mg protein). This mixture was incubated in an
fer. Fractions with a 3.5 ml volume were collected
Eppendorf vial for 1 h at 30 °C. The liberated 14C0
2 and those containing the enzyme (no's 67—76) were
was assayed as in the case of the tyrosine decarboxyl
pooled and used as an enzyme source.
ase.
b) Phenolase
5. Amineoxidase
A pressure filtrate from Berberis stolonifera cells
In a total volume of 300 ul [ring-3H]tyramine
was prepared as described under a). The concentrate
10 nmol (1.2 x 104 cpm) in 50 \IM KP02" buffer and
4 (20 ml) was added to a hydroxylapatite column (Bio-
protein (up to 1 mg) were incubated for 40 min at
Rad, Richmond; 1 x 10 ml, flow rate 20 ml/h). The
30 °C. The reaction was terminated by the addition
phenolase does not bind to the column material
of 0.1 ml IN HCl and the mixture extracted with
under these conditions and was eluted with the wash
400 ul isoamyl alcohol (30 min). After centrifugation
ings (30 ml, 10 mM phosphate buffer, pH 7.5). This
(3 min) a 200 ul aliquot of the organic phase was
fraction was added to a DEAE column (Amicon-
counted (Rotiszint; Roth, Karlsruhe) in order to de
Matrex™Cellufine-AH) where it was again eluted
termine the presence of any transformed [3H]-/?-hy-
with the washings (35 ml). The protein solution was
droxyphenylacetaldehyde.
concentrated (ultra filtration) and used as an enzyme
source.
6. ATP: L-Methionine-S-Adenosyltransferase
The assay system contained in a total volume of c) Transaminase
150 5 [Amol KCl, 5 umol MgCl, 20 umol Tris-
2 Frozen tissue of the species under investigation
buffer, pH 8.5, 1 umol ATP, 10 nmol [S-
was thawed with stirring in 50 mM Na-pyrophosphate
C3H]methionine (60000 cpm) and up to 0.5 mg pro
3 buffer pH 7.0 containing 10~4 M thiamine pyrophos
tein. The reaction (1 h at 37 °C) was terminated by
phate and 10~4 M MgCl. An equal amount of poly
the addition of 50 ul aliquots of the incubation mix 2
vinylpyrrolidone (Polyclar AT) was added. After
ture onto 1 cm2 pieces of filter paper (Whatman-P81
20 min the slurry was filtered through cheesecloth
phosphocellulose). These were dried with a heat
and centrifuged for 10 min (15000Xg). The super
gun, washed in a Buchner-funnel with 4 1 water, and
natant was saturated with (NH)S0 (to 709b) and
placed into scintillation vials containing 5 ml Quick- 42 4
the precipitated protein sedimented. The pellet was
szint 2000 (Zinsser). This method was modified after
resuspended in a small amount of extraction buffer
the procedure of Markham [14].
and used as an enzyme source.
Enzyme purification
d) p-Hydroxyphenylpyruvate decarboxylase
a) Tyrosine decarboxylase
Frozen tissue of Berberis stolonifera was extracted
Frozen cells of Berberis stolonifera (150 g) were as given for tyrosine decarboxylase, however, all buf
thawed under stirring in 0.1 M KPO2- buffer pH 7.5 fers used contained 20 mM ß-mercaptoethancl. The
containing 20 mM ß-mercaptoethanol. After 20 min eluate from the Sephadex G-25 column was immedi
the suspension was centrifuged at 12000 xg ately applied to a DEAE-column (15 x 2.5 cm. Ami-