%

re­ 55

re­

ac­

the

254

Ep­

rutin,

5

from

vcrinı­

oven

Pack­

at

ve

record·

An

TI1e ESI-MS

Reaksiyo11

was

C

esterifica­

synthetic

enzhnatik

higher

tube

yüksek

transesteri­

B,

commercial

rutinose. 300.

of

were

triglikozitleriııiıı

isolated

isocratic

reading

(Hewlett

their

throughout

and

s;

glass

WM

rnL/min.

1

polyhydroxylated

Açilleunıesi

by

HP

1

glikozit/er,

açillenmiştir.

Spectrum)

rnoiety

nıonoesteri

µC

used

spectra

flavonoit

of

ile

yield

Enzymatic

5

chromophores.

rate

ksantornnınin

detector,

distillation

Bruker

was

GKR-51

Mass edilmiştir.

HPLC:

rutinin flavonoit

100

high

triglycosides

the

Eıızinıatik

identified

flow

by

and

elde

üzerinden

5436 Array

substilisin-catalyzed

subtilisin

lH-NMR

Ester

açillenıe

acylation

diglycosidic TSQ-700. Far Büchi

:

with

were

400 the

a

unchanged

40:60;

üzerinden the

verirnle

on

Diode Ionisation

proteaz

glikozitlerinden

galaktoz

Nucleosil

followed

AM

flavonoid

Methods

and

has

TFEC,

düşük

Finnigan

glukoz

Glikozitlerirıiu

petiolaris

nrn;

and

incubator.

procedures:

report

two were

kelinıeler

1040M

Thermornixer

çok

on

times

piridinde

edilirken,

Bruker

Flavonoit compounds

which

as

and

Glycosides

of

regioselective

350

HP

now

RÜEDI***

on

elde

Flavouoit Özet:

Ie

anlıidr sonucunda,

esterleri Anahtar

the

cornpounds3.4. We tion

rutin, Rhamnus

General Material (Electro-Spray

ed

corded fications

ard; tention and HCOOH/MeOH

pendorf

ylated study TFEB wasuscd.

Petcr

of

of

at

ru­

of

loıv

ıoitlı na­

are be­

acy-

ma­

and

Regioselective

sub­

feru­

glııcose

a11d

in

protense

direct

Institut,

witlı

are

Besides

acids

the

sorne C,

its

cerebro­

catalyze

ERTAN**,

bi/oba

the

and

by

particular

g.

B,

occııred

circulation

011

triglycosides.

of

to

of

Department

Department

enzyrnatic

e.

of

enzyme

esters

ones,

addressed.

occııred

quercetin

ARAŞTIRMALAR

it

be

be

Ginkgo different

Mcvlüt

Flavonoid

moieties.

actioıı

distributed

enzyme-mediated

acylatioıı

arterial

obtained of

flavonoid

monoester

But

solvents These

Pharmacy,

Pha;macy,

glycosides,

with

should an

from Ankara-TÜRKiYE.

symptoms

1995 The

activities,

be

two

frequent

p-coumaroyl

of would

of

of

sugar

xaııtlıorlıanınins

poor

proteolytic

catalytic

single

widely

tJıe

the

06100

a

ÖZİPEK*,

/BİLİMSEL

thus

Organisch-G1emisches

of

esters

the organic most ı.2. 55-59,

the

and

selectivity.

f!avonoid

extract are

on

their

cannot

Ankara-TÜRKiYE.

Faculty

Faculty

as

pyridine.

in

20,

by

the

derivatives

giving of

the

moiety

glycosides,

years

cinnamoyl,

corresponde11ce

06100

Sci.,

Meltem

derivatives

extract

are

of

Chemistry, Zürich,

used

effects

Acylated

lation.

found

19.1.1995

29.6.1994

excellent

rutin

AR.TICLES

:

: :

the

the

acylated

the

pharmacological

glucorhamnosides.

Univer.sity,

U11iversity,

aıılrydrous

whom

glycosides

galactose Zürich-Switzerland.

P/ıarrıı.

by

been

to

esterification,

Flavonoid

roitlı have

to

in

often

insufficiency

positions

been j.

In.recent

the

slıoıoing

have to ÇALIŞ*t,

acylglycosides

esters,

has

p-coumaroyl

yield

011

and

words

derivatives

lıaı~

cornpounds

Hacettepe Pharmacognosy,

Universitaet Author Hacettepe Pharmaceutical

CH-8057

RESEARCH FABAD Enzyme-Mediated

t

Acylations Ihsan

Abstract:

tin

lıiglı rııoicty

sııbtilisi11

yield Key

Received **

Accepted

Introduction

ture

these Flavonoid

specific which

the loyl

jor kaempferol

lieved vascular

displayed *** These

interest. tilisin chemical approach

-

= = 1) 1)

% %

in in

%, %,

40 40

µL µL

be­

J J

for for

and and

The The

day, day,

been been

After After

2.9 2.9

spec­

7.55 7.55

50 50

XCBu XCBu

(q, (q,

the the

results results

20.8 20.8

5th 5th

data data

Rutin, Rutin,

had had

TFEB TFEB

(Figure (Figure

day day

and and in in

Hz), Hz),

rpm. rpm.

anhydrous anhydrous

4.61 4.61

NMR NMR

the the

synthesized synthesized

from from

% %

RuBu, RuBu,

% %

mg mg

µL µL

o o

16 16

trifluoroethyl trifluoroethyl

10 10

of of

These These

2 2

: :

by by

Next Next

= =

1400 1400

cinnamate. cinnamate. 75 75

On On

61 61

(30 (30

µL µL

was was

day, day,

days days

%, %,

xanthorhamnins: xanthorhamnins:

mL mL

reported reported

formed formed

Hz). Hz).

1 1

(d,) (d,)

12 12

30 30

day, day,

36 36

with with

8th 8th

tested tested

yield. yield.

with with

the the

and and

to to

CDCI3) CDCI3)

were were

2,2,2-trifluoroethanol 2,2,2-trifluoroethanol

% %

C), C),

characteristics characteristics 45° 45°

the the

7.81 7.81

J=7.5 J=7.5

with with

substrate substrate

After After

same same

was was

at at

23 23

with with

(t, (t,

rutin rutin

and and

On On

It It

MHz, MHz,

and and

respectively. respectively.

added added

µL µL

8 8

Hz), Hz),

the the

trifluoroethyl trifluoroethyl

XBBu XBBu

of of

obtained obtained

B B

with with

50 50

01 01

(80 (80

0.97 0.97

16 16

On On

added. added.

µL µL

was was

shaken shaken

O/o O/o

following following

= =

again. again.

TFEB. TFEB.

RuBu RuBu

yield, yield,

observed observed

was was

cinnamate(TFEC) cinnamate(TFEC)

40 40

XCBu, XCBu,

Hz), Hz),

as as

% %

was was

H). H).

agreement agreement

the the

\ \

mg) mg)

\ \

1 1

RUBU3D.D RUBU3D.D

RUBU3D.D RUBU3D.D

(d,) (d,)

were were

~ ~

i i

acylations acylations

were were

1 1

and and

obtained obtained

' '

1.5 1.5

1 1

' '

added added

(35 (35

lH-NMR lH-NMR

RuBu RuBu

way way

and and

containing containing

)=7.5 )=7.5

) )

6.49 6.49

good good

RuBu, RuBu,

TFEC TFEC

of of

OT OT

6 6 was was

and and

was was

respectively. respectively.

in in

cinnamoylchloride cinnamoylchloride

XBBu XBBu

(m, (m,

(5xArom. (5xArom.

Xanthorharnnin Xanthorharnnin

mg mg

same same

% %

Hz), Hz),

in. in.

days, days,

ginning, ginning,

100 100

2 2 and and

TFEB TFEB

RuBu RuBu yield yield

suspensions suspensions

0.7 0.7 pyridine pyridine

butanoate butanoate

mg mg

the the

7.36 7.36

Enzymatic Enzymatic

Subtilisin Subtilisin

troscopy troscopy

from from

8.6 8.6

1.69 1.69 obtained: obtained:

Trifluoroethyl Trifluoroethyl

TFEB7. TFEB7.

were were

Cm Cm

in in

li­

day. day.

by by

(80 (80

7.8 7.8

dis­

mo­

Hz), Hz),

100 100

100 100

puri­

Their Their

s. s.

12th 12th

4 4

Time Time

follow­

pH pH

7.5 7.5

isolated isolated

3-A 3-A

solvents solvents

was was

the the

Rhaınnııs Rhaınnııs 0' 0'

to to

)= )=

Bacillııs Bacillııs

distillation distillation

\Ru \Ru

The The

on on

lt lt

synthesized synthesized

of of

further further

(t, (t,

IH-NMR IH-NMR

5 5

550, 550,

with with

were were

by by

methods methods

methods. methods.

several several

from from

HPLC HPLC

2.39 2.39

was was

fruits fruits

adjusted adjusted

by by

Sigma. Sigma.

without without

with with

Hz), Hz),

shaking shaking

purified purified

obtained: obtained:

2 2

spectral spectral

2,2,2-trifluoroethanol 2,2,2-trifluoroethanol

Dried Dried

by by

from from

8.4 8.4

spectroscopy. spectroscopy.

used used

protease protease

by by

was was

triglycosides triglycosides

= =

C: C:

254,4 254,4 350,4

solution solution

J J

established established

been been

and and

it it

was was

extracted extracted

B B

(q, (q,

R R NMR NMR

the the

and and

drying drying

had had

RuBu RuBu

N,N-dimethyl-4-pyridinamine N,N-dimethyl-4-pyridinamine

by by B B

chromatographic chromatographic

obtained obtained

of of

4.46 4.46

butanoate(TFEB) butanoate(TFEB)

Aldrich. Aldrich.

3.4.21.14, 3.4.21.14,

identified identified

and and

LC LC

LC LC

were were

grade) grade)

flavonoid flavonoid

o o

of of

froİn froİn

chloride chloride

by by

0 0

(Merek). (Merek).

yield yield

1 1

was was

2

tested tested

(EC (EC

from from

dried. dried.

were were

......

H

. .

(ana!. (ana!.

TI1e TI1e

Boiss. Boiss.

apart apart

major major

methodology6. methodology6.

ete ete

150 150

in in

sieve sieve

and and

2501 2501

200 200

1. 1.

was was

CDCl3): CDCl3):

butyryl butyryl

purified purified

presence presence

aııd aııd

two two

freeze freeze

characteristics characteristics

106° 106°

E E

o: o:

::ı ::ı

Figure Figure

56 56

MHz, MHz,

the the

ing ing

from from

at at

general general

lecular lecular

fication, fication,

Trifluoroethyl Trifluoroethyl

Rutin Rutin

solved solved

Pyridine Pyridine

Subtilisin Subtilisin

cheniforınis) cheniforınis)

structures structures

petiolaris petiolaris and and

Xanthorhamnins Xanthorhamnins

and and Çalış Çalış

Materials: Materials:

and and •;11 •;11 FABAD ]. Plıarnı.Sci., 20, 55-59, 1995

and RuCi was obtained with 3.6/1.8 % yield. How­ blocked in the intersugar linkage, then the selectivity ever, XCCi and XBCi couldn't be obtained. On the for OH-C (3") is expected. in addition, seleclivity is same day, 100 µL TFEB was added again. RuBu was independent of the presence and nature of the agly­ formed in 84 % yield at the end of two weeks. The en­ cone. It has been shown that the presence of a large zyme was removed by filtration, the solvent evapo­ aglycone moiety doesn't significantly reduce the re­ rated and the crude residue purified by silica gel activity of the substrate. in a"nother example, the en­ chromatography (CHCl3: MeOH: HıO;80:20:2 as the zymatic butanoylation of the rhamnoglucoside h~­ solvent). ringin, in which the interglycosidic linkage is bet­ ween C (1"'), of rhamnose and C (2") of glucose, oc­ curred as 6"-0-butanoyl ester with ·subtilisin as ex­ Results and Discussion pected. On the other hand, when rhamnose was re­ placed by another sugar like arabinose, the Lipases can catalyze the enzymatic acylation of pri­ estirification occured on the arabinose moiety in ad­ mary hydroxyl groups in various unprotected mono­ dition to glucose3. This shows that subtilisin cannot glycosides, but only Porcine pancreatic lipase and acylate the rhamnose unit.

Chroınobacteriımıviscosııınlipase are active in pyri­ dine. Porcine pancreatic lipase, which regioselective­ As subtilisin was found to be favourable for acyla­ ly acylales the primary hydroxyl group of monogly­ tions of glycosides in previous studies, we preferred cosides in pyridine, was found to be unreactive with lo use this enzyme in our study. di- and oligoglycosides8. The two flavonoid triglycosides (named as xantho­ Enzymatic acylation of sugars in water is thermody­ rhamnins} used in our investigation have the struc­ namically inconvenient and therefore expensive co­ tures as rhamnazin 3-0-[0-a-L-rhamnopyranosyl­ factors are required asa source of free energy. Before (1--;3)-0-a-L-rhamnopyranosyl-(1--;6) 1-P-D-galac­ the process of acylation, pyridine, which is one ofa topyranoside (rhamnazin-3-0-P-rhamninoside= xan­ few organic solvents capable of dissolving sugars thorhamnin C) and rhamnetin-3-0-[0-a-L-rhamnop­ and the enzyme, are dried to eliminate hydrolysis of yranosyl-{1--;3)-0-a-L-rhamnopyranosyl-(1--;6)] - P­ 2,2,2-trifluoroethyl butanoate. in the case of hydroly­ D-galactopyranoside (rhamnetin-3-0-P-rhamninosi­ sis, the enzymatic" acylations are not possible in wa­ de = xanthorhamnin B). ter7,8,9. The third compound rutin, has the diglycosidic The proteolytic enzyme subtilisin is both stable and moiety rutinose[6-0-(a-L-rhamnopyranosyl)-D-glu­ active in numerous anhdyrous organic solvents in­ cose], which is linked to OH-C(3) of the quercetin ag­ cluding pyridine. it can regioselectively acylate di­ lycone. When a solution of rutin in anhydrous pyri­ and oligoglycosides, nucleosides and related large dine was treated at 45° with an excess of trifluoro­ moleculesıo.in several studies, subtilisin was used ethyl butanoate in the presence of subtilisin, 84 % to introduce a butyryl moiety into carbohydrates, conversion was observed after two weeks. In a previ­ e.g., the acylation with subtilisin occurs at OH-C (6") ous study, 65 % tonversion was observed after 48 h, or OH-C (3") of the glucose moiety. If OH-C (6") is with the sameagents, under the sameconditions 3.

Abbreviations This shows that the yield of product increases de­ RuBu Rutinbutyrate pending on time. in our study, TFEB was added in RuCi Rutincinnamate five portions instead of adding the whole amount at XBBu Xanthorhamnin B butyrate once, as stated in the previous study3. This is another XCBu Xanthorhamnin C butyrate factor that effects the percentage of the conversion, XBCi Xanthorhamnin B cinnamate as well as duration. During the acylation of rutin, the XCCi Xanthorhamnin C cinnamate selcctivity for OH-C (3") of glucose was expected, TFEB Trifluoroethyl butanoate since OH-C (6") is blocked in the intersugar linkage. TFEC Trifluoroethyl cinnamate As a result of the reaction, a single product was

57

OH OH

OH OH

OH OH

~~'H ~~'H

OH OH

o o

C C

0

H

ol ol

OH OH

~O ~O

O O

OH OH

OH OH

3 3

: :

OH OH

C C v

OCH

H~Ofl H~Ofl

1

o o

O~OH~~'H O~OH~~'H

O O

O O

">::: ">:::

OH OH

Xan1horhamnın Xan1horhamnın

Rutin Rutin

) )

(XC} (XC}

{Ru) {Ru)

4

HO HO

H,co H,co

Me0H-d

MHz, MHz,

oıı oıı

3"-0-

Ofl Ofl

(300 (300

011 011

and and

C C

l l

H H

(7.4) (7.4)

(7.4) (7.4)

(6.2) (6.2)

(9.5) (9.5)

(9.7) (9.7)

(3.4/9.5) (3.4/9.5)

(1.6/3.4) (1.6/3.4)

(1.6) (1.6)

(9.2) (9.2)

(7.9/9.5) (7.9/9.5)

(7.9) (7.9)

(8.5) (8.5)

(2.2) (2.2)

(2.0) (2.0)

(2.0) (2.0)

(2.2/8.5) (2.2/8.5)

j(Hz) j(Hz)

(RuBu) (RuBu)

(Ru) (Ru)

d d

t t

t t

' ' t t

ı ı

dd dd

d d

d d

br br

dd dd

dd dd

d d

dd dd

CH, CH,

CH, CH,

~=o ~=o

Rutin Rutin

_J _J

il il

1.69m 1.69m

2.40 2.40

1.11 1.11 0.99 0.99

3.27t 3.27t

3.54dd 3.54dd

3.64 3.64

3.4-3.5 3.4-3.5

5.00 5.00

6.87d 6.87d

3.60 3.60

3.79 3.79

5.22 5.22 3.4-3.5 3.4-3.5

4.51 4.51

3.4-3.5 3.4-3.5

7.61 7.61

7.65d 7.65d

RUBU** RUBU** S(ppm) S(ppm)

6.39 6.39

6.20d 6.20d

OH OH

i1. i1.

5• 5•

.__ı .__ı

for for

.O .O

"": "":

OH OH

l' l'

oH~:H]'~o oH~:H]'~o

d d

3"-0-Butanöylrutin 3"-0-Butanöylrutin

ı· ı·

Data Data

I' I'

of of

3.0-3.7 3.0-3.7

1.01 1.01 30-3.7 30-3.7

3.0-3.7 3.0-3.7

3.0-3.7 3.0-3.7

5.3d 5.3d 3.0-37 3.0-37

3.0-3.7 3.0-3.7

4.3d 4.3d 3.0-3.7 3.0-3.7

3.0-3.7 3.0-3.7

6.Bd 6.Bd

3.0-3.7 3.0-3.7

7.5d 7.5d RU' RU' S(ppm) S(ppm)

6.3d 6.3d

6.2d 6.2d

7.5dd 7.5dd

O O

o o

(RuBu) (RuBu)

MeOH-<4 MeOH-<4

">::: ">:::

DMSO-d6 DMSO-d6

Spectral Spectral

in in

in in

'I'~ 'I'~

-

12-

12-CO 12-CO

3

(RuBu) (RuBu)

3"-0-Buıanoylrulln 3"-0-Buıanoylrulln

ı

HO HO

"' "'

MHz, MHz,

-CI -CI

MHz, MHz,

-Cl -Cl

5'" 5'"

cı cı

6'" 6'"

4'" 4'" l l

6" 6"

l" l" 3'" 3'"

2'" 2'"

3" 3"

2" 2" 5' 5'

5" 5" 4" 4"

6' 6'

2' 2'

8 8

H H 6 6

......

300 300

H-NMRSpectrum H-NMRSpectrum 200 200

Butanoylrutin Butanoylrutin

lH-NMR lH-NMR

1

ete ete

*"" *""

2. 2.

ı. ı.

and and

Butanoyl Butanoyl

Rhamnosc Rhamnosc

58 58

Glucose Glucose

Aglycone Aglycone

Figure Figure

Table Table

Çalış Çalış

,, ,, Nlf,; Nlf,; F!llJJ\D ]. Plrartıı.Sci., 20, 55-59, 1995

formed, which was isolatcd and purifil'd by chroma­ 2. Stichcr, (_)_, I-1aslcr, A., Meİl'r,--13., "Ciııkgolıi/o/ıa­ tographic mcthods aııdidcntİfİl'd as 3"-0-butanoyl­ Eincb1..•sti111ınung",Dc11lschc J\potlıckcr Zt·İII111g, 36, rutin by spectroscopic propcrtics(UV, IR, NMR, ESl­ 1827-1835 (1991).

MS). On comparison with the ıH-NMR spcctra of ru­ 3. Danieli, B., De Bcllis, P., "Enzynıc-McdiatcdRcgio­ tin and rutinbutyrate (RuBu) (Figurc 2) (Tablc 1) thc sclcctivc Acylations of Fl<ıvonoidDisacch;ıridc signal corresponding to H-3" of glucosc for RuBu

Monoglycosides", l lcf'u. Clıiııı./\cin., 73, ·ı837-1841 was found downficld indicating the site of acylation. On the other hand, thc ESl-MS cxhibitcd a pcak at (1990). m/z 704,3 IM+H+Nal+ that supportcd thc proposed 4. Danicli, I3., De I3t'llis, P., C:;.ırrc<ı,c:., Riva, S., "En­ structure. zyınc-McdiatcdAcylation of Flavonoid Monog:ly­ cosidcs", I lctcrocycfc::;, 29, 2061-2064 (1988). Howevcr, the two flavonoid triglycosidcs had vcry 5. Özipck, M., Çalış,L, Ertan, M., Rü~di,P., "Cchrio­ Iow rcactivities in thc subtilisin c!ltalyzcd transestcri­ sidc, Rharnnı.:otinCounıaroyl Rharnninosidc froın fication with trifluoroethyl butanoatc undcr thc !Vıan11ıııspctio/aris", J>hytoclıcnıistry,37, 249-253 same conditions(3 % after 8 days), possibly duc to the presence of the galactose unit. From thc rcsults of (1994). this study and from timse rcported in prcvious com­ 6. Stcglich, W., Hölle, C., "N,N-Dimcthyl-4-pyridiııc­ munications, it has bccn shown that subtilisin. is not aminc, a Very Effcctivc Acylation Catalyst", /\11gcıv. a suitable enzyme for acylations of rhamııoscaııdga­ C/ıenı.Jııt.Ed ., 8, 981 (1969). lactose moieties. Also it was obvious that thc pro­ 7. H.iva, S., Klibanov, A. M., "Enzymochcmical Rcgio­ cesses, which were madc in this study to create cin­ selectivc Oxidation of Steroids without Oxidorc­ namic acylations, were not successful as statcd in the previous study4. ductascs", /.Anı.C/ıem. Sac., 110, 3291-3295, (1988). 8. Therisod, M., Klibanov, A. M., "Facilc Enzymatic Based on the successful results of the rutin acylation, Preparation of Monoacylated Sugars in Pyridine", /. it is considered that the acylation of xanthorhamnins Anı.Clıem. Soc ., 108, 5638-5640 (1986). wilh suitable enzymes will be possible in further 9. Riva, S., Chopineau, J., Kieboom, A. P. G., Klibanov, studies. A. M., "Protease-Catalyzed Regioselective Esterifi­ cation of Sugars and Related Compounds in Anhy­

References drous Dimethylformamide", J. Anı.Che11ı. Sac., 110, 584-589 (1988). 1.. Fünfgeld, E.· W., Ginkgo biloba, Recent Results i11 10. Waldmann, H., "Enzymatic Protecting Croup Tech­ Plıannacologyand Clinic, Springer Verlag-Berlin­ Heidelberg-NewYork-London-Paris-Tokyo (1988). niques", Kontakte, 2, 33-54 (1991).

59