This article was downloaded by: [McMaster University]On: 03 November 2014, At: 13:30Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK

Analytical LettersPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lanl20

4-Bromophenacyl Esters ofGibberellins, Useful Derivativesfor High Performance LiquidChromatographyRoy O. Morris a & Joe B. Zaerr aa Departments of Agricultural Chemistry and ForestScience , Oregon State University , Corvallis, OR,97331Published online: 24 Feb 2007.

To cite this article: Roy O. Morris & Joe B. Zaerr (1978) 4-Bromophenacyl Estersof Gibberellins, Useful Derivatives for High Performance Liquid Chromatography,Analytical Letters, 11:1, 73-83, DOI: 10.1080/00032717808082227

To link to this article: http://dx.doi.org/10.1080/00032717808082227

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all theinformation (the “Content”) contained in the publications on our platform.However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness,or suitability for any purpose of the Content. Any opinions and viewsexpressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of theContent should not be relied upon and should be independently verified withprimary sources of information. Taylor and Francis shall not be liable for anylosses, actions, claims, proceedings, demands, costs, expenses, damages,and other liabilities whatsoever or howsoever caused arising directly orindirectly in connection with, in relation to or arising out of the use of theContent.

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan,sub-licensing, systematic supply, or distribution in any form to anyone isexpressly forbidden. Terms & Conditions of access and use can be found athttp://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

ANALYTICAL LETTERS, All(l), 73-83 (1978)

4-BROMOPHENACYL ESTERS OF GIBBERELLINS, USEFUL DERIVATIVES FOR H I G H PERFORMANCE L I QU I D CHROMATOGRAPHY

KEY WORDS: G i b b e r e l l i ns , 4-bromophenacyl e s t e r s , HPLC, Mass s p e c t r a

Roy 0. M o r r i s and Joe B. Zaerr

Departments of A g r i c u l t u r a l Chemistry and Fo res t Science

Oregon S t a t e U n i v e r s i t y C o r v a l l i s , O R 97331

ABSTRACT

The 4-bromophenacyl e s t e r s o f s i x g i b b e r e l l i ns were prepared

by crown-ether c a t a l y z e d r e a c t i o n between t h e potassium s a l t s o f

the g i b b e r e l l i n s and 4-bromophenacyl bromide under m i l d non-

aqueous c o n d i t i o n s . The d e r i v a t i v e s were s t a b l e , possessed h i g h

UV absorbance and were w e l l separated by HPLC on uCI8 o r cyanopropyl

s i l i c a . The e s t e r s (and the corresponding TMS e t h e r s ) gave w e l l -

d e f i n e d mass spec t ra w i t h e a s i l y i d e n t i f i a b l e mo lecu la r ions. The

combinat ion o f chromatographic and mass spec t roscop ic p r o p e r t es

suggests t h a t t he d e r i v a t i v e s may be very use fu l f o r a n a l y s i s o f

n a t u r a l l y - o c c u r r i n g g i b b e r e l 1 i ns .

INTRODUCTION

Since 1938, when Yabuta and Sumik i l i s o l a t e d g ibbe re

(GA3) f rom c u l t u r e f i l t r a t e s o f t he fungus GibbereZZa fUj

7 3

l i c a c i d

kuroi,

Copyright 0 1978 hy Marcel Dekker , Inc. All Rights Reserved. Neither this work n o r any part may be reproduced o r t ransmit ted in any f o r m o r by a n y means, e lectronic o r mechanical , including photocopying, microfilming, a n d recording, or by a n y information s torage and retrieval system. without permission in writing f r o m the publ isher .

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

7 4 MORRIS AND ZAERR

standard e x t r a c t i o n procedures f o r g i b b e r e l l i n s have coupled b i o -

assays w i t h a d s o r p t i o n t o c h a r c o a l / c e l i t e , s o l v e n t e x t r a c t i o n and

a v a r i e t y o f chromatographic techniques.

duced t h e use o f t h e methyl e s t e r s and showed t h a t gas chromatography

cou ld be used t o f r a c t i o n a t e complex e x t r a c t s . T h i s approach was

extended by B inks e t a2.3 who showed t h a t g i b b e r e l l i n methy l e s t e r s

and the assoc ia ted t r i m e t h y l s i l y l e t h e r s c o u l d be used i n c o n j u n c t i o n

w i t h gas - I i q u i d chromatography and mass spec t romet ry (GLC-MS) t o

i d e n t i f y unknown g i b b e r e l l i n s i n r e l a t i v e l y crude e x t r a c t s .

lkekawa and Sumiki‘ i n t r o -

Recen t l y , h i g h performance 1 i q u i d chromatography (HPLC) has been

used t o concen t ra te4 and f r a c t i ~ n a t e ’ ’ ~ another c l a s s o f p l a n t hormones

the c y t o k i n i n s . The a p p l i c a t i o n of t h e i n h e r e n t l y h i g h r e s o l v i n g

power o f HPLC t o t h e g i b b e r e l l i n s would be advantageous i f s u i t a b l y

UV-absorbing g i b b e r e l l i n d e r i v a t i v e s c o u l d be prepared. Below, we

r e p o r t t he p r e p a r a t i o n o f such d e r i v a t i v e s , t h e g i b b e r e l l i n 4-bromo-

phenacyl e s t e r s w i t h p r o p e r t i e s s u i t a b l e f o r b o t h HPLC and GLC-MS.

MATERIALS AND METHODS

Standard GA were t h e generous g f t o f D r . G . E l son , 3 ,4,5,7,9&13 P l a n t P r o t e c t i o n L t d . , U.K. The 4-bromophenacy e s t e r s were prepared

by t h e method of D u r s t e t d.’

bromide (0 .15 pmole, o r 0.45 pmole f o r GA ) i n d r y a c e t o n i t r i l e

(100 P I ) were mixed w i t h t h e potass ium s a l t o f t h e co r respond ing

g i b b e r e l l i n (0.15 Urnole). The r e a c t i o n was performed i n M i c r o f l e x

tubes (Kontes Glass Co.) a t 6OoC f o r p e r i o d s o f up t o 1 hour and was

c a t a l y z e d by from one- ten th t o equirnolar amounts o f 18-Crown-6

( A l d r i c h Chemical Co.).

Equimolar amounts o f 4-brornophenacyl

13

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

4-BROMOPHENACYL ESTERS OF GIBBERELLINS 75

Sometimes, when examining c rude a c i d m i x t u r e s we found i t more

convenient t o mix equimolar a m u n t s (determined by t i t r a t i o n ) , o f

t h e crude a c i d m i x t u r e , kbromophenacyl bromide, and 18-Crown-6 i n

a c e t o n i t r i l e and then add an excess o f anhydrous potass ium b i ca rbo -

nate. The r e a c t i o n m i x t u r e was heated as b e f o r e w i t h i n t e r m i t t e n t

s ha k i ng . I n bo th cases, t h e e s t e r s were e x t r a c ed i n t o ch lo ro fo rm, washed

w i t h water , d r i e d in uaem and used d i r e c t y f o r HPLC. Where

a p p r o p r i a t e , t h e TMS e t h e r s were formed from t h e e s t e r s by r e a c t i o n

w i t h b is-( t r imethy l s i 1 y 1 ) - t r i f 1 uoracetam i de c o n t a i n i n g 1 % t r i rnethy 1 -

c h l o r o s i l a n e ( R e g i s i l , RC-2; Regis Chem. Corp.) a t 6OoC f o r 2 hours.

INSTRUMENTAT I ON

HPLC was performed on m i c r o p a r t i c u l a t e , o c t a d e c y l - s u b s t i t u t e d

s i l i c a columns (pBondapak/C18, Waters Assoc.) o r on cyanopropyl

s i 1 i c a (Va r ian Assoc.) us ing a Waters Assoc ia tes Model 600 chromatog-

raph equipped w i t h a s o l v e n t programmer.

e l u t e d w i t h a l i n e a r l y i n c r e a s i n g g r a d i e n t o f e thano l (5O-lOO%) i n

0.02 M ammonium a c e t a t e , pH 3.5. The PCN columns were e l u t e d w i t h

a l i n e a r s o l v e n t g r a d i e n t f rom hexane : ch lo ro fo rm (90 : 10 v / v ) t o

ch lo ro fo rm : 2-propanol (I00 : 1 v / v ) .

The uC18 columns were

Mass s p e c t r a were recorded on a V a r i a n MAT-CH7 magnetic s e c t o r

inst rument equipped w i t h a d a t a system (System 150, System I n d u s t r i e s )

A l l s p e c t r a were recorded a t 70 e.v.

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

subsequent ly e l u t e d w i t h a g rad ien

t o 100%. A s expected, t he d e r i v a t

i n approx imate o r d e r o f decreas ing

hyd roxy la ted g i b b e r e l l i n s (GA 3,495,

MORRIS AND ZAERR 7 6

RESULTS

P r e p a r a t i o n and Chromatography o f Es te rs

E s t e r i f i c a t i o n was e s s e n t i a l l y complete a f t e r 15 min. a t 6OoC

i f equimolar amounts o f g i b b e r e l l i n s a l t , 18-Crown-6, and 4-bromo-

phenacyl bromide were reac ted i n a c e t o n i t r i l e . Increased r a t i o s

o f reagent t o g i b b e r e l l i n d i d n o t improve the y i e l d ; indeed, a ten-

f o l d excess o f t he reagent s u b s t a n t i a l l y reduced the y i e l d o f e s t e r .

I n a d d i t i o n , excess reagent appeared on subsequent HPLC and obscured

one e s t e r peak. E s t e r i f i c a t i o n y i e l d was b e t t e r than 95%.

F i g u r e 1 shows t h e separa t i on o f s i x 4-bromophenacyl e s t e r s o f

Samples were a p p l i e d i n known g i b b e r e l l i n s by HPLC on ~ C , ~ - s i l i c a .

0.02 M ammonium a c e t a t e b u f f e r (pH 3 .5 ) c o n t a i n i n g 50% ethanol and

o f e thano l i n c r e a s i n g l i n e a r l y

ves were e l u t e d from t h e column

p o l a r i t y . Thus, t h e more p o l a r

7) were e l u t e d b e f o r e the l e s s

p o l a r GAg.

o f GA13.

I n turn, GA was e l u t e d b e f o r e t h e t r i c a r b o x y l i c e s t e r 9

R e s o l u t i o n o f r a t h e r c l o s e l y r e l a t e d g i b b e r e l l i ns was good.

The i somer i c GA4 and GA which d i f f e r o n l y i n t h e p o s i t i o n o f a

s i n g l e r i n g hydroxy l group, separated w e l l . Reso lu t i on o f GA4 and

&A7, which d i f f e r o n l y i n t h e presence o f a double bond i n r i n g A ,

was s a t i s f a c t o r y . Unlabeled peaks i n F ig . 1 and t h e shoulder on

GA a r e u n i d e n t i f i e d i m p u r i t i e s i n t h e g i b b e r e l l i n s tandards.

5 ’

3 Good r e s o l u t i o n o f t h e s i x e s t e r s a l s o occurred on cyanopropyl

s i l i c a . Using a g r a d i e n t of i nc reas ing p o l a r i t y (hexane t o c h l o r o -

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

4-BROMOPHENACYL ESTERS OF GIBBERELLINS 7 7

h

E c d- Lo N

W 0 z fn LT 0 v)

W

a

m a

0

GA7

F ig . 1. Separat

V C 8 - 5 i

1

L

5 10

TIME (m in )

U

15

3

5

on of 4-brornophenacyl g i b b e r e l l i n e s t e r s on

i ca.

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

MORRIS AND ZAERR 78

GA,, GA and form : 2-propanol ) , t h e o r d e r o f e l u t i o n was GA13 , GA9,

GA (da ta n o t shown). A l though t h i s was e s s e n t i a l l y t he reve rse o f

t h a t on p C 1 8 - s i l i c a , t he columns do n o t a c t i n a s t r i c t l y r e c i p r o c a l

f ash ion . T h i s cou ld be seen w i t h e s t e r s d e r i v e d f rom crude m i x t u r e s

o f p l a n t a c i d s . A s i n g l e f r a c t i o n on VCN s i l i c a (corresponding i n

5

3

m o b i l i t y t o , say, G A ) was reso lved on pc18 s i l i c a i n t o a mu I t

o f peaks spaced w e l l appar t on t h e e l u t i o n g r a d i e n t .

3

The molar absorbance o f t h e 4-bromophenacyl group i s subs

I n t h e e s t e r s , t h e Amax = 256 nm and t h e molar absorbance ( f o r

p l i c i t y

a n t i a l . GA ) = 3

19,100. Such absorbance a l l owed ready e s t i m a t i o n o f sub-microgram

l e v e l s o f g i b b e r e l l i n s ; 5 ng o f GA3 was e a s i l y seen.

Mass Spectra o f 4-bromophenacyl E s t e r s

Mass s p e c t r a were r e a d i l y o b t a i n e d a t a probe temperature o f

250-275°C and mo lecu la r ions were p resen t a t reasonable i n t e n s i t i e s .

A t y p i c a l mass spectrum, t h a t o f 4-bromophenacyl GA4, i s shown i n

F i g . 2 . The mo lecu la r i on i s a doub le t a t m/e 528, 530 due t o t h e

two i so topes of bromine, 79Br and 8 1 B r , p resen t i n app rox ima te l y

equal abundance. Doub le ts a t m/e 183, 185 and m/e 155, 157 rep resen t

t h e fragment ions BrC6H4C0

Major fragments d e r i v e d f rom the g i b b e r e l l a n e nuc leus appeared as

i ons a t m/e 3 1 4 (M-214, l o s s o f OH' and HC02cH2COC6H4Br); 286 (M-244,

l o s s o f HCo,CH,CoC6HqBr) ; 244 (M-306, loss o f HCO2CH2COC6H4Br, C02,

H20), and 269 (M-259).

made by Binks e t d 3 f o r t h e methyl e s t e r s .

major fragment ions, two smal l doub le ts were p resen t a t M - 1 8 ( l o s s

o f H20) and M-62 (loss o f H20, C02).

+ + and B r C H d e r i v e d f rom the e s t e r . 6 4

A1 1 these assignments a r e analogous t o those

I n a d d i t i o n t o t h e

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

4-BROMOPHENACYL ESTERS OF GIBBERELLINS

- - rr N

+-

9

II 10 -4: OD N ," E

7 9

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

80 MORRIS AND ZAERR

T r i m e t h y l s i l y l a t i o n of t he 4-bromophenacyl GA e s t e r s produced

v o l a t i l e d e r i v a t i v e s w i t h accep tab le mass spec t ra . The TMS e t h e r

o f 4-bromophenacyl GA4 had a doub le t M ’ a t 600 and 602 and t h e

expected major fragment ions a t m/e 471, 473, 403, 386, 358, 269,

and 224.

+

To determine t h e u t i l i t y o f t h e method when a p p l i e d t o n a t u r a l

samples, t h e mixed a c i d s d e r i v e d f rom a commercial GA p r e p a r a t i o n

were e s t e r i f i e d and f r a c t i o n a t e d on pCN s i l i c a ( F i g . 3 ) . The r e -

s u l t i n g f r a c t i o n s were no t s u b j e c t t o f u r t h e r chromatography b u t

were analyzed d i r e c t l y by mass spect rometry . The sample con ta ined

the expected GA4 and GA

many minor components were observed bu t i n v e s t i g a t e d f u r t h e r o n l y

4 9 7

and s i g n i f i c a n t amounts of GA3. I n a d d i t i o n , 7

f o r F r a c t i o n 15 ( i n d i c a t e d by t h e arrow).

severa l components,one o f which gave a mass

o f a GA e s t e r . Peaks were observed a t 526,

248 and 221. The spectrum was c o n s i s t e n t w

h i s proved t o c o n t a i n

spectrum c h a r a c t e r i s t i c

528 (Mt) , 283, 267,

t h t h e pub1 ished s t r u c t u r e

o f GA36.

t i o n .

Work i s c u r r e n t l y underway t o s u b s t a n t i a t e t h e i d e n t i f i c a -

D I SCUSS I ON -- The p r e p a r a t i o n o f seve ra l UV-absorbing o r f l u o r e s c e n t d e r i v a t i v e s

of c a r b o x y l i c ac ids has been repo r ted r e c e n t l y . Several techniques

a r e a v a i l a b l e f o r p r e p a r i n g pen ta f l uo robenzy l esters8” .

r e p o r t s concern the f a c i l e s y n t h e s i s o f 4-methoxyanil i des ” and t h e

Other

f l u o r e s c e n t 7-methoxycoumaryl e s t e r s o f f a t t y a c i d s 1 1 .

The 4-brornophenacyl d e r i v a t i v e s desc r ibed he re have some advantage

over t h e pen ta f l uo robenzy l d e r i v a t i v e s i n t h a t t h e e s t e r i f i c a t i o n

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

4-BROMOPHENACYL ESTERS OF GIBBERELLINS 81

I

0 5 10

T IME (m in )

F ig . 3. Separat ion on p C N - s i l i c a o f t h e 4-brornophenacyl e s t e r s

o f crude mixed a c i d s f rom a commercial g i b b e r e l l i n

p r e p a r a t i o n . F r a c t i o n 15 i s i n d i c a t e d by the arrow.

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

a2 MORRIS AND ZAERR

process does n o t seem t o be comp l i ca ted by reagent p o l y m e r i z a t i o n

8 under bas i c c o n d i t i o n s as r e p o r t e d f o r pen ta f l uo robenzy l bromide .

The crown-e he r c a t a l y z e d r e a c t i o n may be performed under m i l d cond i -

t i o n s and, n t h e e s t e r s produced, the mass s p e c t r a i n d i c a t e t h a t

the l a c t o n e b r i d g e across r i n g A o f t h e g i b b e r e l l i n molecule

r e t a i n s i t s i n t e g r i t y . The mass s p e c t r a l f ragmen ta t i on p a t t e r n s

a r e easy t o i n t e r p r e t and a r e analogous t o those p u b l i s h e d f o r t h e

methyl e s t e r s . I n v iew o f t he f a c i l e l o s s o f t h e e s t e r mo ie ty

t h i s i s perhaps no t s u r p r i s i n g . The d e r i v a t i v e s possess an

a d d i t i o n a l advantage i n t h a t t h e mo lecu la r i o n i s r e a d i l y recogn izab le

because o f t h e presence o f t he two bromirle i so topes . Bromine i s o -

tope r a t i o s should l ead t o ready d e t e r m i n a t i o n of the number o f

ca rboxy l groups i n t h e o r i g i n a l a c i d . F i n a l l y , t h e presence o f

3

t he bromine atom shou ld render t h e e s t e r s s u s c e p t i b l e to d e t e c t i o n

on GLC by e l e c t r o n cap tu re . T h i s aspect i s c u r r e n t l y under i n v e s t i g a -

t ion.

ACKNOWLEDGMENTS

Technica l Paper No. 4711 f rom t h e Oregon S t a t e U n i v e r s i t y

A g r i c u l t u r a l Experiment S t a t i o n . The a u t h o r s w i s h t o thank

Karen Southward and Don G r i f f i n for v a l u a b l e ass i s tance .

REF EREN C E S

I. T. Yabuta and Y. Sumiki. J. Agr. Chem. S O C . Japan 14, 1526 ( 1 938).

2 . N. lkekawa and Y. Sumik i . Chem. and Ind. 1728 (1963) .

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014

4-BROMOPHENACYL ESTERS OF GIBBERELLINS a3

3. R.

4. R .

5. N.

6. T.

7. D.

8. 0 .

9. 8.

10. N.

1 1 . w.

B inks, J . MacMi l lan and R.J. Pryce. Phytochem. 8, 271 (1969).

0. M o r r i s , J. B. Zae r r and R . W. Chapman. P l a n t a ( B e r l . ) - 1 3 1 , 271 (1976).

G. Carnes, M. L. Brenner and C . R . Anderson. J. Chromatog. - 108, 95 (1975).

H. Thomas, J. E. C a r r o l l , F. M. R . Isenberg, A. Pendergrass, and L. Howel l .

Du rs t , M. Mi lano , E . J. K i t k a , J r . , S. A. Conne l l y and E . Grushka. Ana ly t . Chem. 9, 1797 (1975).

P l a n t P h y s i o l . 2, 410 (1975).

Gy l l enhaa l , H . B r o t e l l and P. H a r t v i g . J . Chromatog. 2, 295 (1976).

Davis . A n a l y t . Chem. 9, 832 (1977).

E . Hoffman and J . C . L iao. Ana ly t . Chem. 5, 1104 (1976).

Dtnges. Ana ly t . Chem. 9, 442 (1977).

R e c e i v e d N o v e m b e r 2 8 , 1 9 7 7

A c c e p t e d D e c e m b e r 16, 1977

Dow

nloa

ded

by [

McM

aste

r U

nive

rsity

] at

13:

30 0

3 N

ovem

ber

2014