Structure and configuration of the polyprenoid side chain of dihydromenaquinones from Myco- and Corynebacteria

BIOCHIMIE, 1973, 55, 591-603.

Structure and configuration of" the polyprenoid side chain of dihydromenaquinones from Myco- and Corynebacteria. (*)(**) R o b e r t AZERAD a n d M a r i e - O d i l e CYROT-I'ELLETIER.

Institut de Biochimie, Universit~ de Paris XI, 91405 Orsay, France. Summary. - - Dihydromenaquinone-9, the typical vitamin K of mycobaeteria, was obtained from Mycobacterium phlei and M. auium and degraded via a chromanyl derivative. The ehromanyl aldehyde obtained was identical to a reference compourui prepared in the same way from synthetic I I - dihydromenaquinone-3 [MK- 3 ( I I - H2)]. These results which confirm previous assumptions based on mass spectroscopy, establish the structure of myeobacterial menaquinones as I I - dihydromenaquinone-9 [MK- 9 ( I I - H~)]. An identical aldehyde was obtained from the menaquinone of Corynebaclerium diphtheriae, which is thus a MK- 8 ( I I - H0. After ozonolysis of MK- 9 ( I I - H~) from M. phlei, oxidation and methylation, the saturated isoprenoid unit was recovered as 4-methyl, 8-oxo nonanoic acid methyl ester, the optical rotation of 'which was found identical, in the 280-550 nm range, to the rotation of the authentic S (--) acid methyl ester, derived synthetically from R (-[-) citronellol. The absolute configuration of the saturated isoprenoid unit of M. phlei dihydromenaquinone [7'S (--) M K - 9 (II-H.z)] is thus identical to that found in the corresponding isoprenoid units of phytol or phylloquinone (vitamin K~). 2 - m e t h y l 3 - p o l y p r c n y l n a p h t h o q u i n o n e s (vitam i n s K) o c c u r in l i v i n g o r g a n i s m s as t w o m a i n g r o u p s of c o m p o u n d s , d i f f e r i n g o n l y in the l e n g t h a n d e x t e n t of s a t u r a t i o n of t h e p o l y p r e n o i d s i d e c h a i n : phylloquinone (I) f r o m plants, a n a p h t h o q u i n o n e w i t h a m o n o u n s a t u r a t e d C20 s i d e c h a i n , and menaquinones (II) f r o m b a c t e r i a , w i t h a C,20O O CH:

0) O CH3 --14 n

(*) This paper is dedicated to Professeur E. Lederer, on occasion of his 65th birthday. Most of this worl~ has been made possible only by his eo~ntinuons encouragement and the facilities offered in using the spectrometry services of the Institut de Chilrde des Substances Naturelles, Gif-sur-Yvette. (**) Nomenclature and abbreoiations as recommended by the I.U.P.A.C.-I.U.B, Committee (Biochim. Biophys. Acta, 1965, 107, 5).

C4~ c o m p l e t e l y u n s a t u r a t e d all-trans side c h a i n [1, 2]. N e v e r t h e l e s s , it w a s d e m o n s t r a t e d in 1963, by t h e F o l k e r ' s g r o u p [3], t h a t t h e m e n a q u i n o n e of t h e g r a m p o s i t i v e Mycobacterium phlei w a s in f a c t an all-trans d i h y d r o m e n a q u i n o n e - 9 , d i f f e r i n g f r o m t h e t y p i c a l m e n a q u i n o n e s by tile o c c u r r e n c e of a s a t u r a t e d i s o p r e n o i d u n i t in t h e s i d e c h a i n : t h i s s t r u c t u r a l a n o m a l y c o u l d be e a s i l y d e t e c t e d by NMR s p e c t r o s c o p y , a n d a c a r e f u l l e x a m i n a t i o n of the s p e c t r u m r u l e d out t h e l o c a t i o n of t h e satur a t e d J s o p r e n o i d u n i t e i t h e r in the t e r m i n a l p o s i t i o n of t h e side c h a i n , o r as t h e first one, n e x t to the ring. A similar quinone with a shorter side chain, ~ K - 8 (H 2) w a s f o u n d , s h o r t l y after, in Corynebacterium diphtheriae [4] a n d a g a i n t h e a b s o l u t e p o s i t i o n of t h e s a t u r a t i o n i n s i d e t h e c h a i n c o u l d not be d e t e r m i n e d . A s y s t e m a t i c i n v e s t i g a t i o n of m e n a q u i n o n e s in t h e t w o m a i n A c t i n o m y c e t e s g r o u p s , Myco- a n d Corynebacteria ( T a b l e I) s h o w e d that all s t r a i n s t e s t e d y i e l d e d MIK- 9 (H 2) in t h e first g r o u p a n d ~ K - 8 (H 2) in t h e s e c o n d one, e x c e p t t h e CTSC s t r a i n of C. diphtheriae, a n m t a n t p r o d u c i n g o n l y t r a c e s of m e n a q u i n o n e s [5]. In all cases, t h e i s o l a t e d d i h y d r o m e n a q u i n o n e s e x h i b i t e d NMR f e a t u r e s i d e n t i c a l to t h o s e p r e c e d e n t l y d e s c r i b e d for M. phlei a n d C. diphtheriae q u i n o n e s . R e c e n t l y , P h i l l i p s et al. [6] h a v e r e p o r t e d t h e p r e s e n c e , in a Streptomyces s t r a i n , of a g r o u p of 39

592

Robert

A zerad and

Marie-Odile

five n a p h t h o q u i n o n e s differing only by the extent of s a t u r a t i o n in t h e i r side chain, a n d identified as ~ K - 9, MK- 9 (H2) , M'K-9 (H4) , MK= 9 (H 6) and MK - 9 (Hs). TABLE

b r e a k i n g w i t h m i g r a t i o n of 1 h y d r o g e n atom (*). This regular f r a g m e n t a t i o n p a t t e r n is i n t e r r u p t e d after 6 losses of 68 mass units and some u n d i s t i n c t peaks appear i n the region M = 300-350, speI.

Distribution of dihydromenaqninones

Strains

Cyrot-Pelletier.

in Myco- a n d C o r y n e b a c t e r i a .

Main quinones produced

Rderences

Corynebactcria C. diphtheriae PW-8..

MK

- 8

(H 0

o CH~

C. rubrum . . . . . . . . . . . C. dipbtheriae C~SC... IlK - 7 and IlK- $

13 ,, .,~7

5

Mycobacleria M. phlei . . . . . . . . . . . . MK - 9 (H2) )) M. avium . . . . . . . . . . . )) M . [or/uitum . . . . . . . . . M. butyricum . . . . . . . . M. luberculosis var. ho)) minis (Brdvannes).. M. Iubercnlosis var. hominis (Canetti). . . . . M. kansasii . . . . . . . . . . M. smegmalis . . . . . . . .

Mass s p e c t r o m e t r y was used as a first a p p r o a c h to the e l u c i d a t i o n of the position of the saturated unit, in collaboration w i t h Professeur E. Lederer, Dr B. C. Das a n d the Mass s p e c t r o m e t r y service of the Institut de Chimie des Substances Naturelles, Gif-sur-Yvette. It was felt that characteristic fragm e n t a t i o n p a t t e r n s should be obtained, revealing a n y i n t e r r u p t i o n of the regularly u n s a t u r a t e d i s o p r e n o i d side chain. The mass s p e c t r u m of MK-9 (H 2) of M. p h l e i is sho~vn in Figure 1 together w i t h the s p e c t r u m of the c o r r e s p o n d i n g quinol diacetate. The p r e p a r a t i o n of this derivative was made necessary because in most cases the q u i n o n e suffers a d i s p r o p o r t i o n a t i o n i n the heated mass spectrometer i n t r o d u c t i o n d e v i c e ; this gives rise to an i m p o r t a n t M + 2 h y d r o q u i n o n e peak a n d to the c o r r e s p o n d i n g f r a g m e n t ions w h i c h increase w i t h time, o b s c u r i n g the entire s p e c t r u m

[101. Two c o n c l u s i o n s could be d r a w n from the spectra of fig. 1. First, a c o n f i r m a t i o n of m o l e c u l a r weight, 2 mass u n i t s h i g h e r than for M K - 9 (784) a n d m e n a q u i n o l - 9 diacetate (870). Secondly, i n both cases, the o c c u r r e n c e of a regular fragmentation p a t t e r n c o r r e s p o n d i n g to repetitive losses of 68 mass u n i t s (CsH s) i n t e r p r e t e d as a diallylic BIOCHIMIE, 1973, 55, n ° 5.

3 13 13 13 13 n1÷n~ = l

13 unpublished ))

cially at M = 313 for the q u i n o n e and M ---- 310 for the diacetate. A very s i m i l a r p a t t e r n is observed w i t h a l l the d i h y d r o m e n a q u i n o n e s - 9 tested and also with M K - 8 (H2) from C. d i p h t h e r i a e a n d C. r u b r u m w i t h the difference that only 5 losses of 68 mass u n i t s o c c u r r e d (fig. 2). These results p r o m p t e d us to propose, as soon as 1964, w i t h Professor E. L e d e r e r [11], the hypothesis that i n all these q u i n o n e s , the saturated isop r e n o i d u n i t was located in second position from the n a p h t h o q u i n o n i c ring. I n this paper, we report some new c o m p l e m e n tary data about mass s p e c t r o m e t r y of d i h y d r o m e n a q u i n o n e s c o m p a r e d w i t h synthetic models, together w i t h a chemical degradation a l l o w i n g an u n a m b i g u o u s localization of the saturated isopren o i d u n i t i n the second p o s i t i o n of the side c h a i n of d i h y d r o m e n a q u i n o n e s from M. p h l e i , M. a v i u m (*) It is interesting to note that, at the contrary, for other isoprenoid compounds tested (ubiquinones [7J, plastoquinones [8], isoprenoid ketones [9]...), the first fragmentation peak corresponds to a loss of 69 mass units. The origin of this discrepancy is not known at this time.

Structure and Configuration of Dihudromenaquinones.

22s 187

M K.9(H2 ]

(: io)

M-w.6 i

593

M-r~.5

M-~,4

M-ss=3

M-ss.2

204

237

M-68

M*

718

786

271

l~',,,~

i~i' r ,

r ,I

M -4Z~2 788

C: Dihydromenaq.inol- 9

diacetate

I:tol

225

720

167

38O

M -42 448

830

T ,"iTi"~ ~, [

2OO

FIG. 1. - - M a s s s p e c t r a of M. phlei M K - 9

I

,_

4OO

600

,i2

I~

672

I

I eoo

m/e

(H:) ( a b o v e ) a n d its q u i n o l d i a c e t a t e ( b e l o w ) .

225

MK-8[H2] 65Q

718 7i3

187 211

)

2T"I

L

368 378

,I,

446

1

I I

514

L:)ol

582

I

,I

Oihydromenaquinol- 8 diacetate

[:101

187

1,

267

229 225[L

200

l

532

310

~

"

,I 400

Fzo. 2. - - M a s s s p e c t r a of C. diphtheriae M K -

BIOCHIMIE, 1973, 55, n ° 5.

T

$6

,

i

584

iI600

i ~?

7~

li

762

,~

800

8(It~) (above) a n d its q u i n o l d i a c e t a t e (below).

m/e

594

R o b e r t A z e r a d and Marie-Odile Cyrot-Pelletier.

and C. diphtheriae ('*). The absolute configuration of the assymetric centre created in the side c h a i n by the s a t u r a t i o n has been d e t e r m i n e d in M K - 9 ( I I - H e ) of M. phlei. MATERIALS A,ND METHODS.

TLC (solvent A). F r o m 4 kg of wet bacteria, 2.54 g of pure d i h y d r o m e n a q u i n o n e were generally obtained. R ( + ) citronellol (a D = + 3.70°), carefully checked for optical p u r i t y , was o b t a i n e d by courtesy of Dr Teisseire, Roure B e r t r a n d et D u p o n t Ets, Grasse, F r a n c e ) .

GENERAL.

T h i n layer c h r o m a t o g r a p h y (TLC) was c a r r i e d out on silicagel GF 254 or P F 254 precoated plates (0.25-2 ram) w i t h one of the following solvents : A, h e x a n e - m e t h y l e t h y l k e t o n e ( 9 7 : 3 ) ; B, Benzene ; C, H e x a n e - m e t h y l e t h y l k e t o n e (9 : 1) ; D, H e x a n e - m e t h y l e t h y l k e t o n e (8:2) , E, BenzeneMethanol (9 : 1). Silicagel Merck (0.2-0.5 mm) was used for c o l m n n c h r o i n a t o g r a p h y ; elution was c a r r i e d out w i t h h e x a n e c o n t a i n i n g i n c r e a s i n g a m o u n t s of ether. I n some cases silver nitrate-imp r e g n a t e d silicic acid was used [12]. Gas chromat o g r a p h y (GPC) was c a r r i e d out on a SE 30 (3 p. cent) c o l u m n (1.5~) m length) w i t h n i t r o g e n as c a r r i e r gas (2-2.4 bars) and an i o n i z a t i o n flame detector. UV spectra w e r e m e a s u r e d in cyclohexane solutions a n d IR spectra on t h i n films. N,MR spectra were m e a s u r e d i n CDC13 at 60 Mc./sec. w i t h tetram e t h y l s i l a n e as a n i n t e r n a l reference. Mass spectra w e r e d e t e r m i n e d w i t h an AE[ MS 9 spectrometer o p e r a t i n g at 70 ev. : all samples were introduced t h r o u g h the s t a n d a r d heated inlet system at t e m p e r a t u r e s r a n g i n g from 130 to 250°C. Optical rotations in the 28p0~550 n m range were determ i n e d i n ethanol w i t h a Spectropol I r e c o r d i n g p o l a r i m e t e r (Fica, F r a n c e ) a n d corrected for solvent rotation. Optical rotations at 589 n m were m e a s u r e d on ethanol solutions u s i n g a P e r k i n E l m e r polarimeter, model 141, w i t h digital read out. QUINONES. M K - 3 a n d M K - 3 ( H - H z) w e r e p r e p a r e d as p r e c e d e n t l y described and c h r o m a t o g r a p h e d on silver-nitrate i m p r e g n a t e d silicic acid [12!. MK- 9 was a gift of Dr. O. Isler, Hoffmann La Roche, Basel. Bacterial q u i n o n e s were extracted with acetone a n d c h r o m a t o g r a p h e d as p r e c e d e n t l y described [13]; a p r e l i m i n a r y filtration on florisil, i n hexane, was f o u n d useful for an initial purification of the c r u d e extract. In some cases, an ultimate p u r i f i c a t i o n was p e r f o r m e d by p r e p a r a t i v e (**) A preliminary account of part of this vcork has appeared (R. Azerad, M. O. Cyrot and E. Lederer, Bio-

chem. Biophys. Res. Comm., 1967, 27, 249). BIOCHIMIE, 1973, 55, n ° 5.

DEGRADATION OF MK-3.

Menachromenyl-2 acetate (III) : M K - 3 (3 g) and Nail {1.4 g as a 5,0 p. cent dispersion in m i n e r a l oil) were refluxed in dry b e n z e n e (120 ml) u n d e r n i t r o g e n for 3 hours. 4 n d of acetic a n h y d r i d e were added w i t h cooling and the m i x t u r e was shaken for 1 hour, then treated w i t h acetic acid (4 ml) a n d water (100 ml). E x t r a c t i o n with ether yielded an orange oil w h i c h was chromatographed on silicagel (20.0 g) in hexane. U n c h a n g e d q u i n o n e was eluted w i t h 3 p. cent ether in h e x a n e and pure III (2.1 g) w i t h 6 p. cent ether in hexane. A small a m o u n t of m e n a q u i n o l - 3 diacetate could be eluted w i t h 15 p. cent ether 0, max eye,oh..... = 23.3 nm). U.V. ).,,,.~. (e) : 223, 266.5 (39 OO0), 334, 340 (3 900) and 367 n m ; shoulders at 239 and 248 nm. I.R. Vinax

N.M.R.

: 1 765, 1 650, 1 620, 1 595, 1 565 and 1 20'5 cln -1. : see Table H.

Mass Spectrometry : m / e = 418 ; m a i n fragmentations at m / e = 376 ( M - 42, CH e = C = O), 349 ( M - 6 9 , EsH~), 307 ( M - 4 2 d- 69), 267 (M-151, (~11H19) and 226 (M-15.1 q- 42).

Menachromanyl-2 acetate ( V I ) : Pieces of sod i u m (3 g) were a d d e d d u r i n g 1.5 h o u r to a refluxing solution of m e n a c h r o m e n o l - 2 acetate (1.1 g) in dry ethanol (15 ml), u n d e r nitrogen. After cooling, ethanol (25 ml) then acetic acid (6 ml) then water (100 ml) were slowly added. E x t r a c t i o n w i t h ether a n d d r y i n g yielded an oily residue w h i c h was taken up in p y r i d i n e (0.4 ml) and acetic anhyd r i d e (10 ml). After one hour, acetic acid (5 ml) a n d w a t e r (10 ml) were added a n d the m i x t u r e shaken for 15 minutes. A d d i t i o n of w a t e r and extraction w i t h h e x a n e gave a b r o w n yellow oil from w h i c h the c h r o m a n o l acetate (400 rag) was o b t a i n e d by silicagel c h r o m a t o g r a p h y a n d preparative TLC (solvent C ; Rf = 0.4). U.V. k..... (e) : 245 (4 50~0), 306, 314, 322 and 329 nm ; s h o u l d e r at 242 n m .

Structure and Configuration of Dihydromenaquinones. I.R. Vmax

: 1 760, 1635, 1 210 cm-L

N.M.R.

: see T a b l e III.

1600,

1575

595

tate w h i l e the s e c o n d one, e l u t e d w i t h 30 p. c e n t ether in hexane was 9,12-dihydromenachromanyl-2 acetate.

and

9,10,11,12-tetrahydromenachromanyl-2

M a s s - S s p e c t r o m e t r y : m / e = 420 ; m a i n fragqnent a t i o n s at m / e = 378 ( M - 4 2 ) , 26.9 ( M - 1 5 1 , CzzH~,) , 225-7 a n d 186-7.

acetate :

(V) U.V. kn~a~ : 266,5, 2'74 a n d 277 n m .

TABLE H.

N.M.R. Spectra of chromenyl acetates (*). I

i

I

l I

8

I[

I

7

I MenachromenyI-2 acetate (III) I-Dihydromenachromenyl-2 acetate Menachromenyl-8 acetate I-Dihydromenachromenyl-8 acetate I-Dihydromenachromenyl-7 acetate

7.2-8.3 (m) 7.2-8.3 (m) 7.2-8.3 (m) 7.2-8.3 (m) 7.2-8.3 (m)

6.60 and 5.62 5.12 (d) (d) I (t) 6.62and5.63i 5.08 (d) (d) l (t) 6.63and 5.65j 5.12 (d) (d) j (m) 6(d~2andS(.d6? 5.13,m) 5.13 (m)

2.42 (s) 2.40 (s) 2.43 (s) 2.43 (s) 2.45 (s)

2.23 (s) 2.22 (s)

5

2.24 (s) 2.25 (s)

1.45 (s) 1.45 (s) 1.45 (s) I. 43 (s) 1.44 (s)

(*) All spectra in CDCls ; data given in ppm ; s : s i n g l e t , d = doublet, t :

1.96 (m) 2.0 (m) 2.0 (m) 2.0 (m) 2.0 (m)

il.67andl.57 p(s) (s) i l . 6 8 a n d 1.59 1.26 (m) (s) (s) ,il.67 and 1.60 [(s) (s, i l . 6 7 a n d 1.60 1.27 (m) i(s) (s) ilis~8 and 1.60 1.27 (s) (m)

triplet, m :

0.83 (d)

O, 83 (d) 0.83 (d)

multiplet.

TABLE III.

N.M.R. Spectra'of chromanyl acetates. [

I

I

_5

? k

Menachromanyl-2 acetate (VI) I-Dihydromenachromanyl-2 acetate Menachromanyl-8 acetate I-Dihydromenachromanyl-8 acetate I-Dihydromenachromanyl-7 acetate

7.2-8.3 (m) 7.2-8 3 (m) 7.2-8.3 (m) 7.2-8.3 (m) 7.2-8.3 (m)

5.17 (m)

5/)7

5.12 (m) 5.12 (m) '5.13 (m)

2.43 (s) 2.44 (s)

2i2

2i2

.<

<

2.17 (s) 2.17 (s) 2.17 (s) 2.17 (s) 2.18 (s)

2.73 (t)

I n s o m e p r e p a r a t i o n s a n o t h e r p r o d u c t w a s eluted after t h e c h r o m a n y l e acetate ; it w a s s e p a r a t e d in two eonstituants by column chromatography o n Ag NO a- i m p r e g n a t e d s i l i c i c a c i d : the first one, e l u t e d b y 10 p. c e n t e t h e r i n h e x a n e w a s i d e n t i f i e d as 9, 1,0, 11, 1 2 - t e t r a h y d r o m e n a c h r o m a n y l - 2 ace-

BIOCHIMIE, 1973, 55, n ° 5.

2i 2 2i 2 2i 3 2.75 (t)

1.92 (m) 1.90 (m) 1.92 (m)

2.0 (m) 1.90 (m) 2.0 (m) 2.0 (m) 2.0 (m)

1.35 (s)

liy 1.34 (s)

1.68 and 1.60 (s) (s) 1.67and1.58 (s) (s) 1.68 and 1.60 (s) (s) 1.68and1.60 (s) (s)

I [

1.25 (m)

0.85 (d)

1.25 (m) 1.68 and 1.60 1.25 (s) (s) (m)

0.85 (d) 0.85 (d)

I.R. V~ax : 1 760, 1 580 a n d I 210 c m -1. N.M.R.

: see s c h e m e 1.

Mass S p e c t r o m e t r y : m / e = 4 2 4 ; m a i n f r a g m e n t a t i o n s at m / e = 382 ( M - 4 2 ) , 273 ( M - 1 5 1 ) , 230-2 a n d 190-1.

R o b e r t A z e r a d a n d Marie-Odile Cyrot-Pelletier.

596

9,12-dihydromenaehromanyl-2 acetale : (IV)

Mass S p e c t r o m e t r y : m / e : 326 ; m a i n f r a g m e n t a t i o n s at m / e : 2:84 (M-42), 225-7 a n d 186-7.

U.V. ~ma~ : 266.5, 274 a n d 277 n m . I.R. Vm~x : 1 760, 1 5 7 5 a n d 1 2 1 0 em-L N.M.R.

DEGaADATION OF M K - 3 (II- H2).

: see s c h e m e 1.

Mass S p e c t r o m e t r y : na/e = 42,2 ; m a i n f r a g m e n t a t i o n s at m / e = 38.0 ( M - 4 2 ) , 271 ( M - 1 5 1 ) , 229 a n d 189.

U.V. ~'m,~ (e) : 266.5 (4,0'0,00) a n d 340 n m (3 500). : 1 766, 1 6:50, 1 620, 1 59,5, 1 5,65 a n d 1 205 e m -1.

I.R. v_,~,x

OCOCH3 2 3o s

OCOCH$2 "~ s

58,3~

72 -a3

N.M.R.

.~

.(tit). . . . 2 2 o R. . . . . . (I . . .o 735 s

R

125 s R : C1~H~9

OCOCH3 230s

CH) I 99s

(V)

[

II-Dihydromenachromanyl-2 acetate (460 rag) w a s o b t a i n e d , t o g e t h e r w i t h 580 nag of d i h y d r o - 9 , 1 2 - I I - d i h y d r o m e n a c h r o m a n y l - 2 acetate, f r o m 1.2 g of c h r o m e n y l - a c e t a t e . U.V. kmax (e) : 245 n m (47 I)00).

OCOCH~ 2.30 s

208s H ~ C ~ H )

t98

I.R. Vmax

R

125s

125~

SCHF~ME 1.

<< S H O R T

CHAIN

(VII)

ALDEHYDE)):

M e n a c h r o m a n o l - 2 acetate (1.05 rag) d i s s o l v e d i n e h l o r o f o r m e (1 ml) a n d m e t h a n o l (8 ml) w a s t r e a t e d at - - 3 0 ° C w i t h ozone g e n e r a t e d b y a n <) i n s t r u m e n t o p e r a t i n g at 80 volts w i t h 0.3 l / r a i n o x y g e n . E x c e s s ozone w a s r e m o v e d d u r i n g 15 nainutes w i t h n i t r o g e n at - - 1 5 °, t h e n 0,1 m l of d i m e t h y l s u l f i d e w a s a d d e d . After 1 h at - - 3 0 ° t h e n 1 h at r o o m t e m p e r a t u r e , s o l v e n t s w e r e e v a p o r a t e d a n d the r e s i d u e e h r o m a t o g r a p h e d o n p r e p a r a t i v e TLC ( s o l v e n t D). T h e m a i n U.V. a b s o r b i n g b a n d (Rf = 0.8.8) w a s e l u t e d b y ether and chromatographed again in solvent E to give 39 m g of c r y s t a l l i n e a l d e h y d e (VII), m.p. 92-3 °. U.V. ~,,ax ( 0 : 245 (39600), 308, 3'15, 322 329 n m ; s h o u l d e r at 242 n m . I.R. Vm~x N.M.R.

: see T a b l e II.

Mass S p e c t r o m e t r y : m / e = 420 ; m a i n f r a g m e n t a t i o n s at m / e = 378 ( M - 4 2 ) , 363 ( M - 4 2 + 15), 267 ( M - 1 5 3 ) a n d 225.

R'_ C16H~1

260I

l-Dihgdromenachromanyl-2 acetate (1.2 g) w a s o b t a i n e d as p r e e e d e n t l y d e s c r i b e d , s t a r t i n g f r o m 3 g of M K - 3 ( I I - H 2 ) .

: 2 725, 1 760, 1 7215, 1 635, 1 575 a n d I 210 c m -1.

and 1 60'0,

I

2.69 (t) ~ C H 2 - - - C = O ; 2.45 (s) C H 3 - - , C O - - ; 2.17 (s) A r - - C H 3 ; 2.0,6 a n d 1.92 (2t) ~ C H 2 - - C - - O - - - ;

I

1.33 (s) C H a - - C ~ O - - .

I BIOCHIMIE, 1973, 55, n ° 5.

I

<< L O N G C H A I N A L D E H Y D E )> (VIII) (43 mg) w a s o b t a i n e d b y o z o n o l y s i s of 2:30 m g of c h r o m a n y l acetate.

U.V. )'.~x (e) : 245 riD1 (49 000). I.R. v,~lax : 2 725, 1 760, 1 725, 1 635, 1 600, 1 57'5 a n d 1 210 cm-1. N.M.R. : (see F i g u r e 7) : 9.77 (m) - - C H O ; 7.22-8.3 (m) A r - - H ; 2.75 (t) A r - C H z - - ; 2.45 a n d 2.37 (s + m) CH3~CO-- a n d ~ C H ~ - - . C = O ; 2.,18 (s) A r ~ C H 8 ; 1.9,2 (t) ~ C H 2 -

I

G - - O - - ; 1.35 (s)

I

CHz--G---O-- ;

I

/

1.58-1.25 (m) ~ C H 2 ~ C H ~ ;

0.87

I

: (see Fig. 6) : 9.83 (t) ~ C H O ; 7.228.3 (m) A r c H ; 2.78 (t) A r ~ C H , - - ;

1

: 1 760, 1 635, 1 600, 1 575 a n d 1 210 cm-1. N.M.R. : see T a b l e III. Mass S p e c t r o m e t r y : m / e = 422 ; m a i n f r a g m e n t a t i o n s at m / e : 380 ( M - 4 2 ) , 2~67 ( M - 1 5 5 ) , 22.5-7 a n d 186-7).

(d) C H a ~ C - - .

I Mass S p e c t r o m e t r y : m / e = 39,6 ; m a i n f r a g m e n t a t i o n s at m / e -= 354 ( M - 42), 3,2,6 ( M - 4 2 + 28), 22.5-9 a n d 186. DEGRADATION OF M K - 9.

Menaehromenyl-8 acetate (0.4 g) f r o m 1.04 g of MK-9. I.R. v..... : 1 765, 1 660, 1 205 c m -1.

1 650,

1 620,

1 595

and

Structure and Configuration of Dihydromenaquinones. N.M.R. : see Table lI. Mass S p e c t r o m e t r y : m / e : 82'4; m a i n fragmentations at m / e = 8,09 ( M - 1 5 ) , 782 (M- 42), 307, 267 a n d 225.

Menachromanyl-8 acetate (0.2'05 g) from 0.4 g of m e n a c h r o m e n y l - 8 acetate. N.M.R. : see Table I[I. Mass Spectrometry : m / e :

Dihydromenachromenyl-8 acetate (2.6 g) from 4.35 g of M K - 9 (H2). U.V. ~max (e) : 26'6.5 n m (42 06~)). I.R. Vmax

: 1 765, 1 660, 1 650, 1 620, 1 595 a n d 1 205 cm-L

N.M.R.

: see Table II.

Mass S p e c t r o m e t r y : m / e = 828 ; m a i n fragmentations at m / e : 813 (M-15), 786 (M-42), 771 ( M - 4 2 d- 15), 760 (M-6~8), 718 ( M - 4 2 + 68), 3073a9, 267 and 225.

Dihydromenachromanyl-8 acetate (0.6.74 g) from 1.6 g of d i h y d r o m e n a c h r o m e n y l - 8 acetate. U.V. kmax (e) : 245 n m (55 000). I.R. Vm,~x

: 1 760, 1 6'~0, 1 635, 1 6:00, 1 575 and 1 210 cm-L

N.M.R.

: see Table III.

Mass S p e c t r o m e t r y : m / e = 8 3 0 ; m a i n fragmentations at m / e : 788 ( M - 4 2 ) , 762 (M-68), 720 ( M - 4 2 + 68), 310, 267, 229, 225, 186-7. CHAIN

ALDEHYDE

))

(VIII) (92 nag)

was

o b t a i n e d by ozonolysis of 3,05 mg of d i h y d r o m e n a c h r o m a n y l - 8 acetate, together w i t h traces of h i g h e r homologues, separated by TLC a n d identified by mass spectrometry. DEGRADATION OF M K - 8 (H 2) (from C. diph-

theriae). Dihydromenachromenyl-7 acetate (1.26 g) was o b t a i n e d from 1.45 g of MK - 8 (H2). U.V. ~'max (e) : 2~6.5 n m (42 0~)0). Mass S p e c t r o m e t r y : m / e : 760 ; m a i n fragmentations at m / e = 745 (M-15), 718 ( M - 4 2 ) , 703 ( M - 4 2 + 15), 692 (M-68), 677 ( M - 6 8 + 15), 650 ( M - 4 2 + 68), 624 ( M - 6 8 × 2) 267 a n d 225.

Dihydromenachromanyl-7 acetate (0.460 g) was o b t a i n e d fronl 1.26 g of chromenol-acetate, toBIOCHIMIE, 1973, 55, n ° 5.

gether w i t h 0.3 g of 9 , 1 2 - d i h y d r o - d i h y d r o m e n a c h r o m a n y l - 7 acetate. U.V. ~'r~ax (e) : 245 n m (52 000). Mass S p e c t r o m e t r y : m / e = 762 ; m a i n fragmentations at m / e = 720 (M-42), 694 (M-6,8), 652 ( M - 6 8 + 42), 267-9, 225-9 a n d 186-7. <
8,26.

DEGRADATION OF M K - 9 (H2) FROM M. phlei.

¢ LONG

597

Ozonolysis of 6 S, 10-dimethyl undecene-9,2one (XI). The ketone (XI) p r e p a r e d from high p u r i t y R ( + ) citronellol (X) (a n = + 3.7 °) [12] was p u r i fied on a silicagel c o l u m n a n d eluted w i t h 4 p. cent ether in h e x a n e ; a~~° = - - 1.06 ° (e : 4.6). 1.3 g of 6 S-ketone was treated w i t h ozone i n ethylacetate (20 ml) at --3,O°C u n t i l a blue color was obtained. After flushing w i t h n i t r o g e n for 10 m i n u t e s the solvent was evaporated at 20°C ; to the residue diluted w i t h ethanol (3 ml) was added d r o p w i s e w i t h s t i r r i n g at 0°C a solution c o n t a i n i n g KMnO~ (1.1 g) and MgSO~ (1.1 g) i n w a t e r (10 ml). After 1 h o u r s o d i u m hisulfite was added to remove excess oxidant. The m i x t u r e was strongly acidified a n d extracted w i t h ether. The c o m b i n e d extracts were evaporated to a small volume, then treated w i t h d i a z o m e t h a n e at - - 3 0 ° C . The c r u d e p r o d u c t is c h r o m a t o g r a p h e d on a silicagel c o l u m n (50 g) a n d the fractions eluted from 8 to 16 p. cent ether i n h e x a n e (9,18 mg) c h r o m a t o g r a p h e d again on a similar column. The p r o d u c t was u l t i m a t e l y purified by p r e p a r a t i v e TI, C (solvent D, Rf = 0,33, or E) to give 452 mg of pure 4 S - k e t o ester (IX) (one peak in G P C / 1 2 0 ° C ) . U.V. ~Etoa max (e) : 276 n m (36) • s h o u l d e r at 281 nm. I.R. ~max N.M.R.

: 1 710, 1 730 cm -1. : see Figure 11.

[~1 ~" = ~ 0 . 4 ' 5 ( c : 6 , 6 3 ) . Semicarbazone, m.p. ---- 58-59 °.

Ozonolysis of MK- 9 (II - H2), The keto ester (IX) (40 rag) was obtained by a s i m i l a r t r e a t m e n t of M K - 9 ( I I - H2) from M. phlei (1.5 g). 5 g of KMnO 4 a n d 5 g of Mg SO4 were used in the o x i d a t i o n of ozonolysis products. [a]~"

=

--

0.58 ° (c

: 4.8).

RESULTS AND DISCUSSION. In o r d e r to o b t a i n more i n f o r m a t i o n about the f r a g m e n t a t i o n p a t t e r n of I I - d i h y d r o m e n a q u i n o -

Robert Azerad and Marie-Odile Cyrot-Pelletier.

598

nes, c o m p a r e d to completely u n s a t u r a t e d menaq u i n o n e s , we e x a m i n e d the mass spectra of M K - 3 a n d M K - 3 ( I I - H 2) together with their q u i n o l diacetates, o b t a i n e d by reductive acetylation. These c o m p o u n d s p r e v i o u s l y o b t a i n e d by synthetic methods were carefully purified by c h r o m a t o g r a p h y on silver nitrate i m p r e g n a t e d silicagel E12], to eliminate a n y trace of u n d e s i r a ble oversaturated isoprenolog. Only m i n u t e differences could be observed in the mass spectra of M,K-3 a n d MrK-3 ( [ [ - H u ) (fig. 3) : the f r a g m e n t a t i o n peak at 307 (M-6,9) of M K - 3 is replaced in the s p e c t r u m of M,K-3 ( I I - H 2 ) by a group of peaks at m / e : 302-309 w h i c h r e m i n d s the general p a t t e r n of d i h y d r o m e n a q u i n o n e f r a g m e n t a t i o n i n this region (see fig. 1 a n d 2). No more i n t e r p r e t a b l e difference could be observed i n this region of the mass spectra of the quinol diacetates (fig. 4) : a comm o n peak a p p e a r e d at m / e ---- 310 (M- 70), i n d i c a ting that some r e a r r a n g e m e n t was o c c u r r i n g i n the course of M K - 3 fragmentation, a n d s h o w i n g that some caution was to be exercised i n the int e r p r e t a t i o n of mass spectra l e a d i n g to the preced i n g hypothesis. Such an electron i m p a c t i n d u c e d r e a r r a n g e m e n t has been reported for various isop r e n o i d ketones [9] i n c l u d i n g a C43-isoprenoid ketone c o n t a i n i n g one satured u n i t isolated from tubercle bacilli [14], w h i c h led to a different conclusion i n the assignement of the position of the saturated unit.

*2O l

22~ i 242

i

a~

~S426 2 ~ I I,_~,

310

I,

336

aao

i

i

i 100

0

i2

200

300

400

FIG. 4. Mass spectra of menaquinol-3 d i a c e t a t e (above) and I[-dihydromenaquinol-3 diacetate (below). -

-

The m a i n p r o b l e m was to <> the first i s o p r e n o i d u n i t next to the ring, in o r d e r to utilize usual oxidative t e c h n i q u e s for b r e a k i n g the r e m a i n i n g double bonds. This o x i d a t i o n w o u l d thus begin only at the t h i r d i s o p r e n o i d u n i t in the ease of a I I - d i h y d r o m e n a q u i n o n e (such as MK- 3 ( I I - H~)), or at the second i s o p r e n o i d unit, in the case of M K - 3 or any d i h y d r o m e n a q u i n o n e c a r r y i n g a s a t u r a t i o n i n a n y i s o p r e n o i d u n i t more distal than the second one. The key step of this degradation, the <> of the first i s o p r e n o i d unit, was successfully c a r r i e d out t h r o u g h two successive reactions, examplified in scheme 2 for M K - 3 : isome-

225

a~

As no definitive results could be expected from mass spectrometry, w e u n d e r t o o k , u s i n g M,K-3 ( I I - H 2) a n d M K - 3 as model c o m p o u n d s , to design some chemical d e g r a d a t i o n w h i c h w o u l d allow an u n e q u i v o c a l assignement of the position of the saturated i s o p r e n o i d unit.

,2a9

lS7

OhC ~9 z.~20

109

22~

I

• 95 I 28

tLI I T

OAC aTs

~ 2"Ac20/pyrldine

OA¢

2.Me2S L .....

4oo Fro. 3. - - Mass spectra of MK-3 (above) and MK - 3 (II - H2) (below). 100

200

BIOCHIMIE, 1973, 55, n ° 5.

300

O

O CHO SCHEME

~.

!

Structure and Configuration of Dihydromenaquinones. risation by s o d i u m h y b r i d e in benzene [15] to

menachromenol-2 w h i c h was i m m e d i a t l y acetylated, then t r e a t m e n t of the resulting c h r o m e n y l acetate (III) by s o d i u m i n ethanol, in order to reduce the conjugated double b o n d of the chrom e n e r i n g l 16, 17]. After r e d u c t i o n , the c o m p o u n d was reacetylated a n d c h r o m a t o g r a p h e d on a silicagel c o l u u m to eliminate occasionally o c c u r r i n g c o m p o u n d s w h i c h were identified respectively as the 9,12-dih!Idromenachromanyl-2 acetate (IV) a n d the 9,10,11,12-tetrahydromenachromanyl-2 acetate (V), from e x a m i n a t i o n of their spectroscopic data. The U.V. spectra of the d i h y d r o - - - a n d tetrah y d r o - - c o m p o u n d s were indeed very s i m i l a r to the s p e c t r u m of tt-tocopheryl acetate, a dimethylb e n z o c h r o m a n y l a c e t a t e , as a result of a partial or total s a t u r a t i o n of the b e n z e n o i d r i n g (fig. 5). NM.R. spectra showed again m a n y similarities with tocopheryl acetate as illustrated in scheme 1. Molecular weight m e a s u r e m e n t s by mass spectrom e t r y confirmed the p r e s e n c e of a di-or tetrahydrogenated n a p h t h o c h r o m a n e ring. Such a reduction of the u n s u b s t i t u t e d b e n z e n o i d r i n g was not

599

ced with dimethylsulfide [19] ; a c r y s t a l l i n e aldeh y d i c p r o d u c t was obtained w h i c h was identified by NMR, UV, IR a n d mass spec!roscopy as the expected <~short c h a i n aldehyde >> (VII) (fig. 6). W h e n the same reaction sequence was applied to I I - d i h y d r o m e n a q u i n o n e - 3 (scheme 3) the ¢ long c h a i n aldehyde ~ (VIII) was obtained, as confirOAC 0

(vm) SCHEME

3.

reed by UV, IR, NMR a n d mass spectroscopy (fig. 7). The two aldehydes were easily separated either by t h i n layer c h r o m a t o g r a p h y in various solvents or by gas c h r o m a t o g r a p h y on a siliconated support. The clear s e p a r a t i o n a n d identification of the two derivatives was a chief requi-

i E (\/\ ] •

//^(-/

/,

// // / /

/ / ///

\ /

\ '\

/

\

\.

_~"

/

1//

', ! ',~

', I ',i ii

',i

/

t~

u n e x p e c t e d [18] a n d the a c c u m u l a t i o n of the d i h y d r o - d e r i v a t i v e could be a t t r i b u t e d to the migration of a r e m a i n i n g double b o n d i n the 10,11-unconjugated position, Pure m e n a c h r o m a n y l - 2 acetate (VI) was t h e n treated w i t h a limited excess of ozone in chlorof o r m - m e t h a n o l a n d the ozonolysis p r o d u c t redu-

BIOCHIMIE, 1973, 55, n ° 5.

FIG. 5. - - U.V. spectra (in cyclohexane) of menachromanyl-2 acetate, ( - ), 9,12-dihydromenaehromanyl-2 acetate ( ......... ) and a-tocophenyl acetate (-- • -- ). The UV spectrum of 9,10,11,12tetrahydromenaehromanyl-2 acetate, not figurated here, is nearly identical to the spectrum of the dihydro-derivative.

r e m e n t for a good c h a r a c t e r i z a t i o n of n a t u r a l d i h y d r o m e n a q u i n o n e s , because three possibilities could be a n t i c i p a t e d : a r a n d o m l y d i s t r i b u t e d saturated i s o p r e n o i d u n i t l e a d i n g to 85.7 p. cent of s h o r t e r aldehyde a n d 14.3 p. cent of longer aldehyde ; a saturated i s o p r e n o i d u n i t specifically located either i n the second or a n y o n e of the

Robert Azerad and Marie-Odile Cyrot-Pelletier.

600

o t h e r u n i t s , l e a d i n g r e s p e c t i v e l y to 100 p. c e n t of l o n g e r o r 100 p. c e n t of s h o r t e r a l d e h y d e .

i!

®

B e f o r e a p p l y i n g t h e s a m e s e q u e n c e of r e a c t i o n s to n a t u r a l d i h y d r o m e n a q u i n o n e s , it was interest i n g t o e x p e r i m e n t i t w i t h p u r e s y n t h e t i c all-trans

/I

iJ

t ~

Ji

'

i

/ /

/

OAc CH 3

L

1

/ ./ ._~. ......

o. . . .

o cHo

® I

3

4

2

1

Oppm

f

FIG. 6. - - N.M.R. spectrum of the ¢ s h o r t c h a i n aldehyde >>.

OAc

"~-M~-.c.o 5

4

m in.~14-

1=0

I

I

I

i_

i

8

6

4

2

0

FIG. 8. - - GPC of the <> on a SE 30 in~pregnated c o l u m n at 260°C (respective r e t e n t i o n t i m e s : 6.5 mill a n d 16.5 miu) (A) a n d of crude ozo.nolysis products of the e h r o m a n y l acetate of M. phlei d i h y d r o m e n a qulnone ( ) a n d the e h r o m a n y l acetate of M K - 9 ( ........... ) (B). 3

2

1

Oppm

Fro. 7. - - N.M.R. spectrum of the ¢ long c h a i n aldehyde >>.

CHCI3-AcOEt

menaquinone-9. In this case, only the shorter a l d e h y d e w a s o b t a i n e d (fig. 8) ; c a r e f u U e x a m i n a t i o n of e v e r y i n t e r m e d i a t e ( c h r o m e n y l a n d c h r o m a n y ] a c e t a t e s ) b y IVMR a n d m a s s s p e c t r o s c o p y i n d i c a t e d t h a t less t h a n 1-2 p. c e n t s a t u r a t i o n w a s introduced in the side chain by the foregoing reactions. Occasionnaly, uncomplete ozonolysis occurred, a n d it ~vas o b t a i n e d t r a c e s of h i g h e r u n s a t u r a t e d h o m o l o g s of t h e s h o r t e r a l d e h y d e ; t h e y w e r e easily differenciated from the longer aldehyde by TLC, G P C o r m a s s s p e c t r o s c o p y . W h e n t h e s e r e a c t i o n s w e r e a p p l i e d to d i h y d r o m e n a q u i n o n e - 9 of M. p h l e i (4 g of p u r i f i e d q u i n o n e were necessary), only the longer aldehyde (VIII)

BIOCHIMIE, 1973, 55, n ° 5.

112

t

0

(9:1)

Benzene -MeOH

P

I I

1

2

(9:1)

3

~

6

I

I

1

2 3

~ S 6:

Fro. 9. - - TLC in two solvent systems of purified c h r o m a n y l aldehydes 1 : f r o m M K - 3 (II-H~), 2 (and 6) : f r o m M K - 3 , 3 : f r o m M K - 9 , 4 : fro,m M phlez MK- 9 (I-Lz), 5 : f r o m C. diphtheria M K - 8 (H~) ; spot-, visualized by c h a r r i n g w i t h 10 p. cent sulfuric acid.

Structure and Con[iguration o[ Dihydromenaquinones. was detected a m o n g the ozonolysis p r o d u c t s , e i t h e r by GP,C (fig. 8) or by T,LC (fig. 9). An imp u r i t y consistently d e t e c t e d by GPC of the c r u d e ozonolysis p r o d u c t of the c h r o m a n o l s of M K - 9 and MK - 9 (Hz), at a r e t e n t i o n time slightly h i g h e r than the s h o r t e r a l d e h y d e (see fig. 8), h a d a v e r y different m i g r a t i o n on TLC.

tOCH

~COOCHa

6

5

4

r

n o n a n o i c acid m e t h y l ester (IX) w a s easily prep a r e d f r o m o p t i c a l l y p u r e R ( + ) c i t r o n e l l o l (X) t h r o u g h the 6 S-ketone (XI) p r e v i o u s l y d e s c r i b e d [121, w h i c h was ozonized and t r e a t e d as for M K - 9 ( I I - H2) (scheme 4). An i d e n t i c a l ketoester could be o b t a i n e d by ozonolysis of M K - 3 ( I I - I-I~) s y n l h e t i s e d in the usual w a y [12] f r o m the same R

I-cOCH3

-COCH

ppm 7

3

~CCH~

2

Fie,. 10. - - N.M.R. spectrum of the purified ketoester obtained by direct ozonolysis of MK-9 (II- H~) from M. phlei. i

I d e n t i c a l results w e r e o b t a i n e d w i t h the d i h y d r o m e n a q u i n o n e - 9 of M. avium a n d again w i t h the d i h y d r o m e n a q u i n o n e - 8 of C. diphtheriae. These results establish u n e q u i v o c a l l y the structure of m y c o b a e t e r i a l d i h y d r o m e n a q u i n o n e as an all-trans, I I - d i h y d r o m e n a q u i n o n e - 9 and the structure of the c o r y n e b a c t e r i a l d i h y d r o m e n a q u i n o n e - 8 as an all-trans, I I - d i h y d r o m e n a q u i n o n e - 8 . Our results h a v e been e n t i r e l y c o n f i r m e d , for ~ K - 9 ( I , l - H o) of M. phlei by S n y d e r and Rapop o r t [20] w h o r e p o r t e d a p h o t o o x i d a t i v e d e g r a d a tion of p o l y p r e n i c n a p h t o q u i n o n e s in u n p o l a r solvents. D i h y d r o m e n a q u i n o n e s p r e s e n t a slight negative optical rotation, r e s u l t i n g f r o m the c r e a t i o n of an a s s y m e t r i c c e n t r e in the side c h a i n at the saturated s e c o n d i s o p r e n o i d unit ; the e x a c t a value is difficult to measure, as these c o m p o u n d s suffer e x t e n s i v e d e s t r u c t i o n by light [20]. D i r e c t ozonolysis of H - d i h y d r o m e n a q u i n o n e - 9 f r o m M. phlei, f o l l o w e d by o x i d a t i o n a n d met h y l a t i o n yielded l e v u l i n i c acid m e t h y l ester, toget h e r w i t h a d e r i v a t i v e of the s a t u r a t e d i s o p r e n o i d unit, the l e v o r o t a t o r y 4-methyl, 8-keto n o n a n o i c a c i d m e t h y l ester, w h i c h w a s identified by its UV, IR and NMR s p e c t r a (fig. 10), and by c o m p a r i s o n w i t h the s y n t h e t i c c o m p o u n d . 4 S-methyl, 8-keto

BIOCHIMtE, 1973, 55, n ° 5.

601

CH~ (XI)

CH)

(x)

l l.o~ 3.

2. oxidotion methylotion

0

L°3

COOCH

~

3

CH 3

(ix)

0

CH~

CH~

CH~

SCHEME 4.

( + ) eitronellol. T h e ketoester p r e p a r e d M K - 3 (II-He) was 550 n m r a n g e (*) to

optical r o t a t i o n of the S ( - - ) f r o m the ketone or f r o m n e a r l y i d e n t i c a l in the 280that of the d i h y d r o m e n a q u i -

(*) Measurements in the UV region proved very useful in this case as it has been reported that <> [21]. The negative value measured for the molecular rotation of the 4 S-ketoester ([M] ~toa ----- --1 °) is to be compared to the reported value of D (R) 4-methyl alkanoic acids ([M], -------2°). The contribution of the carbonyl group in position 8 cannot be neglected and probably exerts a predominant effect on the sign and absolute value of the optical rotation (as examplified for the 6 S-ketone EtOH [M] D,-n.. ---- --2°) •

Robert Azerad and Marie-Odile Cyrot-Pelletier.

602

n o n e d e r i v a t i v e (fig. 11), e s t a b l i s h i n g t h e a b s o l u t e c o n f i g u r a t i o n of t h e q u i n o n e of M. phlei as a 7'S-II-dihydromenaquinone-9 as i l l u s t r a t e d i n s c h e m e 5.

r e l a t i o n w i t h e v o l u t i o n p r o b l e m s r e l a t e d to b i o synthesis of terpenoid compounds in plants and bacteria.

Ac&nawledgments. The a u t h o r s are g r a t e f u l to Dr D. P a n t a l o n i for the use of the FICA spectropolarimeter. T h i s ~,ork was supported b y a s u b v e n t i o n of the F o n d a t i o n de la Recherche M6dicale, Paris. +8

Appendix : M a s s s p e c t r o m e t r y

of m e n a q u i n o n e

derivatives. +4

400

500

600

It m

I n a d d i t i o n to t h e f r a g m e n t a t i o n i o n (I) c h a r a c t e r i s t i c of t h e m e n a q u i n o n e r i n g [23] t h e f r a g m e n t a t i o n s c o n s i s t e n t l y o b t a i n e d w i t h all h o m o l o g o u s d e r i v a t i v e s w e r e t e n t a t i v e l y a t t r i b u t e d to t h e follo~ving i o n s : (II) f r o m e h r o m e n y l a c e t a t e s , (II), (II[), (IV) a n d (V) f r o m c h r o m a n y l a n d e h r o m a nylaldehyde acetates.

% OH

-4

-8

!i

OAC

I (./,,:.s) FI6. 11. - - Compared optical r o t a t i o n s of the 4 Sketoester (IX) o b t a i n e d f r o m the 6 S-ketone (XI) I-) c : 6.63 (325-550 nm), c ---- 0.66 (285-350 nm) a n d f r o m M. phlei d i h y d r o m e n a q u i n o n e ( ...... } c ---- 3.28 © .... O, c ---- 1.29 • ...... • a n d c ---- 0.24 •

OAC

.

In spite of the apparently inverted stereochemieal denomination, the saturated isoprenoid unit of M K - 9 ( I I - H 2 ) h a s t h e s a m e c o n f i g u r a t i o n as t h e t w o c e n t r a l i s o p r e n o i d u n i t s of p h y l l o q u i n o n e [22] ( s c h e m e 5). T h i s r e s u l t m a y b e of i n t e r e s t i n

.I

(,./o =.,)

OH

CH3

CH3

%

CH 3

7'S- II- dihydromenoquinone-9

oo

O

,v (m/o=.,)

CH 3 CH3

CHs CH 3 7'R,I'I'R_ phylloquinone SCHEME 5.

BIOCHIMIE, 1973, 55, n o 5.

CH 3

CHs

®O V

( m / e =186)

Structure

and Con[iguration

R~SUM~.

La d~gradation de la dihydrom~naquinone-9 de Mycobacterium phlei et M. a v i u m et de la dihydrom6naquinone-8 de C o r n y n e b a c t e r i u m diphleriae, par l'interm6diaire d'un dgriv6 c h r o m a n n i q u e , a p e r m i s d ' o b t e n i r dans chaque eas une ald6hyde identique /l celle pr6parge p a r les m~mes in~thodes /~ partir de la II - d i h y d r o m g n a q u i n o n e - 3 EMK - 3 (II - H2)] synth6tique. Ces r6sultats confirment la localisation spgcifique du c h a i n o n isopr6no~de satur6 comme le deuxi~me /t partir du noyau. L'ozonolyse directe de la d i h y d r o m g n a q u i n o n e de M. phlei p e r m e t d ' o b t e n i r u n d6rivg du chainon satur6, l'ester mgthylique de l'acide m6thyl-4 c6to-8 nona~o)que dont le pouvoir rotatoire est identique, dans la ~6gion 280-550 nm, h celui du S (--) c~toester, synth6tis6 h p a r t i r du R (d-) citronellol. La configuration absolue du chainon isopr6noide de la d i h y d r o m 6 n a q u i none de M. phlei [7'S (--) M K - 9 (II-H~)] est donc identique h celle des chainons isopr6noides satur~s c o r r e s p o n d a n t s du phytol et de la p h y l l o q u i n o n e (vitamine K~). REFERENCES. 1. Crane, F. L. in <> R. A. Morton Ed., Academic Press, London and New York (1965), pp. 183-206. 2. Pennock, J. F. (1966) V i t a m i n s and Hormones, 24, 307-329. 3. Gale, P. H., Arison, B. H., Trenner, N. R., Page, A. C. Jr. & Fo.lkers, K. (1963) Biochem., 2, 200. 4. Scholes, P. B. & King, H. :K. (1965) Biochem. J., 97, 754.

BIOCHIMIE, 1973, 55, n ° 5.

o[ Dihydromenaquinones.

603

5. Imhoff, J. M. a Azerad, R. (1970) Bull. Soc. Chim. Biol., 52, 695. 6. Phillips, P. G., Dunphy, P. J., Servis, K. L. Brodie, A. F. (1969) Biochem., 8, 2856. 7. Doyle Daves, G. Jr., Friis, P., Olsen, R. K. Folkers, K. (1966) V i t a m i n s and Hormons, 24, 427. 8. Das, B. C., Lounasmaa, M., Tendille, C. a Lederer. E. (1965) Biochem. Biophys. Res. Commun., 21, 318. 9. Bhalerao, U. T. ,~ Rap oport, H. (1971) J. Amer. Chem. Soc., 93, 105. 10. Aplin, R. T. ~ Pike, W. T. (1966) Chem. anti Industr!t, 2009. 11. Lederer, E. (1964) I.U.B. S y m p o s i u m Series, 33, 63. 12. Azerad, R. ~ Cyrot, M O. (1965) Bull. Soc. Chim.. 3740. 13. Beau, S., Azerad, R. & Lederer, E. (1966) Bull. Soe. Chim. Biol., 48, 569. 14. Coles, L. ~ Polgar, N. (1968) J. (;hem. Soc. (C), 2376. 15. Wagner, A. F., Wittreich, P. E., Arison, B., Trenner, N. R. ~ Folkers, K. (1963) J. Amer. Chem. Soc.. 85, 1178. 16. Sehudel, P., Mayer, H., Metzger, J., Riiegg, R. s~ Isler, O. (1963) Helv. Chim. Acta, 46, 333. 17. Morimoto, M., Imada, I. ,~ Goto, G. (1969) Ann. Chem., 729, 171. 18. Cameron, D. W. • Hildyard, E. M. (1967) J. Chem. Soc. (C), 2126. 19. Pappas, J. J., Keaveney, W. P., Grancher, E. Berger, M. (1966) Tetrahedron Letters, 4273. 20. Snyder, C. D. ~ Rapoport, H. (1969) J. Amer. Chem. Soc., 91, 731. 21. Kates, M., Joo, C. N., P a l a m e t a , B. ~ Shier, T. (1967) Bioehem., 6, 3329. 22. Mayer, H., Gloor, U., Isler, O., Riiegg, R. ~ Viss, O. (1964) Helv. Chem. Acta, 47, 221-229. 23. Di Mari, S. J., Supple, J. H. a Rapoport, H. (1966) J. Amer. Chem. Soc., 88, 1226-1232.