Bull. SOC. Chim. Belg. vo1.89/no 2/1980

SOLUTION CONFORMATION OF CIS AND TPANS N-ACETYL-L-THIAZOLIDINE-

4-CARBOXYLIC ACID.

wv/ I Frans A.M. BORREMANS, Willy A. NACHTERGAELE, Milos BUDESINSKY'

and Marc J.O. ANTEUNIS Laboratory of Organic Chemistry, NMR Spectroscopic Unit

Rijksuniversiteit-Gent, Krijgslaan 271 (S4bis), B-9000 GENT (BELGIUM)

and

A. KOLODBIEJCBYK and Bogdan LIBEREK Instytut Chemii, Universytet Gdanski, GDANSK 80-952 (POLAND)

(Received: 28 NOV. 1979 ; Accepted: 23 JAN. 1980)

ABSTRACT,

From an extensive ' H and I3C nmr study, the conformation of cis- and trans-N-acetyl-L- thiazol idine-4-carboxyl ic acid in D 0 is revealed to be close to an envelope with Cb as the flap down (b-) in the entire pD range.

2 The pucker around N-Ca remains pronounced (0 -83").

The cis-trans equilibrium is in favour of the isomer (70%) in acidic medium, but slightly i n favour of the cis isomer (54%) in basic solution.

INTRODUCTION. Recently, increasing interest developed into the substitution of proline

by a suitable congener in biologically important (po1y)peptides. Thus when

substituting thiazolidine-4-carboxylic acid (Thz) for proline in oxytocin

(jThz I-oxytocin) the synthetic hormone had a doubled activity (1). The dra-

matic simplification of the 'H nmr spectra, as compared to Pro directed at-

tention to thiazolidine-4-carboxylic acid (Thz) . data necessitates the determination and assignment of 13C-lH coupling con-

stants for a detailed description of the geometry of the thiazolidine ring.

In contrast, there is no loss of nmr parameters relevant to the back-bone con-

formation ( $ , $ ) .

7

The concomitant loss of 'H

At present the study of simple models is important to recognize the (disk

similarities between Pro and Thz with respect to (i) thermodynamics of their

- s-cis and 2-trans peptide bonds: (ii) conformation of the rings (Xi); (iii)

back-bone conformation ( $ , $ ) ; (iv) "long-distance''-interaction that is im-

* On leave from the Czechoslovakian Academy of Science, Institute of Organic Chemistry and Biochemistry, Flemingovo Namesti 2, 16610, Prague 6.

- 101 -

portant in the reinforcement of secundary structurization.

There appears compiling evidence in the literature that Pro and Thz behave

very similar in the solid state. Thus X-ray data exist and confirm this for

L-ProOH (2) and L-ThzOH (3), BOC-L-ProOH ( 4 ) and BOC-L-ThzOH (51, L-Pro-hydan-

toin ( 6 ) and L-Thz-hydantoin (6), Ac-N-L-ProNH2 ( 7 ) and Ac-N-L-ThzNH2 ( 7 1 ,

Ac-N-L-ProNHCH3 (8) and Ac-N-L-ThzNHCH3 (6), c/Gly,L-Pro/ (9) and c/Gly,L-Thz/

(10).

In a recent paper (11) we determined the solution conformation of Thz-hy-

dantoin. We now report a study by ’H nmr spectroscopy at 300 and 360 MHz of

the solution conformation of Ac-N-ThzOH as a function of the pD. We estima-

ted the cis-trans isomeric population around the amide bond (Ac and At) in the

acid (pD = 1.5) and the anionic state (pD = 12.3). Additionally the anionic

species were investigated at 50.3 and 90 MHz by 13C nmr at the natural abun-

dance level at pD = 5.7, and relevant 13C-’H vicinal coupling constants are

discussed in terms of the ring conformation*.

trans c i s

Assignments of signals to 9 and trans isomers. In both the acid (pD = 1.5) and the anionic form (pD = 12.3) all proton

signals of cis and trans isomers are anisochronous, and can be distinguished on the basis of relative intensities of 30/70 for the acid form and 54/46 of

the anionic form. The assignment of the two groups of signals to cis and trans isomers as presented in Table 1 is based on the following arguments :

1) The most reliable criterion to assign Cis and trans isomers about a peptide bond are the 13C chemical shifts, whereas the typical back-to-back pattern

of the a,B (y) and 6 carbon resonances for the and trans isomers of Ac-N-Pro stands as the model (12). Correlations of 13C and ‘H signals in

In describing typical ring conformations we use the symbols E (envelope, Cs) and T (twist, Cp), or alternatively we indicate the position of the flap with the positive sign if at the

same side of the reference group (carboxylate group).

as che flap down is coded as BE or 8-; a twist with 8 down and a up as ‘T. Thus an envelope with the CB

B

- 102 -

the anionic form (at pD = 5.7) were achieved by selective heteronuclear

decoupling experiments. Thus irradiation of respectively the 3.147 ppm H-8

and 4 . 7 3 0 ppm H-6 signals (both belonging to the 46% isomer) affects the C6

carbon at 5 0 . 6 ppm and CB at 34.3 ppm selectively (that are nuclei belonging

to the tranS isomer), leaving the respective inner signals at 49.5 and 35.8 ppm (of the cis isomer) unaffected (Figure 1). Both these two (and other si-

milar experiments prove that in D20 solutions at pD = 5.1 the trans isomer is the minor component. Tracing the proton nmr signals gradually to low pD va-

lues allows the transfer of this cis/trans assignment to the acid form, where

now the trans isomer is in excess.

i1H>13C

6 6

C C

o m Vlv

FIG, 1. Back to back pattern of 13C6 and 13C6 in cis and trans Ac-N-Thz-OH.

2 ) The 30% abundant isomer in the acid form, and the 54% isomer in the anionic

state show a long-range coupling between both the 6 protons and the methyl

group, which is typical of the trans zig-zag path in the cis isomer. This

long-range coupling is not present in the other isomer.

3) The assignments are in agreement with those derived from a previous inves-

tigation on similar compounds but in DMSO-d6. Thus Savrda (13) found the

6 protons of the cis isomer of t shielded relative to those of the tranS isomer, and the B protons of the trans isomer shielded relative to the - cis isomer. These assignments were based on a comparitive study of shifts

in N-acetyl-, and N-formyl-thiazolidine-4-carboxylic acid and their 5,5-

dimethyl analogues, I ‘H} ‘H NOE enhancements and long-range couplings of

the formyl protons.

Relative assignments of ‘H signals to H-BC/H-Bt and H-6C/H-6t

H-BC is assigned to the high field 6 proton for the following reasons :

*We use the codings A and B to designate respectively low- and high field protons. The codiw c and t refer to cis and trans with respect to the carboxylate group taken as the reference.

- 103 -

cow

H

2a h.r

2 b N

1) W e n o t i c e (Table 1) t h a t t h e sum-value of t h e v i c i n a l coup l ings between H-a

and H-B is r e l a t i v e l y s m a l l , L3J (a ,E) < 11 Hz. Th i s corresponds t o a pa r -

t i a l conformation a long x1 where H-a i s s t a g g e r e d between bo th H-E p r o t o n s

( s t r u c t u r e ;a) , w i th t h e l a r g e s t coup l ing t o H - E t , s i n c e t h e r i n g pucker

can ha rd ly exceed 6 0 ' .

should show L3J(a,B) = 1 4 t o 1 7 H z .

A conformation a long X1 as d e p i c t e d i n s t r u c t u r e zb

2 ) I t i s known ( 1 4 ) t h a t i n g e n e r a l and i n t h e absence o f any o t h e r r i n g sub-

s t i t u t i o n , H-BC i s expected a t t h e h i g h e s t f i e l d s i d e , b e i n g sc reened (15 )

r e l a t i v e t o t h e t r a n s p ro ton by t h e a d j a c e n t Ca-C' bond.

3 ) The c l o s e proximity of an i o n i z a b l e group shou ld i n f l u e n c e i n p r e f e r e n c e

t h e c i s v i c i n a l p ro ton . The s l o p e s of s h i f t t i t r a t i o n ( F i g u r e 2 ) i n d i c a t e

t h a t H-EC should t h e r e f o r e indeed be a t t r i b u t e d t o H-BB.

The assignments of t h e 6 pro tons i s less c e r t a i n . Although t h e p re sence o f a

c i s s u b s t i t u e n t i n 8 -pos i t i on u s u a l l y d e s h i e l d s a p ro ton (15 ) t h e a p p l i c a t i o n

of t h i s r u l e i s e s p e c i a l l y r i s k y i n t h e p r e s e n t c a s e because t h e s u b s t i t u e n t

i s n o t s p h e r i c a l l y symmetric and c o n s t i t u t e s a n-system. I ts i n f l u e n c e w i l l

g r e a t l y depend on t h e p rope r o r i e n t a t i o n of t h e rr-plane wi th r e s p e c t t o t h e

p ro ton under c o n s i d e r a t i o n ($-angle around Ca-C'). A s t h e i n d i v i d u a l a s s i g n -

ments of t h e 6 pro tons ( a s 6 c and s t ) were n o t e s s e n t i a l f o r t h e d e s c r i p t i o n

of t h e conformational behaviour , w e have n o t a t t empted any f u r t h e r experiments

i n o r d e r t o c l a r i f y t h i s p o i n t .

s - c i s / s - t r a n s e q u i l i b r i u m .

The cis-amide isomer i s t h e minor compound i n t h e a c i d ( 3 0 % ) b u t i t becomes

t h e m o s t abundant form i n a l k a l i n e medium ( 5 4 % ) . The d e s t a b i l i z a t i o n of t h e

t r a n s isomer i n t h e an ion r e l a t i v e t o t h e uncharged s p e c i e s may be a s c r i b e d

t o i n c r e a s e d r e p u l s i o n between t h e n e g a t i v e cha rge on t h e c a r b o x y l a t e group

and t h e proximate ca rbony l oxygen of t h e a c e t y l group, as proposed (16) f o r

AcNProOH, where similar e q u i l i b r i u m composi t ions e x i s t i n D 0 ( % cis : 2 0

( a c i d ) and 50 ( a n i o n ) ) . The c i s - t r a n s r a t i o i n wa te r i s independent i n ab-

2

- 104 -

12

l i

r" 9 I I I I I

I I

I

I

I I I I I I I I I

1

,

I I

P I I

I I I

I I I

I

I I I I I I I I I I

I I

,s I I

I

I

1

I / I 1

5.2 5.0 L.0 4.6 4.4 3 .L 3.2 3.0 8 FIG. 2: Chemical shift titration curves for the 'H resonances of cis- and tranS

N-acetyl-L-ThzOH in D20. (-) =Cis isomer and (---) = tr= isomer.

solute concentration. Thus a 40-fold decrease in concentration has but slight

influence on the amount of cis isomer in the entire pD range. It seems there-

from that in D20 solutions an intramolecular hy-

drogen bond as in species 2 of the trans-amide isomer is energetically not important. Other- I b wise the trans isomer would become disfavoured

relative to the cis form with increasing propa-

bility for intermolecular aggregation.

The cis:trans ratio in the corresponding ester Ac-N-ThzOMe in CDC13 was also

found (17) identical ( 3 0 : 7 0 ) with Ac-N-Pro-OMe (28:72) (18). Preliminary stu-

dies in our laboratory (19) of Ac-N-L-ThzOH and Ac-N-L-ThzNHCH3 indicate an

increased population of the s-trans isomer at low concentrations in apolar solvents, paralled by a change in $ towards typical values for a y-turn."

%/* 0' .A

3 rv - t y p e

3c This study also implies that in D 2 0 solution AcN-Thz-OH has a $-value excluding any y-bend

like structures characteristic f o r hydrogen-bridged species (structure 3) .

- 105 -

TA

BL

E 1.

'H nm

r 300 M

Hz

sp

ec

tra

l d

ata

(a)

for cis- (

kc)

an

d =

-Ac-

N-T

hz-O

H

(kt)

in

D20 a

s a

fu

nc

tio

n of

pD

and

th

erm

od

yn

amic

e

qu

ilib

riu

m d

ata

fo

r tc

Z &

t in

terc

on

ve

rsio

n.

and

c

ou

pli

ng

s in

Hz.

Sh

ifts

are

in

6

(DS

S (b)

in

tern

al)

pD

C

ompo

und

Sh

ifts

co

up 1 in

qs

( %

cis

AG0(c+t)

a 5'

Bt

(c,d

) -

CH

,CO

(c)

a,BC

a,E

t BC

,Bt

6c,

6t(

f)

Cal

/mo

l

0.1

&c

t t

1.5

&C

t t

2.2

kc

It

2.E5

&c

It

4.9

&C

It

12.3

&C

&t

5.093

4.925

5.0E5

4.921

5.001

4.902

4 .El6

4.82

7

4.69

1 -4.75

4.6E9

-4.75

3.448

3.434

3.2E8

3.279

3.401

3. 268

3. 331

3. 224

3. 280

3.14,

3. 280

3.147

3.46

2 3.435

3.448

3.447

3.444

3.394

3.442

3. 350

3.44

2 3.351

3.42

5

3.419

4.619

& 4.518

4.6E4

& 4.J37

4.625

& 4.

507

4.676

8 4.J34

4.634

& 4.

503

4.671

& 4.

733

4.65

&

4.49

1 4.650

& 4.732

1

4.662

& 4.48*

4.61

& 4.730

5 4.661

& 4.4E4

4.628

8 4.731

2.14,

2.215

2.128

2.116

2.0E9

2.199

2.197

2. 188

2 .0

70

2.174

2 .O

le

2.174

deg

d

eg

3.4

7.1

2.5

6.0

3.5

7.3

2.6

6.1

3.65

7.2

2.6

6.8

3.E5

7.0

3.9

7.15

2.95

7.0

3.0

7.0

4.2

7.2

deg

-1

0.0

29.6

-500

-12.4

-9.2

-12.

2 -10.0

-12.3

-9.1

-12.1

-10.0

33.2

-12.3

-9.1

28.3

-540

-12.

0 -9.9(4

41.5(e)

-12.

15 -9.0(e)

-11.9

-

44.0

+90

-11.9

-9.1

-11.9

-10.2(f)

54.1(f)

+90

-11.9

-9.2(f)

(a) Concentration ca 4

0 mg/ml, taken at 18" in CW-mode.

(b)

Sodium d6-dimethyl-silapentane sulfonate.

(')

In the cis amide the patterns of CH3CO and both

6 protons were broadened to an extent of 0.5 H

z relative to the trans isomer -

by

(dl A

ssignments of 6'

and 6t

have not been done (see text). Assignments of cis and trans protons referring to the carboxylate substituent.

long-range coupling. This was demonstrated by double resonance decoupling at 360 M

Hz.

Taken in FT-mode at 360

MHz

on

a sample

of 2.3 mg/ml the

% cis at pD

= 2.6 was 37.5%; coupling constants

for the cis isomer: 2.3,

6.7, -12.0, -10.0; for the trans isomer: 3.6, 7.2, -12.2 and 9.1

Hz.

-10.1

and for the

isomer 4

.1,

7.2,

-11.9 and -9.2 Hz.

(f) For a sample concentration o

f 2.3 mg/ml the

% cis at p

D 13.2 was 55%; coupling constants were for the

isomer: 2.9, 6.9,

-12.

0,

A similar behaviour was observed for the Pro congener Ac-N-L-ProNHCH3 (20).

The relative high percentage of isomer in Ac-N-L-ThzOH, and especially

the close parallelism in cis:trans ratios in corresponding Pro and Thz amides

in general, contrast to calculations of the relative stability of all cis poly-Thz versus all trans poly-Thz, the latter being favoured by 4.83 kcal/mol (3) per peptide unit, compared to a value of ca. 1 kcal/mol for poly-Pro.

Ring conformational aspects.

A. From 'H nmr data (Table 1)

At variance with most acyclic Pro derivatives (21, 22) or with acyclic Hyp

derivatives (231, the present values of 3J(a,6) reflect a rather biased situ-

ation around the x1 torsion (Ca-CB), and this is the case for the two amide isomers.

in basic solution [trans-amide: 3J(a,Bc) = 3.4 + 4.2 Hz; 3J(a,Bt) = 7.1 + 7.2;

cis-amide: 3J(a,BC) = 2.6 + 3 . 0 Hz; 3J(a,Et) 6.1 + 7.0 Hz].

the findings in c/Gly,Thz/(lO) and in c/D- or L-Phe, L-Thz/ (241 the present

two couplings of 3J(Ha,H5) are widely different, a feature that for Pro pep-

tides has been explained in terms of the occurence of a pyrrolidine conforma-

tion close to a+ ("E) (25-27).

equation (28) we can propose certain discrete interproton torsion angles, as

gathered in Table 2. Any satisfying correlation extracted from Table 2 would

suppose the compound to be present in a unique conformation or an admixture

of forms close to a+, 6 , y , that is in the allowance of a restricted pseudo- rotational libration. If this is so, and in view of a situation presented as

in structure xa, one should find for each set of J-values (J(a,Bc) and cor-

responding J(a,Bt)) a combination of torsion angles with an algebraic diffe-

rence near 120". Actually, straightforward application of the Kopple equa-

tion yields present values of 140-145', whereas data on trans in the solid

state obtained by X-ray analysis (32) show a value of 122O. Apparently the

Kopple equation may predict torsion values that are in error by as large as

20° *. The discrepancy can originate from: (i) the lack of electronegativity

corrections; (ii) breakdown of the three-fold symmetry of projected bond an-

gles at CB (29); (iii) conformational inhomogeneity resulting in the observed

There is a slight systematic change in 3J(a,B), increasing somewhat

As opposed to

Analyzing the a,6 couplings with the Kopple

- +

w A similar discrepancy was found (10) in c/Gly,Thz/ where the application of the Kopple equa-

tion gave a value of 112' in solution, whereas in the crystal structure a value of 127' was

found .

- 107 -

u,B coup l ings t o be averaged.*

TABLE 2.

I n t e r p r o t o n t o r s i o n a n g l e s de r ived from t h e Kopple equa-

t i o n ( 2 8 ) and t h e expe r imen ta l 3 J ( a , B ) v a l u e s ( i n Hz)

Compound S t a t e J(a ,BC) ' d ( ~ . , 8 ~ 1 J ( a , B t ) 9 (a ,B t )

a c i d 2.6 -105 6 .1 4 0

anion 3 . 0 -108 7 .O 33

a c i d 3.4 -1 10 7.1 32

anion 4.2 -117 7.2 31

1"

k t

( a ) For a d i s c u s s i o n see t e x t .

We t h e r e f o r e surmize t h a t x1 i s n e a r +40° i n a c i d i c and +30° i n b a s i c solu-

t i o n s , w i th t h e l a r g e s t pucker i n t h e cis-amide isomer.

mation can e x p l a i n t h e observed a , 0 i n t e r p r o t o n coup l ings , t h i s form i s pro-

bably n o t important s i n c e t h e t y p i c a l 4 J ( H - 6 c , H - 6 c ) long-range coup l ing i s = observed.

due t o long-range coup l ing wi th t h e COCH3 p r o t o n s also favour s a 8- conforma-

Although a y+ confor-

The observed equal broadening of both 6 p r o t o n s i n t h e cis isomer

t i o n .

B. From 13C-lH coup l ing d a t a (Tab le 3) f o r t h e a n i o n i c forms** (pD = 5.7)

The v i c i n a l 13C,lH coup l ing of t h e carboxyl carbon t o t h e 5 t p ro ton

3 ( J(C00H,E-Pt) of 5.7 Hz i n t h e

l y l a r g e r t han t h e corresponding coup l ing t o H-6' (3.1 Hz e n 3.2 Hz i n * and =-amide r e s p e c t i v e l y ) .

pucker from 'H-lH coup l ings .

t h e &-amide aga in i n d i c a t e s a s l i g h t l y more pronounced pucker around Cu-C

r e l a t i v e t o t h e trans isomer.

I n both t h e cis and isomers, t h e v i c i n a l coup l ings o f t h e a c e t y l carbo-

n y l t o H-a and t o b o t h 6 pro tons e q u a l 1.3 Hz.

and 4.8 H z i n t h e =-amide) i s marked-

Th i s c o r r o b o r a t e s o u r conc lus ion about t h e x1 The l a r g e r 3J(~OOH,~-6t)/3J(COOH,H-BC) r a t i o f o r

B

* We do not consider this in contradiction with our conclusion of a unique conformation or highly restricted pseudorotation in 1 (vide supra), e.g. close t o 6-, since a simple calcu- lation shows that only 5% admixture of e.g. a 8' conformation is sufficient to produce the observed discrepancy.

The poor solubility of

slightly changed ]H-'H coupling values however, one may savely accept that the conformatio- nal changes will be too small as to violate the general conclusions.

prevented any detailed I3C study in acidic medium. From the only xx

- 108 -

TABLE 3.

13C nmr (50.29 MHz) spectral parameters of cis- (kc) and trans- Ac-N-ThzOH (kt) in D20(a) at pD = 5.7.

Chemical Shifts(b)

Compound COOH - COCH3 CH3 CCl CB C6

171.3 173.4 23.0 65.9 35.8 49.5

177.6 172.5 22.8 64.6 34.3 50.6 1" It

Coupling constants(c)

Compound 'J(Ca,Ha) 1J(C8,H8C't) 'J (C6,HsCrt) 3J(CS ,Ha)

IC 145.8 148.2, 145.7 157.3, 157.3 4.5

kt 146.7 147.9, 145.5 156.5, 156.5 3.4(e)

Compound 3J(C6,HBC) 3J(C6 ,HBt) 3J(cOOH,H5t) 3J(~OOH,H0c)

3.5 1.3 5 . 1 3.1

It 3.4(e) 2.0(e) 4.8 3.2

1"

Compound 2J(FH,Hd 'J(cOCE3) 3J(cOCH3,@) 3J(COCH3,H6C't) 'JgEd

1.3, 1.3 129.0

1.3, 1.3 129.0

kC 3.1 6 .O 1.3

4.0 6 .O 1.3 kt

Saturated solution at 303 K.

6 in ppm downfield relative to TMS, taking 1,4-dioxane at 67.4 ppm as the internal standard.

Coupling constants in Hz.

Not assigned couplings: on Ca of @: 1.0, 2.4, 2 . 1 and 2.7; on Ca of At: badly resolved pattern, approximate couplings 2.0, 2.1, 2.1 and 2.6; on

C5 of Lc: 2.2, 2.7 and 3.9; on CB of Lt: 2.7, 2.7 and 3 . 3 Hz.

Interchangable values, badly resolved pattern, J-values are approximate.

The ratio of 1.0 for 3J(CH3C0,g-&C)/3J(CH3C0,E-6t) points to xi, 2 0 (torsion

angles HGCCG-NCOCH3 Also, and

especially for an a+ form, a value fo r 3J(CH3C0,E-a) larger than 1.3 Hz is ex-

pected since this form virtually eclipses both coupling nuclei (expected coup-

ling 3.5 Hz). From the existing Karplus-type relation for this fragment (30)

we derive, assuming an sp2 hybridised nitrogen, a 9 value of-83'.

The x2 torsion can be appreciated from the high ratio of 3J(CS ,g-BC) /3J (C6 ,E-f34

= 3.5/1.3 = 2.7 (9 amide) which is not far from the value 5.2/1.85 = 2.8 ob-

served for the C,H couplings over sulfur, observed (31) in the rigid six-mem-

HGtC&-NCOCH3 5 60') i.e. a 0- conformation.

- 109 -

bered ring homolog thiapipecolic acid derivatives (cf. 2 ) . ling of 3.5 Hz to the Bc proton corresponds to an angle of ca. 150' for the

HBCC6-S C6 torsion (X2 2. -30').

Although the couplings for 3J(CS,fl-Sc) and 3J(CB,E-6t) are not assigned in the

present study, any combination of the 3 couplings observed for CB (see Table

3) produces a ratio <1.5 revealing for x 3 a small torsion as compared to x2. Finally the relatively large value of 3J(C&,g-a] of 4.5 HZ (cis amide) proves the torsion angle x5 to have a distinct neg-ative value (cf. 8 ) . For a positi- ve torsion x5 < HaCa-NC6 would be close to 90' giving a 3J(C6,ga) value close to zero.

The larger coup-

The pucker around x5 therefore seems less pronounced in the trans amide ('J(C6,Ha) % 3.4 Hz). Together with the observed decreased x1 pucker in comparison with the cis isomer, we conclude that the trans amide possesses so-

mewhat more LT character.

Table 4 smarizes the main conclusions for each of the torsion angles of

the cis isomer in the anionic state.

TABLE 4

Approximate torsion angles for Cis N-acetyl-L-thiazolidine-4- carboxylic acid in the anionic state in D20 solution (pD=5.7)

+30" -30' 10' to 20' 0 -loo to -20' -03

CONCLUSION.

AcN-L-ThzOH is found to adopt a B- conformation in water solution, in the

acid as well as in the anionic forms, with a 0 value of ca -83". Within the

limits of the applied methods this conformation is identical to the solid sta-

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te (32) conformation of its trans isomer.

EXPERIMENTAL SECTION.

IH nmr spectra as a function of pD were run in CW mode (40 mg/ml) on a VARIAN HR-300 in- strument with decoupler unit -SC 8525-2. were acquired in FT-mode on a Bruker WH-360. The analysis of the patterns was checked by si- mulation with the SIMEQ 16/11 program (HR-300) and with the SIMULATION-ITERATION program sup- plied by BRUKER on the ASPECT 2000 computer (WH360). WP200 instrument operating at 50.3 MHz on saturated solutions. Heteronuclear {'H}l3C double resonance experiments were performed on a WH-360 (BRUKER) at 90 MHz. Selective decouplings of

H5,Ea and CH3 allowed assignments of two- and three- bond 'H couplings to c6, SOOH and COCH3 carbons, as well as the intercorrelation of lc and At I3C and 'H signals.

Some spectra at low concentrations (up to 0.6 mg/ml)

I3C spectra were obtained on a Bruker

- -

ACKNOWLEDGEMENT. Two of us (A.K. and B.L.) are indebted to the Polish Academy of Science for a grant No.

MR-1.12.1.6.l/4. M.B. thanks the "Ministerie voor Wetenschappelijke programtie" for a post- doctoral fellowship. The same institution is thanked for extensive financement of our nmr fa- cilities installed at the University of Ghent, Belgium. W.N. is a scholar of the I.W.O.N.L.

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- I l l -

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...-... (1980)(subsequent paper).

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