a thesis submitted for the degree of doctor of ......2.2 the mechanism of action of a) dcc and b)...
TRANSCRIPT
c W e A A ATHE USE OF CR OWN E T HERS IN P E P T I D E SYWTfflSSdkS-.
A Th es i s sub m i t t e d for the D e g r e e of
DO C T O R OF P H I L O S O P H Y
in the
F A CULT Y OF S C I E N C E
U N I V E R S I T Y OF L O ND ON
by
C A R O L Y N B A R K E R HYD E
School of Pharmacy, D E C E M B E R
29/39 B r u n s w i c k Square,
London WC1N 1AX.
1989 .
-1-
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A B S T R A C T .
The fo rmati on of amino acid fatty es ters u s i n g
18- Cro wn-6 is dis c u s s e d and comp a r e d wi t h p r e vious
methods. Initial resu lts indicate that the m e t h o d w o u l d
be viable. Thus the use of the K* salts of N - p r o t e c t e d
amino acids, 18 -Crow n- 6 and the a p p r o p r i a t e alkyl h a li de
gives rise to the N - p r o t e c t e d amino acid ester in
mo de ra te to good yield, p r o v i d i n g there is no steric
h i n d r a n c e .
The use of 18 -C rown- 6 as an N -t ermina l p r o t e c t i n g g r o u p
in p e pt id e synthesis is studied. In so lvents such as as
MeCN or C H C 1 a , o l i g o m e r i s a t i o n occurs. This is shown to
be due to an in t r a m o l e c u l a r h y d r o g e n bond b e t w e e n the
NHa + g r o u p and the imine n i t r o g e n of the O - a c y l i s o ur ea
der i va t i v e .
The use of a d i p e pt ide co mpl ex inhibits the f o r m a t i o n of
this h y d r o g e n bond to a c e rtai n extent. C o m b i n i n g the
use of a d i p e ptide co mplex with a po la r solvent
inhibits o l i g o m e r i s a t i o n but gives rise to
unw a n t e d amino acid d e r i v a t i s a t i o n . T h er e is r e a c t i o n
b e t w e e n the co mp lex and the DMS O at the N - t e r m i n a l end.
The carb o x y l a t e ion g r ou p acts as ca t a l y s t in the
r e a ction be t w e e n DCC and DMS O to give an int erm ediate.
The m e c h a n i s m of this r e a ct ion is proposed.
S u b s t i t u t i o n of DMF as solvent leads to s u p p r e s s i o n of
both o l i g o m e r i s a t i o n and d e r i v a t i s a t i o n . T he re fore,
a d d ition of a d i p e ptide complex and DCC s o l u t i o n to a
-2-
s o l ut ion c o n t a i n i n g an ami no acid ester and TEA at 0°C
leads to pept ide bond formation. O p t i m i s a t i o n of the
con di tion s is still required, ho we ver the s y n t hes is of
an E n k e p h a l i n d e r i vati ve has been achieved.
-3-
A C K N O W L E D G E M E N T S
I would like to express my thanks and a p p r e c i a t i o n to:
Paolo Mascagni
for his s u p e r v i s i o n and help throu ghout my P h . D . .
and Ann Coll ins
for all her help and support.
I would also like to thank everyon e else for their
e n cour ag em ent .
-4-
CO N T E N T S
page n ° .
Title. 1
A b s t r a c t . 2
Ac kn o w l e d g e m e n t s . 4
Cont ent s . 5
List of Figures. 13
List of Schemes. 13
List of Tables. 14
N o m e n c l a t u r e and Ab brevi at ions. IV
Ch ap te r One: I n t r o du ct ion
1.1 p e p ti de synthesis.
1.1.1 H i s t o r y 18
1.1.2 P r o t e c t i n g Gr ou p s 18
1.1.3 P e p t i d e Bond F o r m a t i o n 26
1.1.4 C o u p l i n g Rea g e n t s 28
1.1.5 S o l u t i o n Phase Peptide Sy n t h e s i s 30
1.1.6 Sol id Phase Peptide S y n t hesis 32
-5-
1.2 C r o w n Ethers.
page n ° .
1.2 C r o w n Ethers.
1.2.1 His tory 34
1.2.2 C h a r a c t e r i s t i c s 35
1.2.3 T h e r m o d y n a m i c s 37
1.2.4 Kin e t i c s 40
1.2.5 M o l e c u l a r R e c o g n i t i o n 41
1.2.6 A p p l i c a t i o n s to O r g a n i c C h e m i s t r y 44
1.2.7 P h a s e Tr a n s f e r C a t alysis 47
1.2.8 Host - Guest C o m p l e x a t i o n 49
1.3 B a c k g r o u n d .
1.3.1 A m i n e Pro tection. 52
1.3.2 Est er Fo r m a t i o n 53
C hapter Two: R e s u l t s .
2.1 Ester Formation.
2.1.1 I n t r o d u c t i o n 54
2.1.2 R e a c t i o n s in A c e t o n i t r i l e 55
2.1.3 R e a c t i o n s in N , N-d irnet hy 1 f o r m a m i d e 59
2.2 O l i g o p e p t i d e Formation.
t2.2.1 I n t r o d u c t i o n 60
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page n ° .
1.2 C r o w n Ethers.
1.2.1 H i s t o r y 34
1.2.2 C h a r a c t e r i s t i c s 35
1.2.3 T h e r m o d y n a m i c s 37
1.2.4 Ki n e t i c s 40
1.2.5 M o l e c u l a r R e c o g n i t i o n 41
1.2.6 A p p l i c a t i o n s to O r g a n i c C h e m i s t r y 44
1.2.7 P h a s e T r a n s f e r Ca t a l y s i s 47
1.2.8 Host - Guest C o m p l e x a t i o n 49
1.3 B a c k g r o u n d .
1.3.1 A m ine Prote ction. 52
1.3.2 Este r F o r m a t i o n 53
Ch apt er Two: Results.
2.1 Est er Formation.
2.1.1 I n t r o d u c t i o n 54
2.1.2 R e a c t i o n s in A c e t o n i t r i l e 55
2.1.3 R e a c t i o n s in N ,N - d i m e t h y l f o r m a m i d e 59
2.2 O l i g o p e p t i d e Formation.
2.2.1 I n t r o d u c t i o n 60
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page n°
2.2.2 R e a cti on s usi ng DCC 63
2.2.3 Rea c t i o n s us in g M e t h y l a m i n e HC1 68
Coinpl ex
2.2.4 R e a ct ions using Vil s m e i e r R e agent 69
2.2.5 R e a c ti on s usi ng 6 - A m i n o h e x a n o i c 71
Acid C o m pl ex
2.3 D i m e t h y l s u l p h o x i d e D e r i v a t i s a t i o n .
2.3.1 I n t r o d u c t i o n 73
2.3.2 React io ns at 0.02 M C o n c e n t r a t i o n 73
2.3.3 Rea c t i o n s at 0 . 2M C o n c e n t r a t i o n 74
2.3.4 R e a c ti ons us ing NSu 76
2.3.5 R e a c ti on s us in g M e t h y l a m i n e HC1 77
Co mp le x
2.3.6 R e a c tions u s in g G l ycine C o m p l e x e s 78
2.3.7 Other R e a c tions 83
2.4 P e p t i d e Synthesis.
2.4.1 I n t r o d u c t i o n 87
2.4.2 Reac ti ons of D i p e p t i d e Co m p l e x e s 89
with Amino Acid Esters
2.4.3 Syn t h e s i s of [a - a m i n o d e c a n o y 1 J5- E n k e p h a 1 in 91
0-Benzy l D e r i v a t i v e
2.5 F o r m a t i o n of O l i g o p e p t i d e Complexes. 92
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Ch ap te r three: Discussion.
Opage n
Ch ap te r three: Discussion.
3.1 Este r formation. 94
3.2 O l i g o p e p t i d e formation. 99
3.3 D i m e t h y l s u l p h o x i d e D e r i v a t i s a t i o n . 106
3.4 R e a c t i o n s in D i m e t h y 1 f o r m a m i d e . 119
Chapter four: M a t e r i a l s and Methods.
4.1 Materials. 122
4.2 Pr ep arati ons.
4.2.1 Boc 1 - 2 - a m i n o d e c a n o i c acid 123
4.2.2 Ami no a c i d - c r o w n ether co mpl exe s 123
4.2.3 D i p e p t i d e - c r o w n ether com pl exes 125
4.2.4 0 1 i g o p e p t i d e - c r o w n ether com pl exes 126
4.2.5 Vil s m e i e r Rea ge nt 127
4.2.6 Boc Di p e p t i d e esters 128
4.2.7 1 - 2 - a m i n o d e c a n o i c acid methyl ester 129
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page
4.3 React i o n s .
4.3.1 Boc Amin o A cid Ester F o r m a t i o n using
18-crown-6
4.3.2 R e a c t i o n of amino acid compl ex es with DCC
in CHC1a or MeCN
4.3.3 R e a c t i o n of Amino acid comp lexes with
V i l smeier Reagent
4.3.4 R e a c tio ns us in g M e t h y l a m i n e 14C1 Co mp lex
4.3.5 R e a c tio ns of D i p e p t i d e Co m p l e x e s with DCC
in DMSO
4.3.6 Rea c t i o n s of M e t h y l a m i n e HC1 Co m p l e x
in DMSO
4.3.7 Re action s of G l y c i n e Com p l e x e s in DMSO
4.3.8 Re ac tions of Boc P r o t e c t e d Co mp ounds
in DMSO
4.3.9 M i s c e l l a n e o u s Re a c t i o n s in DMSO
4.3.10 Reac tions of Di p e p t i d e C o m p l e x e s in DMF
4.3.11 Rea c t i o n s of D i p e p t i d e C o m p l e x e s with
Amino Acid Esters in DMF
4.3.12 The S y n thes is of an E n k e p h a l i n D e r i v a t i v e
4.4 Ot he r R e a cti on s used.
4.4.1 R e s o l u t i o n of d,l N - a c e t y l - 2 - a m i n o d e c a n o i c
acid
4.4.2 Rem oval of an Ester G r ou p
n .
129
132
134
135
135
136
137
137
139
139
140
142
144
144
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page n .
4.4.3 Remo val of a Boc G r o u p 145
4.4.4 P u r i f i c a t i o n P r o c e d u r e for C o m p o u n d 5. 145
Ch ap te r Five: Conclusions.
5.1 Este r F o r m a t i o n 147
5.2 O l i g o m e r i s a t i o n and Peptide S y n t h e s i s 148
Appendlces. 15 2
A p p e n d i x One. *H NMR Data from the R e a c t i o n s of
Di p e p t i d e C o m p lexes in DMSO
A . 1 . 1 Tos Di Cpx + DCC 0 . 02M 153
A . 1 . 2 Tos Di Cpx + DCC + NSu 0 .02M 154
A . 1 . 3 Tos Di Cpx + DCC 0 . 2M 155
A . 1 . 4 Tos Di Cpx + DCC + NSu 0 . 2M 156
A. 1 . 5 HC1 Di Cpx + DCC 0 . 02M 158
A . 1 . 6 H C 1 Di Cpx + DCC 0 . 2M 159
A. 1 . 7 H C 1 Di Cpx + DCC + NSu. 0 . 2M 160
Appe ndi x Tw o . XH NMR Data from the R e a c t i o n s of
M e t h y l a m i n e HCi C o m pl ex in DMSO
A . 2 . 1 NH 3 CHa Cpx + DCC 0 . 2M 161
A. 2 . 2 NH 3 CHa Cpx + DCC + AcOH 0 . 2M 161
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page n°
A . 2 . 3 N H 3C H 3 Cpx + DCC + c. AcOH 0 . 2M 162
Ap pendi x Three. *H N MR Data from the
G l y c i n e C o m plex es in
R e a c t i o n s of
DMSO
A. 3 . 1 Tos Gly Cpx + DCC 0 . 2M 164
A . 3 . 2 Tos Gly Cpx + DCC + NSu 0 . 2M 165
A . 3 . 3 HC1 Gly Cpx + DCC 0 . 02M 166
A. 3 . 4 HC1 Gly Cpx + DCC + NSu 0 . 02M 167
A . 3 . 5 HC L Gly Cpx + DCC 0 . 2M 168
A . 3 . 6 HC1 Gly Cpx + DCC + NSu 0 . 2M 169
Ap pendi x Four. *H NMR Data from the R e a c t i o n s of
Boc P r o t e c t e d Co m p o u n d s in D MSO
A . 4 . 1 Boc Ala OH + DCC 0 . 01M 170
A . 4 . 2 Boc AlaOH + DCC 0 . 05M 171
A. 4 . 3 Boc Al aO H + DCC 0 . 2M 172
A. 4 . 4 Boc G l y G l y O H + DCC 0 . 02M 173
A. 4 . 5 Boc G l y G l y O H + DCC 0 . 2M 174
A . 4 . 6 Boc Gly OH + DCC 0 . 2M 175
Ap pen d i x Five. *H NMR Data from M i s c e l l a n e o u s
Rea c t i o n s in DMSO
A. 5 . 1 18C6 + DCC + AcOH + NIlz Bu 0 . 2M 176
A. 5 . 2 DCC + AcOH + NH 2 Bu 0 . 2M 177
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page n ° .
A . 5.3 NSu + DCC 0.2M 178
A. 5. 4 NSu + DCC + AcOH 0 . 2M 179
Ap p e n d i x Six. *H NMR Data of the Pepti de s 180
Synthes i s e d .
A p p endix Seven. Mass Spectral Data. 181
Ap p end ix Eight. S t r u ctu re s of all N u m b e r e d 182
C o m p o u n d s .
A p p e n d i x Nine. Copies of the Papers Published, or 188
Accep t e d for.Publ ication, from
Thi s W o r k .
References. 219
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List of F i g u r e s .
page n°
1.1 S t r u c t u r a l El emen ts of Cr own Ethers. 35
1.2 C r o w n Ethers Be ha viour in D i f f er en t 37
Sur round i n g s .
1.3 C o n f o r m a t i o n s of 18-Crow n-6 with and without 49
K +S C N " .
2.1 P r o p o s e d C o n f o r m a t i o n a l E q u i l i b r i u m for 62
C o m p l e x e d 18-Crown-6.
2.2 The m e c h a n i s m of ac tion of a) DCC and b) the 70
V i l s m e i e r Re agent with the C o m p l e x lb.
2.3 The Pr o d u c t s O b t a i n e d wit h the V i l s m e i e r 71
R e a g e n t .
3.1 Th e Ion A g g r e g a t e P r o p o s e d by DeTar for the 104
R e a c t i o n B e t we en C a r b o x y l i c Acids and DCC.
3.2 I n t e r m e d i a t e B e t w e e n DCC and DMSO as Pr op osed 115
by Mo f f a 1 1 .
List of Schemes.
1.1 P o s s i b l e Pr o d u c t s from the C o u p l i n g of Two 19
Amino A c i d s .
1.2 Two M e c h a n i s m s by Which an Amine Will 27
D i s p l a c e a L e a v i n g Group.
1.3 P r o p o s e d M e c h a n i s m of A c tion for DCC. 29
-13-
page n .
1.4 Steps Involved in Solid Phase P e p tid e 33
Sy nt he s i s .
3.1 The R e a c t i o n p a t hwa y P r o p o s e d for Ester 95
Format i o n .
3.2 The P r o p o s e d M e c h a n i s m for the F o r m a t i o n of 101
the V i l s m e i e r A d d u c t .
3.3 Two P o s s i b l e P a t hways for the F o r m a t i o n of 102
O l i g o p e p t i d e s .
3.4 M e c h a n i s m P r o p o s e d for the F o r m a t i o n of N-Acyl 107
Urea Derivat ives.
3.5 S o l v a t i o n of the ’N a k e d ’ Ion by Water, and the 111
Su b s e q u e n t H y d r a t i o n of DCC.
3.6 Acid - Base E q u i l i b r i a Pres ent in the D M S O 113
R e a c t i o n Solution.
3.7 The P r o p o s e d M e c h a n i s m for D MSO 114
D e r i v a t i s a t i o n , Using G l yci ne Complexes.
3.8 F o r m a t i o n and H y d r o l y s i s Mech a n i s m s of the 116
Ac t ive E s t e r s .
3.9 The P r o p o s e d M e c h a n i s m for the Re moval of the 119
Boc G r o u p with D M S O - D C C Adduct.
List of T a b l e s .
1.1 N - t e r m i n a l P r o t e c t i n g Groups. 21
1.2 C - t e r m i n a l P r o t e c t i n g Groups. 24
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1 . 3
2 . 1
2 . 2
2 . 3
4 . la
4.1b
4.2a
4 . 2b
4 . 3
4 . 4
4 . 5
page
E x a mples of some Or g a n i c R e a c ti on s in whi ch
C r o w n Ethers are Utilised.
C h e mical Shift as a Fu n c t i o n of T e m p e r a t u r e
for the C o m p l e x (la) in CDCla.
R e s u l t s from the C o m p a r a t i v e S t udy of Three
Co m p l e x e s v a r y i n g the Time of R e a c t i o n and
the DCC C o n c e n t r a t i o n in M e C N .
Yi el ds O b t a i n e d of the T r i p e p t i d e s
Sy n t h e s i s e d .
Q u a n t i t i e s Used in the P r e p a r a t i o n of the
Amino Acid Compl ex es
E l e mental A n a lysis and NMR Data for the
Amino Acid Com p l e x e s Synthesised.
Element al An a l y s i s Data for the Di p e p t i d e
C o m p l e x e s Synthesised.
NMR D ata for the D i p e p t i d e Co mp lexes
Sy n the s i s e d .
NMR Data for the O l i g o p e p t i d e C o m pl ex es
Syn t hes i s e d .
N MR Data, Rf and Yi el d O b t a i n e d of Boc
D i p e p t i d e Esters Synthesised.
P r o d u c t s O b t a i n e d in the Boc Amino Acid Ester
S y n t h e s e s using 18-Crown-6.
NMR Dat a for Boc Ami no Acid Esters
S y n t h e s i s e d usin g 18-Crown-6.
46
61
67
88
124
124
125
126
127
128
130
131
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page
4.7 C o m p l e x e s Used and Produ c t s O b t a i n e d from the
R e a c t i o n of Amino Acid C o m p lexes with DC C in
M e C N / C H C l 3 .
4.8 NMR Data from the Reactio ns be t w e e n the
V i l s m e i e r Re agent and C o mp le x lb.
4.9 Q u a n t i t i e s used in C o m plex Rea c t i o n s with DCC
in DMSO.
4.10 Q u a n t i t i e s used in Boc P r o t e c t e d C o m pounds
Re a c t i o n s with DCC in DMSO.
4.11 Q u a n t i t i e s used in the M i s c e l l a n e o u s
R e a c ti on s in DMSO.
4.12 Q u a n t i t i e s Used in the R e a cti on s in DMF.
4.13 Free Acid Com p o u n d s O b t a i n e d after Ester Gro up
R e m o v a l .
4.14 P e p ti de s O b t a i n e d After Removal of the Boc
G r o u p .
133
134
138
138
139
141
145
146
-16-
N o m e n c l a t u r e and Ab brevia t ions
18- C r o w n -6 1 , 4 , 7 , 1 0 , 1 3 , 16 - H e x a o x a c y c l o o c t a d e c a n e .
AcOH Acetic Acid
Ala 1-Alanine
Boc T e r t i a r y B u t y l o x y c a r b o n y 1 gro up
Ci o 2-Amino De ca noic Acid
Ci i 2-Amin o T e t r a d e c a n o i c Acid
C H C 1 3 C h l o r o f o r m
DCC N , N ’- D i c y c l o h e x y l c a r b o d i i m i d e
DCU N ,N ’- D i c y c l o h e x y l u r e a
DMF N ,N - D i m e t h y 1formam ide
DMS O D i m e t h y 1s u 1phox ide
EtOAc Ethyl Ac etate
EtOH Ethanol
Gly Gl yc ine
MeCN Aceto nit rile
NSu N - H y d r o x y s u c c inimide
OCia Oct a d e c y l ester
Phe 1-P h e n y l a l a n i n e
TE A T r i e t h y l a m i n e
Tos p - T o l u e n e s u l p h o n a t e ion (a)
Tyr 1-Tyrosi ne
V a 1 1- V a 1ine
Wscdi l - ( 3 - D i m e t h y l a m i n o p r o p y l ) - 3 - E t hy 1
C a r b o d i i m i d e H y d r o c h l o r i d e
-17-
C H A P T E R O N E . I N T R O D U C T I O N .
1 .1 P E P T I D E SYNTHESIS.
1.1.1 History.
The first simple pe pt ide d e r i v a t i v e s were s y n t h e s i s e d by
C u r t i u s in 1881 (Curtins 1881) , but it was not until
1902 that it was r e c o g n i s e d that the structur e of
pr o tei ns was best r e p r e s e n t e d by chains of amino acids
linked th rou gh amide bonds (Hofm eister 1902) . Th es e
e n d e a v o u r s were in an attempt to r e p roduc e the work of
nature. However, the aims of peptide syn th es is be cam e
more p r a g m a t i c and syn thet ic pep ti des were p r e p a r e d for
the study of the s p e c i f i c i t y of p r o t e o l y t i c enzymes.
The is ol at ion of pure ox y t o c i n by du V i g n o a u d et al .
(Pierce 1952), fo ll owed by the d e t e r m i n a t i o n of its
st r u c t u r e (du V i g n e a u d 1953a,b) and its total sy nt hesis
(Tuppy 1953) gave u n p r e c e d e n t e d impetus to the
d e v e l o p m e n t of syn th etic procedures. Similarly, the
e l u c i d a t i o n of the st ructur e of insulin by Sanger show ed
that peptide synthes is would be useful to m e d i c i n e in
the study of pe ptide hormo n e s which reg ul at e the life
p r o c e s s e s (Sanger 1953).
1.1.2 P r o t e c t i n g Groups.
Simple amino acids have two funct ional grou ps and
either can react g i v i n g a large nu mbe r of products,
unless one fu nction is "d eactivate d" or "protected" (see
-18-
1932 (Bergtnann 1932). This gro up could be r e move d by
c a t a l y t i c h y d r o g e n a t i o n at room t e m p erat ur e and
a t m o s p h e r i c p r e ssure and gave r e l a t i v e l y har ml ess b y
pr o d u c t s whilst leavin g the peptide bond intact. The
oth er me th ods of re m o v i n g this g r o u p are sod ium in
liquid a m m onia or a c i d o l y s i s (Sifferd 1935; B e n - Isha i
1952). The b e n z y l o x y c a r b o n y l gro up is reg ar de d as the
c or ners tone in pe pt ide synthes is h i s tory bec ause i t has
the a b i l i t y to protect the chix*al integri ty of the amino
acid du ring the synthesis. It s t i m u l a t e d further
r e s e a r c h into both acid- and b a s e - l a b i l e p r o t e c t i n g
groups to ove rc om e speci fi c pro bl ems in the syntheses of
co m p l e x peptides. The most important a c i d - l a b i l e groups
d e v e l o p e d are the tert. b u t y l o x y c a r b o n y l (Boc) group,
the o - n i t r o p h e n y l s u l p h e n y l (Nps) gr oup and the
b i p h e n y l i s o p r o p y l o x y c a r b o n y l (Bpoc) gr ou p (Carpino
1957a; b; A n d e r s o n 1957; M c Kay 1957; G o e r d e l e r 1959;
Zervas 1963; Sieber 1968a; b). The most important base-
labile g r o u p is the 9-f luoreny line thy l o xy carbony l ( Fmoc )
gr ou p wh ich has becom e much used in the synthe si s of
pe p t i d e s c o n t a i n i n g up to 74 ainino acids (Carpino 1970;
1972; Wu 1987).
There are five main types of ami no p r o t e c t i n g grou ps
(listed in Table 1.1) and which type is used is
d e p e nd en t on the amino acid to be protected, the
sequenc e of the pe ptide to be s y n t h e s i s e d and the
protocol to be followed.
-20-
schem e 1.1) . Fur the r c o m p l i c a t i o n s occur when the side
chai n also carries a functional group. However, groups
w h ic h " deacti va te" one or other of the func tional groups
in a way w h i c h req uir es h a rsh con dit io ns to remove them
are of no use in pe pt ide synthesis. Hence p r o t e c t i n g
gr oups w h i c h are inert to the reacti on con d i t i o n s of
pe pt id e synthesis, and yet can be removed easily u s in g
mild conditions , without b r e a k i n g the pe pt id e bond, are
required. The first pepti d e s pro du ce d had b l o c k i n g
gr ou ps which could not be removed without the
d e g r a d a t i o n of the pe ptide bond and thus the first
p r o b l e m to ov ercom e was that of re movable p r o t e c t i n g
g r o u p s .
Sc heme 1.1 P o s sibl e Produ ct s from the C o u p l i n g of Two
Amino Ac i d s .
NH2CH2CONHCH2COOH
n h 2c h 2cooh NHoCHCONHCHCOOH
c h 3n h 2c h 2c o nh c h co o h
n h 2chc o o h
c h 3n h 2c h c o n h c h 2cooh
Polymers
This was done with the d i s c o v e r y of the
benzy 1 o x y c a r b o n y 1 (Z) g r ou p by Berginann and Zervas in
T a b l e 1.1 Amine P r o t e c t i n g Groups
G r o u p Type IEx am pl e A p p l i c a t i o n s and C o n d i t i o n s
A:C a r b o x y l i c Acid
F o r m y 1 (For )
S u f f i c i e n t l y stable towards bot h alkali and a n h yd rous acids. U s e ful in the synthes is of some small pe ptides and i n t e r m e d i a t e s not of val ue in the synthe si s of larger peptides.
T r i f l u o r o acetyl (T F A )
Introduced via the anhydride. C l e a v e d by mil d alkali or N a B H 4 . Gives in te r e s t i n g rea ct io ns and is not r e c o gn ised for its use in higher peptide synthesis.
P h t h a l o y 1 (Pht )
Stable to acids and h y d r o g e n o - lysis thus h y d r a z i n o l y s i s only meth o d of removal. Pept i d e s thus pr o t e c t e d cannot be treated wit h alkali due to ring opening g i ving a pro duct which is not easily cleaved. Int rod uced u s in g N - c a r b e t h o x y p h t h a l a m i d e in w ea k alkaline solution.
Aceto- a c e t y 1 (Aca )
Introduce d by re ac t i o n with ketene then s a p o n i f i c a t i o n of the esters. Stable to acids but cl e ave d with eg. h y d r o x y l a m i n e . Not found general appl icati on.
M a 1e o y 1 Not sui tab le for use in rout in e synthesis, useful in re a c t i o n wit h thiol groups. Intr od uced by reactin g N - c a r b o x y m a l e a m i d e in mild al k a l i n e conditions. Stab l e towards TFA. cleaved by tr e a t i n g wit h dilute alkali followed by aq ueo us acid.
C h 1oro- acetyl (Cla)
Introduced by c h l o r oa cetyl chloride. C l e a v e d with 1, 2 - p h e n y l e n e d i a m i n e or thioure a L i m ited signi ficance.
Di thia- succ i n o y 1 (Dts )
Stable to acids and photolys is. S e l e c t i v e l y cl ea ve d by re d u c t i v e proc e d u r e s in pre se nce of 0 . 5M t r i e t h y l a m i n e . Intr od uced u s ing e t h o x y t h i o c a r b o n y l amino acid t-butyl esters and chloro- c a r b o n y 1s u p h e n y 1 chloride.
-21-
T a ble 1 . 1
B:Ure thane Ty pe
cont i n u e d .
B e n z y l o x ycarbonyl(Z)
In tro duced via the c h l o r o f o r m a t e C l e a v e d by acid o l y s i s and h y d r o g e n o l y s i s , S u b s t i t u e n t s in the ring help wit h purific ation. Some give increas ed s t a b i l i t y to acids and so are useful for side ch ai n protection. Some render the g r ou p more sen si ti ve to acid
9-f 1uorenyl me thyloxy carbonyl (F m o c )
Base labile p r o t e c t i n g group.Can cause s o l u b i l i s a t i o n problems. E l i m i n a t i o n is a c c o m p l i s h e d at room tem p e r a t u r e us in g piperidine. Stab le to acid Of more use in solid phase than in s o l ut io n phase.
Fur f u r y 1 oxy-c a r b o n y 1 (Foe )
Introduce d by r e a c t i o n of furfurol with i s o c y a n a t o c a r b o n i c acid esters. C l e a v e d by TFA or catalyt ic hy dr ogenation.
Di iso- p r o p y 1- me thyloxy carbonyl (Dmc)
Stable to all common cle av age m e thod s used for N - p r o t e c t i n g groups. C l e aved by liquid HF at 20°C in pr es ence of anisole. Has received scant attention.
I so-b o r y l o x y carbonyl ( I be )
Acid labile co m p a r a b l e to t-Boc. Has found some use in synthesis.
t - b u t y 1- oxy-c a r b o n y 1 (Boc )
Acid labile. Most co m m o n l y used group along with Z group. Introdu ce d w ith the ditert.- b u t y l - d i c a r b o n a t e .
2- ( 4-bi-pl (B p o c )
le ny ly 1)p r o p y 1(2 ) o x y c a r b o n y 1 useful for q u a n t i t a t i v e d e p r o t e c t i o n of large pep ti des be c aus e the methyl protons are ea si ly de t e c t a b l e by NMR. Introduce d by the azide or active ester. C l e a v e d by T FA in DCM or by hydrog enolys is .
2- ( 4-pyr i<iy1)p r o p y 1(2 ) o x y c a r b o n y 1 The presen ce of the n i t r o g e n increases the p r o t e c t i n g gr oup s s t a bility to acids, the subst i t u e n t s also in fluence it. Cl e a v e d by Zn in AcOH, e l e c t r o lytic reduction.
-22-
Tabl e 1.1 continued.
C:S u l p h u r - / P h o s p h o r u s - C o n t a i n i n g groups
2 -ni tro- p h e n y l - s u l p h e n y 1 (Nps )
IIntr od uced via 2 - n i t r o p h e n y 1- | sulphenyl chloride. C l e a v e d in we ak ly acidic medium, when t-butyl residues are unaffected. Has found wide applicatio n. Also cl e ave d by thiolysis.
4 - toluene s u l p h o n y 1 ( T o s y l , Tos )
One of the ea rlies t N - p r o t e c t i n g groups used. C l e a v e d by HI and P H 4 I in AcOH at 60°C and Na in liquid ammonia. Intr od uced by 4 - t o l u e n e s u l p h o n y 1 chloride. Partial d e c o m p o s i t i o n of Arg,C y s , T rp and h y d ro xyl amino acids occurs d u ring cleavage of this group. Impor tant for side chain protection, e s p e c i a l l y for Arg as the t o s y l g u a n i d i n e bond is clea ved by liquid H F .
Di p h e n y l - ph o s p h i n e (D p p )
D i p h e n y 1ph osphin e chlori de used to prep are the p r o t e c t e d ami no acids. Can be used with all me t hod s of condensation. S l i g h t l y more acid labile than Boc g r o u p .
D :AlkylGroups
T r i p h e n y l me thyl (Trt )
Introduce d us ing trityl chlo ri de O nl y alkyl g r ou p of impor tance for Na protection. Ca uses steric h i n d e r a n c e to the car b o x y l i c g r oup but c o u p l i n g using DCC, D C C - H O S u or D C C -HOBt give good yields. C l e a v e d by h y d r o g e n a t i o n but it is p r e f e r a b l e to use w ea k ac i d s .
E:Oxo compoun d der ivat i v e s .
Benzyl- idene d e r i v s .
Introduce d via the b e n z a l d e h y d e . T h e y are transient p r o t e c t i o n for ^ - a m i n o gr oups in the p r e p a r a t i o n of N a - p r o t e c t e d d i a mi no acids. C l e a v e d by treatment with b r o min e water or n i t rous acid in ac etic acid. If Tyr present, the a p p l i c a t i o n of Bromine water is not feasible.
Table com piled from Ge ige r 1981 and ref e r e n c e s therein.
In so 1u t i o n-ph as e pe pt ide synthesis, the carboxyl
protect in g g r ou p also pr om ot es s o l u b i l i t y of the amino
-23-
acid or peptide and helps in the p u r i f i c a t i o n of the
pro ducts. The choice of which p r o t e c t i n g gr oup to use
for the c a r b oxylic end is depen de nt on the overall
pr o t o c o l that is g o ing to be used. Ta ble 1.2 lists the
v a r i o u s group s used for p r o t e c t i o n and the po ssib le
c l e a v a g e methods.
Tabl e 1.2. Car bo xyl P r o t e c t i n g Groups and The ir M e t ho ds of C l e a v a g e .
Pr o t e c t ing G r o u p
Cl ea va ge Me thod
Uses and D i s a d v a n t a g e s
T r i m e t h y l s i ly 1 e s t e r s
H y d r o l y s i s in a c i d i c , bas i c or neutral aqueous media
Te m p o r a r y p r o t e c t i o n to aid s o l u b i l i s a t i o n and prevent carboxyl ion reaction. Formed in situ.
t - B u t y 1 esters
mi Id acid conditions. HC1 in organi c s o l v e n t s , T FA or HBr in AcOH
In c o m b i n a t i o n with amine p r o t e c t i n g groups which are cleaved by hydro genation. Stable to the mild c o n d i t i o n used to cleave the B p o c , Nps and Trt groups. E s p e c i a l l y used if peptide has sulphur c o n t a i n i n g amino acids.
Di p h e n y 1 met h y 1 esters
mild ac i d conditions, TFA h y d r o g e n o l y s i s
Used only sparingly. Pr e p a r e from d i p h e n y l d i a z o m e t h a n e - this is a d e t e rrant to its use .
T r i m e t h y lbenzylesters
mild ac i d , 2N HBr in AcOH, TFA
More stable to acid than t e r t . butyl esters. Use d in c o m b i n a t i o n with more acid labile amine p r o t e c t i n g g r o u p s .
Ph t h a l - i m i d o me thy 1 esters
mild acid, 2N HBr in AcOH
P r e p a r a t i o n involves several steps, the re fore they are not g e n e r a l l y used though they have great versat ility.
.............. - .............. . . . . . . . ...
-24-
Tab le 1.2 continu ed
Benzyl and p - N i t r o - benzyl es ters
1h y d r o g e n o l y s i s . Zn in A cO H for p- n i t r o b e n z y l esters when benzyl esters are p r e s e n t .
Benzyl ester not c o m p l e t e l y stable under cond i t i o n s used for removal of Z group, p - n i t r o b e n z y 1 esters more stable to acidolysis. Benzyl esters used for side cha in p r o t e c t i o n on G lu and Asp.
4-pi col ylesters
H y d r o g e n o l y s i s . E l e c t r o l y t i c reduction, Na in liquid a m m o n i a , sapon- i f icat i o n .
h y d r o g e n o l y s i s can be slow leading to side reactions. Useful g r o u p for p u r i f i c at ion e n h a n c e m e n t of i n te rmedia te s and products.
Me t h y 1 esters
tr eatment with a l k a l i , non- basic nu cleo- p h i 1e s .
many side r e a c ti ons can occur when us in g alkali, but they can be minimised.
Phenylesters
pe roxid e anion no r a c e m i s a t i o n is detected, solvents such as MeOH and EtOH should be av o i d e d to pre ven t t r a n s e s t e r i f i c a t i o n .
Pol y-ethyle neglycol
saponi ficatio n has some a d v a n t a g e s of SPPS and allows p u r i f i c a t i o n by di ff e r e n c e s in mo l e c u l a r size.
13-p- t o 1uene sulphonyl e t hy 1 ester
^- elimi nation, treatment with cyanide ion.
only one of this type to have been used in a c o m plex synthesi s .
P h e n a c y 1 esters
sodiu m thio- phenoxi d e ,Zn in Ac O H
P r e p a r e d from ph ena cyl br omide and a c a r b o x y l a t e salt. Used in SPPS to a t tach the first amin o acid to the r e s i n .
9 - a n t h r y 1- met h y 1 e s t e r s
C HaS Na in hexa- me t h y 1p h o s ph or - ami de .
not been put to use in s y n t h e s e s .
2 - t r i - me t h y 1- s i l y 1- e t hy 1 esters
quat ernary a m m o n i u m fluor ide in DMF
the Z g r oup tends to form hy da n t o i n s under these conditions. Benzyl esters p ar t i a l l y cl e a v e d and d i s u l p h i d e s d i s p r o p o r t i o n a t e Useful in some instances.
-25-
T a bl e 1.2 continued.
2 , 2 ’- pho tolysis this m e t h o d of c 1eavage anddinitro- thus this type of group hasd i p h e n y 1- d e v e l o p e d only s l o w l y .me t h y 1ester
Ta bl e compi l e d from Ro esk e 1981 and r e f e re nces therein.
All these types of p r o t e c t i n g gr oups give an
o v e r w h e l m i n g choice for the chemist when se tt ing out on
the synth esis of a co mplex peptide, on how to achieve
the best po ssibl e results and the sim plest p u r i f i c a t i o n
p r o c e d u r e .
1.1.3 Pept ide Bond Formation.
The main react io n in peptide synth esis is a c y l a t i o n of
the amino g r o u p of one amino acid by the car boxyl g r ou p
of ano ther to form an amide bond. The p r e sen ce of
several functional groups in an amino acid and the
n e c e s s i t y to m a i n t a i n the integrity of the chiral
centres duri ng co u p l i n g are what makes pept id e synt he sis
muc h more c o m p l i c a t e d than simple c a r b o x a m i d e formation.
Many method s have ther efore been d e v e l o p e d for p e p tide
bona formatio n under mild conditions. All these methods
are based on the e l e c t r o p h i 1 ic a c y l a t i o n m e c h a n i s m of
the N H 2 -R i n v o lv in g a c t i v a t i o n of the a - carbon yl
f uncti on al ity of N - p r o t e c t e d amino acids and peptides.
The elec trophi li city of the carboxyl c a r b o n is u s u al ly
enhan ced by e l e c t r o n e g a t i v e groups a t t a c h e d to it.
T h es e reduce its low elect r o n densi ty still further' and
-26-
come in two categories: 1) repl ac ement of the h y d r o g e n
and 2) r e p l ace me nt of the hydroxyl group. C a t e g o r y 1)
is typical of active esters, anh ydrides, i m i n o a n h y d r i d e s
and acetals. C a t e g o r y 2) is typ ified by azides,
d i i m i n e s , azoles, a c y l a m i n e s , thioesters, acid halides
and cyanides. The react i v i t i e s of the m a i n types of
a c y l a t i n g agents are in the order: R C O N R ’< R C O O R ’<
R C O O C O R ’< RCOHal and w i t h i n one type the r e a c t i v i t y is
d e p e n d e n t on R.
Sc heme 1.2 Two M e c h a n i s m s by w h ich an Amine Will
Di spl a c e a L e a v i n g Group.
Gene ral ly, the a c y l a t i n g power of RC O X inc reases with
the a b i l i t y of X to depar t and is the refor e depe nd ent
on the acid st r e n g t h of H X . There are two m e c h a n i s m s by
whi ch the in co ming amine g r ou p dis p l a c e s the leaving
g r ou p X (see scheme 1.2); one being a one step pro ce du re
and the other a two step. The i n t e r med ia te in the two
a) Con certed.R-C-0J -------=» RCONHR + HX
H-N-HI.R'
b ) T w o - S t e p . 1R-C-X•=> RCONHR'
+ HX
RCOX +NH?R'
R'
-27-
step m e c h a n i s m has never been d i r e c t l y d e t e c t e d or
trapped, but ki ne tic evide nce points to its e x i stenc e— Q - j owith a lifeti me of 10 to 10 seconds (B od anszk y
1976; Jakubke 1966; Jo hnson 1967) .
1.1.4 C o u p l i n g Reagents.
Re activ e inter m e d i a t e s such as acid chlo ri des and acid
a n h y dr id es were used for amide f o r mat io n befor e the
birt h of pe ptide synthesis, and so were used i n i tially
in the f o r m a t i o n of pe ptide bonds. However, al ong with
the new meth ods of p r o t e c t i n g the functional groups, new
methods of bond foi'ination were evolved. C u r ti us
de v e l o p e d the classic al m e t h o d usi ng azide d e r i v a t i v e s
which is still in use today (Curtius 1902), whilst
Fi scher used the acid chl or ide m e t h o d which is rarely
used now (Fischer 1903 ) . The most po pular met h o d was
the use of acid anhydrides. Mixed a n h y drides were
pr op os ed by many investigato rs, all using di fferen t
co mp onents to ac tivate the amino acid but using the same
general procedure. Goo d results were o b t a i n e d using an
alkyl carbo ni c acid - p r o t e c t e d amino acid m i xed
anhydride, the best being the sec. b u t y l c a r b o n i c
an hydride (Wieland 1951; B o i s son na s 1951; Va u g h a n 1951;
1952).
A no the r m e th od of a c t i v a t i n g the amino acid, p r o p o s e d in
1955, was that of usi ng an ac t i v a t e d ester such as the
nit ro ph enyl ester or the N - h y d r o x y s u c c i n i m i d e ester
(Bodanszky 1955; A n d e r s o n 1963; 1964).
-28-
The most attr a c t i v e ap p r o a c h though had to be the use of
"c oup li ng reagents" which could be added to a m i x tu re of
both p a r t i a l l y p r o t e c t e d amino acids or peptides (one N-
protected, the other C - p r o t e c t e d ). The most success fu l
of these mus t be N ,N ’- d i c y c l o h e x y l c a r b o d i i m i d e (D C C )
which was intr od uced by Sh e e h a n and Hess (Sheehan 1955 ) .
Th er e have bee n att em pts to replace DCC wit h more
effici ent or less dra sti c materials, but it is still
leading the field along with its water sol uble variants.
The more recent 1- e t h o x y c a r b o n y l - 2 - e t h o x y - 1,2-
d i h y d r o q u i n o l i n e (E E D Q ) and 1 - i s o b u t o x y c a r b o n y 1-2-
i s o b u t o x y - 1 , 2 - d i h y d r o q u i n o 1 ine (IIDQ) are used to form
mixe d an hydrid es in situ. The y are readily prepared,
e asi ly stored and cause no d i s c e r n i b l e r a c e m i s a t i o n
(Belleau 1968; Kiso 1973).
Scheme 1.3 Pr o p o s e d M e c h a n i s m of A c tio n for DCC
H®R'N=C:NR' ----- R'NH=C=NR'
(RC0)?0(b)
R'NHCNHR'110
a = O -acylisourea b= Acid anhydride
c= N -acylurea
d = Peptide
RCOO
RCOOH R'NHC^NR'
.00
R'NHCNR'
/ i ° z C 0a i R
(c) R-C=0
NHoCHCOOCH1R"
rco nhchco o ch
(d) R”
03 + R'NHCNHR'
-29-
DCC has be come the most co m m o n l y used co u p l i n g reagent
for p e p ti de syn th es is be cause as a d e h y d r a t i n g agent it
aids the e l i m i n a t i o n of water be tween two amino acids.
Its b y - p r o d u c t N ,N ’- d i c y c l o h e x y l u r e a has low solu b i l i t y
and thus can be e a si ly re mo ved from aq ue ou s media. Its
m e c h a n i s m of r e a cti on is thought to follow scheme 1.3
(Kurzer 1967 and r eferenc es therein).
1.1.5 S o l u t i o n Phase P e p tide Synthesis.
S o l u t i o n phase peptide synthesis can be done in two ways
stepwis e a d d i t i o n and fragment conde nsation. The
latter allows for more f l e x i b i l i t y in the choice of
p r o t e c t i n g groups and cou p l i n g methods used and has been
applied s u c c e s s f u l l y to the syntheses of pepti de s up to
about 60 amino acids long (Gross 1979). Larger peptid es
made by this m e thod are impeded by an increase in the
occ u r r a n c e of r a c e m i s a t i o n , poor s o l u b i l i t y and low
couplin g speed, thus i n c r ea si ng the p o s s i b i l i t y of
irreversib le side reactions (Sieber 1970; 1977; Ge ig er
1969; Ivanov 1976).
Stepwise ad d i t i o n of amino acids to the pept ide chain
permits the use of excess a c y l a t i n g com po nent to drive
the re a c t i o n close to completion. However, with
incremental chain elongation, proof of the pe pt ide
ho m o g e n e i t y becomes less accurate.
There are four co mm on me th ods of stepwise synthesis:
A: The pe pt ide is a t t a c h e d to a soluble po ly mer w h e r e b y
the most important feature of solid phase, the
-30-
f a c i l i t a t i o n of the s e p a r a t i o n of int ermediates, is
co m b i n e d with the important feature of s o l ut io n phase,
ha v i n g the reacti on s o c c u r r i n g in a h o m o g e n e o u s phase.
The best poly mer found was p o l y e t h y l e n e glycol with a
m o l e c u l a r weight from 2,000 to 20,000 d a lton (Mutter
1972 ) .
B: Incor p o r a t i o n of an ionisable gr ou p into a pro te cted
p e p tide makes it p o s si bl e to ad sorb it onto an ion-
excha ng e column. Thus i n t e r media te s can be pu r i f i e d by
washing. This is k n own as the " h a n d l e ” m e t h o d (Young
1973). Two gr oups pr o p o s e d for this were 4-
d iine t h ylami no -4 ’ - h y d r o x y m e t h y l a z o b e n z e n e (p-dimethyl-
a m i n o a z o b e n z y 1 alcohol) and 4 - h y d r o x y m e t h y l p y r i d i n e (4-
picolyl alcohol). E s t e r i f i c a t i o n of a peptide carboxyl
group with one of these alc oh ols foll owed by p r o t o n a t i o n
gives a cati onic centre wh ic h will bind to cation
exchange resins such as s u 1f o e t h y 1s e p hadex (Wieland
1968; Camble 1968; 1969). This m e th od can be ap pli ed to
segment c o n d e n s a t i o n as well (Bratby 1979).
0: ’In s i t u ’ synthesis. This is si mil ar to the solid
phase method in that the i n t e r m edia te s are p u r if ie d by
p r e c i p i t a t i o n from the r e a ct ion s o l ution by a d d i t i o n of
a ca ref ully ch osen solvent in which the p e p tide product
is insoluble (a " n o n - s o l v e n t " ). For a simpler e x e c ution
of this met ho d a ce nt r i f u g e tube with a st andard taper
joint was used so that both co u p l i n g and d e p r o t e c t i o n
could be carr ied out in this "reactor" w h ic h also
-31-
p e r m i t t e d the removal of solvents by e v a p o r a t i o n in
v a cuo (Bo danszky 1973; 1974).
D: S y n t hes is without i s o l at io n of the int er m e d i a t e s
a t t r a c t e d wide interest. E a rly schemes used the water -
sol ubl e c a r b o d i i m i d e s for the rapid synthesis. A
p r o t e c t e d h e p t a p e p t i d e was secu red by this m e th od
(Sheehan 1965) . The use of N - c a r b o x y a n h y d r i d e s made
e x c e p t i o n a l l y rapid cha in e 1ongat i on possi bl e
(Hirsch ma nn 1967). However, the is ol ation of the
in te r m e d i a t e s does lead to a better u n d e r s t a n d i n g of the
me t h o d s used and an insight into the p o s sible m e c h a n i s m s
involved, even though it is u s ua ll y a slow and
r e p e ti ti ous process.
The re is an upper size limit be yond w h ich amino acid
an aly s i s will fail to show whe ther cou p l i n g of a
f r e q ue nt ly o c c u r r i n g amino acid did occur or not. For
prac ti ca l purposes, this size limit is b e t w e e n 15 and 20
r e s i d u e s ,
1.1.6 Solid Phase Pe p t i d e Synthesis.
The re petit iv e c h a r acter of the chain e l o n g a t i o n process
p r o mp ted s p e c u l a t i o n about the m e c h a n i s a t i o n of the
p r o c ed ur e (Bodanszk y 1960). The r e a l i s a t i o n of these
ideas came s i m u l t a n e o u s l y from M e r r i f i e l d and from
L e t singer (Merrifi eld 1963; L e t s in ge r 1963). In both
methods, an amino acid was a t t a c h e d to an insolub le
p o l y me ri c support and the chain e l o n g a t i o n was then
carried out (see scheme 1.4). It is only the v e r s i o n
-32-
pr opose d by M e r r i f i e l d that has de v e l o p e d into a major
dis c i p l i n e - Sol id Phas e P e p ti de S y n the si s (SPPS). The
polymers used as the solid support are gels rather than
real solids. The re actions occur inside the beads as
well as outs ide and so the solven ts must be such that
Scheme 1.4 Steps Inv olved in Soli d P h a s e peptide
Synthe s i s .
O R(c h 3)3co cnhchco o " + c ic h 2
EtOH. 80°C
0 11(CH3)3COCNHCHCOOCH2
R
TFA/DCM v Deprotection
CF3C00G NHfcHCOOCH2-< ) - {p )
CYCLE
R
Et3N/DCM m Neutralisation
0ii(CH3)3COCNHCHCONHCHCOOCH
n h 2c h c o o c h ;R
BocAAOH/DCC/DCM
v Coupling
R' R
HF Cleavage v
PEPTIDE
-33-
these beads swell so as to allow the r e a ctants to
di ffuse in and out of them. The d e s ir ed i n t e r m e d i a t e s
and pr od u c t s can easily be s e p a rated from the b y
pr odu ct s and excess reagents. Since its in troductio n,
this m e th od has given en ormou s stimuli to the field as a
whole and has been used to prepare m any hundre ds of
pe ptide s of v a r y i n g size and function. The m a i n
a d v a nt ag es of this met h o d are: high speed; no
i n solu bi li ty problems; c o n v e n i e n c e of o p e r a t i o n and the
a b i li ty to aut om ate the whole procedure. The ma i n
d i s a d v a n t a g e comes from product m i c r o i n h o m o g e n e i t y
caused by either repeated inco mplete coup li ngs or
d e c o m p o s i t i o n of the product during the final clea va ge
(Sharp 1973; Mar sh al l 1973).
1.2 CROWN E T H E R S .
1.2.1 History.
M a c r o c y c l i c po l y e t h e r s were first s y n t h e s i s e d as e a rl y
as 1937 by Liittringhaus and Ziegler ( 1937 ), but it was
not until 1967, when P e d e r s e n pu b l i s h e d a d e t a i l e d paper
on the syn thesis and pr op e r t i e s of C r own compounds, that
their use in many fields of c h e m is tr y st ar ted (Pedersen
1967a). Peder s e n ob t a i n e d 2 , 3 , l l , 1 2 - d i b e n z o - l , 4 , 7 , 1 0 ,
1 3 , i 6 - h e x a o x a c y c l o o c t a d e c a - 2 ,11-diene (dibenzo 18-Crown-
6) as a by -produ ct in the r e a ct io n of mon o p r o t e c t e d
catechol and d i c h 1 or o e t h y 1 ether. This was found to be
due to some non p r o t e c t e d catechol pre sent in the
-34-
re a c t i o n solution. S t u d y i n g the p r o p erties of this
c o m po und showed that there was a m a rked increase in its
s o l u b i l i t y in me thano l in the pr es en ce of NaOH. The
co mplex thus formed was shown to be stable and sol ubl e
in non polar organic solvents. It was p o s t u l a t e d that
these com plexes were formed as a result of ion- di pole
in te ra ctions b e tw ee n cations and the n e g a t i v e l y ch arged
ox ygen atoms s y m m e t r i c a l l y placed about the pol y e t h e r
r ing .
1.2.2. Cha ra cter is ti cs.
To test this hypothesis, P e d e r s e n s y n t hesise d 49
m ac r o c y c l i c p o l y e t h e r s and st udi ed their comple x
f or mation with var iou s metal salts and put forward the
important c h a r a c t e r i s t i c s of crown comp ou nds whi ch have
been used as the basis for man y studies since.
1. Many of the m a c r o c y c l i c po ly e t h e r s h a vi ng 5 - 1 5
ox ygen atoms, formed stable co mplexe s with most metal
s a l t s ;
Figure 1.1 St ructura l Elem en ts of C r o w n Ethers,
LIPOPHILIC
HYDROPHILIC
-35-
2. The sta b i l i t y of these com plex es d e p e n d e d on the
r e l a t i o n s h i p be t w e e n the ionic radius of the ca ti on and
the cavity in the m a c r o c y c l i c polyether;
3. Various inorganic salts of metal ca ti ons were
soluble in man y organic solvents, inc l u d i n g non polar
ones, in the p r e se nc e of the m a c r o c y c l i c polyethers.
These com po unds were n a me d cro wn co mp ounds be c a u s e of 1 )
their chemical struct ur e and 2) the a p p e a r a n c e of the
complex, w h ich resembles a cro wn placed on an ion. T h e y
consist of a series of li poph i1 i c and h y d r o p h i 1 i c
stuctural elem ents (see Fi gure 1.1). The b e h a v i o u r of
this type of co mpo un d in h y d r o p h i l i c m e di a can be
likened to that of a fat droplet in water (an
e n d o l i p o p h i 1 ic ca vity is formed) see Figure 1.2a. In
lipophilic media, p o l a r i s a t i o n is reversed, a s s u m i n g a
degree of fl ex i b i l i t y in the backbone, and the compoun d
behaves like a water dr oplet in oil (an e n d o h y d r o p h i l i e
cavity is formed) see Figure 1.2b (Vogtle 1979; Web er
1981) . In most cases, the lipoph ilic c a v i t y wo ul d be
too small for the uptake of a lipo philic guest molecule.
However, the h y d r o p h i l i c cavity is idea lly su ited to the
uptake of cations. The ligating o x y g e n atoms in this
cavity are located in a regular, coplanar manner and
they contact the cati on located in the ce ntre at an
a r i t h m e t i c a l l y c a l c u l a t e d interato mic d i s t a n c e (as shown
by the K*--18-crown-6 complex, see Fi gure 1.1).
This means that they are stable to en tr opy w h ich is not
the case for the linear polyethers. However, the
st abili ty and se le c t i v i t y of crown co mplexes is
dep en de nt on the dyna mic beh av io ur of the system,
compose d of the ligand, the cation and the solvent.
Figure 1.2 Cr ow n Ethers Beh av iour in D i f f ere nt
S u r r o u n d i n g s .
a) In H y d r o p h i l i c Media.
ENDOLIPOPHILIC CAVITY
b) in Lipo philic Media.
ENDOHYDROPHILIC CAVITY
1.2.3. T h e r m o d y n a m i c s .
C o m p l e x a t i o n is not a simple one step r e a ction be tw ee n
cation and ligand. It often includes a series of steps
such as removal of one (or several) solvent m o l e c u l e s
-37-
from the c o o r d i n a t i o n sphere of the catio n and
c o n f o r m a t i o n a l re a r r a n g e m e n t s of the ligand,
p a r t i c u l a r l y when the ligand is a inultidentate one
(Webe r 1981 ) .
The c o m p l e x a t i o n re ac tion of crown co mpounds is
d e s c r i b e d by the equation:
(L).oiv + (M*+ ,mS) *— ~ c-d» (L,Mn *).oiv + mS
(Hi raoka 1982)
where L rep r e s e n t s the crown compound, M n + the ca tion
and S a so lvent molecule. An imp ortant value for these
r ea c t i o n s is the s t a b il ity constant, which is n o r m a l l y
gi ven b y :
K - Kt hf if w = [L ,M n *]
fc [ L ] [ M n + ]
Many d i f f e r e n t me th od s of m e a s u r i n g the s t a b il ity
co nstan t have been used, and they include NMR (Tiinko
1974), c a l o r i m e t r i c ti tra tio n (Izatt 1969; 1971; 1976)
and e l e c t r o n i c s p e c t r o m e t r y (Frensdorff 1971; Wong 1970;
Shchori 1975). It has been shown that for mono v a l e n t
cati ons and crown ethers the values of log K depen d on
the c a t i o n diameter, and the ma x i m u m value is found with
K* whose di a m e t e r is closest to the ca vi ty size of 18-
crown eth ers (Izatt 1976). The solv ent plays an
important role in complex stability. Fr e n s d o r f f
m e a s u r e d the s t a b i l i t y co nst ants for alkali metal
c o m pl exes of va ri ous crown com po unds in methanol and on
-38-
c o m p a r i n g these values with those ob t a i n e d for the same
co mplex es in H 2 O, they were found to be 1,000 to 10,000
times larger (Frensdorff 1971). This is because
met hanol is a weak competitor’ for c o o r d i n a t i o n compar ed
to HzO. It is less able to di ssolve a ca tio n in the
a b s en ce of the crown ether than H 2 O. However, the
r e l a t i o n s h i p b e twee n cavity size and ca tio n di ameter is
sim ilar to that found in HzO for crown ethers with ring
size 14 to 18 atoms.
The re are few st a b i l i t y co nstants r e p orte d for solven ts
other than methanol and water. It may be e x p ected that
K values will increase for small cations in low polar
solvent s be ca use of dec r e a s e d c o m p e t i t i o n b e t we en the
solvent and the crown compound. H o w ever , the si tuatio n
is comp l i c a t e d by the effect of the c o u n t e r - i o n because
of ion pair formation. M a t s u u r a and c o-worke rs
d e t e r m i n e d the K values of d i b e n z o - 18 - c r own-6 co mplexes
of alkali metal salts by e l e c t r o c o n d u c t i v i t y
meas ure ments. They used solvents such as dimethyl
s ul pho xid e (D M S O ), N ,N - d i m e t h y l f o r m a m i d e (D M F ) and
p r o p yl en e carb on ate and found that there was no
c o r r e l a t i o n o b s e r v e d b e tween the K value and the
d i e l ec tr ic constan t of the solvent (Matsuura 1976).
They also found that the K value d e c r e a s e d with an
in creas in g number of donors in the solvent.
In the c o m p l e x a t i o n of pr ima ry a m m o n i u m ions and NHa*,
hy d r o g e n bonds play an important part and hence have an
-39-
effect on the s t a b i l i t y constant of the complexes. Cram
et al. d e t e r m i n e d the K value in C H C 1 3 for;
L + Bu * NH 3 + . SCN ” -----— > Bu * Nil s + . L . SON ~
Some crown compo un ds with rigid cavitie s c o n t a i n i n g
donor atoms in the correct arra n g e m e n t for the h y d r o g e n
bonds had K values to 1 0 , 0 0 0 times greate r than that of
p e n t a g l y m e (Timko 1974; 1977).
1 . 2 . 4 . Kinetics.
A l t h o u g h much is know n about the th e r m o d y n a m i c s inv olv ed
in c o m p l e x a t i o n reactions, the same cannot be said for
the k n o w ledg e of the kinetics. In general, the
reactions of alkali and alk al ine earth metal ions
pr oce ed rapidly and d i f f u s i o n is c o n s i d e r e d to be the
rate d e t e r m i n i n g step. M e a s u r e m e n t s have been made of
the c o m p l e x a t i o n rate con stant ( k f ) and the d i s s o c i a t i o n2 3 3 0 8 7rate constant ( k d ). By usi ng N a - , K-, R b - a n d
1 3 3 Cs-NMR, the mov em ent of the cation in and out of the
co mp le x can be seen directly. Shchori et a l . e m p lo yed
this metho d in their i n v e s t i g a t i o n of the k i n eti cs of
c o m p l e x a t i o n b e tw ee n d i b e n z o - 1 8 - c r o w n - 6 and Na* in
dimethyl ether. They found that the chemic al shift of2 3Na in the com ple x was ne arly equal to that of solv at ed
Na , but that the widt h of nu clea r q u a d r u p o l e r e l a x a t i o n
of the co mp le x was 25 times greater than that of a
solvated Na because of the steric a s y m m e t r y around the
-40-
ion. Thus they were able to o b tain values for kt and kd
from the mean lives of both species of Na* in so lu tion
(Sh cho r i 1971).
T h ere is a close r e l a t i o n s h i p betw een the a c t i v a t i o n
energy and the c o n f o r m a t i o n of the complex. This has
been shown by Shchori (1973) when they o b s er ved
u p p r o x i m a t e l y the same valu e for the. a c t i v a t i o n energ y
of the d i s s o c i a t i o n of d i b e n z o - 1 8 - c r o w n - 6 complex in
DMF , methanol and dimethyl ether (12.6 ± 1 . 0 kcal\mol).
This value is in ag ree men t with that r e p or te d by Tr uter
et al. for the a c t i v a t i o n e n ergy of the co nf or m a t i o n a l
change in the crown ring d u r i n g the c o m p l e x a t i o n (Bright
1970; Bush 1971).
1.2.5. M o l e c u l a r Recognition.
All this must be taken into account when trying to use
crown compoun ds for m o l e c u l a r recognition, where a
ligand must select and bind a specific substra te out of
a gro up of pos si ble substrates. This bi nding makes use
of all kinds of in t e r m o l e c u l a r interactions;
e l e c t r o s t a t i c i nter ac tions are the most important in the
fixation of cat ion ic species such as metal cations, but
when the substr at e is some other form of cation,
hydrogen bonding, van de Waals forces, short range
repulsion s and steric hind e r a n c e s also have to be taken
into a c c o u n t ,
Cry p t a n d X.
The sim pl es t m o l e c u l a r ca ti on is the a m m o n i u m ion and
this cannot be d i s c r i m i n a t e d from the p o t a s s i u m ion by
size very eff ectively. However, there is a d i f f eren ce
in the charge dis tribution ; the a m m o n i u m ion is
tetrah edr al and the po t a s s i u m ion is spherical. It has
been found that the cr.yp lurid X is the optimal receptor
for the NH-»* ion as it can sit in the cavity. The
t et ra hed ral a r r a n g e m e n t of the n i t r o g e n bi nd ing sites is
ideal for the fo rm at ion of hyd r o g e n bonds with the
N H < ' ion (Lehn 1978a; 1978c). However, a pr imary
a m m o n i u m ion cannot sit in the ca vity of c r y pt an d X for
steric reasons, This cation requires a trigonal
a r r a n ge ment of h y d r o g e n bond a c c e pt ors as found in 18-
ero wn- 6 and the crown co mpound Y (Lehn 1977b) . Both of
these can d i s t i n g u i s h b e t we en p r i ma ry and se c o n d a r y
a m m o n i u m ions. This p r o p e r t y has been used by Barrett
et a l . (1978). The y s e l e c t i v e l y ac y l a t e d second ar y
-4 2-
amine s in the pre se nce of p r i m a r y ones by cornplexing the
p r i m a r y amines with 18-crown-6.
H
0
N
0
N
18-Crown -6 Cr ow n C o m p o u n d Y.
Crown compounds do not just com plex with cat ionic
molecules, neutral m o l e cu les can be complexed. This is
found in nature in such systems as the base pa i r i n g of
nucleic acids (Jones 1980) and the in te r a c t i o n b e t w e e n
enzyme and substrate. The first comple xe s of this type
were repor te d by P e d e r s e n (1971). The y were of thiourea
and rel ated m o l ec ules and cr ow n ethers. The
s t o i c h i o m e t r y of these comple xes with neut ral guests
lies be tw een one mo l e c u l e of po l y e t h e r and one to six
m o l ec ul es of the other components. The exact nature of
these compl exes is unclear. In these com plexe s the
neu tral m o l e cu le s c o n t a i n an NH g r o u p which h y d r o g e n
bonds with the oxyg ens of the crown compounds. H o w e v e r
there are polar organic molecu le s w h ich do not c o nta in
amine groups, but are still able to form co mplexes wi t h
species. Even without buildi ng c o m p l i c a t e d host
-43-
cro wn compounds. For example, Gokel et a l . (Gokel
1974; 1977) pr e p a r e d the comp lex of a c e t o n i t r i l e and
18-crown-6, but the X - ray an al ysis of the crystals
formed was dif fi cu lt due to the ch a n g i n g stoichio metry .
This has been found to be a good m e th od of pur i f y ing 18-
c r o w n - 6 because it remains pure upon d i s t i l l a t i o n of the
a c e t o n i t r i l e in vacuo. Another neutral, polar or ga nic
c o m p o u n d which has been shown to comple x with 18- cro wn ~6
is d i m e t h y l s u l p h o x i d e (Marji 1987) wh ic h is act i v a t e d by
the crown co mpound in its reaction with aromati c amines
to form q u i n o n e i m m o n i u m dyes.
1.2.6. A p p l i c a t i o n s to Or g a n i c Chemistry.
Co n s i d e r a b l e a t t e n t i o n has been paLJ to crown com pou nd s
since their spe cif ic characteris'tics may play a
si g n i fi cant role at the interface of s y n thetic c h e m is try
and life science. The re are four pos si ble functions
which can be ut i l i s e d and these are:
1) se lectiv e capture, sepa r a t i o n and transport of a
ca t i on ;
2) s o l u b i l i s a t i o n of inorg anic salt;
3) a c t i v a t i o n of the anion, and
2 ) s o l u b i l i s a t i o n of alkali metal.
Any one of these four functions has been ap pl ie d in
fo ur te en main areas of chemistry; organic, syntheses,
a n a l y t i c a l chemistry, drugs, poly mer synthe ses and
e l e c t r o c h e m i s t r y to name a few.
-4 4-
Spe cif ically, in organic chemistry, all four functions
are used in studies of re a c t i o n m e c h a n i s m s and kinetics;
whilst in organic synthesi s funct io ns 2), 3) and 4) are
used in phase transfer cataly si s and syn th eses in volving
inorganic salts. O r g a n i c synthesi s is the area where
most pr ogr ess has b een made in the a p p l i c a t i o n of crown
co mpo unds beca use of fu nctions 2) and 3) . The
n u c 1e o p h i 1icity and b a s i c i t y of the anion are in cre ase d
beca use it is not solvated, but a ’n a k e d ’ , ac t i v a t e d
ion. Thus the ani on can at ta c k a s t e r i c a l i y hinde r e d
rea ct ion site which a sol vated anion cannot due to its
size. The a d v a ntag es of using crown compoun ds are:
1) cheap inorganic salts can be used in or ga nic
syntheses; 2) no n- a q u e o u s h o m o g e n e o u s re act ion s can be
achi ev ed by s o l u b i l i s a t i o n of inorganic salts or alkali
me tals in non polar or low polar solvents; 3) react ions
pr oceed under mild cond i t i o n s and 4) re actions with
st ericaliy hin de red s ubstr at es can be carr ied out.
The main d i s a d v a n t a g e s for the use of crown co mp ounds in
orga nic sy ntheses a r e : 1) some crown com po un ds are toxic
and 2) they are rel a t i v e l y expensive. D e s pi te this,
crown com poun ds have found use in many types of organic
syntheses, though many are still in the expe r i m e n t a l
stages. Some rea ct ions in whi ch crown compoun ds have
been used are d e s c r i b e d in Tabl e 1.3.
-4 5-
T able 1 3 ^ Exa mp le s of some Or ga ni c R e a c t i o n s in whichC r own Ethers are Utilised.
Reacti on Type
R e a c t i o n Example and Condi t i ons
Comment s Ref .
S a p o n i f i - cat ion
2,4, 6 -tr ijnethy 1- benzoic acid t-butyl ester. KOH/ DC18C6 in toluene 110°C, 5 h r s .
S t e r i c a l l y h i n d e r e d ester gives no reactn when reflux ed for 5 hrs wit h xs KOH in n P rO H
P e d e r sen1967
Oxida t ion toluene to benzoic acid. KMnC>4/DC18C6 in benzene, room temp.
r ea ctions can be done using s o l i d - l i q u i d phase transfer catalys i s .
Sam1972
fluorene - fluore none O 2 , KOH, 18C6 in b e n z e n e .
very good yield obtained.
Hi raoka 1982
Re duc t ion ketone to alcohol N a B H 4 , DC18C6 in toluene, reflux 5 hr s
the ad d i t i o n of crown e ther increased the fo r mat io n of the alcohol to g r eate r extent than eg digl yme
Mat suda 1973
Subs t i t- ut ion
a) E l e c t r o p h i 1 i c . r a c e m i s a t i o n of o p t i c a l l y active cpds
Almy1970 G u t hrie Ro i tman Wong1971
b) N u c 1e o p h i 1 i c .Ce H x 7 Br to CttHi7 I ,M I ( a q . ), .01 eq crown ether, 8 0 0 C
yield: wi th c .e 80-100%; wi tho ut <4%
Landini1974
RBr to R O A c . KOAc 2eq 18C6 ( c a t . ) in MeCN/ benzene
R = b e n z y l , nCeHx j , n C a H n , 2 — C a H 1 7
Lio t ta 1974
E l i m i n at io n
a) PhCH=CHa to 1 ,1 - d i c h l o r o - 2 -phenyl- cyclopropane. C H C 1 3 , NaOH (5 0%) ,DB18C 6 (1 moT/o) , room temp.
Makos za 1974
-46-
Tabl e 1.3 contin ue d... . .!
Elimin- a t i on
I13) elim. of T o s O H by both syn and anti elim. pathways. tBu OK t B u O H , 18C6
+0T*» onit
... !ant i elim. p r e d o m in ates in p r e se nce of c .e syn elim. p r e do m i n a t e s in its a b s e n c e .
Bar t sch 1974
C o n d e n s - a t i on
PhC Hz COCH a to P h C H (B u )C OCH 3 . n B u B r , D C 1 8 C 6 , N a O H ( a q ) , 80° 1 . 5 h r s .
Landini1974
1somer- i sa t ion & r - » *6*
t B u O K / 18 C 6 , D M S O .
a d d i t i o n of t e t r a g 1yme instead of 18C6 had no e f f e c t .
Mastor- ni ck. 1972
Pe p t i de synthes i s
B o c T r p G l y O E t to B o c T r p ( Z ) G l y O E t .PhC H zOCOOCelU NOz l^eq KF 2 e q , 18C6 leq, t-PrzNNE, M e C N , 4 h r s .
no r a c e m i s a t i o n o c c u r r e d under these conditns
C h o r o v1976 Klaus- ner1977
Cl eav ag e of pe pt ide from resin.18C6/KCN l-10eq, D M F , 25 0C , 8-16 h r s .
gave better yield s than N a O H in dioxane -wate r or PhSN a i n D M F .
Tam1977
P o l ym er - i sa t i on
using acryl ic acid.80 ° C , K O A c , 18C6 init
o b t a i n e d degree of p o l y m . <14.
Y ama da 1976
Fo rm ed by s u c c es sive n u c l e o p h i l i c a d d i ti on s of ac r y l a t e anion whose n u c 1e o p h i 1 icity was i n c r ea se d by c o m p l e x a t i o n of K .
1.2.7. Phase T r a n s f e r Catalysis.
In a d d i t i o n to ho mo g e n e o u s liquid phase reactions which
e mpl oy one or more e q u i v a l e n t s of a high m o l e c u l a r
weight crown ether to an inorganic salt, ca ta lytic
amo unt s of crown com po un ds can be used in phase tra ns fe r
catalysis. Not only can cro wn compou nd s act as phase
transfer cat alysts in the usual 1 i q u i d - 1iquid transfer
b e t w e e n an aqueous so lu tion of an inorgan ic salt and an
-47-
or ganic p h a s e , but also in s o l i d - l i q u i d cata ly sis
be tween solid inorganic salt and an or ganic phase. This
type of so li d - l i q u i d phase transfer catalysis, where the
use of an aque ous sol ut ion is not r e q u i r e d , is specifi c
to cro wn co mpounds and cannot be p e r f o r m e d by well kno wn
phase transfer cata lysts such as q u a t e r n a r y a m m o n i u m
salts and phosphoniuin salts.
Phase transfer ca ta lysis usi ng these salts was first
d e s c r i b e d by Starks (1971; 1973), M a k o s z a (1973) and
B r a n d s t r o m ( 1969) . An exam ple of this type of re ac tion
is cyanation. The re ac tion hardly pro ce ed s when two
liquid phases (an aqueous so lution of NaCN and an
organic so luti on of ha loalkane) are he ated and s t i r r e d .
Add i t i o n of small am ounts of phosphoniuin salt, however,
leads to rapid cyanation. In the overall reaction, the
rate d e t e r m i n i n g step is the s u b s t i t u t i o n re a c t i o n in
the organic phase. L i q u i d - 1 iquid phase transfer
catalysis using crown compounds follows a similar
scheme. However, s o l i d - l i q u i d phase transfer cat al ys is
utilises the a b ili ty of a crown c o m p o u n d in an or ga nic
solvent to dissolve n o r m a l l y insoluble, solid inor ganic
salts in the solvent by the f o r m at ion of a complex.
This proc ess can be appl ied to c o m poun ds which are
hydrolysed, or react with w a t e r , b e c a u s e it proceeds under
a b s o l u t e l y anhydrous conditions. Not only is the
rea ct ion simpler, but so is the work-up.
1.2.8. H o s t -Gues t C o m p l e x a t i o n .
One of the main q u e s ti on s in h o s t - g u e s t c o m p l e x a t i o n is:
"To what extent does a guest orga ni ze its host upon
c o m p l e xa tion? " A l t h o u g h the oxyg ens of a p o l y e t h y l e n e
glycol ether host are collected, they are
c o n f o r m a t i o n a l l y d i s o r g a n i z e d until they co mplex with a
suit abl e guest. When the ch ain is formed into a ring
though, the number of p o s sible c o n f o r m a t i o n s is g r e a t l y
reduced. Thus it is that cr own com po unds form com pl ex es
several kcal mol 1 more stable than their open cha in
Figure 1.3 C o n f o r m a t i o n s of 18- Crow n- 6 with and without
K + SCN '
U1
° o
KSCN f K
" 0 0 I . 0
counterparts. This r e o r g a n i z a t i o n of the host m o l ecule
has been shown by D u nitz (Dunitz 1974a; 1974b; Do bler
1974a; 1974b; 1974c; Seiler 1974) with the crystal
st r u ctures of 1 8 - c r o w n - 6 and of its K + SCN complex ( see
figure 1.3). Cram and c o - w o r k e r s have used this ability
of crown c o m p o u n d s , — to select the com po und for
in c l usion, — to study many related areas in m o l e c u l a r
-4 9-
r e c o g n i t i o n and ho st-gue st chemistry. One of their main
areas of re search is into o p t i c a l l y active hosts to
d i s c r i m i n a t e b e t w e e n optical isomers of bio lo gi cal
co mpo un ds such as amino acids (Cram 1981; Kyb a 1973a;
H i r a o k a 1982; Sousa 1974; 1978). They u t i l i s e d the
op tical isomerism by rest ricted rotatio n (atrop
isomerism) which is caused by the b inapht hy l gr ou p of
crown com po un d Z. In the c o m p l e x a t i o n be tween a chiral
crow n ring and an e n a n t i o m e r i c guest, the st a b i l i t y
const ant is larger for the co mplex with the dia s t e r e o
isomer that has a more fitted c o m p l e x a t i o n and thus the
s e l e c t i v i t y arises. This work has been a p p lied to
0 - '
1-0
Cro wn Co mp ou nd Z .
s e p a r a t i o n techn iques such as l i q u i d - l i q u i d
c h r o m a t o g r a p h y and solid -liquid c h r o m a t o g r a p h y where
the f u n c t i o n a l i s e d cro wn compo und s are i m m o b il is ed on a
silica gel or p o l y s t y r e n e support. The y have also
a pp lied this work to the synthesis of enzyme models and
to as ym m e t r i c a l r e a c t i o n s , for example, the sy nthesis of
an o p t i c a l l y active crown compo un d which models the
binding site of subs tr ates to enzymes (Kyba 1973b).
Recent work by Sasaki and cow or ke rs (1985) has taken a
simi lar ap p r o a c h by using f u n c t i o n a i i s e d crown ethers as
an enzyme model for pep tid e synthesis. T h e y have used a
crown ether b e arin g one thiol and one thio ester w ith a
N -p r o t e c t e d amino acid, as it was shown that thio-
b e a r i n g crown ethers enh an ced the rate of the thio lysis
of a- a m i n o ester salts. This is based on the fact that
the N H a + gro up of an amino acid will co mpl ex with the
f u n c t i o n a i i s e d crown ether and thus br in g the ca rb o x y l i c
group into close p r o x i m i t y with the thiol g r o u p on the
side chain. The next step is the S to N acyl transfer
which occurs at an enhanced rate due to the
i n tram ol ec ular na tur e of the reaction. The y have
sy nt h e s i s e d a t et rapept id e by this m e t h o d (Sasaki 1985).
This shows that amino acids complex re adily with cro wn
e t h e r s .
Another ap proa ch to the use of crown ethers in the study
of b iolog ic al systems has been used by Te mu ss i and c o
workers (C a s t i g 1 ione-More 1 1 i 1987; T e muss i 1987; Pasto re
1984). They have st udied the c o n f o r m a t i o n in s o l ut ion
of op ioi d pep ti des by Nu cl ea r M a g n e t i c Re s o n a n c e
Spectr osc opy. It is g e n e r a l l y b e l i e v e d that these
pe pti des und ergo a c o n f o rmatio na l o r d e r i n g when they
change from the aqueous e n v i ro nm ent of the transport
fluid to the apolar one of the m e m b r a n e lipids.
H o w e v e r , there is d i f f i c u l t y in st u d y i n g these pept ides
by NMR S p e c t r o s c o p y in apolar solvent s be cause of their
ins ol ub ility and a polar solvent such as water, al th ou gh
-51-
r e pres en ta tive of the transport media, does not have the
propertie s h y p o t h e s i z e d for the active sites of these
peptides. Temussi and co-w or kers have ov e r c o m e this
problem by using an e q u i valent amount of 18-crown- 6 in
the C D C I 3 so lu ti on of the peptide. This also over co mes
any a g g r e g a t i o n problems. The NH3 + g r o u p of the peptide
co mp le xes with the 18-crow n-6 and thus the pe pti de is
taken into solution. The y believe that this mimics the
op ioid recept or site because all models based on the
p r o p ertie s of rigid n o n - p e p t i d e ag onists point to a
h i g h l y hy dr o p h o b i c pocket c o n t a i n i n g a spe cific anio nic
subsite. Thus the CDCls mimics the h y d r o p h o b i c pocket
and the 18-crown-6 the anioni c subsite (together with
the coun ter-ion ) . There are some l i m i t ations to this
m e thod due to the imposed c o n f o r m a t i o n on the residu es
closest to the crown ether, but they have been able to
show that Enkephal ins, for example, form folded
structure s in apolar solvent s and therefore p r o p o s e . t h a t
these folded stru ctures are also found in the receptor
site. This compar es well with other work on these
peptides using energy c a l c u l a t i o n s (Isogai 1977; D e C o e n
1977; Momany 1977).
1.3 BACKGROUND.
1.3.1 Amine Protection.
The ap p r o a c h we d e c ide d upon was to use the simplest
crown compoun d whi ch would complex with an amino acid
-5 2 -
and use this as a p r o t e c t i n g gro up in pe pt ide synthesis.
Thus we chose 18-crown- 6 whose ca vi ty is the corr ect
size for the pr i m a r y am m o n i u m ion and it is av a i l a b l e
c o m m e r c i a l l y in a pure form without being too expensive.
The use of DCC and its water - solu ble der i v a t i v e
ena bled us to com pare our work with that in the
litera tur e because there has been c o n s i d e r a b l e inroads
into the m e c h a n i s m of ac ti o n of DCC with many compoun ds
(see for exam ple DeTar 1966a; b; c; B u r d o n 1966; L e r c h
1971; Pf it zner 1965).
1.3.2 Ester Formation.
The use of C r ow n Ethers in the for m a t i o n of N - p r o t e c t e d
amino acid esters was carried out f o l lo wi ng a similar
pro ce dure to that of Ro eske (Roeske 1976) , to see if
the method could be a p p lie d to the f o r m at ion of fatty
esters. The se us ua ll y require harsh con d i t i o n s and give
low to mo derat e yields. We chose to use 18- Cr ow n-6 here
because it gives a stable comp lex with the K* ion.
-53-
CH A P T E R TWO. R E S U L T S
2 . 1 Ester Formation.
2.1.1 Introduction.
Durst has shown that the use of 1 8 - C rown -6 in the
reactio n sol ut ion enhance d the e s t e r i f i c a t i o n of
po t a s s i u m salts of carbox yl ic acids (Durst 1974).
The p-b roinophenacy 1 esters were o b t a i n e d in 90 to 98%
yield when using 0.05 e q u i val en ts of 18-Crown-6. The
m e t h o d was also used for the s y n t h e s i s of acid
a nh yd rides and an oxa z o l o n e (Hiraoka 1982; Deh m 1975).
S tu dies by Roeske and cowo rkers (Roeske 1976) have
shown that crown ethers could be used in the a d d i t i o n of
N - p r o t e c t e d amino acids to c h l o r o m e t h y l a t e d resins. The
work was bas ed on the effects that a c o m p l e x b e t w e e n the
amino acid p o t assiu m salt and the crown ether would have
on the re ac t i v i t y of the c a r b oxyla le ion. The form at ion
of the amino acid K * salt enables the c r o w n ether to
tor-m a complex, thus a l l o w i n g s o l u b i l i s a t i o n in orga nic
solvents. This enhances the n u c l e o p h i l i c i t y of the
carboxyl anion as it is "naked" in or ga ni c solvents. In
a polar solvent such as water, the n e g a t i v e charge on
the car boxy li c group is p a r t ially n e u t r a l i s e d by a shell
of water molec ul es aroun d the anion. The ne g a t i v e
charge cannot be shared by the g e n e r a l l y n o n - pola r
organic solvent molecules; thus the anio n is said to be
-54-
"naked". This faci l i t a t e d the reacti on with
c h l o r o m e t h y l a t e d resins: there was no s o l v a t i o n e n er gy
barrier to overcome.
It was believed that this a p p ro ach could be a p p li ed to
the fo rma tion of N - p r o t e c t e d amino acid esters,
e s p e c i a l l y those of long alkyl cha in esters. These are
difficult to obtai n because many e s t a b l i s h e d meth ods
rely on ha ving the alco hol component present as the
solvent and are therefore not readi ly a p p l i c a b l e to long
chain solid alcohols. Othe r methods require the use of
N - p r o t e c t e d amino acids and co up ling reagents. The
yields ax'e mod ex* ate to good, but a further’ step of N~
depro lection is requ i red and the reaction coxxdi Lions
need to be anhydi'ous. Direct ac i d-c a t a 1 y s ed
es terif ication using m e t h a n e s u l p h o n i c acid in a fatty
alcohol melt is the procedu re propose d by Pe nney et a l .
(Penney 1985) which gives moderat e to high yields for
s t e a r y 1 e s I e r's .
F o l l o w i n g the results ob taine d by Roeske, a study was
c ar ried out using a c e t o n i t r i l e or N ,N - d i m e t h y 1 fo rma mide
as the solvents in an attempt to form various types of
ester of dif fe rent amino acids.
2 . 1 . 2 R e a c t i o n s in A c e t o n i t r i l e .
A : B o c V a 1 .
Pi’e-format ion of the K* salt of the amino acid was
a c c o m p l i s h e d by n e u t r a l i s a t i o n of a sol ut io n of Boc
Va lO H in aque ous ethanol, using 1 e q u i v a l e n t of IN K O H .
The salt was t h o r ou ghly dried by l y o p h i 1 isat i o n , then
s us pe nded in MeCN and one equ i v a l e n t of 18-C ro wn-6
added. Once the so lut i o n had become clear, an excess (5
equ iva lents) of methyl iodide was added. Upon r e f l u x i n g
overnight, a p r e c i p i t a t e formed which was later
c h a r a c t e r i s e d as the KI - 1 8-Cro wn -6 complex. After
e x t r a c t i o n of the re a c t i o n residue, in E t O A c , with acid,
alkali and water, an *H NM R spect ru m showed the p r e sence
of only one product, c h a r a c t e r i s e d by signals at
8 = 0.87 and 0.94 (valine m e t h y l s ) , 5 = 1.43 (Boc
methyls), 5 = 2.10 (CpH), 5 = 3.71 (methyl ester),
8 = 4.20 (C a H ) and 5 = 5.02 (N H ) . This produc t was
ob t ain ed in 92% yie ld by weight (38ai, see A p p e n d i x
Eight ) .
R e p e t i t i o n of the re a c t i o n using p - n i t r o b e n z y 1 chloride,
gave a crude yield of the ester of 98% by weight. The
*H NMR spect r u m c h a r a c t e r i s e d the produc t as Boc
Val0B zN0 2 by signals at 5 = 0.9 and 1.0 (Valine
methyls); 8 = 1.5 (Boc methyls); 5 = 2.1 (CpH); 5 = 4.3
( C « H ) ; 5 = 5.0 (NH); 5 = 5.2 (Benzyl C H 2 ) and S = 7.5 -
8.0 (p - s u b s t i t u t e d a r o matic r i n g ) . The re was a trace
of the starring material present, as shown by larger
integrals for the peaks at 8 = 5.2 and 5 = 7.5 -8.0
(38aii, see Ap pe n d i x Eight).
The use of tertia ry butyl chl or id e as the e s t e r i f y i n g
co mponent gave no yield. Both *H NMR s p e c t r o s c o p y and
TLC showed no signs of reaction. Even when the tertiary
-56-
butyl chloride was used as the solvent and was therefore
pr ese nt in large excess there was no ester formed. This
was pro ba b l y due to the steric hi n d r a n c e b e t w e e n the
te rt ia ry butyl meth yl s and the Valine methyls.
Fo l l o w i n g these results, the syn the si s of a fatty ester,
n a m e l y the oc tad ecy l ester of Boc ValOH, was attempted.
One equ ival en t B r o m o o c t a d e c a n e was add ed to a s o l u t i o n
of the salt and the s o l utio n then re fl uxed for 4 hours.
The solvent was e v a p orated in vacuo, the resi due taken
up in EtOAc and e x t ra cted with acid, alkali and water.
After dr yi ng over NazSOa and removal of the solvent,
the residue gave a crude yield of 27% by weight. *H NMR
analysis of the resi due showed that some s t a rt ing
b r o m o o c t a d e c a n e remained, a c c o u n t i n g for 9%, in the *H
NMR s p e c t r u m . This was shown by the presence of a
triplet at 5 = 3.41 from the - C H a B r , and s l i gh tly larger
integrals for the peaks at 5 = 1.62, 1.31 and 0.89 from
the CiaHav-. The product was identi fi ed by the
f o 11 ow i ng s i gna Is: 5 = 5.02 (1H d NH); 5 = 4.20 (1H
m C« H); 5 = 4.12 (2H m O C H 2 ); 6 = 2.12 ( 1H m CpH); 5 =
1.6 2 ( 2H m CH 2 CH 3 ) ; 5 = 1.4 3 ( 9H s Boc CHa's); 5 = 1.31
( 3 OH m CH 2 ’ s ) ; 5 = 0.97 (3H d Val CHad) and 0.89 ( 6H m
Val CHau + C H 3 C H 2 -) (38aiii, see A p p e n d i x Eight).
Ha vi ng obt ai ned a m e t h o d by which ester formation, in
the absence of steric hindrance, p r o c e e d e d with good
yield, except for the stearyl e s t e r , it was de ci de d to
attempt the formatio n of an active ester. However, when
a re ac ti on with 1 - f l u o r o - 4 - n i tr ob enzen e was carried out,
there was no f o r m ation of the ex p e c t e d Boc V 0 I O B Z N O 2 .
On l y d i - n i t r o p h e n y 1 ether was o b t a i n e d from the rea ct ion
mixture. An iH NMR sp e c t r u m of the product showed
signals at 5 = 8.30 and 7.18, typical of a par a- di-
s u b s t i t u t e d benzene ring. The Mass S p e c t r u m gave M + m/z
= 260 and Elemental An a l y s i s gave C 55.37 ( 55.39) , H
3.16 (3.09), N 10.68 (10.76) cons i s t e n t with the formula
Ci 2 H a N 2 0 6.
B : Boc P h e .
F o l l o w i n g the same p r o ce du re as for Boc Val, the
for’mat ion of the fatty ester was attempted. Thus 1
equivalen t of the amino acid salt and 1 eq uivalen t of
18-Crown-6 were d i s s o l v e d in MeCN and 1 eq ui valent
br omo oc tadecane (Ci a H ^ I a d d e d . The so l u t i o n was brought
to bo iling and al lowed to re flu x overnight. After
e x t r a c t i o n of the re a c t i o n residue in EtOAc with H 2 O,
Brine, NaOH , Brine and H 2 O, the y i el d of the crude
product was 55% by weight. The *11 NMR s p e ctrum of the
ex t r acted material showed the pr odu ct by signals at
5 - 0.87, 1.30, 1.76, 1.85, 3.4 0 and 3.53, a t t r i b u t e d to
the C113 and Cli2 ’s respect ively, of the fatty alkyl
chain. The integrals ind ic ated the pr es ence of st a r t i n g
br o m o o c t a d e c a n e in the residue. This was q u a n t i f i e d by
the two triplets at 5 = 3.4 0 and 3.5 3 in the ratio 1:1
-58-
from the s t a rtin g b r o m o o c t a d e c a n e and the product,
respectively. The other signals are: 6 = 1.43 (Boc
methyls); 5 = 4.06 (C.H); 5 = 5.0 N H ; 5 = 7.12 - 7.25
(Aromatics) (38biii, see A p p en dix Eight).
The for ma tion of the methyl and p - n i t r o b e n z y 1 esters
fol lo wed the same trend as ob t a i n e d for Boc V a l O H
reactions, but wi t h lower overall yields. Hence the
methyl ester was formed in 78% yield by weight, wit h an
lH NMR s p e ctrum sh owing signals at 5 = 1 . 4 (Boc
methyls); 5 = 3.1 ( C p H 2 ); 5 = 3.7 (OMe); 5 = 4.5 (C«H);
5 = 5.0 (NH); 5 = 7.0 - 7.4 (Aromatics) (38bi, see
Appen d i x E i g h t ) . Crude p - n i t r o b e n z y 1 ester was formed
in 71% yield by weight but again, the 1H NMR sp e c t r u m
showed that this was c o n t a m i n a t e d wit h s t a rt in g m a t er ia l
to 47%, thus g i v i n g an overall yield of 37.6% of the
ester (38bii, see A p p e n d i x Eight).
2.1.3 Re ac tions in N ,N - d i m e t h y I f o r m a m i d e .
As the f o r mat io n of amino acid fatty esters gave rather
poor yields in aceto nitri le , the re ac tion was r e p eated
i n D M F .
The p r e - f o r m a t i o n of the Boc VulO K* salt p r o c e e d e d as
before, g i v i n g 100% yield. When this salt was s u s p e n d e d
in DMF and 1 eq ui val ent 18 -Crown-6 added, the sol u t i o n
cle ared after ] hour of stirring at Room T em pe rature.
One equivale nt of Cialb vBr was then added and the
so lutio n ref lu xed for 4 h o u r s . U p o n removal of the
solvent and ex t r a c t i o n of the r e a ction residue in EtOAc
with acid, alkali and water, a crude yie ld of the ester
of 70% by weight was obtained. Ana ly sis of the re act i o n
residue by NMR s p e c t r o s c o p y showed no start i n g
material remaining.
R e p e t i t i o n of the reactio n using Boc P h e O H gave a 7 4%
yield of the ester by weight and agai n lH NMR ana ly si s
showed that there was no CisHa 7Br pre sent in the
ex t r a c t e d rea ct ion residue. The XH NMR data for both of
these reactions is identical to that ob t a i n e d from the
MeCN reactions, except for the ab sen ce of st arting
material signals.
2.2 O l i g o p e p t i d e Formation.
2.2.1 Introduction.
Our study into the use of cro wn ethers, nam el y
18-Crown-6, as p r o t e c t i n g groups for the amine function
of arnino acids was ca rried out to see if this could be
used in pept ide synthesis. This would give a synthe si s
method which requires very mild co n d i t i o n s for the
p r o t e c t i o n and removal of the amine p r o t e c t i n g group. A
change in pH and was hing with a KC1 s o l ution would
remove 1 8 - c r o w n -6 effectively.
Ta bl e 2.1 Chemic al Shift as a F u n c t i o n of T e m p e r a t u r e for Co mplex (la) in CPC13.
T e m p . 0 C NHa + O c K 18 C r 6 C p H 3 OH
-75 7 . 27 4 . 35 3 .53 1 . 57 ---
-50 7 . 25 4 . 42 3 . 54 1 . 54 ---
-30 7.26 4 . 48 3 .71 3 . 57 1 . 59 ---
-5 7 . 24 4 . 57 3 . 60 3 . 69 1 . 61 10 . 70
3 7 . 23 4 .56 3 . 65 1 . 61 10 . 74
21 7 . 22 4 .61 3 . 66 1 . 61 10 . 70
27 7 . 22 4 . 62 3 . 66 1 . 61 10 . 62
35 7.22 4 . 65 3 . 67 1 . 61 10 . 38
45 7 .21 4 . 68 3 . 67 1 . 61 10 . 40
50 7 .21 4 . 69 3 . 67 1 . 61
Firstly the for mat io n of a complex was acc omp lished. In
the case of Alanine, this was done by d i s s o l v i n g 1
equival en t of the amino acid (5 g , 0.112 moles) in
aqueous EtOH and ad din g 1.1 e q u i valents of the
appr o p r i a t e acid (either HC1 or T o s O H ) . The salt formed
was dried, suspend ed in C H C 1 a , 1 equival ent of 18-Crown-
6 (29.6g) added and the sus p e n s i o n stir red until it
became clear. E v a p o r a t i o n of the solvent gave a w h ite
powder, the H NMR sp ect r u m of which showed one set of
signals at 5 - 7.11 (3H, s, N H a + ); 4.42 (1H, m, Call);
3.59 ( 24H, s, 18 - C r - 6 ) and 1.61 ( 3H , d, CpHo) ,
consisten t with the ex pecte d stru cture for the complex.
To see if these signals were the a v e ra ge of two sets of
signals c o r r e s p o n d i n g to the c o m p le xe d and free forms of
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the components, a t e m p er at ure study was ca rri ed out. As
shown in Table 2.1 , the amin o acid signals re mained
sharp and unique, even at -75°C. However, the crown
ether gave two signals at this temperature; one being a
m u l tiplet and the other a broad singlet.
As there was no c o r r e s p o n d i n g s p l i t t i n g in any of the
other1 signals, it was a s su med that the e q u i l i b r i u m
be tw ee n the free and co mplexe d forms was faster than the
NMR time scale even at -75°C: The spl i t t i n g seen for
the C r ow n Ether s i g n a 1 was put down to a c o n f o r m a t i o n a l
e q u i l i b r i u m between two types of cavity; one where all
the oxy gens po in ted inwards and one where three of the
m e t h y l e n e groups are turned s l i ghtly inwards. This is
shown in Figure 2.1
Fi gur e 2.1 Pr op osed C o n f o r m a t i o n a l E q u i l i b r i u m for
Com p l e x e d 18-Crown-6.
As the comp lex ap pe ared to be a stable entity, it was
reacted with the co upling reagent, DCC, in solv ents such
I
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as MeCN and C H C I 3 , botia of w h ich are c o m monly used in
peptide synthesis. It was hoped that under these
conditi on s pe ptide bond fo rmatio n could be carried out.
However, the first task was to find out wh et her the two
compoun ds would react in the way p r o po sed for ’n o r m a l 1
N - p r o t e c t e d amino acids, ie, via the O-acyl isourea
i nt er mediat e (Jones 1979).
2.2.2. React io ns using DCC.
When one equ ival en t of DCC was added to a s o l uti on of
complex (lb) in MeCN, p r e c i p i t a t i o n o c c urred readily.
Fil t r a t i o n of the r e a ction s o l ution y i e l d e d 33mg (57.5%)
of white powder, later c h a r a c t e r i s e d as d i c y c l o h e x y l
urea (DCU ) by c o m p a r i s o n of the IR and *H NMR spec tra
with those of an au th entic sample. Thus the *H NMR
spectrum gave the follow in g in C D C 1 3 : 5 = 1 - 2 (CHz ' s
from cyclohe xyl rings); 5 = 3 - 3.5 (C H ’s from
cyclohexyl rings) ; 5 = 5.0 (Nil’s) . The IR sp ect r u m
showed NH stretch at 3350c m 1 , CH s t retch at 2 930 and
2850cm , C=0 st re tc h at 1630cm 1 and NH d e f o r m a t i o n at
15 7 5cm 1 .
Upon standing, 15mg of al an ine di peptid e comp lex (26% of
the s t a rting amino acid) p r e c i p i t a t e d from the clear
re a cti on solution, as shown by the *H NMR sp ect ru m with
signals at 5 = 8.71 (NH), 5 = 4.26 (C.H, A l a 2 ), 5 = 3.84
(C«H, Alai), 5 = 3.52 (18-Crown-6) and 5 = 1.36
( Cpll 3 ’ s ) . The di pep t ide was fil te red off and the
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so l uti on solvent evaporated. Analysi s of the r e a cti on
re sidue showed that all DCC had been consumed, as
indic ate d by the abs enc e of an infrared signal at 2120
crn 1 (N = C = N) . The residue was di s s o l v e d in DCM and
e xt ra cted with water. L y o p h i 1 isation of the water
ex tra cts gave (35mg) of a solid w h ic h was shown by *11
NMR s p e c t r o s c o p y to be t etrape pt ide (80%, a c c o u n t i n g for
68% of the st arti ng amino acid) , the re mainin g 20%
con si sted of other oli go mers whi ch could not be
c h a r a c t e r i s e d by NMR. The di-, Lri- and te t r a - p e p t i d e s
isolated acc o u n t e d for 95% of the st a r t i n g amino acid.
The reaction was rep ea te d in CUCla and fol low ed di r e c t l y
by *H NMR s p e c t r o s c o p y by using the d e u t e r a t e d form of
the solvent. The same work up p r o c ed ure as used for
MeCN was e m p loyed here.
O l i g o p e p t i d e s thus obtai n e d were i d e n tif ie d by FAB Mass
S pe ct rom etry, as the trimer [M* less CL 496J; the
le trainer [M less Cl 5 67 J and the hexumer IM + less C 1"
709J. It could be that other o l i g o p e p t i d e s were present
in the mixture, but they remained u n d e t e c t e d by FAB Mass
Spect ral Analysis. This was p r o b a b l y due to the
r e m a in in g traces of DCU present in the water fr a c t i o n of
the re act io n residue. This interferes in the analysi s
beca use it can be ionised more re adily than pep ti des
under the conditi on s used.
Both the above reactio ns were repea Led using the alan ine
co mp lex (lft) to e s t a b l i s h w h e ther the
c o u n t e r - i o n had any effect on the sta b i l i t y or
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r e a c t i v i t y of the complex. By using the same r e a cti on
conditions, it was shown that the amou nts of s t a rti ng
co mplex re ma ining at the end of the reac ti ons was larger
than with co mp lex (lb), thus i nd icatin g that the Cl had
a d e s t a b i l i s i n g effect.
The r e a ctio n of co mplex (la) with DCC in MeCN yi elded no
DCU p r e c i p i t a t e in the first % hour. The re act i o n
re sidue when e x t racte d after 16 hours with water gave a
m i x tu re co n t a i n i n g 80% of the st artin g material, 17% of
the d i p ep ti de co mple x and 3% of the tripeptide complex,
as shown by the 1H NMR spectrum. It must be e m p h a s i z e d
that all p e r c e n t a g e s qu oted are a p p r o x i m a t e because of
the prese nc e of i m m e asura bl e traces of highe r oli g o m e r s
in the *H NMR spectra, as indicated by a series of small
NH s i g n a 1s .
The re act i o n in CHC1 3 also gave no p r e c i p i t a t e after
hour re action time. Upon e x t r a c t i o n of the reac t i o n
residue with water after 24 hours and l y o p h i 1 i s a t i o n , it
was found that 66% of the sta rt ing mat erial had not
reacted. The *H NMR sp e c t r u m also showed 16% of the
d i p ep tide complex, 14% of the tripeptide com ple x and 4%
of the tetrapeptide.
Ha ving shown that the tosylate ion stab ilised the
ala nin e - crown ether complex, (la) was used in a stu dy
where the amount of DCC was varied. It was hoped that a
change in the product ratios would be seen.
The re ac ti on betw een co mpl ex (la) and )£ equiv al ent DCC
in MeCN was c o m pared with the re action c o n t a i n i n g 1
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equi valen t each of DCC and comp lex (la) . Both r e a ct ion
solutions were filtered after \ hour to remove the
pr e c i p i t a t e d DCU . The rea c t i o n c o n t a i n i n g % e q u i va le nt
of DCC yi el ded 7% of the e x p ec te d DCU, whereas the
re a c t i o n with 1 e q u i valen t DCC gave 32.5% of the
e x p ec ted DCU. Both reacti on s were left st irring for 22
hours before being dried, taken up in CHC1 □ , e x t rac te d
wit h water and lyophilised. XH NMR analysi s of the
water fractions showed that there was 30% of the
d i p e pt id e and 7% of the t ri pe ptide present with 63% of
the st ar ting material left, in the r e a c t i o n using
eq uivalent of DCC. An alysis of the resi due from the
re ac ti on c o n t a i n i n g 1 equi va lent DCC by *H NMR
sp ec t r o s c o p y showed that there was 38% of the st ar ti ng
materi al remain in g and two products were present: 32%
d ip ep tide and 30% tripeptide.
A further reaction, c o n t a i n i n g c o m p l e x (la) and 2
eq uival en ts of DCC showed, when analysed, no r e m a ini ng
s ta rt ing material. This was indica te d by the lack of
the comp lex N H a + signal, a broad singlet at 5 = 7.27.
Ther e were several o l i g omers of va r y i n g length, al t h o u g h
these were diff icult to c h a r a c t e r i s e and quantitate,
because of ex ten siv e signal overlap.
Thus the use of \ eq uivale nt of DCC re duc ed the extent
to which o l i g o m e r i s a t i o n occurred, but did not elimin at e
i t .
A further study was ca rri ed out in MeCN and u s ing
compl ex&s to compare the effect of the counte r - ion
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and r e a ct io n time on the product ratios as ob ta i n e d from
the NMR spectra of the water washes. The results
from this study are shown in Table 2.2. Some ano ma lies
were found, in these results, for when using com plex (lb)
and \ equ ival en t DCC, there was more st art i n g ma ter ial
re m ain in g in the re ac tion left for 24 hours than there
is in the r e a ct io n left for % hour. This can be
ex p l a i n e d by the pr es ence of water in the reactions,
most p r o b a b l y from the solvent.
T a b 1 e 2.2 Result s from th e com p a r a five S t u d y of Thr ee C om p l e x e s v a r y i n g the T i rne o f _R eact_i on and DCC
C o n c e n t r a t i o n in MeCN.
Cpx % e q , % h r .. ...... ..... r •1 eq, \hr. j \ eq, 2 4 hr. 1 e q , 2 4 h r .
1 a 42% mono mer 34% dimer 23% trimer >100% urea
33% mo nom er 4 4% dimer 22% trimer 70% urea
69% mo no mer 2 3% dimer 8% trimer
>100% urea
9% mo no mer 63% dimer 27% trimer 58% urea
lb 52% mo nomer 36% dimer 12% trimer 13% urea
25% mo no me r 4 5% dimer 30% trimer 33.5% urea
53% m o n omer 35% dimer 11% trimer 1 9 % urea
21% m o n o m e r 46% dimer 32% trimer 33.3% urea
3bi
I ..
9 3% s .m .7% 6a
12% urea
87% s .m . 13% 6a 12 % urea
>9 9% s .m . <1% 6a 19% urea
86 % s .m . 14% 6a n . a .
To try to ex plain the results o b t a i n e d in m e c h a n i s t i c
terms, the re ac tion b e tw ee n methyiainine h y d r o c h l o r i d e -
crown ether complex(2)and DCC was followed. This, it was
hoped, would show the part (if any) played by the
c o u n t e r - i o n in the re a c t i o n leading to DCU formation.
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2.2.3 Re a c t i o n s us in g M e t h y l a m i n e H y d r o c h l o r i d e
C o m p l e x .
Three re actions were ca rr ie d out. Firstly, 1 e q u i va lent
of co mplex (2) and \ equ iv alent DCC were d i s s o l v e d in
C H C 1 a and the re a c t i o n followed by IR spectroscopy.
There was no. p r e c i p i t a t i o n or formati on of DCU, even
after 24 hours stirrin g at room temperature, as shown by
the un c h a n g e d intensit y of the N = C = N signal at 2120 cm
in the IR spectrum.
To half of the solution, 1 equ iva lent benz oic acid was
then added. W i t h i n 25 minutes, there was p r e c i p i t a t i o n
of DCU, which was c o l l ec ted after 24 hours (30mg, 28%) .
XH NMR analysis of the re actio n residue after 48 hours
st ir ri ng at room temp eratu re showed that the main
product was the benz oic anhydride, with 7% of N-
m e t h y 1b e n z a m i d e . The main product was c h a r a c t e r i s e d by
c o m p a r i s o n of T L C , and NMR s p e c t r o s c o p y with an
authentic sample.
The results from this reaction showed that the
o l i g o m e r i s a t i o n of amino acid co mp lexes seen in the
previous experime nts , n e c e s s i t a t e d that the ainine and
carb ox ylic groups be lo ng to the same molecule. This led
to the rea ctions d e s c rib ed in the next two sections.
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2.2.4 Re ac ti ons using the V i l s me ie r Reagent
It was as sum ed that the o l i g o m e r i s a t i o n reacti on was
oc c u r r i n g via the O-acyl isourea d e r i v a t i v e p r o p o s e d as
the interme di ate of DCC me d i a t e d peptide bond formation.
It was also thought that the d e p r o t o n a t i o n of the N H 3 +
group, a m e c h a n i s m re q u i r e d to o b t a i n the free N H 2
gr ou p and hence for ma tion of amide bonds, wo ul d occur
via an hy d r o g e n bond be twee n the DCC n i t r o g e n and the
N H i + (see Figure 2.2a).
ChU © ,H ,N=C
Vils me ie r Reagent. qj_j 0 "q |3 Cl
The o l i g o p e p t i d e sy nth esi s reaction was repeated, usi ng
the Vil smei er Reagent instead of DCC as the c o u pli ng
reagent. The Vi ls meier Reagent contains one imide
fu nct io n be a r i n g a positi ve charge. It is similar to
DCC and should react in a similar* way but the p o s it iv e
charge on the nit r o g e n would not all ow the fo rm ation of
the hy d r o g e n bond b e t w e e n the prot o n a t e d amine gro up and
the imine n i t roge n (see Figure 2.2). There would be
el e c t r o s t a t i c re pulsio n be tween the two p o s i t i v e l y
charged nitrogens.
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Figur e 2.2 The M e c h a n i s m of A c t i o n of a) DCC and b) the
Vilsmei er Reagen t with the C o mp le x lb.
© NhUCHCOOH X©Xa )
b)X©
(CH3)v'© tH J N = C//"Nil \ * ^(CH3r 0 cciCl
NHUCHCOOH■>
X©
When an aliquot of V i l s m e i e r Reagent (see 4.2.5) was
added to a CDC1 3 so l u t i o n of co mplex (lb) at room
temperature, there was prompt f o r m a t i o n of an amide
product. This was shown by the a p p e a r a n c e of a doublet
al 5 - 8 . 6 and a mul t i p l e t at 5 - 10.34 in the XH NMR
spectrum. The d i s a p p e a r a n c e of the broad signal at 6 =
7.6, coming from the N H 3 * of the complex, ind ic ated that
r e a c t i o n had taken place at the amine end. The pr o p o s e d
st ruc tu re of the product is shown in Fi gur e 2.3a. This
was c o n f irmed by d e c o u p l i n g the doubl et of dou bl et s a 5
= L0.34 due to an N H . Both the doublet at 5 = 8.60 (due
to the -CH=N) and the mul t i p l e t at 3 = 4.82 (due to the
C a H ) collapsed.
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Fig ure 2.3 The Pr oduct s O b t a i n e d with the Vil s m e i e r
R e a g e n t .
.0© XH -, ^
0-C=N_ C1H CH3
Whe n the re action was repeate d at - 4 4 ’C (Fujisawa 1983) ,
there was form ation of the ex pec t e d carboxyl d e r iv ed
adduct (Figure 2.3b) as shown by a singlet at 5 = 8.2
and a broad singlet at 5 = 7.6 in the NMR sp e c t r u m of
the reaction. This c o m po und remai ne d in sol ut i o n
without d e c o m p o s i t i o n for at least 12 hours at room
t e m p e r a t u r e .
This reaction showed that the p r e se nce of a posi ti ve
charge in the coupl in g reagent stops the o l i g o m e r i s a t i o n
reactio n of the comp lexes by p r e v e n t i n g the f o r mation of
the H y d r o g e n bond of Figure 2.2a.
2.2.5 Re action s usi ng 6-Am i n o h e x a n o i c Acid Complex.
Since it was found that the o l i g o m e r i s a t i o n reaction was
initiated by the f o r ma tion of the h y d rog en bond (as
shown in Figure 2.2), the rea ct ion using 6 - a m i n o h e x a n o i c
acid complex was tried. If the h y p o t h e s i s was correct,
incr ea sing the di sta nce b e t ween the two functional
groups - the amine and the c a r b o x y l i c acid - would
prevent the i n t r a mole cu lar hy d r o g e n bond and hence
CH3 H © £ ©' n = c x * n h 3c h c ;
a) c h 3' © ' n h c h c o o h b) 1 CH3 'C H o
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oli gomer is at ion. As was shown in sect ion 2.2.3, there
was little for ma tion of amide bonds when the two
functional groups are in differ en t molecules. However,
when the two functiona l groups are at tac he d to the same
c a r b o n atom, i n t e r m o 1 e c u 1 ar amide bond fo r m a t i o n readily
o c c u r s .
Thus 1 equi va lent of complex ( 3fc) and 1 equiv al ent of DCC
were th or oughly mixed in MeCN and left s t i rr ing for 24
hours at room temperature. A m a x i m u m of 10% of the
ex p ect ed DCU was reco ve red after this time. E x t r a c t i o n
with water and l y o p h i 1 i sa tion gave a residue which, as
shown by *11 NMR spectroscop y, co n t a i n e d 8 6 % of the
st artin g complex and 14% of the N-acyl urea d e r i v a t i v e
(6 a). The 1H N MR sp e c t r u m showed signals at 5 = 1.31,
1.55 and 1.72 from the (C H 2 ) 3 and some DCU rem a i n i n g in
the r e a ction residue after ext raction; 5 = 2.23 due to
the CaHz ; 5 = 3.57 from the 18- Cr ow n-6 ; 5 = 7.89 from
the N H 3 * and 5 = 8.32 due to the NH of the N-acyl urea
d e r i v a t i v e ( 6 a ) . The other signals from the N-acyl urea
d e r i v a t i v e o v e r l a p p e d with those from the s t a rt in g
material. There were no signals c o r r e s p o n d i n g to the
anhydride. This could be due to the rearr a n g e m e n t of
the co mp lex ion pair or c o n c e n t r a t i o n effects. This is
d is c u s s e d fully for the dip eptid e com p l e x e s in DMSO in
cha pter Three, S e c t i o n 3.3, and shown in Scheme 3.7.
The results f rom this ex pe riment showed that i n c r e a s i n g
the dis ta nce be tween the two functiona l groups of the
amino acid e lim in ates the o l i g o m e r i s a t i o n reaction.
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2.3 DMSO D e r i v a t i s a t i o n
2.3.1 Introduction.
From the above results, it was de ci ded that a change in
the solvent used for the reactio n was required, if
s t a b i l i s a t i o n of the DCC-coinplex O-acyl isourea
de ri v a t i v e was to be accomplish ed. In particular, it
was thought that by i n c r ea sing the p o l a r i t y of the
solvent, the forma ti on of the d e s t a b i l i s i n g
in t r a m o l e c u l a r h y d r o g e n bond would be re du ced in favour
of i n t e r m o l e c u l a r , solvent - solute h y d r o g e n bonds. It
was also be li ev ed that the comp lex ne eded to be of a
d i p e pt id e rather than an amino acid. This followe d from
the results o b t ained with the 6 - a m i n o h e x a n o i c acid
complex. The r e f o r e the reactions of G l y c y l G l y c i n e
complexes with DCC in DMSO were c a r ri ed out. Rea c t i o n s
using G l y c i n e com plexe s with DCC in D M S O were also done
for c o m p a r i s o n purposes. All the re actions in DMS O were
followed di r e c t l y by 1H NMR spec t ros copy us ing the
d e u t e r a t e d form of the solvent and usi ng the solvent
peak as internal standard.
2.3.2 Re ac ti ons at 0.02M Conc en trati on.
There was no d e t e ctable c o n s u m p t i o n of DCC 20 hours
after 0.01 mmoles of G l y c y l G l y c i n e co mp le x were pl aced
in an NMR tube w i t h 1 equ ivale nt of DCC in 0.5ml
D M S O - d e . After 25 hours there was a new spe cies in
solution, as shown in the sp e c t r u m by a triplet at
5 = 8.60 and a doublet at 5 = 8.38 with the same
int eg rate d area. The ratio b e t w e e n this new produc t and
the startin g mat erial reached 3:5 and re mai ne d thus for
the next 70 hours. When the re a c t i o n was repeat ed on a
larger scale, the pr od uct was iso lat ed by HPL C and shown1 3by Mass S p e c t r o m e t r y and C NMR to be the N-acyl urea
d e r i va ti ve of the dip e p t i d e co mplex (6b). The presence
of one, large nOe at the DCC N-C- N car bon upon
i r r a d iati on of the NH do ub let at 5 = 8.38 and a Mass
Sp ect ral base peak at m/z 214 c o n f i r m e d this structure.
Th ese results were ob t a i n e d w h e n the c o u n t e r - i o n was the
T o s y l a t e ion; however sim ilar results were ga ine d when
the chl oride ion was used instead, the only ap pr e c i a b l e
d if f e r e n c e being a faster for m a t i o n of the N-acyl urea
d e r i va ti ve (20 hours, as comp a r e d to the 70 hours with
the tosylate ion).
It was co nc luded that at this co nc entration, the
c o m b i n a t i o n of polar solvent and di p e p t i d e complex was
suf fic ient to suppre ss o l i go me risati on .
2.3.3 Re actio ns at 0.2M C o n c e n t r a t i o n ,
The rea cti on c o n c e n t r a t i o n was incre ased tenfold
because there was no carboxyl a c t i v a t i o n at 0.02M. This
was shown by the rea ctions invol vin g NSu whi ch have no
ester formation. This is d e s c ri be d in detail in s e c ti on
2.3.4.
The re was immediate p r e c i p i t a t i o n of DCU on the a d d i t i o n
of 1 equivale nt of DCC to a DMSO-de s o l uti on c o n t a i n i n g
0.1 mmoles of co mpl ex (4a). However, a pept ide product
did not appear in the spectru m until the r e a c t i o n had
been ru nni ng for 20 minutes. This was i n d i cated by the
a pp e a r a n c e of two triplets with the same i n t e ns ity at
5= 8.3 8 and 6.47. The c o n c e n t r a t i o n of this prod uct
incre ase d in the follo wing hour, though not at the same
rate as the c o n s u m p t i o n of DCC. The product ce ase d
fo rming when all the DCC had been co nsu m e d and resume d
upon ad d i t i o n of anot her equ ivale nt of DCC. When this
loo had been consumed, there was 20% of the s t a rting
mat er ial remaining, the rest hav in g been c o n v e r t e d to
a major product (70%) and d i k e t o p i p e r a z i n e (< 10%).
Upon repetit ion of the reaction on a larger scale the
product was isolated and shown by Mass S p e c t r o m e t r y to
be the dip eptid e - DMS O adduot (5a) with a base peak at
m/z 193 ( L M e z S NHCHzC ON HCHzCOO H) * ) .
X (CH3)2S®NHCH2CONHCH2COOH
( 5 )
When the re action was repeate d using co mplex (4b) , it
was found that there was again immed iate p r e c i p i t a t i o n
of DCU, but this time there was no fo r m a t i o n of any
pe pt ide product for the first \\ hours of the reaction.
As the last of the DCC was consumed, two
triplets of equal i n t ens it y ap p e a r e d at 5=8.8 8 and
7.40. They were from a co m p o u n d later i d e n ti fi ed as
the D M S O - p e p t i d e adduct (5b) . There was no further
change in the re ac tion until a second e q u i va le nt of DCC
was added. This induced p r e c i p i t a t i o n of DCU and a
parallel decrease in the st a r t i n g c o m pl ex con ce ntration.
At the end of the reaction, there was 20% of the D M S O -
di p e p t i d e adduct (5b) and traces of d i k e t o p i p e r a z i n e
(10), the N-acyl urea d e r i v a t i v e (6b) and the
t e t r a pe ptide - DMS O d e r i v a t i v e (11b) , with 70% of the
st artin g complex remaining.
2.3.4 R e a ctions using N - h y d r o x y s u c c i n i m i d e .
These react ion s were done 1) tp see if there was
carboxyl a c t i v a t i o n and 2) if a c t i v a t i o n took place
wh ether it wou ld compete wit h the fo r m a t i o n of the DMSO-
d i p e pt id e adduct. When the c o n c e n t r a t i o n was 0.02M,
there was not f o r m atio n of the ex p e c t e d ester. This
show ed that a c t i v a t i o n of the c a r b o x y l i c g r o u p did not
take place. When the c o n c e n t r a t i o n was inc re ased to
0 . 2M , and DCC was added to a solut ion of complex (4a)
plus NSu in DMSO-de, there was fo r m a t i o n of a peptide
pro duc t wit hi n 5 minutes. This was shown by the
a p p e aranc e in the XH NMR sp ec tr um of a triplet at 5 =
9.00 and a singlet at 5 = 2.71. These sig nal s c o n t in ue d
to increase in i n t e ns ity for the next 45 minutes. At
this t ime t he ir i n t e nsit ie s st ea di ed and two more
triplets appea re d at 5=8.39 and 6.38. The se increased
in i n t ensit y and after anot her 30 mi nutes an a d d i ti onal
two triplets a p p e a r e d at 5=8.75 and 6.39. DCU was
pr o duc ed at the same rate as the peptide products. The
prod uc ts ob t a i n e d in this reaction were is olated and
shown by Mass S p e c t r o m e t r y to be the succ i n i m i d e ester
of the co mp lex (7, m/z 494, 18%, NH 5 = 9.00, C H u ’s 5 =
2.71); the D M S O - d i p e p t i d e adduct (5, m/z 193, 17%, N H ’s
5 = 8.39, 6.38) and the s u c c i ni mi de ester of the DMSO-
d i p e pt id e adduct (8, m/z 290, 15%, N H ’s 5 = 8.75, 6.39)
(see A p p en dix Eight for S t r u c t u r e ) .
Similar results were o b t ain ed when complex (4b) was
used .
In both cases, when two e q u i valents of NSu were used,
there was increase d q u a n titie s of the esters to 25%,
though the r e a ction pro d u c e d the same products.
2.3.5 R e a ct io ns u s ing M e t h y l a m i n e H y d r o c h l o r i d e
C o m p l e x .
A series of re ac tions were done us in g co mplex (2) to
eluc id ate the part p l a y e d by the c a r b ox yiic g r oup in the
formation of the D M S O - d i p e p t i d e addu cts (5).
One equ ival en t of D CC was added to a 0 . 2M so lu ti on of
the comple x (2) in DMSO-de. There was no DCU f o r m at io n
and no d e t e c t a b l e c o mplex derivatives, even after 40
-77-
days. When 1 e q u i vale nt of acetic acid was added,
however, a quartet at <5 = 6.83 and a dou blet at 5 = 2.67
a p p e a r e d in the XH NMR sp ect ru m w i thin 10 minutes.
Af ter 26 hours, this was the major compone nt of the
r e a ct io n s o l uti on and shown by Mass S p e c t r o m e t r y to be
the D MSO -m eth y l a m i n e adduct (9) (see Ap p e n d i x Eight for
Structure).
The results from these e x p e rim en ts indic ate d that the
c ar bo xylic gro up was n e ed ed for the for m a t i o n of the
DMSO adduct and that it was acting as a catalyst in the
reaction.
2.3.6 React io ns using G l y ci ne Complexes.
Th ese reactio ns were carried out for c o m p a r i s o n
purposes. It was thought that o l i g o m e r i s a t i o n wou ld
occur, but to a lesser extent than ob t a i n e d in MeCN or
CHC1 a .
The reactions were carried out in a sim ilar way to those
a l read y described, for example, 1 equi v a l e n t of co mplex
(33b) was d i s s ol ve d in DMSO-de (0.5ml) and 1 e q u i valen t
DCC added. The re a c t i o n was followe d by 1H NMR
spe ctr oscopy, in itially at a c o n c e n t r a t i o n of 0.02M and
wi th in 3 minutes two signals a p p ea re d at 5 = 7.23 (d)
and 5 = 3.96 (m) . After 10 minutes, a triplet a p p eared
at 5 = 7.54 in the XH NM R spectrum. The se two sets of
signals were c h a r a c t e r i s e d as co ming from the N-acyl
- 7 8 -
urea d er ivativ e (6d) and the D M S O - g l y c i n e adduct (34b),
respect i v e l y .
One hour after the a d d i t i o n of DCC, another triplet
a p p ea re d at 5 = 8.69 and a doublet at 5 = 4.11 which
were a s s igned to the NH and the CoHz, respectively, of
the D M S O - G l y c i n e N-acyl urea derivative. There were
smaller peaks whi ch were not identified. C o m p a r i s o n of
their chemical shifts with those from authen ti c samples
showed that these were not the dipept id e nor its D M S O
adduct. Fur thermore, the rea ct ion c o n t a i n i n g 1
e qu iva len t of NSu gave no active ester in dicatin g that
there was no carboxyl act ivation. Thus it seemed
u n l ik ely that the d i p e pti de and its deri v a t i v e s were
be i ng f o r m e d .
The react io n c o n t a i n i n g 1 e quival en t of NSu at
0 . 02M c o n c e n t r a t i o n gave rise to the fo rma tio n of a
c om po und wi thin 10 minutes. This was indic ate d by the
a ppear an ce of a triplet at 5 = 8.69 and a doublet at 5 =
3.81 in the XH NMR s p e ctrum and was p r o po sed to be the
N-acyl urea d e r i vativ e of the D M S O - g l y c i n e adduct. The
intensity of these signals incr ea sed s l i gh tly before
be co mi ng constant. After 20 minutes, a doublet a p p eare d
at 5 = 7.15, which was due to the N-acyl urea
derivative. After 55 mi nutes of re a c t i o n time, two more
signals a p p eared in the spectrum; a triplet at 5 = 8.31
and a broad signal at 5 = 7.53. These signals incr eas ed
in inte nsity at the same rate and were due to the
reaction product of d e h y d r a t i o n of three m o l ecules of
-79-
NSu (30) and the D M S O - g l y c i n e adduct (34), respectiv ely.
The re were no signals from the dipeptide. The f o r ma ti on
of the N-acyl urea d e r i v a t i v e of D M S O - g l y c i n e adduct
before the f o r mati on of either the D M S O - g l y c i n e adduct
or the N-acyl urea d e r i vati ve of the comp lex indi ca ted
that the NSu was c a t a l y s i n g the reaction. Both these
compoun ds formed first when no NSu was present in
s o 1u t i o n .
These react ions showed that at a c o n c e n t r a t i o n of 0.02M,
the o l i g o m e r i s a t i o n reaction was g r e a t l y reduced, and
that the a d d it ion of NSu stopped it altogether.
The reactions were rep ea ted at a c o n c e n t r a t i o n of 0 . 2M
usi ng both complexes.
W ith in three minutes of the a d d i t i o n of 1 e q u i va le nt DCC
to a DMSO-de so lutio n c o n t a i n i n g 1 e q u i valent of co mplex
(33a) and thorough m i x i n g there was the a p p e a r a n c e of
two triplets at 5 = 8.63 and 6.50 w h i c h had di ff erent
int eg ra ted areas. The triplet at 5 = 8.63 re m a i n e d
con st ant thro ugh out the re ac ti on period, wherea s that at
5 = 6.50 inc re ased in intensity. The latter was found
to be from the D M S O - g l y c i n e adduct (34) (see A p p e n d i x
Eight for Structure) .
After 10 minutes, a further triplet a p p e a r e d at 5 = 8.40
which slowly increased in intensity. The two minor
products, with signals at 5 - 8.63 and 5 = 8.40, were
c h a r a c t e r i s e d as the d i p e pt ide and the D M S O - d i p e p t i d e
adduct (5) respectively.
-80-
When the re actio n was repeat ed with NSu in the sol ut ion
there was immediate f o r ma tion of the gl yc ine complex
active ester as shown by the a p p e a r a n c e of a signal at 6
= 8.07. The re was also the f o r ma tion of the DMSO-
gl y cin e adduct wi thin 5 minutes, as ind ic ated by the
a p p e a r a n c e of a triplet at 5 = 6.50 and a doublet at 5 =
4.44. After 25 minutes re a c t i o n time, the c o n c e n t r a t i o n
of the co mp lex active ester (35) began to decre as e and
a p p eared to be reactin g p r e f e r e n t i a l l y to the start in g
complex. The re was for m a t i o n of the d i p e p t i d e ester, as
indicated by the a p p e a r a n c e of a triplet at 5 = 9.00.
There was also f o r m at ion of the D M S O - G l y c i n e adduct
ester, ind icated by the triplet at 5 = 7.10, partia ll y
ov e r l a p p i n g with the tosylate proton doublet.
The re act ion s were aga in repeated, this time using
complex (33b) at a c o n c e n t r a t i o n of 0 . 2M .
Three minu tes after the a d d i t i o n of DCC to the r e a ction
solut io n a doublet app ea red in the 1H NMR sp e c t r u m at
5 = 7.28 and was a c c o m p a n i e d by a smal ler triplet at
5 = 7.67. The c o n c e n t r a t i o n of these pr o d u c t s in creased
over the entire r e a ct ion period. The produ ct s were
later' character* ised as the N-acyl urea d e r i v a t i v e (6d)
and the D M S O - g l y c i n e adduct (34), res pectively .
Other signals ap p e a r e d duri ng the reaction. A triplet
at 5 = 8.75 was later i de nt ified as c o m i n g from the N-
acyl urea d e r i vativ e of the DMSO-Glycirre adduct. The
rest of the signals, double ts and triple ts co ming
be twe en 5 = 8 and 9 were not c h a r a c t e r i s e d as it had
-8] -
been shown that the cond itions were not those required.
It may be that some of the signals are from the
d i p ep ti de and its derivatives.
The reacti on c o n t a i n i n g NSu showed the immediate
fo r m ation of the complex active ester (35) . It was
a s s umed that the broad signal at 5 = 9.36 was from (35)
through c o m p ariso n with the spectra of similar esters.
It was shown that the products were the D M S O - g l y c i n e
adduct (34, triplet at 5 = 7.25) and the active ester of
the D M S O - g l y c i n e adduct (36, triplet at 5 = 8.92). The
active ester (35) remained constant for the rest of the
reuclion. This may be at tr i b u t e d to either: the
pr es en ce of water in the reactio n solution, whi ch
h y d r o l y s e d the ester as it was formed or the
pr e f e re ntial reacti on of the ester to form the dipepti de
and its derivatives. Ten minutes after the a d d i t i o n of
DCC to the reaction, a triplet ap p e a r e d at 5 = 8.39 in
the XH NMR spectrum, comin g fr.om the d i p e ptide complex.
After 23*> hours, there were signals from N H ’s at 5 =
8.82, 8.23 and 7.98 w h i c h are most p r o ba bly from
ol ig om ers al t h o u g h their struc tures were not proven.
These react ions showed that the use of a polar solvent
largely inhib ite d oligomerisa ti on, but gave rise to
d e r i va. t i s a t i on of the amino acids by the solvent.
-82-
2.3.7 Other Rea ct ions Ca r r i e d out in DMSO.
The fo ll owing reactio ns were done so that a c o m p a r i s o n
could be drawn between the re act ion m e c h a n i s m s of the
c om plexes and that of Boc amino acid and dipeptides.
A. Boc Amino Acids with DCC in DMSO-da
Whe n 1 equiva le nt of DCC was added to a DMS O-da so lu ti on
of Boc Al anine at a c o n c e n t r a t i o n of 0.01M, there was
fo rm at ion of the c o r r e s p o n d i n g anhydride. This was
in di ca ted by the a p p e ar an ce of a doublet at 5 = 7.47 and
a m u l t iple t at 5 = 4.11 in the XH NMR spectrum. There
was also the f o r m at io n of the N-acyl urea derivative,
(DCC-NH at 5 = 8.12 and amino acid NH at 5 = 6.86).
After 56 hours, the N-acyl urea was the majo r prod uct ,
with all Boc Alan ine being consumed.
The rea ct io n was repeat ed at two diff erent
c o n c e n t r u t i o n s , 0 . 05M and 0.2M. The results were
similar, except at these conce ntrat io ns, there was slow
f o r m at io n of a second pep tid e product, which after 19
hours had reached 7.5% of the total product s present.
By c o m p a r i s o n with the D M S O - g l y c i n e sample, it was
believed that the sig nals at 5 = 7.79 and 4.87 were due
to the D M S O - a l a n i n e adduct. The removal of the Boc
gi'oup may have been a c c o m p l i s h e d by the a c t i v a t e d DMSO-
DCC derivative. (See Chapter T h r e e , Se ction 3.3 for the
d i s c u s s i o n on these results. )
- 8 3 -
The re act ion of a Boc amino acid with DCC at a
c o n c e n t r a t i o n of 0 . 2M was repeated using Boc G l y O H to
give a better c o m p a r i s o n with the com pl ex es and the Boc
d i p e p t i d e reactions. Thus Boc G l yO H ( 1 7 ing , 9.7x10
moles) was dis so lved in DMSO-du and 1 eq uivale nt DCC
added. The s o l ut ion was mixed and pl ac ed into an NMR
tube. As expected, two signals were seen in the
sp e c t r u m of the start in g material, co mi n g from the E and
Z forms of the Boc amino acid. Within 5 m i n ut es there
was form ati on of Boc G l y ci ne anh yd ride and N-acyl urea
derivative. The ap p e a r a n c e of a triplet at 5 = 7.23 in
the 1H NMR spe ct ru m indicated the pre sence of the
anhydride, whilst the doublet and triplet at 5 = 8.14
and 6.70 res pectively, ind icated the N-acyl urea
d e r i v a t i v e . After 45 min utes r e a c t i o n time, the
a n h y dride c o n c e n t r a t i o n had steadied and the second form
of Boc Gly (E) had begun to react to form its N-acyl
urea derivative. After 2 hours, it was cle arl y vi sible
at 5 = 8.26, 4.31 and 4.05. At the end of the re act io n
27% of the starting Boc Gly remained, the rest having-
been conver te d to the N-acyl urea der i v a t i v e and what
was assu med to be the oxazolone. This has been shown
to be a po ss ible product from the reacti on of Boc amino
acids with water - soluble carbodi imides (Benoiton
198 1 ) . As thenawas no a n h y dri de r e m a in in g at the end of
the reaction, it was assume d that a second mo le cule of
DCC had reacted with the anhydrid e to give the N-acyl
urea and the oxazolone.
-84-
When Boc G l y c y l G l y c i n e was used, the re act ion s at 0 . 02M
and 0 . 2M both gave the same results, the only d i f f e r e n c e
b e in g the p e r c entag e of p r o duc ts obtained. At the lower
co ncentr ation, there was more N-acyl urea d e r i v a t i v e
pr o d u c e d than at the highe r conc en trati on. Thus the N-
acyl urea der i v a t i v e gave signals at 5 = 8.34, 7.88 and
3.91, the second NH triplet o v e r l a p p i n g with the
sta rt ing material at 5 = 7.00. After 8 days there was
86% of the N-acyl urea in the 0 . 02M reactio n and 68% in
the 0 . 2M reaction. Again, all the a n h y d r i d e had re acted
to give the N-acyl urea and what was as su me d to be the
oxazolone, as shown by the broad signal at S = 6.39.
Th ese rea ctions showe d that the for m a t i o n of the N-acyl
urea d e r i vative in DMSO was not via the 0-acyl isourea
intermediate, instead some, if not all, was formed via
an independen t m e c h a n i s m (see C h a pter Three, scheme
3.4) .
B. R e a c t i o n s of DCC wit h other moieties.
The fo l l o w i n g reactions were ca rried out in order to
verify the mode of a c t i v a t i o n of the DCC and the DMSO.
The first reaction followed was that be tw een DCC and
NSu. This was to ensure that the NSu was not c a t a l y s i n g
the rea ct io n by a c t i v a t i n g the DCC.
Four hours after the ad d i t i o n of DCC (20.7mg, 1x10 4
moles) to a DM SO-d a s o l uti on of NSu (11. 5mg), there was
the ap p e a r a n c e of a.small signal at o = 5.38, i n d i c a t i n g
that DCU was being formed. The re was no sign of any
-85-
pr od uc t from the NSu. However, after 17 hours, there
were signals at 5 - 8.31, 3.19, 2.73, 2.65 and 2.58 in
the XH NMR spectrum, in the ratio 1: 1:3:2. 5:5:5. The
product pr o p o s e d was the product o b t ai ned by the
d e h y d r a t i o n of 3 m o l e cu les of NSu wit h one of them ring
o p e n i n g (30) , as this has been shown by Gross and Bilk
to be a product from the r e a ct ion of DCC and NSu (Gross
1968; Rich 1979). This occurs e s p e c i a l l y if active
ester f o r mati on is slowed by steric hindrance. The
a d d i t i o n of AcOH to a r e a ct ion so l u t i o n like above led
to the immediate fo rma tio n of the s u c c i n i m i d e ester of
AcOH. Thus in the *H NMR spectrum, there app e a r e d
s i gna Is at 5 = 2.76 (s CH z ) and 5 = 2.30 (s CH 3 ) . Thi s
was the only product at the end of the rea ct io n and
acc o u n t e d for 24% of the st a r t i n g material.
The second reactio n was be tween DCC , AcO H and a base,
n - b u t y l a m i n e , in DMSO-da with and wi thout the pre sen ce
of l B - C r o w n - 6 ,- 4One equivalent DCC (21.4ing, 1x10 moles) was added to a
DMSO-d a so lution of AcOH ( 5 . 7 jj 1 in 0.5 ml), followe d by
1 e q u i va lent N H 2 BU (9.9 p l ) and 1 eq u i v a l e n t 18 -Crown- 6
I26.7mg) when necessary.
Both re actions showed the slow for m a t i o n of the N-acyl
urea de ri v a t i v e of AcOH ( 6 c ) , with signals a p p e a r i n g at
5 = 8.31 and 1.97 in the H NMR spec: 11* urn. The r e a c t i o n
without 1 8 - C r o w n -6 also showed the slow form at ion of the
amide at 5 = 7.88. The se re act ions in dic ate d that 18-
86-
C r o w n - 6 acted as a p r o t e c t i n g g r ou p for the amine g r o u p
so that amide fo r m a t i o n did not take place.
2_ .4 Rea c t i o n s of Co m p l e x e s in D M F .
2.4.1 Introduction.
From the results ob t a i n e d using MeCN and CHCI3, it was
d e c i d e d that a more polar solvent was required. As
d i s c u s s e d in sect ion 2.3.1, a polar solvent would favour
sol vent - solute h y d r o g e n bond formation. This wou ld
stabili se the co mp le x when r e a cting with DCC. However,
with the use of DMSO as solvent, a l t h o u g h
o l i g o m e r i s a t i o n was inhibited, d e r i v a t i s a t i o n of the
umino acid with the solvent took place. Havi n g
e s t a b l i s h e d that the r e a ction m e c h a n i s m lead ing to this
d e r i v a t i v e involve d the a c t i v a t i o n of solvent by DCC
lt;ee chapter Three) , a polar solvent with out a po ten tia l
react ive site was required. To this end, N,N-
d i me t hy 1 f orniam i de was c h osen be cause it is a polar
solvent com mo nly used in p e p ti de synth es is and it
co nta ins an amide funct i o n which is not as p o t e n t i a l l y
reacti ve as the s ul phoxid e group.
Initially, the stabi li ty of the comp lex wit h DCC in DMF
was tested. Thus 1 equ iva lent of DCC (2 06.3mg) was
added to a solutio n of complex (4a) ( 5 6 8 . 7mg, 1x10
moles) in DMF (5ml). This gave a c o n c e n t r a t i o n of 0.2M.
Withi n % hour of reaction, pr'ecipitution of DCU
-87-
occurred. When the r e a c t i o n was repeate d without
stirring, there was no p r e c i p i t a t i o n of DCU for the
first hour. After 18 hours, the solvent was removed
from the second reaction, the residu e taken up in DCM
and filtered before the 1H N MR sp ectru m was run. This
showed the presence of two products; the D M F - d i p e p t i d e
adduct [12b, 17%, signals at 5 - 3.83, 4.04 ( C a H z ’s); 5
= 8.68 (NH); 5 = 2.71, 2.87 ( D M F - M e ’s) and 6 = 8.23
(DMF-CH)J; and the N-acyl urea deri v a t i v e (6b, 18%).
The rema inder of the mater ial was st a r t i n g complex.
Table 2.3 Yields O b t a i n e d of the T r i p e p t i d e s Sy n t he s i s e d .
Product Yield % By wt .
Yield % By NM R
Rxn No . *
G l y G l y G l y O E t 45 . 0 58 . 5 1
•• 67 . 0 63 . 8 2
•• 70 . 0 66 . 7 3if 43 . 0 21.1 4• I 33 . 0 18 . 7 5» -- 50 . 0 6
" -- 11.1 7
G l y G 1yPheOMe 33.0 33 . 3 8
Gl y G l y C i a O M e 47 . 0 _ _ 9
G l y G 1y C i 4 OMe 64 . 0 100 . 0 10
Gl y P h e C i 4 0 M e 81 . 0 -- 11
Gl y P h e C i o O M e 82 . 0 -- 12
* See Se ct ion 4.3.12, Table 4.12.
-88-
2.4.2 R e a ct io ns of D i p e p t i d e C o m p l e x e s with Am ino Acid
E s t e r s .
As comp le x (4a) was not d e r i v a t i s e d by DMF to the same
extent as with DMSO, the reacti on of the co mplex with
various amino acid esters was attempted.
The first re ac ti on was be tw ee n co m p l e x (4a) , DCC and
G l y O E t . One equi va lent of DCC (206.7mg, 1x10 3 moles)
was added to a DMF s o l ut ion c o n t a i n i n g comp lex (4a)
(568.6mg). A second DMF so l u t i o n c o n t a i n i n g 1
e q u i va le nt H C 1 . G l y O E t (13 9. 6mg) and 1 equ iva le nt TEA
(140jii) was added after 5 minuLes, c a u sing immediate
pr e c i p i t a t i o n of DCU. After 2 hours stirring, the
so l uti on was fil te red and the sol vent removed. The
1H NM R spectra showed that the react io n residue
c o n t ai ne d 40% of the st ar ting complex, 27% of the
starting ester and 33% of a pe pti de product, later
id en tified by Mass Sp ec t r o m e t r y as the ester com plex
(13a, M + 483 L N H 3 +C H 2C O N H C H *C O N H C H 2 C O O C H z C H 3 J ) .
The r e a ct io n was re pea te d using v a r y i n g ratios of
co mp le x to ester, the results of which are shown in
Table 2.3. From these results, it was con cl uded that a
ratio of 3:1 d i p ept id e co mplex to ester gave the highest
pe r c entage of the crude desire d t r i p ep ti de (70%). In
order to facilit ate i s o l at ion of the pro duct from the
s t a rting material and hence ca rr y out a qu i c k e r
de t e r m i n a t i o n of the reactions ch ar act e r i s t i c s , we used
an aromatic or a fatty amino acid ester to replace the
Gly OEt ester. The use of these amino acid esters w o uld
f a c i li ta te the HPLC p u r i f i c a t i o n p r o ce dure by c h a n g i n g
the c h r o m a t o g r a p h i c p r o p er ti es of the tripeptides.
In the first instance, the ester ch ose n was Phe OMe( HCl).
This was rea cted with co mpl ex (4a). There was fo r m a t i o n
of the tr ip eptid e (33%), but also for m a t i o n of trace
am ou nts of the DMF deri v a t i v e and a high p e r c e n t a g e of
the s t a rtin g material remaining.
In an attemp t to overcome the f o r m at ion of the u n w a n t e d
by-products, two c on dition s were changed. Firstly, the
re a c t i o n temp erature was lowered to 0°C whi ch gave rise
to fewer by-products. Secondly, the use of the water
soluble d e r i v a t i v e of tiCC- 1 - ( 3 - d i m e t h y l a m i n o p r o p y l )-3-
ethyl ca rbo d i i m i d e h y d r o c h l o r i d e (wscdi) - with
h y d r o x y b e n z o t r i a z o l e (HOBt) also inc rease d the pu rity of
the reaction and gave less st arting mat er ia l at the end
of the reaction.
A decrease in the re a c t i o n t e m p eratu re meant an increase
in the re action time from 1-2 hours to a mi n i m u m of 6
hours. However, even with the improv ed conditions, the
fo r mat io n of a tri pe pt ide usi ng Phe as the ester m o i e t y
gave low yields (30%) . This was most p r o b a b l y due to
steric hin dr ance b e t we en the 0-acyl isourea and the
benzene ring of the pheny lal anine.
When the fatty amino acid ester was used in the m o d i f i e d
synthesis procedure, the product was ob t a i n e d in 64%
yield by weight and the *H NMR of the r e a ct ion residue
showed it to be pure.
-90-
W hen the di pe ptide complex was ch an ge d to the Gl yPhe
complex, the yields by weight improv ed (re aching 82%),
al t h o u g h the product was not pure and HPLC was requi re d
for p u r i f i c a t i o n purposes. This showed that there was
no steric h i n dr an ce caused by the P h e n y l a l a n i n e when it
co n t a i n e d the ca rboxyl ic group, only when it con t a i n e d
the amine group.
These rea ctions also led to the finding that by
d i s s o l v i n g the re act i o n residue in DCM and e x t r a c t i n g
with a sa tu ra ted s o l ut io n of KC1 , the crown ether was
removed from the complex. This is a simple and mild
d e p r o t e c t i o n step.
Furthermore, the react io ns ca rr ied out showed that
sele ct ive pep tid e bond formation was po ss ib le when 18-
Crown-6 was being used as an amine p r o t e c t i n g group. To
v e rify this, the syn thesis of an E n k e p h a l i n der i v a t i v e
was carried out.
2.4.3 Sy nt hesis of [T y r - o b z J 1-(a - a m i n o d e c a n o y 1 ] 5 -
E n k e p h a l i n Derivative.
One eq uivalent of T F A .G 1y P h e CioOMe (26.6mg, 5.12x10
moles) and 1 equiva lent TE A (7.1jal) were d i s s o l v e d in
DMF ( 2 m l ) . One equi va lent wscdi (10,6mg) was added to a
DMF soluti on (2m'l) c o n t a i n i n g 1.4 e q u i v a l e n t s of Com ple x
(23) and 1 equi valent HOB t (7.8mg) and cooled to 0°C.
The two solut ions were mixed and left st ir ri ng
overnight, slowly wa rm ing to room temperature. Removal
-91-
of the solvent after 24 hours and e x t r a c t i o n of the
re sidue in DCM wit h H 2 O and KC1 so l u t i o n gave a 1:1
mi x t u r e of the s t a rt ing ester and the d e sired
p e n t a p e p t i d e , as shown by 1H NMR s p e c t r o s c o p y after
p u r i f i c a t i o n by Cie reve rse phase H P L C . The
p e n t a p e p t i d e product gave by Mass S p e c t r o m e t r y m/z 716
( I T y r (B z )G l y G l y P h e C 1 o O M e ]) and had an 1H NMR sp ec tr um
sh ow in g the NH sig nals at: 5 = 8.73 C 1 0 , <5 = 8.48 Phe ,
5 = 7.33 G l y 4 and 5 = 5.51 G l y 3 . The signal from the
NHa * of the Tyro s i n e was too broad to be seen.
Thus it has been shown that 18- Cro wn -6 can be used as
^ amine p r o t e c t i n g g r oup in peptide synthesis.
2 . 5 F o r m a t i o n of O l i g o p e p t i d e Coinp 1 exes .
The fo rmati on of amino acid and d i p e p t i d e co mplexes has
been shown to be facile (Mascagni 1987; Hyde 1989).
Work by Temussi et a l . (Pastore 1984) on NMR St ud ie s of
O p ioid Rec ep to rs in a S i m u l a t e d R e c e p t o r Environm ent ,
led to the attempt to form c o m p lexes of larger
pept i d e s .
This could also be of use if this m e t h o d of pep tide
sy nth esis was ap plied to fragment conden sat ion.
The fo rma tio n of three pept ide com pl exes was under taken.
Firstly, HC1 A l a G l y G l y was co mp lexed with 1 8 - C r o w n -6.
This gave a clear solution of the co mple x in C D C 1 3 , one
set of signals in the NMR spe ctrum and an E l e mental
Analysis of the co mplex c o n t ain in g 1.5 mol e c u l e s of H 2 O.
-92-
The f o r m a t i o n of a clear sol ut ion in CDCla ind ic ated
that c o m p l e x a t i o n had occurred. Sim ilarly, when the
tosylate salt was used, a clear s o l ut ion was obtained.
Two ba tc hes of the acetate salt of M e t 5- E n k e p h a 1 in were
c o m p l e x e d w i t h 18-Crown-6. The first bat ch gave a
cl o u d y s o l u t i o n and the second gave a clear solution.
This was due to the pre se nce of water in the secon d
sample, most p r o bably present as water of
cr ystal l i z a t i o n . This en ab led the C r o w n Ether to
s o l u bilis e the peptide in CDCI3 readily; 13mg of the
c o mple x d i s s o l v e d in 0.8 - 0.9ml solvent.
The 1H NMR data for these complexe s is shown in T a ble
4.3.
-93-
C H A P T E R THREE. DISCUSSION.
3.1 Ester Formation.
There have been reports of ester if ication reactions
which are enhanced by the use of crown ethers. For
example, in 1974 Durst reported the fast e s t e r i f i c a t i o n
of p o t a s s i u m salts of ca rb o x y l i c acids (Durst 1974). He
used 5 mol % 18-C rown- 6 in a c e t o n i t r i l e or benzene, when
f or ming the p - b r o m o p h e n a c y 1 ester. The d e s ire d product
was o b t ai ne d in 90 to 98% yield when reflu xed for 10 to
30 minutes. A similar m e thod has also been used to
o b t a i n acid a n h y dr id es and also a lactone ( Hi r a o k a
1 982; Dehm 1 975 ) .
The rea ct io n is thought to go through an SNi pathway, as
shown in Scheme 3.1. The reaction occur s with ease
bec aus e the anion is "naked". In apolar solvents, a
charge is said to be "naked" when there are no mol e c u l e s
whi ch can asso ci ate wit h it and ’s h a r e ’ the charge.
Thus the an ion has inc re as ed n u c 1e o p h i 1 icity and this
e nha nces the reactivity.
This me th o d was ap pl ie d to the a t t a chm en t of the first
amino acid to c h l o r om ethyl p o l y s t y r e n e resin for use in
solid phase peptide synt hesis by Roeske (Roeske 1976) .
This had been a c h ieve d prev iously by using one of the
fol lowing methods: 1) the t r i e t h y J a m m o n i u m salt of amino
acid de ri v a t i v e s in r e f l uxing EtOH; 2) the tetramethyl
am mo ni um salt in DMF at room temperature, and 3) the
-94-
ces ium salt of Boc amino acids in DMF at 50°C. Onl y the
use of cesium salts gives q u a n t i t a t i v e ester i f i c a t i o n .
This is requir ed so as to e l i m in ate the possi ble side
reactio ns of reagents or amino acid side chains with any
reactive chlo romethy l group s remaining. G i s i n found
that the cesium salt of am in o acids was the most
reac ti ve with the c h l o r om et hyl resin (Gisin 1973).
Scheme 3.1 The R e a c t i o n P a t h w a y P r o p o s e d for Ester
Format i o n .
The method d e v ised by Roe ske used the p o t a s s i u m salt of
Boc amino acids, 18-Crown -6 and the c h l o r o m e t h y l resin,
initially in various solvents at 100°C for 18 hours.
The use of the p o t a s s i u m salt was s u g g e s t e d by its high
a s s o c i a t i o n constant with 18 -Crown-6 (logK« - 6.10), as
o p p osed to the ce si um salt ( logK. = 4.62) which is the
most reactive with the c hlo ro methyl resin. The yield s
ob ta in ed by Roesk e ranged from 91% in EtOAc to 100% in
DMF. When the temperature was lowered to 50°C, the
yields ranged f rom 5% in EtOH , to 96% in DMF. Thus the
best results o b l ain ed were in DMF at 100°C and using 2
0 R (H3C)3C0CNHCHi
0 R 0 R111 11
+
(H3C)3C0CNHCHC0C-R2
-95-
eq u i v a l e n t s of amino acid salt and 18-Crown -6 to 1
eq u i valent of resin.
The aim of our w o r k was to apply this m e t h o d to the
sy nthes is of amino acid esters, e s p e c i a l l y fatty ones.
The latter are difficu lt to prepare in good yi el d and
using mild c ondit io ns (Penney 1985). Since pu b l i s h e d
wor k in dicated that MeCN and DMF were the best so lve nts
for this type of reaction, we carrie d out the sy nthesis
of esters, firstly in MeCN, as this solvent can be
re mo ve d easily at the end of the reaction. The
f o r m at io n of the methyl ester of Boc ValO H was carried
out to ensure that the m e th od could be ap pl ie d to
esters. The yield ob ta ined was 92%, in d i c a t i n g that the
p ro c e d u r e could cer t a i n l y be ap pl ied to simple ester
formation. R e p e t i t i o n of the reacti on usin g Boc Phe
gave a yield of 78%. This lower yield may be e x p l ai ned
by the steric h i n dra nc e of the benz ene ring on the
a p p r o a c h of the anion to the alkyl halide. The yields
of crude p - n i t r o b e n z y 1 esters were similar to those for
the methyl esters.
When the form ation of the t e r tiary butyl ester of Boc
Va .OH was attempted, there was no reaction. This was
most probab ly due to two factor's. Firstly, steric
hi n d r a n c e b e t ween the tertiary butyl and the valine
me thyls would stop the a p p r o a c h of the anion to the
alkyl halide (see Scheme 3.1, R 1 , R Z , R 3 = CH3).
Secondly, the s t a b il ity of the tertiar y butyl ca tion
would inhibit the r e a ction because of the energy
-96-
require d to revert this stable en tity to the ester.
Even when the terti ar y butyl chlori de was used as
solvent, and was ther efore in large excess, there was no
reaction. As this did not occur, it was assu med that
the en ergy barrier to ester form at ion was too great to
be overcome. As the results ob t a i n e d were good, if
there was no steric hindrance, the m e t h o d was a p p li ed to
the sy nthesis of fatty esters. Both Boc ValOK and Boc
Ph eO K were reac ted in turn with b r o m o o c t a d e c a n e and
1 8- Cr own-6 in MeCN. We ob ta i n e d yields of 27% and 55%
res pectively. The low yie]d s were put down to the
charue I erisLi cs of the solvent, therefore we repoutod
both rea ctions in DMF. Here we o b t a i n e d yields of 70%
for Boc ValOCia and 74% for Boc PheOCia.
Penney et al. pr op osed a simple procedur e for the
s yn thesis of amino acid fatty esters (Penney 1985) .
This was achie v e d by the ine t hanesu 1 phon i c acid c a t al ys ed
r e a c t i o n of amino acids in an o c l a d e c a n o l melt. This
gave yields in the range 40 to 90%, d e p e n d i n g on the
amino acid. Met hods pre vio us to this de p e n d e d upon the
alco hol compo nent b e ing present as the solvent, and
therefore were not readily a p p l i c a b l e to solid, long
chain alkanols. (Penney overc am e this by using a melt
of the fatty alkanol . ) There were two p o t e n t i a l l y high
y i e l d i n g methods, one of which involved the use of the
cesium salt of the amino acid and the a p p r o p r i a t e alkyl
halide (Wang 1977). The other involved the use of the
amino acid and alkanol in the presen ce of a co u p l i n g
-97-
rea gent and catalyst (Dhaon 1982). The main d r a w b a c k to
these me th ods is the need to use h a rsh conditions. A
further d e p r o t e c t i o n s t e p , w h i c h gives the added ha za r d
of r a c e m i s a t i o n , is also required.
The results we ob ta in ed co m p a r e d f a v o u r a b l y with
P e n n e y 's results. Our m e th od re q u i r e d milder re action
condi t ions and the use of only one equ i v a l e n t of the
alkyl halide. P e n n e y ’s m e t h o d has the ad v a n t a g e of not
re q u i r i n g a further d e p r o t e c t i o n step, but it does
require an excess of the alkanol. C o m p a r i s o n of the
pro po sed m e th od with that of Wang et al . showed our
me t h o d lo be inferior. The yields were lower and the
r ea ct ion required ele vated temperatures. The cesium
salts of N - p r o t e c t e d amino acids or peptides react
re adily with alkyl halides in DMF al room t e m p e r a t u r e ,
but rea ction times vary from 30 mi nutes to 17 hours.
The a d v antag e of our m e t h o d over this one is that the
P o l a s s i u m stilts of amino acids are easier to pr od uce
(Wang 1977) . The use of carbodi imides and 4-
(diinethylamino) - pyridi ne in ester f o r m a t i o n has been
shown to give mo de r a t e to high yield s of a v a r i e t y of
esters, but in some cases, rac emi sa t ion occurred. This
was great es t with Boc A s pO H and Boc G l u O H (Dhaon 1982).
Our me th od of ester form at ion is of limited use as it
cannot compare, as yet, with e s t a b l i s h e d methods for the
sy nt hesi s of methyl or benzyl esters. However, it does
pr ovide a way of maki n g fatty esters in mo der a t e to good
yie ld without the use of ha rs h conditions. There is
-98-
po tenti al to improve the me th od so that it becomes the
simplest way to sy n t h e s i z e fatty esters.
3.2 O l i g o p e p t i d e F o r m a t i o n .
In the reactions car rie d out in an attemp t to find the
c o n d it io ns requi red lo use lB-Crown-6 as an amine
p r o t e c t i n g group, DCC was used as the co u p l i n g reagent.
The me ch a n i s m s of react i o n b e tw een DCC and amino acids
are well documented, (for example, Kurzer 1967; DeTar
1966a, b, c; Burdon 1966 ). Thus any r e a c t i o n done with
the com ple xe s could easily be com pa re d with those of
’n o r m a l l y p r o t e c t e d ’ amino acids.
Once the complex is in solution, the pr ot o n a t e d amine
g r oup is in e q u i l i b r i u m between the free and compiex ed
forms. We tried to det e r m i n e the ex ch ange rate but even
at -75°C, we did not detect any e x p ec te d change in the
11 NMR spectrum. The s p l it ting of the signals into two
dis ti nct peaks was not seen. This is indicat ive of a
slow exchang e rate. However, there was slight
b r o a d e n i n g of the signals, indic ative of a me di u m to
fast exc hange rate. Thus in the first app roxima ti on, it
could be c o n cl ud ed that the exchange rate between the
f roe and com pi ex ed forms was faster than the NMR time
scale at 300MHz at room temperature. H a v i n g c o n c luded
this, the comp lex was reacted with DCC in MeCN or
CHCi 3 .
-99-
P r e v i o u s work has shown that DCC reacts with N - p r o t e c t e d
amino acids to give the anhydride, N-acyl urea and
dicyc lohexyl urea. It has been pr o p o s e d that the
intermedia te is the O-acyl isourea (Jones 1979; See
Scheme 1.3). The results ob tained from our work have
been i nt er preted under the a s s u m p t i o n that the O-acyl
isoarea was the int er mediat e obtai n e d du ri ng the
re ac ti ons of the complexes w ith DCC in MeCN or C H C 1 3 .
These rea ctions led to oli gom er isation . The f o r m ation
of di- and tri-peptid e de ri v a t i v e s showed that under
these conditions, the 18-Crown-6 was not acting as an N-
protec ting group. It was acting as a solubiliser,
maki n g the amino acid av ai lable in s o l ution to react
with the c a r b o d i i m i d e . The complex r e a ct ion with DCC
would thus give the 0 -acy"l isourea which would then
react with one of the n u c l e o p h i l e s in the react io n
solution. In the ’n o r m a l ’ c on dition s of peptide
synthesis, the most pro mi ne nt of these n u c l e o p h i l e s is
the amine group from the second amino acid. Since, in
the condi ti ons we employed, we o b t ained oligomers, even
in the absence of a second amino acid component, the
free arnine gro up had to be sup pl ied by the complex.
Thus the first q u e s t i o n we tried to answ er was: how does
deprotonation, and hence d e p r o t e c t i o n occur? One
hypothe si s was that one of the other n u c l e o p h i l e s
present in the solutio n was d e p r o t o n a t i n g the amine
group. These other species were the c o u n t e r - i o n and the
DCC. Thus a study of their effects in the reaction
- 1 00-
c o n d it io ns was c a r ri ed out. Firstly, we c o n s idered the
counter- ion ; being "naked" in the reaction solution, it
had inc reased n u c 1e o p h i 1 i c i t y . To ensure that the
c o u n t e r - i o n was not re a c t i n g with DCC and then
d e p r o t o n a t i n g the amine group, reaction s using the
m e t h y l a m i n e com ple x (2) were ca rried out. As there was
no change in the a b s o r b a n c e peak of N=C= N at 2120 cm 1 in
the IE spectrum, it was co n c l u d e d that the c o u n t e r - i o n
was not re acting wit h DCC. However, when benzoic acid
was added to the re ac ti on solution, the prompt for ma tion
of DCU and benzoic anhyd ri de sh owed that the ca rb o x y l i c
acid inoiety reacted with DCC in the predict ed fashion.
Sch eme 3.2 The P r o p o s e d M e c h a n i s m for the F o r m a t i o n of
the V i l s meier Adduct.
e $ ©9H3ci > n h 3c h c o o h
c h 3
NHoCHCOOH
CpI i'
_ H H^CI®© I C l ©
N=C-NCHC00HCl HCH3
N=CHNHCHC00H ♦ HCl
-101 -
Sch eme 3,3 Two P o s sible Pa th wa ys for the F o r m a t i o n of
O l i g o p e p t i d e s .
O
C T - 0
cr-ocr-o
c r-or © i ^
cr-o
S ® 2 - h :cr-o
o ocr-o -•
o o x “
©_ro cncr-o
cr-o
- J 0 2 -
T h er e was also a small amount of N - m e t h y l b e n z a m i d e
o b t a i n e d which indicated that d e p r o t o n a t i o n of the amine
had also occurred. However, either the e q u i l i b r i u m
b e t w e e n the free and c o m piexed forms of the amine lay
towards the co m p i e x e d form or the f o r m a t i o n of an hy dride
was faster than amide bond formation. It was con c l u d e d
from these results that the m e c h a n i s m by which
o l i g o m e r i s a t i o n o c c urr ed c o n t a i n e d a step invo lv ing both
the amine and the carboxyl ic functions of the same
mo l e c u l a r species. If it was otherwise, then more N-
m e t h y 1 be n z amide would have been formed.
The results obt ai ned from the rea ctions of the Vil s m e i e r
Reagent with co mple x lb at -44°C indic ated that the
o 1 i g o m e r i s a t i o n process invol ved the N H 3 * and the imine
of the DCC in an hydro g e n bond. This hy d r o g e n bond
cannot form when the V i l s m e i e r Reagen t was used because
the imine ni t r o g e n is p o s i t i v e l y charged.
At room temperature, the N-a dd uct from the Vi lsmeier
Reag ent (see figure 2.1a) was formed because the
e q u i l i b r i u m be twe en the free and c o m p i e x e d forms of the
amine gro up lay more towards the free form than at
-44°C. The m e c h a n i s m pr opo s e d for the fo r m a t i o n of the
N -a dd uct of the Vil s m e i e r reagent is shown in Scheme
3.2.
The m e c h a n i s m leading to the oligomers, involvi ng an
i n t r a mole cu lar hy d r o g e n bond, was further v e r if ied by
the re act ion s using 6 - a m i n o h e x a n o i c acid co mp lex (3 ) .
The only product o b t ai ned here was the N-acyl urea
- 1 0 3 -
de rivat i v e , whi ch ind i ca t ed that the two
functio nal groups had not only to be on the same
molecule, but also a t t ached to the same ca rbo n for
o l i g o m e r i s a t i o n to occur. This is be ca use the m e c h a n i s m
goes through an O-acyl isourea d e r i v a t i v e which then
forms an i n t r a m o l e c u l a r h y d r o g e n bond. When the usual
routes of amide bond form ution and acid anhydri de
f o r ma ti on are inhibited, then the N-acyl urea d e r i v a t i v e
forms slowly. The m e c h a n i s m of the rea cti on is shown in
Scheme 3.3
DeTa r and co - w o r k e r s have shown that simple c ar boxylic
acids form ionic dimers or higher a g g r e g a t e s in solvents
such as MeCN (DeTar 1966 ) . The DCC would react with the
ion pair to give the ionic ag g r e g a t e shown in Figure
3.1. This has been a p p lied to the comp lex and DCC
reactions, as shown in Scheme 3.3.
Figure 3.1 The Ion A g g r e g a t e P r o pos ed by DeTar for the
R e a c t i o n B e t wee n C a r b o x y l i c Acids and DCC.
0:®:0 H-0%c = o/
C o m p a r i s o n of this method with the one propose d for
’n o r m a l ’ N - p r o t e c t e d amino acids, showe d that DCC
rea cted in a similar way. Aft er fo rmation of the O-acyl
isourea, the p r e senc e of an amine fun ct ion gave rise to
ol ig om erisat io n. As the 6 - a m i n o h e x a n o i c acid comp lex
gave only slow formatio n of the N-acyl urea derivative,
it was c o n cl uded that the 1 8- Cr own-6 cou ld pro tect the
amine group of an amino acid in the p r e se nc e of DCC.
This was only possible, though, if the two functional
group s were not a t t ached to the same carbon atom. For
peptide synthesis, this would require the use of at
least a dipeptide. The work c o n tinued usi ng a dipeptide
c o m pl ex because this co n t a i n e d the same number of atoms
in the backbone as 6 - a m i n o h e x u n o i c acid arid it was hoped
that it would react in a simi lar way.
As a co rollar y to this, a tri- and a p e n t a p e p t i d e were
both c o m p ie xed with 18-Crwon-6, to show that the method
used for co mplex for mat io n was not limited to amino
acids and dipeptides. Both the tri- and the
p en t a p e p t i d e formed 1 : 1 co mplexes and gave clear
so lu tion s in C D C I 3 . This could be useful if app lied to
fragment c o n d e n s a t i o n m e t h o d of peptide synth esis
(Sieber 1970, 1977; Ge ige r 1969; Ivanov 1976). This
method often has problems with s o l u b i l i t y of the larger
peptide fragments and 18-Crown- 6 may be a way of
over c o ruing this.
3.3 DM S O De ri vatisati on.
From the results obtain ed in MeCN or CHC1 3 , it was
c o n c l u d e d that an i n t r a m o l e c u l a r h y d r o g e n bond was the
cause of the o l i g o m e r i s a t i o n reaction. It was
thought that the use of a polar solvent, such as DMSO or
D M F , may stop this since both solvents favour the
fo r m a t i o n of solvent - so lu te h y d r o g e n bonds, over
solute - solute ones. The use of a dip e p t i d e complex,
as de s c r i b e d before, should also el im inate the
intram o l e c u l a r h y d r o g e n bond by the in cre ased distance
between the two functiona l g r o u p s . Thus a c o m b i n a t i o n
of a di peptide comple x and a polar solvent (DMSO) shoul d
e l i m in at e the o l i g o m e r i s a t i o n reaction. However, amino
acid co mplexes were also re acted with DCC in DMS O to see
if just a change in solvent was suf fic ient to elimin at e
the oligo meris at ion.
The reactio n of di p e p t i d e c o m p lexes with DCC in DMSO at
a c o n c e n t r a t i o n of 0.02M gave rise to the N-acyl urea
d e r i v a t i v e after 20 hours, as judged by *H NMR
spectroscopy. It was thought that the N-acyl urea could
not be coming from the r e a r r a n g e m e n t of the 0 - a c y ’l
isourea because there was no carboxyl a c t i v a t i o n at this
concentration. This was indicated by the lack of es ter
for m a t i o n when the n u c l e o p h i l e NSu was add ed to a 0 . 02M
c o n c e n t r a t i o n sol ut i o n of the dip e p t i d e co mpl ex and DCC.
The m e c h a n i s m shown in Scheme 3.4 was propo se d which
-106-
gave rise to the N-acyl urea via a c o n c e r t e d mechanism,
not depend en t on the O-acyl isourea intermediate.
Scheme 3.4 M e c h a n i s m P r o p o s e d for the F o r m a t i o n of
N-Acyl Urea Derivatives.
xe > nh®c h 2c o n h c h 2coo,h -
R'N-C=NR’
XG>NH®CH2CONHCH2COR'N-C-NHR' 11
0
■*» X® NH®CH2CONHCH ct ©/ R'N-Cp^R’Lai
H
©S © ,0)X >NHoCHoC0NHCH2C ^
1NHR'
Thus it app ea red that at this c o n c e n t r a t i o n (0.02M) ,
o l i g o m e r i s a t i o n had been suppressed. However, peptide
synth es is is us ua lly carried out at high c o n c e n t r a t i o n
to reduce the risk of side reactions. Thus as there was
no apparent a c t i v a t i o n at 0.02M concentr ation, the
r e ac tions were repea te d at a c o n c e n t r a t i o n of 0.2M to
see if a c t i v a t i o n would take place. The rapid fo rmatio n
of only the DMSO adduct (5) when co mp le x (4a) was
reacted with DCC in DMSO, showed that o l i g o m e r i s a t i o n
rem ained supp re ssed at higher conc en trati on. However,
p r o t e c t i o n of the amine function was still not feasible
-10 7-
because of the re ac tion between the solvent and the
amine function.
The influence of the c o u n t e r - i o n on co mp lex stabi li ty
was ev a l u a t e d by the use of the chl or ide ion as well as
the tosyla te ion. Thus when comp lex (4b) was used in
the reac t i o n at a c o n c e n t r a t i o n of 0.2M, DCU was formed
with out any a c c o m p a n y i n g f o r mat io n of pept ide products.
O nly as the last of the DCC was co nsumed did any
p r o d u c t s other than DCU appea r in the *H NMR spectrum.
This was due to water present in the r e a cti on solution,
and will be di s c u s s e d fully later.
The for m a t i o n of the D MS O adduct indicated that the
crown ether was not o f f e r i n g p r o t e c t i o n . However, the
a d d i t i o n of the n u c l e o p h i l e NSu into the reaction
solu Lion showed that the ca rboxyl ic grou p was be h a v i n g
as a ’n o r m a l ’ amino acid, because there was prompt
f o r m at io n of the active ester. Thus at this
c o n c e n t r a t i o n (0 . 2M ) , the c a r b ox ylic g r oup was being
activated. The active esters never reached large
p ro po rtions in the r e a cti on solutions, most proba bl y
due to the pre se nce of water. This is; d i s c ussed more
fully later. To ensure that the lB -Crown-6 was not
a c t i v a t i n g the amine gro up in some way, the reactions
using n - b u t y l a m i n e , acetic acid and DCC were carried out
with and without 1 8 - C r o w n -6. The results showed that
the 18 - C r o w n - 6 was d e f i n i t e l y acti ng as a p r o t e c t i n g
group , not an activator' to the amine function. This was
shown by the slow form at ion of the amide* when no crown
- 108-
ether was present but there was no such re ac t i o n when
the crown ether was present.
As seen in the o l i g o m e r i s a t i o n react ions in MeCN or
C H C I 3 , the car bo xylic group had to be on the same carbon
for the d e p r o t e c t i o n of the amine group to occur.
T h e r e f o r e to see if the ca rboxyli c group was
p a r t i c i p a t i n g in the d e p r o t e c t i o n of the amine g r ou p in
some way in the re ac tion in DMSO. they were repeated
using the m e t h y l a m i n e h y d r o c h l o r i d e com ple x (2) . There
was no re ac tion between the c o mp le x and DCC , and no
deri v a t i s a t i o n o c c ur re d in the r e a ct ion s o l u t i o n until
a cet ic acid was added. At tlx is time, the D M S O - u d d u c t
(9) formed. This proved that the car b o x y l i c group was
ne ed e d for d e r i v a t i s a t i o n to occur.
Ha vi ng found that dipe pt ide com pl exes were derivati sed,
amino acid complexes were reacted under the same
c on di tions to see if o l i g o m e r i s a t i o n would still occur.
Fir stl y they were reac ted at a c o n c e n t r a t i o n of 0.05M to
see if there would be carbox yl act ivation. The produ cts
ob t a i n e d were the N-acyl urea d e r i v a t i v e (6d), the DM S O
adduct (34) and later the DMSO adduct of the N-acyl urea
de r i v a t i v e (6f). This showed that only a change in
solven L was sufficien t to elimi na te o l i gome ri sation.
The inclusion of NSu in the re a c t i o n at 0.0 2M
concentration, stopped the f o r m ati on of the N-acyl urea
derivatives, but not that of the; DMSO adduct (34). The
fo rma tion of the N-acyl urea d e r i v a t i v e s was p r e v en ted
by gi vin g the a c t iv at ed DCC an a l t e r n a t i v e pathway. The
-109-
p r e s e n c e of NSu means that the a c t i v a t e d DCC can react
with either the comp lex or the NSu. It could be that
c o m p o u n d 30 was being formed, but only in minute
amounts. The re was no carbox yl a c t i v a t i o n because there
was no ester formation.
The se react ion s were rep e a t e d at the higher
c o n c e n t r a t i o n (0.2M), where there was facile f o r m at io n
of the d i p e ptide (4) , its D MS O adduct (5) and the DMS O
adduc t of the co mp lex (34) when no NSu was present. The
use of co mplex (33b) also gave the tr ip eptid e and its
DMS O adduct. This is e x p l a i n e d later. When NSu was
included in the re a c t i o n solution, there was prompt
f o r mation of the active ester, followed by the DMSO
adduct (34), the d i p e ptide (4), its DMS O adduct (5) and
traces of higher oligomers. He; re, it app e a r e d that the
NSu had incre as ed the o l i g o m e r i s a t i o n reaction. This
was due to the active esters reacting with the free
amino acid. (.NSu d e r i v a t i v e s are oft en used as the
active esters in peptide syn th esis (An derson 1964;
B o d a n s z k y 1984 ). j
All these results can be e x p l a i n e d using the work of
DeTar with carb ox ylic acids and c a r b o d i i m i d e s as a
model. DeTar showed that or ganic acids form ion pairs
in organic solvents (DeTar 1 9 6 6 b ) . He p r o posed the
forma ti on of ion pairs in or ganic sol ve nts in pr eferen ce
to a six-cent red reac tion becau.se of the increased
re ac tivity which acco m p a n i e s the increase in acidity.
- 11 0 -
In DMSO, the complexes are s u r r ou nd ed by a solvati on
shell, whi ch gives rise to solvent sep a r a t e d ion pairs.
The DCC will react with the ion pair as before to give
the ion aggregate, shown in Figure 3.1, but the solvent
s e p arated version. The n u c l e o p h i l e s present in the
reactio n solution then react with this ion aggregate.
The re are four n u c l e o p h i l e s present - water, ainine , DMSO
and NSu. Thes e are di s c u s s e d in turn.
Scheme 3.5 S o l v a t i o n of the ’N a k e d ’ Ion by Water, and
the Su bs equent H y d r a t i o n of DCC.
RCOOH + R N-C=NR*A
v
RCOO + R'Nt?-C=NR'K— ' C—
0-H H -0
H x® H
H H '0'
H® R — R’NHCNHR'
eX = Tos/C l
W a ter* . This was present in the r e a ction soluti on as an
impurity, coming from either' the complex or the solvent.
In the case of complex (4b) , the water’ came from the
R'NHC=NR'
h i -
c o m pl ex where it was presen t as water of
cr ystal li sa tion, as shown by Elem en tal Analysis. This
water impurit y was p r e s u m a b l y a s s o c i a t e d with the
"naked" anion and formed a s o l v atio n shell around it.
The water m o l e cu les became p o l a r i s e d by the ne g a t i v e
charge and were therefo re more able to attack the
e l e c t r o p h i l i c ca rbon of the a c t i v a t e d DCC, gi vi n g DCU.
As more DCU was formed for co mp lex (4b) before any
p e p tide pr oducts appeared, it was c o n c luded that a)
there was more water present, and b) the water was
re a c t i n g with the DCC pre ferenti al ly. This is shown in
Scheme 3.5.
Am i n e . This was presen t in the react i o n sol ut ion
because of one of the acid - base e q u i l i b r i a shown in
Scheme 3.6. As this was a component requi red in peptide
formation, the product o b t ai ne d from its rea ct io n with
the ion aggregate would be a peptide. Under the
conditi on s of the re ac tion using the d i p e p t i d e com plexes
though, the other n u c l e o p h i l e s were more reactive and so
only traces were found of higher oligomers.
Furthermore, the amine react ed with the ac t i v a t e d DMSO
(discussed under DMSO) to form the DMS O adducts. The
form ation of these adducts was a fast reaction cap t u r i n g
any free amine in solution. This acted on the
e q u i l i b r i u m to give more free amine, and thus the DMSO
adduct became the major product of the reaction.
- 112 -
Scheme 3.6 Acid - Base E q u i l i b r i a Presen t in the DMSO
R e a c t i o n Solution.
A t\ —o
X
x xo oo oo oCM CMX Xo oX XX Xo oo oCM CMX X
°© CO @_co
\s\s\s\s
o ©X X
oX
- 11 3 -
S che me 3.7 The P r o p o s e d M e c h a n i s m for DMSO
D e r i v a t i s a t i o n , Using Gl y c i n e Complexes.
Me2s x© 0
SMe, SMe, SMe,Q " "
h?n c h 2c; ° ho; cch2nh® \ x® nMe2'OH 0" 2 3 s 0
0 0 oII II
S M & SMe^ SMe^
SMe2 SMe2 SMe?n L i\ 1
Me2S xe MHfNCH2C ^ H H°:CCH2NH? - X®SMe2 Me2 0 0
:2 SMe2
0
II0
SMe.0 Me, 0II cSMe.
©0SMe2.©- ©- -',0 0 NH3
Me2S X 0 NH,CH,C' n i CH,C00H" OHS X® 2OSMeo
11 0 SMe. Me2
R'N=C=NR'DCC
Me.
Me?0 S n e " ©
s x " o NH3CH2C'0® n NHfcH2C00HHMe;Me2 Me,
^N-C-NR . R /
0=SMe2
X®Me2 S 2 0
S X 0 J-UNCHX00H ■< H?NCHX00H *■ Me J 1 n 1 1 c0
R'fsL ^ k)R'N' 2
H M e X XX °
sMe. ©
Me2S=NHCH2COOH ©
■H20 Xsolvent-separated
ion pair11 'i
D M S O . Work by Moffa t t et al . showed that DMS O reacted
with DCC in the pre se nc e of acid (Pfitzner 1965;
F e n s e l a u 1966; Bu rdon 1966; Ler ch 1971). They showed
that the ox yge n from the DMSO was q u a n t i t a t i v e l y
transferred to the DCU. The i nte rm ediate they pr op osed
is show n in Figure 3.2.
In their work, this i n t e r mediate was atta c k e d by an
alkano'l and an aldehy de was obtained. In our work, it
was pr op osed that this i n t e r medi at e was at tacked by the
ffee amine which would lead to the D MS O adduct, as shown
by Sche me 3.7.
Fvide nc e that the propos ed i nterme di ate was involved in
the m e c h a n i s m came from the Mass spect ru m of the
reaction residue. A peak at m/z = 285 c o r r es po nds to
structure (40) (see Figure 3.2).
Figure 3.2 Intermedia te Be t w e e n DCC and DM S O as
Pr o p o s e d by Moffatt.
N - h y dr ox y succ iniinide . The prese nc e of this in the
r e a ction solutio n gave the act i v a t e d carboxyl g r oup an
a l t e r n a t i v e pathway. The for ma tion of the ester was the
fastest reaction. Thus the active ester was pro du ce d
R'N=C-NHR'I0
H3C CH3
- 1 1 5 -
first. This meant that the water impurit y was still
present and was th ere fore able to react with the active
ester, h y d r o l y s i n g it back to the acid. This is shown
in Scheme 3.8.
Scheme 3.8 F o r m a t i o n and H y d r o l y s i s M e c h a n i s m s of the
Ac t i ve E s t e r s .
0-0
Z 0ZX
Xo o oCNJ
XoXz o oCNJ
XoX
0
LOo00X D CO
X
The for ma ti on of the DMSO adduct co n t i n u e d as before,
though some m o l e cules were present as their active
e s t e r .
To ensure that the DCC was not ac t i v a t e d by NSu, the
r e a ct ion of these two co m p o u n d s was carr ied out. The
only prod uct seen in the *H NMR s p e ctrum after 17 hoursI
was (30), ob t a i n e d from the d e h y d r a t i o n of three
m o l e c u l e s of NSu with one mo l e c u l e u n d e r g o i n g ring
opening. This product has been shown to be formed whe n
activ e ester forma ti on was slowed by steric h i n dr ance
(Rich 1979). Thus some c o m p o u n d (30) could be seen in
Lhe re actions of gl yci ne complex es with DCC and NSu.
This was due to the steric hin d r a n c e of the crown ether
rings of the ion pair. However, for the d i p e pt ide
complexes, NSu mo le cules could be in c o r po rated into the
sol v a t i o n shell and the refore come into closer contact
with the ion pair or aggregate. The re act ion s of
'normally p r o t e c t e d ’ amino acids with DCC in DMSO have
not been well documented, the refore the rea ctions usi ng
Boc Glycine, Boc Al ani ne and Boc G l y c y T G l y c i n e with DCC
were carried out for comparison. The results ob ta ined
were as exp ec ted but led to a further question: why was
the anhydr id e formed with Boc co mp ounds but not with the
comp le xe s? This may be e x p l aine d by the r earra ng ement
of the complex ion pairs. Once the DCC had been
a c t i va te d by the c ar boxyli c group, the ion pair1 could
form in a head to tail conf orrnat ion , due to the
positiv el y charged amine group. There may be salt
-11 7 -
br idges formed in the dimeric form. As d e s c ri be d
before, in DMS O the ion pairs were solvent separated.
This would lead to the facile f o r ma tion of the DMS O
adduct. The Boc compou nd anh y d r i d e s rea cted further
to prod uce the N-acyl urea d e r i v a t i v e and the o x a z ol one
be cause there was excess DCC present in the reac ti on
solution. (Only \ equi valen t of DCC is r e q ui re d to form
the acid anhydride. ) The o x a z ol on e was an e x p ec ted
pr oduct from the rea ct io n c o n t a i n i n g a pro t e c t e d
d i p e pt id e but not from the react i o n c o n t a i n i n g a
p r o t e c t e d amino acid. It was ob t a i n e d beca use amide
bond formatio n was h i n d e r e d (Be noiton 1981). The
d e p r o t e c t i o n of Boc Alanin e at a r e a ction c o n c e n t r a t i o n
of 0 . 2M may have been brought about by the D M S O - D C C
adduct. This contains a po si tive charge which could
attract the NH to donat e its electrons. The produ ct s
would be the DMSO adduct, C O 2 and a te rti ar y butyl
d e r i v a t i v e of DCU. A m e c h a n i s m for this re act i o n is
p r o po se d in Scheme 3.9.
This w or k showed that the use of a polar solvent and a
d i p ep ti de comp lex was enoug h to eli mi na te
oli go meri sa ti on. However, the solvent needed to be
a l t er ed to one which did not co nt ain a reactive centre
like the sulpho xide g r ou p of DMSO. A polar solvent
c om mo nly used in peptide synth esi s is DMF and this does
not contain as p o t e ntiall y reac t i ve ctgroup as DMSO. It
was hoped that o l i g o m e r i s a t i o n would remain s u p p re ssed
- .1 1 8 -
with the change in sol vent and also no de r i va t i sa t i on
would occur.
S che me 3.9 The P r o p o s e d M e c h a n i s m for the Removal of
the Boc G r o u p with the D M S O - D C C adduct.
0 R (H3O 3COCNHCHCOO
(h3c )2s ^ - c = n r ' N
H R
© P(H3C)2SNHCHCOO
0
0 0 (H3C)2SOCCHNHC^C(CH3)3
R
* R'NHCONHR'
h3c;,ch3H N " '0
R - ^ 0
♦ CO.0+ (H303C©
- H
(H3C)2C=CH2
3.4 R e a c tio ns in D M F .
The study was co ntinue d in DMF, f o l l ow in g the results
ob t a i n e d in DMSO. The rea ct ion of comp Lex (4a) with DCC
in DMF overnight led to 17% of the DMF adduct and 18% of
the N-acyl urea de r i v a t i v e (6b) . This showed that the
two compo und s were r e l a ti ve ly stable towards each other ,
at least for the first hour. It was believ ed that the
- 1 1 9 -
DMF adduct was being formed by the same m e c h a n i s m as for
the D M S O ad duct (see Schem e 3.7). It was co nc luded that
if an amino acid ester was added to a s o l ution of
co mp le x and DC C with i n the first 15 minutes, p e ptid e
bond f o r m a t i o n may occur. The f o r m at io n of a pe pt ide
bond is a faster re action than the f o r m ation of either
the DMF adduct or the N-acyl urea derivative. W he n the
a d d i t i o n of an ester and TEA so l u t i o n to a co mp le x and
DCC s o l u t i o n was carr ied out, there was prompt f o r ma ti on
of DCU, i n d i c a t i n g that pe ptide bond f o r m ati on had taken
p l a c e .
The o p t i m u m c o n d ition s for the reaction were found to be
3 e q u i v a l e n t s of comp lex and DCC to 1 e quivale nt of
ester and TEA. It was belie ve d that there was an
e q u i l i b r i u m present.
When the c o n c e n t r a t i o n of the ester, and hence TEA, was
incr eas ed twof old there was a decreas e in the yield of
the d e s ir ed product. This could be due to the fact that
the free ester was c o-prec ip i tating with the TEA. HC1
salt as the so l u t i o n was too con cen trated.
Thus it a p p e a r e d that se le ctive peptide bond f o r m at ion
had been ac h i e v e d u s i n g 18-C rown- 6 as the N - t e r m i n a l
p r o t e c t i n g group.
The m e th od was tried usi ng vari ous c o m b i n a t i o n s to see
if it would have general application. Ho wev er the use
of pheny 1 a 1 an i ne inethyl pster gave a low yield. This
was most p r o b a b l y due to the sleric h i n drance b e tw een
the benzene ring and the ion aggregate. The presen ce of
-12 0 -
two cro wn rings and the cyclohe xy l m o i et ie s in the ion
a g g r e g a t e would inhibit the a p p r o a c h of the bulky amino
acid esters to the r e a ctio n site.
F o r m a t i o n of tripept id es other than those with the
p h e n y l a l a n i n e as the ester, p r o c e e d e d well. E s p e c i a l l y
when m o d i f i c a t i o n s to the pr oc edure were made. The use
of the water soluble carbodi imide (wscdi) improved the
pu r i t y and the yield of the reaction. This has been
shown before in the fo rm ation of s y m m e tr ical a n h y d r i d e s
(B od anszky 1984). Whilst the inclus io n of 1-
h y d r o x y b e n z o t r i a z o l e inhibits any rac em isat i o n . This
a d d i t i o n inhibits r a c e m i s a t i o n because the lifetime of
the re act ive O-acyl isourea or sy mm e t r i c a l a n h y dr id es
is reduced. The HOBt c o n c e n t r a t i o n remains almost
constant throughout the reaction and this a c c e l e r a t e d
the entire process. It also con ve rt s the h i ghl y
a c t i v a t e d carbodi imide i n t e r med ia tes to the less
reactive ester's of HOBt which are less likely to give
side reactions. Thus it provi des a l t e r n a t i v e pathways
to the same product (Bodanszky 1984).
The successful synthesis of an E n k e p h a l i n de r i v a t i v e
showed that the p r o cedur e usin g 18-Crown -6 as a
pr ot e c t i n g group was viable, though ob v i o u s l y
refinemen ts are required before the m e thod could compete
with est a b l i s h e d p rocedur es . The stro ng est points in
its favour so far- though, are the simple p r o t e c t i o n and
de p r o t e c t i o n steps. Repea t e d was hing with a satu ra ted
so l u t i o n of KOI removes the crown ether effectively.
C H A P T E R FOUR. M A T E R I A L S A N D ME T H O D S
4.1 M a t e r i a l s .
1 8 -Cr own-6 was su pp lied by Al d r i c h and pu r i f i e d u s ing
the m e thod d e v e lop ed by Gokel (Gokel 1974 ) . The crude
ether was d i s s o l v e d in MeCN with warming. On cooling
the crysta ls were filtere d off and pure 18- Cro wn-6 was
o b t a i n e d by wa rm ing under re duc ed pressure on a rotary
e v a p o r a t o r .
DMSO was d i s tilled under v a c u u m (31°C, 0.5mmllg) and kept
in the dark over type 4A m o l e c u l a r sieve.
D M S O - d e was suppl ie d by Al d r i c h and stored in an
airtight containe r over type 5A m o l e c u l a r sieve.
DCC was di s t i l l e d under va cuu m (92°C, 0.3mmHg) and kept
in a desiccator.
D M F , HPLC grade was su pplied by A l d r i c h and used
without fur the i' pur ification.
MeCN was stirred with CaH 2 which was added in small
portions until H 2 e v o l ut ion ceased. The solut io n was
then de ca nted off and f r a c t i o n a l l y di.stilled, the
fraction di s t i l l i n g at 8 1 - 8 2 0 C / 7 60mm Hg was collec te d
and stored over type 4 A m o ]ecu 1 nr sieve.
NSu was sup plied by A l d rich and used as supplied.
All solvents used for HPLC were HPLC grade, filtered
through a 5pin filter and de gassed before use.
-12 2 -
4.2 P r e p a r a t i o n s
4.2.1 Boc i - 2 - a m i n o d e c a n o i c acid (29).
One eq ui valent 1 - 2 - a m i n o d e c a n o i c acid (1.8727g, 0.01
moles) was d i s s o l v e d in a mi xtu re of t e r tia ry butanol
and water (1:1) and the pH ad j u s t e d to 12 -13 using 5N
N a O H . 1.5 equi v a l e n t s of d i - t e r t i a r y - b u t y l - d i c a r b o n a t e
( 3.2738g, 0.015 moles) in ter t iary-but anol (lOinl) was
add ed with s t i rr in g and the pH kept be t w e e n 12 and 13
for \ hour. The so lution was st irred for 3 hours before
the pH was a d j u s t e d to b e t we en 4 and 5 using citric
acid. The so l u t i o n was e x t r acted with ethyl acetate
( 3 x 3 0 m l ) , dried over sodium sulph ate then e v a p o r a t e d to
dryness. The crude product wras obtain ed as an oil.
lH NMR a n a ly sis of the product gave the fol low in g data:
5 = 0.88 (t CHa), 5 = 1.27 (bd Oil 2 ' s ) , 5 = 1.4 5 (Boc
Me ’ s ) , 5 =1.84, 1.68 ( C p H a ’s), 5 = 4.30, 4.09 ( C . H ’s), 6
= 5.13, 6.50 (Nil’s). There are two sets of signals due
to the two stereo isomers of Boc Ami no Acids, ie E and
Z .
4.2.2 Amino acid - Crown Ether Complexes.
A gen eral met ho d for the for ma ti on of amino acid - crown
ether com pl exes is as follows:
-12 3-
Tab le 4.1a Q u a n t i t i e s Used in the P r e p a r a t i o n of AminoA ci d Complexes.
Amino Acid Wt (g) Acid Wt (g) 18C6 Wt :No. Moles iComplex
Alanine 1.0000 Tosylic 2 . 1350 2.9670 1 . 12E-2 1 a
Alanine 1 . 0000 HC1 0.4090 2.9670 1 . 12E-2 lb
N H 3 C H 3 .HC1 1.0000 -- -- 3.9146 1 .48E-2 2b
Caproic Acid 1 . 0000 HC1 0.3056 2.0149 7 . 62E-3 3b
Ta bl e 4.1b Ele ment al Analysis and_ N M R data for AminoAcid Com p l e x e s Synthesi sed.
CpxE.A. (theoretical value in brackets) NMR (5 ) Yield
la C 50.02 ( 50.27 ) , H 7.53 (7.48 ) ,N 2.76 (2.67 ) .
7.8 2H d 8 .3Hz Tos A r o m ; 7.27 3H bd NHs * ; 7.12 2H d 8 . 3 H z Tos Arom; 4.25 1H m Cull; 3.65 34H s 18C6; 2.31 3H s Tos M e ; 1.6 3H d 6.9 Hz CpHa
6 6 . 6 |I
lb C 46.26 (46.21), H 8.33 (8.27),N 3.47 (3.59)
11 1H bd O H ; 7.11 3H bd N H a * ; 4.42 1H m C a H ; 3.59 30H s 1 8 C 6 ; 1 .5 3H d 8 .8 Hz C p H s
96 . 41iIi
3b
1
C 50.05 (50.05) H 8 .84 (8.87)N 3.31 (3.24)
1.42, 1.53, 1.68 6 H 3m C M 2
2.54 2H t CHaCOOH, 2.74 2H m C H z N H a . 3.61 1 8 C 6 , 7.12 3H s N H 3 * . 10.08 Hi bd OH
87 . 6
2b Not available due to hydroscopic nature of compound
2.38 3H q CIl3 , 3.54 29H s 1 8 C 6 , 7.63 3H bd NHs
100
One e qu iv al ent of ainino acid was d i s s o l v e d in H z O / E t O H
(5:1) and one equ ivale nt of’ the a p p r o p r i a t e acid added.
The solvent was removed and the salt tho rough ly dried.
The salt thus o b t ai ned was suspen de d in C H C 1 3 , 1
equ iv al ent 18 Cro wn 6 added and the s o l ution stirred
until it became clear. The solvent was removed in
vacuo, giving a white solid. For c o m pl exes using p-
t o 1. u ene s u 1 phon i c acid, the solid o b t ained was
r e c r y s ta llise d from hot FtOAo - E t O H . The complex es
-124-
u s ing h y d r o c h l o r i c acid requir ed no further
pu rificat ion. See Table 4.1a for the q u a n t i t i e s used in
each synt he sis and Table 4.1b for the E l e me nt al Analysis
and 1H NMR data.
4.2.3 Di p e p t i d e - C r ow n Ether Complexes.
One e qu iv al ent of the d i p e pt id e was d i s s o l v e d in H 2 O and
1 eq ui v a l e n t of the a p p r o p r i a t e acid was added. The
so l uti on was stir red for 30 min ut es before
1 i oph i 1 i sa t i on . The crude salt was then taken up in
ethanol. 1 equ iva le nt 18- Crown-6 added and the
te mp era tur e raised to 30 -4 0° C with s t i rr ing to aid
solubility. To the cool solution, EtOAc was added
dropwis e until the so lutio n became cloudy. On standin g
the complex c r y s t a l l i s e d out. See Table 4.2. for
Elemental Analysis and 1H NMR data.
T a b 1 e 4.2a Ele m en ta 1 An a y s i s Data fjj r___D ip ept i d eC o m p le xe s Synthesised.
Dipeptide I wt g )
Acid (wt g )
....18C6 1 wt g )
...... 1" ■ - 1 -1 -- f— - -----1Elemental Analysis jYield %i
j(Cpd) j
Glygly 5 . 0000
TosOH 7.1985
9.9912 Theory: C 48.58 H 7.09 N 4.93 j 93 j Found: C 48.59 H 6 . 9 7 K 5.05 j (4a) j
Glygly 1.0000
HC1 0.2760
2.0005 Theory: C 41.79 H 7.62 N 9.15 Found: C 41.78 H 7.88 N 6.09
(with 1.5 H 2 O present)
180 | i(4b) j
Glyphe 0.1300
TosOH 0 . 1113
0 . 1546 Theory: C 54.70 H 7.04 N 4.25 Found: C 54.48 H 7.22 N 4.21
67 |(20) i1
TyrGly TFA Bz0 . 0635 of salt
0.0379 Theory: C 54.39 H 6.42 N 3.96 Found: C 54.38 H 6.60 N 4.07
(23) |
- 1 2 5 -
4.2.4 O l i g o p e p t i d e - Cr ow n Ether C o m plex es
One e qu iv alent of the o l i g o p e p t i d e was d i s s o l v e d in
H a O / E t O H (4:1) and a c i d i f i e d with the a p p r o p r i a t e acid.
The solvent was removed, the residue sus p e n d e d in EtOli,
then 1 equ ival en t of 18-Cr ow n-6 was added. The sol ut ion
was stirred with EtOAc being added until the so lu tion
became clear, befor e evap o r a t i o n of the solvent. The
H NMR data is shown in Table
4.3.
Table 4 . 2b H NMR Data Pi p e p tide C o m p l exesSy n I h e_s_i sjj/P
Cpx H NMR Data (5) 1S o l v . |
■ ■ ■ 1. - --- --14a 8 . 65 1H t NH G 1 y DMSG
7.47, 7.11 4H 2d Ar Tos3 . 86 2H d C 0 H 2 Gly3 .61 2H s C 01H 2 Gly3 . 52 24H s CH 2 18C62 . 30 3H s CHa Tos
i4b 3 . 50 24H s CH 2 18C6
IDMSO
3 . 58 2H s C.H: Gly3 . 52 2H d C 0 H 2 Gly7.79 3H s bd N H d * G 1 y 28 . 91 1H t NH Gly 1
20 9 . 30 1H d NH Phe CDC137 . 83 2H d Ar Tos7 . 27 13H m Ar/NHa Phe/Tos CHC14 . 65 1H m C«H Phe3 . 93 1H m C cH2 Gly3 . 58 g CHa 18C6 i 13 . 13 2H m C p H 2 Phe !2 . 34 3H s CH 3 Tos
23 9 . 61 1H t NH Gly CDC137 . 38 5H m Ar Bz7.15, 6 . 92 4H 2d Ar Tyr4 . 05 2H m C.H * s Tjfr + Gly3 .69 m CH 2 ’ s Gly + 18C63 . 00 2H m C p H 2 Ty r
1 . .
Tab le 4.3 *H NMR Data for the _0l_i_gopeptide Co m p l e x e sSynthes i s e d .
Complex Yield 1H N M E Data (5
Enkephalin 100 9 .81 NH 3 . 85 C o H GlyComplex 8 . 29 N H 3.73 C o H Gly
7 . 94 NH 3 . 54 CHz 18C67 .13 Ar + m 3 3 . 14 C p H z Phe Tyr6 . 85 Ar 2 . 80 CpHz Me t4 . 43 C a H Tyr 2 . 56 C Hz Me t4 . 25 CoH Phe/M 2 . 00 CHz Met4 . 03 C„H Met 2 . 40 C H z Ace t . i.... j
Tos 96 . 4 1 . 54 3H d CpHz Ala 7 . 13 2H d Ar TosA la G lyGly 2 . 30 3H s CH a Tos 7 . 19 3H sbd NHz * AlaComplex 3 .63 s CHz 18C6 7 . 73 2H d Ar Tos
3 . 90 5H m CaHs All 7 . 94 1H t NH Gly9 . 46 1H t NH Gly
- -\HC1 100 8.71 1H t NH Gly 3 . 84 5H m C o H s All
A laGlyGly 8 .31 1H t NH Gly 3 . 52 24H s CHz 18C6C omp l ex 8 . 10 3H s NH a * Ala 1 .39 3H d CpHz Ala
4.2.5 Vilsiueier Reag ent (28).
PC'l b ( 3g ) was added in small port ions to an excess of
N , N -dime thy 1 for inamide ( DMF ) at 0°C with stirring. The
s o l u t i o n was left at 0°C for )£ hour with no stirring. A
white p re ci pi tate formed which was the V i l s m e i e r
Reagent. When required, it was filtered, washed with
ether and i m m e di at el y trans ferred to the re act ion vessel
with a glass sp at ula (Hepburn 1974). The 1H NMR
sp ectru m in CDC1 a gave:
6 = 2.59, 2.74 6H 2s =NMe a , 5 = 7.8-1 1 H s N = CH
- 1 2 7 -
4.2.6 Boc Dip ep tide Esters
One equ ivale nt of the releva nt Boc amino acid was
d i s s o l v e d in DCM and 1 equi va lent DCC added. The
s o l ut ion was cooled to 0°C wit h stirring.
T ab 1 e 4 .4 Data for the Boc Pi pep t i de Es t e r s Syn t he s i s ed .
TLC solvent sy stem = 9 parts DCM to 1 part M e O H .
D ipeptide E s t . S . M . ’ s Wts .
Ester Wt j Y i e 1d N o . m o l e s |% w/w
1
Rf 1H NMR Data (5)
BocGlyGlyOEt B o c G 1 y GlyOEt 61 . 0 0 . 64 8 . 64 t 1H NH1.0000 0.7 97 5 i1 6 . 60 t 1H NH
5 . 714E-3 3 .86 d 2H C a H 23.61 d 2H C 0 H 2
3 . 53 s 3H OMe1 . 37 s SH Boc j1
BocPheC140Me BocPhe Cl40Me 83 . 4 0 . 23 0 . 87 t 3H CH 3 1C 1 « j
0.2730 0.3025 1 . 23 s 2 OH C H 2 s Cl 4 |1.029E-3 11.41 s 9H Me Boc |
1 . 63 s 2H CpHz Ci * |3 . 07 t 2H CpHz Phe |3 . 69 s 3H OMe4 . 33 m 1H Call C 1 44 .52 m 1H CaH Phe4 . 99 bd 1H NH C 1 4
1 6 .31 d 1H NH Phe7 . 24 m 5H A r Phe
Boc Tyr . BzGlyOEt BocTyr GlyOEt 85 . 9 0 . 63 1 .26 t 3H CH 3 EtBz 1 . 39 s 9H CHa Boc
0.5000 0.1880 3 . 01 d 2H CpHz Tyr1.346E-3 3 . 97 m 2H C a H 2 Gly
4 . 19 q 2H CHa Et4 . 34 m 1H CaH Tyr4 . 95 bd 1H NH Tyr5 . 03 s 2H CH z Bz6 . 35 t 1H NH Gly6 . 91 d 2H Ar Tyr
i 7.13 d 2H A r Tyriii
7 . 38 m 511 Ar Bz
One equivalent of the relevan t amino acid ester salt was
di s s o l v e d in a m i n imum of DCM with 1 equiv alent of TEA.
The two solutions were mixed and the re actio n left
s t i rr ing overnight. E v a p o r a t i o n of the solvent gave a
residue which was taken up in E t O A c , e x t r ac te d with HC1
(0.1N, 2 x 3 0 in 1 ) , Brine ( 2x30ml ) , NaOH (0.1N, 2x30ml),
Brine (2x30ml) and H 2 O (2x30ml) and dried over Na2.S04
before evaporation.
The If NMR data, Rf and yield by weight are shown in
Tab le 4.4.
4.2.7 1-2-Aininodecanoic Acid Methyl Ester (18a).
MeOH ( 5 1111 ) was cooled in an ice bath and 2.5 equi valent s
of S O C I 2 (0.49ml, 6.7x10 moles) added slowly down the
flask. wail. One equ iv al ent of 1 ~2-arninodecanoic acid
(5 00mg, 2.67x10 J moles) was added, the solut io n left
st irr in g until it became clear and then refluxed
o v e r n i g h t . E v a p o r a t i o n of the solvent yielded a white
solid which was r e c r y s t a l l i s e d from hot EtOAc, yi e l d i n g
766mg, 71%w/w. 111 NMR ana lysis of the solid was
c o n s is te nt with the exp ec ted str uc ture of the ester.
5 - 0 . 8 3 (311 t CHa) 5 = 1.25 (1211 bd CHz's)
5 = 1.92 (211 m CHa CHa) 5 = 3.7 7 (311 s OMe)
5 = 4.0 6 (1H in CoH) 5 = 7.2 5 ( 3H bd NH a * )
4 . 3 R e ac t i o n s .
4 . 3 . I Boc Amino Acid Ester format ion using 1 8 - C r o w n - 6.
A general metho d for this reaction Is as f o l l o w s :
One equivalent of the N - p r o to ctnd ami no acid was
d i ssolved in aqueous E L 0 H c o n t a i n i n g one equ ivalent of
1M KOH . The s o l ution was stir red for 30 minutes, the
solvent removed and the salt thoroughly dried. This was
then su spended in a c e t o n i t r i l e or N ,N - d i m e t h y 1 forma mid e
with stirring, 1 eq ui valent of 18-C rown-6 added and the
s o l u t i o n stirre d until clear. An excess of the
a p p r o p r i a t e h a l o g e n a t e d compo un d was added and the
s o l u t i o n bro ught to boiling. The r e a ction was follow ed
by T.L.C. (solvent: 9 D C M , 1 M e O H ).
Table 4.5 P r o d u c ts o b tai_n e d 1 n t he Boc Am i no Ac id Es terS yn t he s e s us ing 18-C ro w n - 6.
N -protected Amino acid RX
Yield(%) Produc t Solv . Cpd .
Boc Val OH Mel 5 92 BocValOMe M e C N 38aipNOjCeHjF 2 -- ( 0 2N'C8H4 ) :0 MeCNC 7 H o N 0 2 C 1 13s 98 Boc Va l 0B z N0 2 MeCN 38ai iMe a CC1 2 -- M e C N
xs -- ’ Me 3 CC1C x a H 3 7 Br 1 27 BocVaIOCia MeCN 3 0 a l i i
- 70 •• DMF -
Boc Phe OH Mel 5 78 BocPheOMe MeCN 38biC 7 H b N 0 2 C 1 1% 71 BocPheOBzNOz MeCN 3 8 b i i
Ci»Ha 7Br 1 55 BocPheOCia MeCN 3 8 b i i i74 •• DMF ••
_____On completion, the solvent was removed and the residue
taken up in E t O A c , the u n d i s s o l v e d residue being
fil ter ed off; the solutio n e x t r ac ted with HC1 (1M,
2x2 0ml), 11 a 0 (2x20 ml), NaOH (2x20 ml) and brine (2x20 ml)
dried over sodium su lphate and e v a p o r a t e d to d r y n e s s .
Products and yields ob La ined are shown in Table 4.5, the
11 NMR data in Table 4.6.
-130
T able 4.6 *H NMR S p e c t r o s c o p i c Data for the Boc AminoAcids Esters Synthesised.
Product *H NMR Spectroscopic Data (5) |
Boc Val OMe 0 . 87 3H d 6 . 1Hz1
V a l m e t h y l |3 8a i 0 . 94 3H d 6 . 1Hz Val methyl j
1 . 43 9 H s Boc methyls2 . 10 1H m C p H
3.71 3H s Methyl ester4 . 20 1 H m C o H ;
5 . 02 1H bd NH |
Boc Val OBzNO 2 0 . 90 3H d 6.1Hz Val methyl38ai i 1 . 00 3H d 6.1Hz Val methyl
1 . 50 9H s Boc methyls2 . 10 1H m C p H
4 . 30 1H m C o H
5 . 00 2H bd NH + impurity J5 . 20 2H dd Benzyl C H : j
7.5 - 8 . 0 d p-subst. benzenering H ' s
Boc Val OC l a 0 . 89 6H m C . . + C H a C H 2 -
38ai i i 0 . 97 3H d C Ha1 .31 30H m CH 2 ' s1 . 43 9H s CHa Boc1 . 62 2H m CH 2 CH a .2.12 1H m C PH3.41 t -CH a Br SM4.12 2H in -CH z 0- Product j4 . 20 1H m C a H
5 .02 1H d Nli
Boc Phe OM e 1 . 40 9H s Boc methyls 138bi 3 . 10 2H m C p H 2 j
3 . 70 3H s Methyl ester + F. t O A c 14 . 50 1H m C a H |
5 . 00 1H bd NH |7-7 . 4 5H d + in Aroma t i c H ’s j
Boc Phe OBzNO 2 1 . 40 9H s Boc me thy1s38bi i 3 . 06 2H d C p H 2
5 . 00 1H bd C a l l
5 . 17 2H s Benzyl CH:7 . 55 1H d NH7.11- 8 . 17 10H Aroma tic H ’s
Boc Phe OC i a 0 . 87 4H t Cia methyl + imp.38bi i i 1 . 38 48H s Cia CH 2 ’s + i m p .
1 .43 9H s Boc me thy1s1 . 76 + 1 .85 2H 2 in Ci a CH 2 CH a3 . 06 1H bd t C o H SM or P r o d .3 . 40 + 3 .53 1H each 2 1 BrCH 2 - or -OCHa-4 . 06 1H t CoH SM or Prod.5 . 00 1H d NH7 . 12- 7.25 311 ni Aromatic H 1s
Ci z H a N 2 0 5 | 8.30 d 8.7 5Hz p- s u b s t . aromatic ring| 7.18 d 8.75Hz H ’sj_____________________________________________________________________________________________________________. . . I
- 1 3 1 -
4.3.2 R e a c t i o n of Amino Acid Co m p l e x e s with DCC in CHCla
or M eC N .
The ami no acid co mp lex was d i s s olved in the appr o p r i a t e
so lvent and 1 eq uivale nt of DCC added with stirring.
After \ hour, the s o l ut ion was filte red to remove any
precipi tat e, and left sti rr ing for the al l o t t e d time
hour or 24 hours ) . The solvent was e v a p or at ed and the
residue, taken up in C H C 1 3 (5ml) whi ch was washed with
H 2 O (2x5ml). The water fraction was ltjophilised and the
CHC1 □ fr actio n was dried over sodium sul phate and then
ovap o r a te d to drynes s .
H NMR ana lys is of the 112 O fractions led to the data
shown in Table 4.7. A typical lH NMR spe ct rum is shown
in the example below of the 112 O fra ct ion from the
rea ct ion b e t ween co mp lex la and DCC in C H C 1 3.
5 = 1.2 m CpH-/ s , 5 = 1.8 m Trace Imps., 5 = 2.2 s Tos
Me, <5 = 3.5 s 18-Ci’own-6, 5 = 3.9 - 4.0 m Call’s, 5 = 4.0
- 4.3 m Call’s, 5 = 7.1, 7.5 2d Tos Ar, 5 = 7.9 2 bd s
N H / ’s prods, 5 = 8.1 bd s NH-/ S.M., 5 = 8.2 t NH tri /
tetrapep, 5 = 8.6 d NH tripep., 5 = 8.7 d NH dlpep,
5 = 8.8, 8.4 m NH tetrapep.
Tfie CHCls fractio n gave the follo wing Ml NMR data:
5 = 1 - 2 m CH 2 DCU , 5 = 5.2 d Nil DCU , 5 = 3.5 s
1 8 - Cx'o wn - 6 .
- 1 3 2 -
4 _-_7_ C o m p l e x e s u s ed and P r o duc t s Ob t; a. in ed fr om the R e a c tions in Me CN a n d C H C 1 a L
Cpx So 1 v . % DCU 30 mins
j % Prod I(in H s0)
DCC eqs .
Time ( hr )
N o . moles
1 a MeCN 17 Di 3 Tri
80 SM
16 4 . 566x10
32 . 5 22 1.908x10 32 Di
38 SM
22 1.906x10 30 Di 7 Tri
63 SM
CHC1 2 4 1 . 903x10 16 Di 14 Tri 4 Tetra
6 6 SM
MeCN 34 . 2 2 4 21 Di 18 Tri 61 SM
30 . 7 24 46 Di 33 Tri 21 SM
19.2 24 35 Di 12 Tri 53 SM
33 . 5 43 Di 29 Tri 28 SM
13.1 33 Di
5 6 SM
lb MeCN 66 . 4 2.565x10 58 Di 29 Tri
>100 30 Di 20 Tri 50 SM
lb j MeCN 57 . 8 24 2.565x10 64 Di 28 Tri 8 SM
>100 2 4
7 0 SM
3b MeCN 2 4 2.315x10' [ 13 NAUrea8 7 SM
11.7 12 NAUrea 88 SM
12.3 17 NAUrea 8 3 SM
3 7 . 2
-13 3 -
T a b le 4.8 1H NMR Da t. a_ 1'rqm t. h e lie a c 1 i oris _ be L w e e n theV i Is m e ier R e agent and Co rap 1 ex __ lb .
A: C o m p l e x lb at Room Temp erature.
5 10 . 33 10.24 '8 . 60 r ■8.19 ...4 .79 ' ■" — 3 . 35
r ■" — 3 . 08 ’ " "3 . 02. ...
2 .81 1 . 54
Int 4 7 3 . 5 15 3' 85 20 44 44 8
Ees NH OH CH CH CaH CHz CH* c h 3 c h 3 CpHsdd s d s m s 2s s s d26 COOH 26
.DMF 26 18C6
___26 DMF DMFL. . J
26L . . . .
B: C o m p l e x lb at -44°C.
5 10 . 52 "1 8 . 90 8 . 28 "7 . 80 5 . 02 4 . 33.. ■■ i3 . 33 j
13.19 3 . 00 1 .83 1.24'
Int 2 2 12 15 2 5 r6 j 3 4 3 iy 5 1- - :
Res NH h" ...CH CH NH3 + CaH..... _| CoH !C H j | CHa C H 3 CpHa CpH 3 ;
bd d s s m m 2s| s s d d jN add N add Oadd Oadd N add Oudd N add 1 Oadd Oadd N add Oadd |
26 26 27 27 26 27 26 |— ----- 1_
DMF DMF| 26 . —
27!1 . --1
4.3.3 Amino Acid C o m p l e x e s with V i l s m e i e r Reagen t in
C D C L 3 .
One e qu iv al ent of com ple x (lOOmg, lb = 2. 56 5x 10 4 moles)
was di ss ol ved in 3 ml CDClu and taken to the appr o p r i a t e
temperature. An aliq uot of Vi l s m e i e r Reagent was added.
A sample was taken for 1H NMR ana ly sis at the end of the
reaction. The data thus obt a i n e d is tabul ate d in Table
4.8.
-13 4-
4.3.4 Methylainine H y d r o c h l o r i d e C o m p l e x with DCC and
Be nz oi c Acid in C H C 1 3.
One e quiva le nt of Methylainine H y d r o c h l o r i d e complex was
di s s o l v e d in CHC13 and 1 equ ival en t DCC added. The
r e a ct ion was follow ed by l.R. Sp ectroscopy. Ther e was
no change over the entire re a c t i o n period. The final
sp e c t r u m showed the data: 3200 - 3100 cm 1 NH stretch,
3000 - 2800 cm 1 CH stretch, 2120 cm 1 C = N stretch DCC,
1615 cm 1 NH deformatio n, 1470 cm 1 CH deformation, 1450
cm C H C I 3 , 13 50 cm 1 CH d e f o r m a t i o n , 1100 cm 1 C H C I 3 ,
1300 - 1200 cm 1 C-0 stretch.
To the above so lu ti on was added Benzoic Acid. The
r e a ct io n was followed by lH NMR spectroscopy. The
fo llowing data are from the spect ru m taken at the end of
the reaction:
5 - 7.93 211 m Benz oic acid ortho H ’s, 5 = 7.49 1H
be nzoic acid para H, 5 = 7.33 2H m Benzoic acid meta
H ’s, 5 = 7.18 Benzam id e Arornatics + NH , 5 - 7.00 3H bds
Nils + cpx , 5 - 3.48 18-Crown-6, 5 = 2.80 CHa Benzamide, 5
- 2.29 3H q C H 3 cpx.
Rat io of st artin g co mplex to product is 16:1.
4.3.5 Dipepti de C o m p le xes with DCC in D M S O .
These reactions were carr ied out a 1. two co ncentratio ns,
both in the NMR tube and on the bench. The q ua nt ities
used in each case are tabulated in Table 4.9.
- J 3 5 -
The general pr oc edure for Ihe NMR reactions was as
f o l l o w s :
One equiva le nt of the pu r i f i e d dip ept id e complex was
d i s s o l v e d in dry DMSO-do (0.5 - 0.6ml) and treated with
1 e q u i valen t freshly d i s t i l l e d DCC. The soluti on was
th or o u g h l y mix ed and 1H NMR spectra taken every 5
mi nutes for the first hour, then at regular intervals
for the next day. *11 NMR data from these e xperim en ts
are tabu lated in A p p en dix One, along with the reactions
in vol ving N S u .
An equ iv al en t me thod was em ployed when the reactio ns
were repeated on the bench, as shown in the follo wing
general method:
One equiva lent of complex war; d i s s ol ved in DMSO (5 or
10ml) and treated with .1 e q u i va le nt of DCC (freshly
d i s t i l l e d ) . The sol ut ion was mixe d t h o r oughly and left
to stand for the re qu ired length of time. The sol utions
were not stirred in an attempt to du plicate the
co nditi on s ob ta ined du rin g an 1H NMR experiment.
When NSu was present in the r e a ct ion it was dis s o l v e d
with the complex before a d d it ion of the DCC.
4.3.6 Met hylamine Co mp lex with DCC i n D M S O .
•• 4One equivalent of Methylainine complex (34.7mg, 1x10
moles) was diss ol ve d in dry DMS0--d« (0.5ml ) in the NMR
tube and 1 equ ivalent DCC (20.6mg) added. The solution
- 1 3 6 -
was thor o u g h l y mixed, then 1H NMR. spectra taken every 15
in inu tos .
The same pr ocedu re was fol low ed when a) cat al yt ic
am oun ts of AcOH ( 2 . 5 jj1 , 4.33 x10 moles) and b) 1
e qu iv alent AcOH (5.7jal) were added to the react io n
solution. The results from these e x p e ri ments are
tabul at ed in Ap p e n d i x Two.
4.3.7 Gl yc ine Com p l e x e s with DCC in DMSO.
A similar procedur e to that used in s e ction -1.3.7 was
employed, fox' example:4One equivale nt of complex (33a, 1x10 moles, 24. ling)
was d i s s olved in di’y DMSO-de (0.5ml) and 1 equivalent
DCC (20.6mg) added. The s o l ution was t ho roughl y mixed
then 1H NMR spectra were taken every 10 minutes. The
q u a n t i t i e s used are listed in Table 4.9. The H NMR
data ob tai n e d from these reactions, and those using NSu
in the reaction sol ut ion are Labulated in App end ix
T h r e e .
1 .3.8 Boc Protec te d C o m pou nd s wi th DCC in DMSO.
A generaL method for these reactions i s shown be 1o w .
The qu antiti es used in each reaction are given in Table
4 .10.
One equi valent of the Boc amino acid or di pe ptide was
dissolv ed in DMSO-do (0.5ml ) and either 1 or* %
137-
e q u i va le nt DCC added. The r e a ct io n was followed by H
NMR spectroscopy. The data obtai ne d from these
re actio ns is tab ul at ed in A p p e n d i x Four.
Tab le 4.9 Q u a n t i t i e s used in t h e C o mplex R e a ction s ind m s o T
NMR Reaction Bench Reaction
Concent rat ion 0 . 02 0 . 2 0 . 02 0 . 2 M
N o . moles 0.. 00001 0 . 0001 0.0001 0 . 002
Volume 0 . 5 0 . 5 5 10 m 1
Cpx 4b 4 . 3 43.3 43.3 865.8 mg
Cpx 4a 5 . 7 56 . 8 56 . 8 1137.3 mg
DCC 2 . 1 20 . 6 20 . 6 412 . 6 mg
NSu 1 . 2 11.5 11.2 460 mg
Cpx 2 -- 34 . 7 -- mg
AcOH -- 5 . 7 -- — pl
Cpx 33a -- 24 . 1 -- -- mg
Cpx 33b 3.8 37 .7 -- -- mg
Ta b le 4. 10 Quant it ie_s_U se_d in_ the R eact ions b e t w een Boc
Pi -o lec ted C o m p o u nds and DCC in DMSCP
, ! , , ! ,Boc compound Volume
m 1Concn
MN o . m o 1 |
iW t .mg
Wt. DCC jmg }
Boc AlaOH 0 . 50 0 . 10 i5 . 29E-5 j 10.0... j
10.0 j1
Boc AlaOH 1 . 10 0 . 08 5 . 72E-5| 10 . 9 111.3 |i
Boc AlaOH 1 . 10 " 0.05 ' _ i ■5 . 7 2 E - 5 j 10 . 9 '5.6 j
Boc AlaOH 1 . 10 0 . 05 2 . 2E-4j ..... "141.6 ' ■ i4 5.4 |
Boc GlyOH 0 .75 0 . 20.. ...
5 . 71E-5 ji
10 . 0.. . j
11.8 |!
Boc GlyOH 0 . 50 0.19 19.7E - 5 j 17 . 0 i21.0 jJ
Boc GlyGlyOH 0 . 50 0 . 02 r ■ i 1.0 E - 5 j 2 . 3 12.1 jIBoc G lyGlyOH 0 . 50 0 . 20 i1.O E - 4 !
i23.2
■ 1 ..... j2 0 . 6 j
- 1 3 8-
4.3.9 M i s c e l l a n e o u s R e a ction s in DMSO
A) All the reactions were foLlowed by H NMR
s p e c t r o s c o p y and were carri ed out using the general
m e t h o d below:
One eq ui valen t of DCC was d i s s ol ved in DMSO-de and 1 eq u i valent of the other reagents added. The reagents
and the q ua nt ities used of each are shown in Table
4.11. The data o b t ain ed from these reac tio ns are
tabulated in A p p e n d i x Five.
Tab 1 _e 4 . 1 1 Quant i t i e s of Rea g en t s sed in the M i s c e l l a n e o u s R eac t ions in D M S O - d e at a Con c e n t r a t i on ofo .2m 7 ".. ' ...........
Reagent Reac t ion
DCCmg
AcOHgl
NH2Bugl
18C6mg
NSumg
1 20 . 9 5 . 7 9 . 9 26.7 1" I
2 21.4 5 . 7 9 . 9
3 20 . 7 -- 11.5
4 20 . 7 5 . 7 ' 11.5
4.3.10 Dip ep ti de Co m p l e x e s and DCC in DMF.
One equ iv alent Co m p l e x 4a (568.7mg, 1x10 moles) and 1
eq uivalen t DCC ( 2 06 .3mg) were d i s s o l v e d in DMF (5 m l ).
S t i r r i n g the re action so lu tion af l o r d e d a prec ipi tale
after \ hour, whilst non -sti rr ing did not. The reaction
was left ove rnight before e v a p o r a t i o n o 1' the solvent.
The residue was taken up in DCM and filtered. The 1 fl
NMR spectrum of the residue gave the following:
13 9-
5 = 1 - 2 m CH 2 ’ s DCU, 5 = 2.2 9 s* Tos Me, 5 = 2.71,
2 . 87 2s DMF Me ’ s , 5 - 3 . 52 s 18- Cro wn-6 , 5 = 3. 66 ,
3.87 2d CcHz S.M., 5 = 3.83, 4.04 2d C a H a ’s product, 5
= 5.5 8 bd NH DCU, 5 = 7.10, 7.4 7 2d Tos Aroma tics, 5 =
7.6 6 bd NH'j + S.M., 5 = 7.94 s DMF C H , 5 = 8.13, 8.58,
8.2 3 truces of by-produ cts , 5 = 8.68 t NH S.M. +
Product, 5 = 9.23 bd NH product.
Po s s i b l e products: N-acyl urea de r i v a t i v e (6b),
D M F - d i p e p t i d e product (12).
See Appe nd ix Eight for Structures.
4.3.11 Rea ct io ns of D i p e ptid e C o m plex es with Amino Acid
Esters in DMF.
The general procedu re emp lo ye d for these rea ctions is;
de s c r i b e d using the example below.- 3One equ iva lent of the comp lex 4a (5 68 .6 mg, 1x10 moles)
and 1 eq uivalent of DCC (2 06.7rng) were d i s so lv ed in DMF"
( 3 m 1 ) .
One equ iva lent HC1 . G l y O E t and 1 e q u i val en t TEA were
d is s o l v e d in DMF (2ml).
The 2 solutions were combine d and Left stirrin g for 2
hours before removal of the solvent. The residue was
taken up i rj DCM, filtered and dried down before an *11
NMR sp ectr um was run. The rea ctions curried out are
J is Led in Table 4. 12, along wi th the ■ yields and the
quant it ie s used .
1 I 0
M o d i f i c a t i o n s of the above pro ce dure were also used.
They included:1) The re a c t i o n was carried out at 0°C instead of Roo m
Teinpe rature ;
2) 1 - ( 3 - d i m e t h y 1a m i n o p r o p y 1 ) - 3 - e t h y 1c a r b o d i i m i d e HC1
(wscdi) replaced DCC as the c o u plin g reagent;
3) The re action time was increa se d to a m i n i m u m of 6
h o u r s ;
'1 ) HOBt esters, p r e pa red in situ, were used to prevent
racem i sa t i o n .
'J'abi e /3-12 C h e m i c a ls a n d t h e Q u an L i ties_ u s e d in I h eRea c t. ions i. n D M F to f o r m Tri p e pt i d e s .
Rxn No .
Cpx Eq mo 1 Wt .mg
.Ester Eq. lW t . j Coupl mg j R e a g .
Wt . |mg j
T EA | Prod
1 4 a 1 1.OE-3 568 . 6 14 1 139.6 DCC 2 0 6 . 7 |140 . 0 | 1 5a j
2 4 a 2 2.OE-3 113 7.0 14 1 140.0 DCC 4 1 2 . 6 |1 4 0 . c ; 15a
3 4 a 3 3 . OE-3 1706.7 14 1 141.4 D C C | 6 1 6 . 9 | I140 . U| i '1lSd
4 4 a 1 1.OE-3 568 . 5— ....
14 2 280 . 6 DCC 1206.7 j1
j2 8 0 . 0 | 15a 1
5 4a"
1 1.OE-3 570 . 9 14 3 418.8 DCC 1206.7 j1
|4 2 0 . 0 | 15a
6 4 a 3 3 .OE-3 1705 . 0. .. 14 1 h .....1 39 . b DCC 309.5 j1 4 0 . 0 | 15a
7 4b 3 3 .OE-3 1298.7 14 1 139.6 DCC 3 0 9 . 5 |139.0!j-
15b
8 4a 3 3.OE-3 1706 . 0 16 1 215.7 DCC 3 0 9 . 5 |140 . 0 | 17a
9 4a 1 1.OE-3 568 . 6 18a 1 294 . 0 DCC|
2 0 6 . 1 ||
1 4 0 . 0 |I 19a
10 4a 1 1.OE-3 568 . 3 18a 1 2 93.6 wscdi 2 0 7 . 8 |1 4 0 . 0 | 19a
11 '20 1 7. 6E-5 51.2 18a 1 22 . 3 wscdi I15 . 1 j 111 . 0 I 21a
12 20 1 2 . 3E-4 148 . 3 18b 1 53.2 wscdi 51 . 1 ii
31 . 0 j 21b
1 he H NMR data ob taine d from these reac tions is
tabulated in Appen di x Six.
-14 1-
An ex ample ol' this is:
One eq ui valent of comple x 4a (1x10 J moles, 5 6 8 . 3mg) and
one equ iv al ent wscdi (207.8mg) were dis s o l v e d in DMF
(3ml) at 0°C with one equi val ent HOB t (135.1mg). One
eq ui va lent 18a (293.6mg) and one equi va lent TEA (L40pl)
were dis s o l v e d in DMF (3m.L) and added to the first
solution. The so l u t i o n was allo wed to stir ov ernigh t
before the solvent was removed, the residue taken up in
DCM and extracte d with H 2 O (2x5ml) and KC1 (saturated,
2x5ml), dried over NazSO* and e v a p o r a t e d to dryness.
4.3.12 The Sy nthesi s of an E n k e p h a l i n Derivative.
One equi valent of T F A .G 1y P h e C 1 0 OMe (2 6.6mg, 5.12x10
moles) and 1 eq ui val ent TEA ( 7 . 1 nl ) were d i s s o l v e d in
DMF (1 - 2ml) . 1.4 equivalent:; T F A - C r o w n - T y r ( Bx ) G1 y
C o m plex (5 0.8mg, 7.2x10 F" moles), 1 equ ivalent wscdi
(lO.Grng, 5.12x10 & moles) and 1 equi va lent HOBt ( 7 . 8mg ,5. 12x10 ** moles) were dis so lved in DMF (1 - 2ml) and
cooled to 0°C. The two solut ions were com bined
and left stirri ng overnight. Upon e v a p o r a t i o n of
the solvent, the residue was taken up in DCM and
extracted with H 2 O (2x10ml) and KC1 so lut i o n (saturated,
2 x 5 m 1 ) . The combin ed w a s hi ng s were then extracte d with
DCM ( 1 x5nil ) . The c o m bi ne d DCM solutions were dried over
NanSO* before evaporation. Weight ob t a i n e d = 50 . 8ing
(9 0 . 7 % w /w ) .
- 1 4 2 -
1H N MR analysi s of the re acti on residue showed a mi xture
c o n t a i n i n g both st ar ti ng materials, the desi red
peri La pep tide and traces of by-products.
Pu ri f i c a t i o n was; a c h iev ed by 2 runs down a Dynamax 300
Ci a column on an 11PLC system. Two grad ie nts were
e m p lo yed with the 2 solvents as H 2 O with 0.1% v / v TFA
and M e C N .
Gr adi e n t One: 0% Me C N to 100% MeCN in 30 minutes.
C o l l e c t i o n of the main peak at 2 3 mi nu tes gave a 1:1
mi x t u r e of the p e n t a p e p t i d e and the starting tripeptide
ester. R e r u n n i n g this frac tion using Gr adi en t Two: 20%
MeCN to 1.00% MeCN in 1 hour and col lec Ling the peak at
27.5 minutes gave the pure pentapeptide.
The 11 NMR Sp e c t r u m in DMSOae gave:
5 - 8.7 3 bd NH Cro, 6 = 8.4 8 d Nil Phe, 5 = 8.18 s ?, 5 =
8.10 m Bz , 5 = 7.77 d Tyr Ar, <5 = 7.63 t Bz/Phe Ar , 5 =
7.41 m Bz/Phe A r , 5 = 7.33 t NH Gly4, 5 = 7.20 m Phe A r ,
S ~ 6.96 d Tyr Ar , 5 - 5.51 t NH G 1 y 3 , 6 - 5.07 s Bz
Clh , <5 = 4.6 3 m CaH Tyr, 5 - 4.2 2 m CaH Phe, 5 = 3.9 9 bd
Call C 1 0 , 5 = 3.80 m C « H 2 ’s Gly, 5 = 3.62 s OMe, o = 3.00
111 CpHz Tyr, <5 = 2.82 m CpHz Phe, 5 = 1.56 m CH 2 CH 3 C 1 0 ,
S - 1.2 4 bd CH 2 ’s C 1 0 , 5 = 0.8 4 t C H 3 C 1 0 .
The Mass S p e ct rum gave:
m/z 716 M T y r (B z )G 1y G 1y P h e C i o O M e ,
m/z 626 M'-Bz,
m/z 3 68 G 1 yl-'heC 1 oOMe and Ty r ( Bz ) G 1 yG J. y .
4.4 O t h e r R e a c t i o n s used.
4.4.1 R e s o l u t i o n of d,l N - ac e t y 1 - 2 - am i riodecano i c acid.
d , 1- N - a c e t y 1- 2 - a m i n o d e c a n o i c acid (4. 9 20 2g, 2.00 57x10
moles) was su sp ended in 250 ml H 2 O c o n t a i n i n g the
indicator phenol red. LiOH solut io n was add ed dro pwise
to the st irring s o l ution until it became red and clear.
Acylase I (grade II, 133mgs) was added to the sol ut ion
wh ic h was then a l lowed to stand in a water bath at 38°C
for 5 days. D u ring the reaction the pH was m o n i t o r e d
and re-a d j u s t e d with LiOH solution. Al iquots of Acylase
1 were also added at a rute of 10 mgs a day. The
so lut i o n was filtered after 5 days and the solid
o b t a i n e d was impure 1 - 2 - a m i n o d e c a n o i c acid. 100% yield
(1.8728g). Re c r y s t a 11 i s a t i on from AcOH gave the pure
acid, m e l ti ng point = 2 2 5 0 C (decomposes). Id J = 30.8°.
The sol ut io n was aci di fied with 1M HC1 and then
filtered. The solid was dried and was the d - N - a c e t y l - 2 -
a in i 11 o d e c a n o i c acid. 92.1% yield ( 2 . 2 6 6 7 g ) .
4.4.2 Removal of an Ester Group.
The crude protect ed compound was cooled to 0°C and KOH
(3N in H 2 0 / E t OH 1:1, 20ml) added. After 5 minutes
ci tric acid was added until the pH was 4 to 5 . The
sol uti on was extr ac ted wi th DCM (2x1 0ml) whi ch was then
dried over NazSO* and ev aporated down. The free acid
compou nd was recrystal iised f rom MeOH/llzO, filtered,
1 4 4
washed with 11*0 and dried thoroughly . The free acid
com po unds ob t a i n e d and the yields are shown in Tabl e
4.13.
Ta b 1 e _4^ 1 3 Fr ee_ _Ac id Compourjds^ 0 b t w ined A f ter _Es terR e m o v a l .
Starting Material Wei ght Produc t Yield 4H NMR Data (5)(mg ) {%) DMSO d a
BocTyrt B z )GlyOEt 549.80 B o c T y r (B z )GlyOH 99 . 9 8 . 20 t NH Gly7 . 38 m Ar Bz7 . 18 d Ar Tyr6 . 88 d Ar Tyr6 . 82 d NH Ty r5 . 07 s C H z Bz4.11 m CaH Tyr3 .79 m C a H , Gly2.91 in CpHz Tyr2 . 66 m C p H 2 Tyr1 . 27 s C H s Boc
BocGlyGlyOEt 870.00 B oc G lyGlyOH 88 . 0 8 .01 t NH6 . 96 t NH3 .75 d C a H 23 . 56 in C <* H ?.
. . .1 . 37 s CH3 Boc
4.4.3 Removal of a Boc G r o u p .
The crude pro Looted peptide was di s s o l v e d in 60% TFA in
DCM and left sti rr in g for %_ hoax'. The solvent was
rem ove d using a rotary eva p o r a t o r with etha nolic K.0H in
the trap. The peptide s ob t a i n e d from this rea ct ion are
listed in Table 4.14 along with the H NMR data.
4.4.4 P u r i f i c a t i o n Pr o c e d u r e for C o m p o u n d 5.
The crude re act i o n residue was taken up in acre tone and
fi 1 te red 1. o remove DCU. The solvent was eva p o r a t e d off
and the residue taken up in MeCN and filtered again to
remove DCU. EtOAc was add ed dx'opwise to the so lutio n
- 1 4 5 -
until it remain ed cloudy, it was then cooled until it
bec ame clear. The solution was de can t e d oft. The
res idu e was taken up in ace ton e and warmed. The residue
wo uld not go into sol ution so the solvent was again
de cante d off and the residue t h o r o u g h l y dried.
Ele me ntal analysis gave: C 39.83 H 6.27 N 7.32.
T h e o r e t i c a l values: C 39.89 H 5.98 N 7 . '16 .( in cluding
1.5 HzO molecules)
lH NMR data: 6 = 8.5 1H t SNH, 5 = 6.56 1H t CONH,
5 = 3.84 4H m C . H z ’s, 5 = 2.08 3H s NCHa, 5 - 2.00 3H s
NCJla . There is also the c o u n t e r - i o n present in twofold
excess at 5 = 7.47, 7.10 8H 2d Tos Ar, S = 2.29 6H s Tos
CHa .
Mass S p e c t r o s c o p y Data: m/z 193 [M + H J + , m/z 133
NH a *C H 2 C O N H C H 2 C O O H .
Table 4_._ 14 Pep t i d e s o b t a i n ed from the Remo val of the Boc G r o u p .
Compound 1 H NMR Data (5)1I
P h e C 1 j OMe 7 . 2 0 m Ar Phe 3.13 m CpHz Phe j7.13 d NH C 1 4 1 .56 m Chi C 1 4 i4.31 m CaH Phe 1 . 2 0 m CH 2 Cl4 j4.18 m CaH Ci 4 0 . 80 t CHz Cl , 13.59 s CHa OMe i
P h e C 1 oOMe 1as a b o v e . j
T yr ( Bz ) Gl y OH 8.79 t NH Gly 4 . 00 m C a H Tyr |7 . 4 0 m Ar Bz 3 . 8 6 d C a H 2 Gly |7.19 d Ar Tyr 3.05 m CpHz Tyr |6.96 d Ar Tyr 2 . 8 8 m CpHz Tyr 1
5.08 s C H 2 Bz ii
14 6-
C H A P T E R F I V E. C O N C L U S I O N S ,
5.1 Ester Formation.
For the general synthesis of amino acid esters, our
m e thod compar es u n f a v o u r a b l y with those from literature,
for it requires the use of more expens iv e reagents such
as 18-Crown -6 and N - p r o t e c t e d amino acids. The method
pr o p o s e d by P e nney requires the use of free amino acids,
thus ma ki ng it more versatile.
Other literature methods, which use N - p r o t e c t e d amino
acids, also have an a d v a nt ag e in that bett er yields are
ob t ain ed at lower react io n temperatures.
H o w e v e r , the me thod we have de v e l o p e d has been shown to
give good yield in the synthes is of fatty esters. The
yields o b t ain ed with o c t a d e c y 1 halide compare f a v o urab ly
with those obtai ne d using other' methods. The advant ag es
of our method are:
1) the use of a re action solvent. In the meth od
pr o pos ed by Penney, a melt of the fatty alka nol was used
as solvent and reagent. In the m e th od here pr op osed we
use DMF as reacti on solvent. This means that elevated
temperat ur es are not required in order to melt the fatty
alkanol, and
2) enhanced react iv ity brought about by the crown ether
which gives shorter reaction times.
However, a further study is required on this pro ce dure
to optimise the con di tions and improve the yields.
-117 -
Some pa r a m e t e r s whi ch could be in ve s t i g a t e d are:
1) Tem perature. A study could be carried out to
co r r e l a t e r e a ct io n t e m p e ra tu re with re a c t i o n yields.
2) Catalysis. This would show whet her the Crown Ether
could be pres ent in smaller amounts and act as a
catalyst. This would most p r o bably increase the
re a c t i o n time, but if shown to be more effecti ve would
make the m e th od more e c o n o m i c a l l y attractive.
3) No n - p r o t e c t e d amino acids. This would be
d e s i ra bl e beca use it would remove both p r o t ec ti on and
d e p r o t e c t i o n steps. This too would make the me thod more
e c o n o m i c a l l y viable.
4) Other esters. The study could be continu ed into the
formation of other esters by this method. This would
entail the o p t i m i s a t i o n of co n d i t i o n s and fin ding a way
to o v e rcome steric h i n dr an ce when it occurs.
5.2 O l i g o m e r i s a t i o n and Pe pt id e Synthesis.
The study into the use of C r o w n Et hers as N - t e rm inal
pr o t e c t i n g groups has led to a kn o w l e d g e of the
m e c h a n i s t i c pathways involved in the reac tions of amino
acids compl exe s with c o u pl ing reagen ts such as DCC.
It has also given rise; to a pro c e d u r e by which
protection with 18-Crow n-6 occurs. The optimal
c on di tions required for* peptid e synth esis were DMf' as
react ion solvent at 0°C for 18 to 24 hours at a
c o n c e n t r a t i o n of 0.2M. The subs eq uent d e p r o t e c t i o n was
-14 8-
found to be rep ea ted wa s h i n g of the rea ct ion residue in
DCM with a saturated KC1 solution.
This work couid be c o n t i n u e d in many ways, as listed
below:
1) The o l i g o m e r i s a t i o n rea ct io n of amino acid com ple xe s
with DCC in MeCN or CHCla may find a p p l i c a t i o n in the
fo r m a t i o n of polymers, if a way of c o n t r o l l i n g the
extent of the o l i g o m e r i s a t i o n can be found;
2) In ve s t i g a t i o n Lo find the optimu m c o n d itions for the
ste pwi se synth esis of peptides. The cond itions found
for the sy nth esis of a tr ip ept ide may need m o d i f i c a t i o n s
for the syn th esis of higher oligomers. This could be
looked into by the syn thesis of a series of
larger pep tid es (for example, penta- or d e c a p e p t i d e s ) to
see if any change is required irx the reaction
condi t i o n s ;
3) R e p e t i t i o n of the tripep tide syn th esis using other
co u p l i n g reagents', to attempt. Lo improve the yield and
pur i ty of reaction, It has been found that by
s u b s t i t u t i n g the DCC with its water soluble derivative,
an improvement in the yield was ob t a i n e d in the
synthesis of GlyPheCi xOMo L r i p«'p tide. (The yield
increased from 47% to 64%. ) The reaction residue was
c 1 e an or .
4) The complex format io n m e t h odolo gy could be app lie d to
larger peptides and ut ilised in the fragment
c o n d e n s a t i o n p r o c e d u r e . Here the crown ether may
i net'ease the solubi lity of the larger pe ptide fragments
- I 1 d
and hence increase the p o s s i b i l i t y of the r e a ctio n
occurring. The mai n p r o blem for fragment c o n d e n s a t i o n
of large segments is the low molar c o n c e n t r a t i o n of the
c o m p o n e n t s being coupled. For example, the sy nthesis of
the 104mer (the "S"- protei n of R i b o n u c l e a s e A) involved
the c o n d e n s a t i o n of a 44mer and a 60iner whic h gave low
y i eld and there was diff i c u l t y with purif ication
(Hi rs ch mann 1969) . It has been shown that using the
p ri n c i p l e of excess increases the yield though for large
segments this may not ,always be possible, whi ch is when
Lite use of a Crown Ether may enhance the reaction.
6) M o d i f i c a t i o n s to the Crown Ether, by the inclusion
of functiona l groups, may have an a d v a n t a g e o u s effect on
the c o u pl ing reaction. This may include en ha nc ed
reactivity. It may also be of use during the work up
procedure, by in creasing the in so l u b i l i t y of the metal
ion - crown complex.
6) The me thod could be appl ied to the M e r r i f i e l d
proto co l of solid phase pept ide synthesis. The complex
could be used in a similar way to Boc or Fmoc amino
acids and any excess would be washed out of the resin at
the end of the reaction. A study into whether' amino
acid complexes react the same as dipep tide comp lexes in
DMF would have to be carried out to make this possible
use of the method more v e r s atile and viable.
7) The ad dition of a c h r o mopho re onto the crown ether
could lead to use of the proced ur e in the co ntinuo us
flow peptide syn thesis method as d e t e c t i o n would then be
-15 0-
possible. It may be that the p o t e n t i a t e d co nd u c t a n c e
me t h o d of on-lin e m o n i t o r i n g could be a p pl ie d to the
crow n ether r e a ction (Walker* 1989). It has been
shown that the complexutiori of K* iori by d i c y c i o h e x y l -
18- cr ow n-6 in met hanol leads to a r e d u c t i o n in
e l e c t r o c o n d u c t i v i t y (Frensdorff 1971a).
This work has g i ven rise to a p r o c ed ure for the
s y n t he si s of peptid es using amino acids n o n - c o v a l e n t l y
p r o t ec te d with crown ethers. Both Lhe "protecti on" and
"d e p r ot ection" steps are carr ied out in e x t r e m e l y mild
conditions. The p r o t e c t i o n occurs in E t O H / E t O A c
so lu ti on and Lhe deprole e L i on occurs wi Ih repeated
wa s h i n g of Lhe reaction residue in DCM wi th a sat ur ated
so l uti on of KC1 - a very mild d e p r o I e c t i o n step indeed!
-15 1 -
A P P E N D I C E S .
N o te s
The data tabul at ed w i thin the first five a p p e ndice s are
r e f e r e n c e d against DMSO-da.
The signal from i n c o m p l e t e l y d e u t e r a t e d DMSO-de was used
as internal standard.
The sp ect ra f o l l ow ing each of the tables are
r e p r e s e n t a t i v e of the reactions tabulated.
Si gn al s from the CH a ’s of DC C / D C U ap pear be tw een 5 = 2
and 1 and were immeasurable. T h e r e f o r e they have not
been included in the tables.
The time is in minutes unless other’wise stated,
h = h o u r s .
d = day s .
-1. r> 2 -
A p p e n d i x One.
A. 1.1 Tos Di Cpx + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 0 2 M .
5t
8 . 66 8.6 8 . 37 7 .81 7 . 48 7.11
4 .00 3 . 88 3 . 62 3 . 52 2 .29
10 0. 92 2 . 5 -- 1 . 83 1.83 20 . 8 2 . 17
1 h 1 .00 vis vi s -- 2 . 5 0 . 17 1 . 67 1 . 67 X 2 . 17
27* h 0 .71 0.28 0. 28 — 3 . 28 0 . 57 1 .43 2 . 00 X 2 . 86
52 h 0 . 28 0.43 0.43 1.71 2 . 57 0 . 86 0. 57 1. 43 X 2.00
71* h 0.20 0 . 50 0. 50 2 . 50 3 . 00 1 . 00 0 . 60 1 .60 X 2 . 30
NH NH NH NHa Ar C„Ha C.Ha C a H a CHa CHat t d bd d d d d s s
S.M. 6 b 6 b Both Tos 6 b S.M. Both 18C6 Tos
S p e c t r u m taken after hours.
[osCCC/OCUCHj’s
18C6
Tos
NH NH’s
-153-
A. 1.2 Tos Di Cpx + DCC + NSu Reaction at a
Concentration of 0.02M.
6t
9.06 8.71 8.03 7 .79 7 . 52 7 .14
4. 44 3 .87 3 . 54 2 .82 2 . 57 2.3
* 5 / X / / X / X X / X X
11 h 0.25 2 0 . 5 8 . 5 3 0 . 5 5 X 1 4.5 ni
413* h 0.14 1 . 67 0. 38 5.71 7 .19 0. 28 3 .71 X 0.52 4 . 04 6 . 19
NH NH NH* NHa Ar Co H a C.Ha CHa CHa CHa CHat t b d b d d d d s s s s7a SM 7a SM Tos 7a SM 18C6 7a NSu Tos
* no integ rals available.
S p e c t r u m taken after 11 hours.
Tos
TosNSu
NH OCC
-154-
A. 1.3 Tos Di Cpx + DCC Reaction at a Concentration of
0 . 2M .
t / 5 8 . 62 8 . 38 7 . 41 7 .00 6 . 47
5 7 . 25 — 41 . 50 16 . 75 —
15 5 . 33 -- 31 . 80 13.20 —
2 0 5 .00 0 . 2 0 31 . 20 13 . 00 0 . 2 0
25 4 . 50 0.67 29.70 13.00 0. 83
30 4.20 1 . 0 0 27 . 70 13 . 00 1 . 0 0
35 3 . 80 1. 33 26 . 70 13 . 00 1.33
45 3.00 2 . 0 0 25 . 20 13 .00 2 . 40
55 2 . 8 8 2.44 25 . 70 14 .40 2 . 60
65 2 . 2 0 2 . 80 2 2 . 60 14 . 60 2.80
85 1.70 3 . 30 2 2 . 60 15 .10 4.00
* 105 1.37 4.25 23 . 50 17.00 5 .25
« 145 1 . 1 2 4.75 22 . 50 16 . 0 0 5 .00
* 165 1.25 5 . 00 23 . 00 16 .70 5 . 00
1 2 % h 1.43 4 . 50 2 0 . 1 0 14 . 50 4 . 80
NH NH NHa* Ar NHt t + d tSM 5a Tos Tos 5a
S p e ctrum taken after 35 minutes.
* integrals not
a c c u r a t e due to
b r o a d e n i n g of
sign als on p r e c i p
itat ion of D C U .
N o t e s .
1. The C a H 2 signals
are at 5 = 3.90
to 3 . 52 and cannot
be m e a s u r e d due to
over l a p p i n g .
2. 18-Crown-6 is at
5 = 3.52.
3. Tos Me is at 5 =
2.25.
Tos
TosTosAr *NH-
NH Sa
-155-
A. 1.4 Tos Di Cpx + DCC + NSu Reaction at a
Concentration of 0 , 2M .
t\s 9. 00 8.75 8 . 61 8.39 7 . 8 8 7 . 36 7 . 00 6 . 38 4 . 30 4 . 23 2.71 2.48
3 0 . 5 -- 16 . 5 — 8 6 . 7 32 . 0 — 1 . 6 -- 3 . 1 59 . 91 0 1 . 5 — 13.0 — -- 79.5 32.0 — 2.5 -- 7 . 5 55.0IS 2.0 — 13.0 — -- 80 . 0 32 . C — 4.0 -- 11 .0 52 . C20 3.0 -- 12 . 7 — vi s 80.3 32.0 — 5.6 -- 12 . 2 50 . 825 3 .1 — 11. 4 — vis 81 . 0 32 . 0 — 5.2 — 13 . 4 39.730 3 . 5 -- 11 . 5 — vis 78 . 5 32 . 0 — 6 . 5 — 16.0 48.0
35 4.0 -- 11.0 — vis 78.0 32 .0 -- 6.5 -- 17. 5 45.041 4.0 — 11.0 — vis 78. 0 32.0 -- 7.0 — 18.0 44.045 5.0 -- 11.0 -- 0 . 5 81 . 0 32 . 0 -- 8 . 0 — 20.0 43.050 5 . 0 — 9.0 — 0.5 78 . 0 32 .0 -- 6 . 0 — 20.0 42.055 4.5 -- 9.0 — 0.5 78. 0 32.0 — 8.5 — 21.5 40.060 4.2 -- 8 . 4 0 . 8 1.7 83 . 3 32 . 0 -- 10. 5 — 19.4 39.565 4 . 6 -- 8 . 4 1 . 3 0 . 8 80 . 4 32 . 0 1 . 7 10.1 -- 2 0 . 6 38 . 780 3 . 4 0.9 6.9 1 . 3 0.4 74 . 3 32 . 0 2 . 2 6 . 0 1 . 7 22 . 1 37 . 2
135 3.3 1.9 5.7 2.8 0.9 6 3 . b 32 .0 5.2 7 . 5 4.7 28 . 2 38 . 6240 3 . 3 1 . 9 5 . 3 2 . 4 1 . 0 63 . 0 32 . 0 5 . 3 5 . 3 4.3 27 . 7 38 . 2
163*h 2 . 8 2 . 3 5 . 6 2 . 8 2 . 0 64 . 5 32.0 4.8 7 . 1 5 . 6 24.4 38 .1
NH NH NH NH NH NHa Ar NH CaH» C-Ha CHa CHat t t t b d + d t d d s s
7 a 8a SM 5a 1 0 Tos Tos 8/5a 7a 8a 7/8a SM
1 o s A r o ma tic do ub let at 5 - 7.00 was used as internal
st an da rd due to o v e r l a p p i n g of the D M S O signal with NSu
st ar ti ng mat er ial signal. The signals b r o aden ed due to
DCU precipitat ion.
- 1 5 6 -
S p e ct rum taken after 16% hours
Tos Ar * NH]
TosTos
DCC/OCUCtf2's
-157-
A. 1.5 HC1 Di Cpx t DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 .0 2 M .
t/5 8.73 8 . 62 8 ! 38 7 . 95 3 . 95 3 .85 3 .52
5 2 . 0 0 — -- bd — 4 .00 X
15 2 . 0 0 -- -- bd -- 4 . 00 X
2 0 2 . 0 0 — — bd -- 4.00 X
30 i . 94 — -- bd vi s 4 . 00 X
45 1 . 8 8 0.06 0.06 5 . 35 0 . 1 2 3 . 94 X
60 1 . 87 0 . 06 0.06 5 . 94 0 . 1 2 3 . 81 X
1 1 0 1 . 76 0.09 0.09 5.76 0 . 17 3 . 76 X
NH NH NH NH, CaH, CaH, CH,t t d bd d d s
SM 6 b 6 b SM 6 b SM 18C6
Spe c t r u m taken after 110 minutes.
OCC/DCU
- 1 58-
A. 1.6 HC1 Di Cpx + , DCC R e a c t i o n at a C o n c e n t r a t i o n of
0. 2M.
t\S 8 . 94 8 . 65 7 .76 7 . 22
leq DCC 90 26.00 1 .70 84 . 70 2 . 0 0
2eq DCC 5 18.50 1 .25 56 . 50 1 .25
2 0 17 . 50 1 . 0 0 54 . 50 1 . 0 0
13*ir 17 . 30 5 .00 56.00 5 . 30
NH NH NH» NHt X s t
SM 5b SM 5b
There was only DCU fo rmatio n up to \\ hours with 1
equival en t of DCC.
P r e c i p i t a t i o n of DCU bro a d e n e d signals, ma king the
integrals inaccurate.
Spe c t r u m taken uf ter 13 hours with 2 equi v a l e n t s DCC.
NHj18C6 OCC/DCU
r-- I-- I--- II T ~
-15 9--
A. 1.7 HC1 Di Cpx + DC C + NSu R e a c t i o n at a
C o n c e n t r a t i o n of 0.2M.
t\s 9 . 43 9.10 8 . 94 8 . 64 8 . 0 0 7 . 64 7 . 14 4 .36 4 . 27 2 .75 2 . 54
5 0 . 5 — 20 . 5 — — 59 . 5 — 1 . 0 -- 2 . 0 71 . 0
25 1 . 5 -- 19.0 — vis 65 . 0 vis 4 . 5 -- 5 . 5 6 6 . 0
30 2 . 0 — 18 . 0 0 . 5 0 . 5 64 .0 0.5 4.0 -- 6 . 0 65 .0
35 2 . 0 16 . 7 0.7 0 . 3 82 . 7 1 . 0 4 . 7 vis 8 . 0 85 . 3
40 1 . 0 vis 16.0 0 . 8 0.5 57 . 0 1 . 0 2.5 0.5 7.0 63 . 5
50 1.5 vis 15 . 0 1 . 5 0 . 3 55 . 5 1 . 5 2 . 8 0 . 5 7.0 63 .0
60 1 . 5 1 . 0 1 1 . 0 1 . 5 0.5 56 . 5 1.5 3 . 0 0 . 8 8 . 0 62 . 5
65 1.5 1 . 3 14 . 5 1 . 5 1 . 0 54 . 0 2 . 0 3.0 1 . 3 9.0 57 . 5
75 1.3 1 . 5 13 . 5 2 . 5 1 . 5 55 .0 2 . 5 3.5 2 . 0 1 0 . 0 55 . 0
175 1 . 0 1 . 5 10.5 3 . 5 1 . 3 41 . 5 4 . 0 3 . 0 2 . 5 9.5 58.0
300 1 .3 1 . 5 11.5 4.0 1 . 0 42 . 5 4 . 0 3.0 2 . 5 9.0 55.0
Addi tion of second equivalent DCC
415 1 . 3 1 . 3 10 . 7 5 . 3 1 . 3 41 . 3 5 . 3 2 . 3 2 . 7 11.3 64 .0
420 1 . 0 1 . 7 1 0 . 0 6 . 3 1 . 7 32 . 3 6 . 0 2 . 7 4 . 0 1 2 . 0 65 . 3
425 1 . 0 2 . 0 8 . 7 6 . 7 1 . 3 33 . 3 6.7 2 . 7 3.3 13 . 3 62 . 0
1 9hr 0.5 1 . 5 4 . 0 6 . 5 2 . 0 15 . 0 6 . 0 1 . 5 4 . 0 1 0 . 0 40 . 0
NH NH NH NH NH NH 3 NH C a H 3 C.H: CHa CHat t t t bd 2 1 d m s s
7b 8 b SMl
5b 1 0 SM 5/ 8 b 7b 5/ 8 b 7/8b NSu
S p e c t r u m taken, after 19 hours.
NSu
NSu 7 b/8b
DCC/DCU
SbjSb
-160-
A p p e n d i x Two.
A. 2.1 C o m p l e x 2 + DCC R e a c t i o n at a Co nc ent rat ion of
0 . 2M .
Sp e c t r u m taken after 110 minutes.
18C6DCC/ DCU ,
t\5 7 . 67 3 .17 2 . 38
0 39 4 36
15 34 2 0 30
25 33 2 1 30
35 35 2 1 30
1 1 0 33 2 1 30
N H a + C H C H a5 01 s
cpx D C C cpx
A. 2. 2 C o m ple x 2 + DCC + AcOH R e a c t i o n at a
C o n c e n t r a t i o n of 0.2M.
t \ 5 1 2 .0 0 8 . 40 7 . 67 6 . 83 3 . 17 2 . 67 2 . 38 1 . 92cU 1 0 . 0 0 . 5 17 . 0 — 9 . 0 1 . 0 15.0 35 .0
1 0 1 1 . 0 0 . 5 17.0 vis 8 . 0 1 . 0 14.0 34 .0
2 0 1 1 . 0 0 . 5 14 . 0 1 . 0 7.0 2 . 0 13.0 34.0
30 1 1 . 0 0 . 5 1 2 . 0 1 . 5 7.0 3 . 0 1 2 . 0 33 . 0
4 5hr 1 2 . 0 1 . 5 25 . 0 7 . 5 -- 17.0 23 . 0 39 . 0
OH5
AcOH
N Hd
6 c
N H as
Cpx
N Hq9
C Hm
D C C
C H s d 9
C H as
Cpx
C H as
AcOH
S p e c t r u m taken after 45 hours
AtOH18 C 6
OCC/OCU
A . 2.3 C o m plex 2 + DCC + cat. AcOH R e a c t i o n
Concentration of 0.2M .
t\5 1 2 . 0 0 7 . 54 6 . 8 8 2 . 69 2 .42 1 . 92
1 0 14.0 36 . 0 -- 1 . 0 34 . 0 47 . 0
2 0 14 . 0 35 . 0 -- 2 . 0 33 . 0 46 . 0
30 14 . 0 34 . 0 1 . 0 3 . 0 32 . 0 45.0
45 14 . 0 31 . 0 1 . 5 4 . 0 28 . 0 43 .0
60 16 . 0 31.3 2 . 0 6 . 0 28 . 0 49 . 3
7 5 13 . 0 34 . 0 4 . 0 8 . 0 30 . 0 52 . 0
90 16.0 28 . 0 4 . 0 8 . 0 24 . 0 46 . 0
105 13 . 0 25.0 4.0 9.0 2 1 . 0 46.0
1 2 0 18.0 28 . 0 5.0 1 2 . 0 24 .0 52.0
150 18 . 0 27 .0 6 . 0 16 . 0 23 . 0 --285 1 2 . 0 1 2 . 0 5 . 3 13 . 3 1 2 . 0 37 . 3
360 11 . 3 12 . 7 4.7 14.0 11 . 3 38 . 7
OH N H a N H C H a C H a C H as s q d s s
AcOH Cpx 9 9 Cpx AcOH
at a
-162-
S p e c t r u m taken after 6 hours.
CH,
AcOH
ort/ocu
nh;
-16 3 -
A p p e n d i x Three
A. 3.1 Tos Gly Cpx + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0.2M.
t\5 8 .63 8.40 7.40 7 .00 6 . 50 3 .77 3 . 57 2 .19
3 1 . 0 -- 57 . 3 27 . 3 1 . 7 4 . 7 23 . 3 39.3
1 0 1 . 0 1. 3 42, 7 27 .0 5.3 / / 36 .7
2 0 1 . 0 1 . 8 57 . 6 36.8 6 . 8 / / 51 . 6
30 1 . 0 1 . 8 56.8 37 . 2 6 . 8 / / 51 . 2
40 1 . 0 2 . 0 56. 4 36.8 6 . 8 / / 50 . 4
1 2 0 1 . 0 2 . 0 59.0 39.5 7 . 5 / / 54.5
92h 0 . 5 1 . 3 30 . 3 2 0 . 0 4 . 3 . / / 32 . 0
NHt
6 f
NHt
4a
NHa +Ar
+ 34a
Ard
Tos
NHt
34a
C.Had
6d
C.Had
SM
CHas
Tos
S p e c t r u m taken after 30 minutes.
Tot
OCC/OCU
NH's.
-164-
A . 3.2 Tos Gly Cpx + DCC + NSu R e a c t i o n at a
C o n c e n t r a t i o n of 0 . 2 M .
t \ 5 8 . 63 8.07 7 . 54 7 . 00 6 .50 4 . 44 4 . 27 2 . 75 2. 48
5 vis 4.7 33 . 3 — 0 . 3 0.3 3 . 3 9 . 3 44.7
1 2 vis 7.0 17 . 5 — 0 . 5 1 . 5 5.0 14 .0 26.0
2 0 vis 8 . 3 2 0 . 0 — 1 . 0 4.0 7 . 3 20.7 32 .0
25 vis 8 . 0 21 . 3 — 1 . 0 4.0 6 . 7 21 . 3 31 . 3
30 vis 7.7 2 0 . 0 — 1 . 0 4.0 6.7 21 . 3 31.0
45 vis 7 . 3 2 0 . 0 — 1 . 0 4.0 6 . 0 2 1 . 0 31. 3
60 vis 6 . 7 1 2 . 0 — 1 . 3 4.0 5 . 3 21 . 3 32 .7
180 vis 4 . 5 14 . 0 — 0 .8 3.0 4.0 13 . 3 24.7
26 . 5h vis 1 .6 1 0 . 6 — 0 . 6 2 . 0 1 . 8 1 0 . 0 25 .0
NHt
6f
NHabd
35a
NHa bd
SM +Ar + 34a
Ar + NH d
Tos/36a
NHt
6d
C.Hid
35a
C.Has
34a
CHas
7/35/36
CHas
NSuSM
S p e c t r u m taken after 263*> hours.
18C6
DCC/OCU
-1 6 5 -
A. 3. 3 HC1 Gly Cpx + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 0 2 M .
t\5 8 .69 7 . 54 7 .23 4. 11 3 . 96 3 . 46 3.11
3 _ _ -- vis -- vis 56 4
1 0 -- vis 0.5 — 0.5 56 4
15 -- 0 . 2 0.4 — 0.4 51 . 2 3.6
2 0 _ _ 0.4 0.4 -- 0.4 51 . 2 3 . 6
25 -- 0.4 0 . 6 — 0 . 4 51 . 2 3 . 6
30 -- 0.5 1 — 1 61 4
50 -- 0 . 5 1 -- 1 64 4
60 vis 0.75 1.25 — 1.25 65 4.25
80 vi s 0 . 6 1 . 2 vis 0 . 8 52 3.2
1 1 0 0 . 16 0.5 1 0.16 0 . 83 42 . 7 2 . 3
NH NH NH CoHa CaHa CHa CHt t + NHa d d s m
6 f 34b 6 d + SM 6 f 6 d 18C6 DCC/U
S p e c t r u m taken after 110 minutes.
18C6
A. 3. 4 HC1 Gly Cpx + DCC + NSu R e a c t i o n at a
C o n c e n t r a t i o n of 0.02M.
t\5 8.69 8 . 31 7 . 91 7 .53 7 .15 5 . 56 3 .81 2 . 60
4 7 . 50
9 0 . 16 0 . 25 0 .16 7 . 16
2 0 0.33 vis 0.33 0.33 7 . 16
31 0 . 31 : - - — — 0.08 0.46 0 . 36 6 . 77
43 0.50 -- -- 0 . 16 0 . 67 0 . 50 7 . 16
54 0 . 50 0.08 — 0.08 0 . 16 0 . 83 0.67 7 . 33
65 0 . 50 0.16 vis 0 . 16 0 . 25 1 . 0 0 0.83 7 . 16
76 0 . 50 0 . 16 vis 0.16 0 . 25 1 . 16 0. 67 7 . 16
87 0 . 50 0.16 1 . 33 0 . 16 0.25 1 . 16 0 . 67 7 . 16
NH NH NHa NH Nli NH CaH CHat t bd bd d d d s
6f 30 cpx + 34b 6d DCU 34b NSu
S p e c t r u m taken after 87 minutes.
18C6 OCU
DCU
-167-
A. 3. 5 HC1 Gl y Cpx + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 2M.
t\5 9.00 8.85 8 .75 8 . 55 8 . 43 7 . 67 7 . 50 7.28 4.20 3 . 96
3 -- vis * vis — 3 . 00 — 3 .00
13 — 0 . 67 vis vis -- 2 . 70 — 4.00 — 3 . 33
2 0 vis 0.33 vis vis -- 3 . 30 vis 6 . 0 0 vis 4 . 67
25 vis 0.33 vis 0 .33 — 3 . 30 vis 6.70 0.67 5 . 33
30 vis 0. 67 vis 0. 33 — 4 . 00 0 . 67 6.70 1 . 0 0 5 .33
35 0.33 0.67 0.33 0.67 — 3 .30 1 . 0 0 6.70 0 . 67 6 . 0 0
40 0.33 0. 67 0 . 67 0 . 67 — 4 . 00 0 . 67 7 . 30 1 . 0 0 5.33
45 0 . 37 0. 50 0 . 37 0. 37 0 .25 3 . 50 1 . 0 0 6.70 1 . 0 0 /
50 0. 67 0.67 0.33 0.50 0 . 67 5 . 30 1 . 33 9 . 00 1 . 16 /
55 0 . 67 0 . 50 0 . 33 0 . 67 0 . 67 4 . 67 1 . 33 8.70 1 .16 /
60 0.67 0.50 0.33 0.50 0. 33 4 . 67 1 . 50 5.30 1.16 /
135 1 .33 0.50 0 . 50 0.67 1 . 16 3 . 67 1. 67 1 0 . 0 0 2.50 /
NH NH NH NH NH NH NH C-H CaHm t t t t t d m m7 7 6 f 7 7 34b 7 6 d 7 6 d
S p e c t r u m taken after 135 minutes.
ocu
NH DCU
-168-
A. 3. 6 HC1 Gl y Cpx + DCC + NSu R e a c t i o n at a
C o n c e n t r a t i o n of 0 . 2 M .
t\5 9 . 36 8 . 92 8.39 7 . 75 7.57 7 . 25 4 .33 3.80 2 . 81 2 .56
2 vis 1 . 0 0 -- bd -- 0 . 50 1 . 0 0 2 . 0 0 1 . 50 25.00
1 0 vis 1 . 50 0 . 33 bd 0 . 67 1 . 0 0 3 .30 1 . 30 26.70
15 0.50 3 . 00 1 . 50 1 2 . 0 0 1 1 . 0 0 1 . 0 0 2 . 0 0 8 . 0 0 3 . 50 39 . 00
2 0 0 . 50 3 .00 2 . 0 0 1 2 . 0 0 1 1 . 0 0 1 . 0 0 2 . 0 0 8 . 0 0 4.00 39 .00
25 0. 50 3 . 50 1 . 50 1 2 . 0 0 1 1 . 0 0 1 . 0 0 1 . 0 0 9.00 3 . 50 37 .00
30 0. 50 3 . 50 2 . 0 0 1 1 . 0 0 1 2 . 0 0 1 . 0 0 1 . 0 0 1 0 . 0 0 3.00 37 .00
45 0 . 50 4 . 00 2 . 00 1 1 . 0 0 14 . 00 1 . 00 1 . 50 1 1 . 0 0 2 . 50 40 . 00
NH NH NH NHa NHa NH CaH 3 C.Ha CHa CHat t t bd bd t m m s s7 35b 30? 35 SM 6d 35b SM 35b NSu
The r e a c t i o n was fol lowed for l o n g e r , but the integrals
were immeasurable. This was due to the p r e c i p i t a t i o n of
DCU b r o a d e n i n g the signals. Other signals a p p eared at
5 = 8.82, 8.23, 7.98 and 2.78 whi ch could be due to
oligomers, their esters and DMSO derivatives.
S p e ct ru m taken after 45 minutes.
NSu
OCC/OCUNSu
I I I
-169-
Ap p e n d i x Four.
A . 4.1 Boc A l aO H + DC C R e a c t i o n at a C o n c e n t r a t i o n of
0 . 0 1 M .
t \ s 8 . 1 2 7 . 47 7 . 06 6 . 8 6 5 . 50 4 .31 4 .11 3 . 90
15 0 . 50 0 . 50 3 . 00 0.50 1 . 0 0 0 . 50 0 . 50 3 . 00
60 1 . 2 0 0 . 80 1 . 60 1 , 0 0 1 . 60 1 . 0 0 0 . 60 3 . 00
56h 5 . 00 -- — 4 . 00 1 . 0 0 5,00 — --
NH NH NH NH NH C a H C a H C a H
d d d d d 01 m ro6s 3**a SM 6s D C U 6 s 37a SM
Sp e c t r u m taken after 1 hour.
n* i6?-
- 1 7 0 -
A. 4. 2 Boc Al aOH + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 0 5 M .
t\5 8 .17 7 .79 6 . 92 6 . 58 6 .25 4.29 3 .87
2 — — 2 . 70 0.42 -- -- 3 . 17
80 0.15 -- 2.46 0 . 42 -- 0 .15 3 .07
130 0 . 30 -- 2.69 0 . 34 -- 0 . 30 3 .21
19 . 5h 1 . 30 0 . 1 0 2,80 0 . 2 0 0 . 1 0 1 . 30 3 . 60
NH NH NH NH NH C a H C - H
d d d m m
6 g Z 6 g E S M Z S M E 6 g E 6 g Z S M
S p e c t r u m taken after 1 93*> hours.
DillNH.- NH.
-171-
A. 4. 3 Boc Ala OH + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 2M .
t\5 8 . 1 2 8 . 0 0 7 .79 7.23 7.00 6 . 87 5 . 25 4.87 4 . 31 3 . 87
3 0 . 50 — — 0 . 50 22 . 50 22 . 50 — — 0 . 50 24 . 50
14. 5h 8 . 0 0 — 2 . 0 0 2 . 0 0 12 . 50 5 .50 1 . 50 2 . 0 0 6 . 50 21. 50
1 9h 8.50 0 . 50 2 . 0 0 2 . 0 0 13 . 50 — 2 . 50 2 . 50 7 .00 18 . 50
N H N H N H O H N H N H C a H C - H C - H
d d d bds d d m m m6 g o 2 2 3 1 a H 2 0 S M D C U 2 2 6 g S M
S p e c t r u m taken after 19 hours.
NH NH-> CCt/CCI
-VI 2-
A . 4.4 Boc G l y G l y O H + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 0 2 M .
t\5 8 . 34 8 . 03 7 . 8 8 7 .00 5 . 57 3 .91 3 .74 3 . 55 3.1915 -- 1 . 67 -- 1. 50 -- — 3 .33 3 .33 3 . 5030 vis 1 . 42 vis 1 . 28 — 0.14 2 . 8 6 2 . 8 6 3 . 6060 0 .14 1 . 28 0. 14 1 .14 0.28
...2 . 8 6 2 .71 2 . 8 6
155 0 . 23 1.35 0.23 1 .35 vis 0.47 2 . 65 2 . 65 2.822 1 0 0.29 1 .23 0.29 1 . 23 0.06 0.58 2.47 2.76 2 . 4125h 0 . 88 0.70 0.82 1 .35 0 . 17 2 . 58 1.41 2 . 94 vis8d 1 . 64 0 . 27 1 . 64 1 . 54 0 . 18 4 . 18 0 . 36 3 . 54 vis
NH NH NH NH NH C.H> C«Ha C«Ha CHd t t t d d d d m
6 h SM 6 h SM DCU 6 h SM SM DCC/U
Sp ectru m taken after 25 hours.
DCU
-173-
A . 4.5 Boc G l y G l y O H + DCC R e a c t i o n at a C o n c e n t r a t i o n of
0 . 2M .
t \ 5 8 . 34 8 . 0 0 7 . 8 8 7 . 34 7 . 00 3 . 92 3 .77 3 . 53 3 . 32
5 vis 8 . 50 vis — 7 . 00 — 17 . 50 18 . 0 0 --
15 0 . 50 8 . 0 0 0.50 vis 7 .00 1 . 0 0 16 . 50 16 . 0 0 0 . 50
30 0 . 57 8 . 60 0 . 57 0 . 29 8 . 0 0 1 .43 18 . 60 18 . 60 1 . 14
45 0 . 75 7 . 50 0 .75 0 . 25 7 . 50 1 .75 15 . 00 15 . 00 1 .75
60 1 . 33 9 . 67 1 . 33 0. 67 9 . 33 3 .00 19.67 19 . 67 1 . 67
22 . 5h 5 . 00 1 0 . 0 0 5.00 1 . 0 0 13.00 14 .00 2 0 . 0 0 29.00 4 .00
8 d 5 . 25 2 . 50 5 . 13 — 6 . 63 16.25 4 . 75 15 . 25 --
NH NH NH NH NH C.Ha C.Ha C.Ha C.Had t t t X d d d d
6 h SM 6 h 39 SM 6 h SM SM 39
S p e c t r u m taken after 22% hours.
-174-
A. 4. 6 Boc G 1 y OH + DCC R e a c t i o n at a Co nc ent rat ion of
0.2M.
t\5 8 . 26 8 .14 7.23 6 . 91 6 . 79 6 . 70 4.31 4.16 4 . 05 3 . 81
5 -- 0. 50 0.50 20 . 50 — 0 . 50 1 . 0 0j
1 1 -- 1 . 0 0 1 . 0 0 1 2 . 0 0 -- 0 .70 2 . 70
17 — 1 . 0 0 1 . 30 11 . 70 — 1 . 0 0 4 . 00
30 -- 2 . 0 0 2 . 0 0 10 . 67 — 1 . 67 5 . 30
45 -- 2.70 2 . 0 0 1 0 . 0 0 — 2 . 0 0 — vis vis 6 .70
1 2 0 vis 4 . 00 1. 50 7 .00 — 3 . 00 vis vis vis 8 . 0 0
250 1 . 0 0 9 . 00 1 . 30 1 0 . 0 0 -- 3 . 00 0 . 50 0 . 50 1 . 0 0 12 . 50
24 . 5h 2 . 50 13 . 00 -- 4 . 0 0 2 . 0 0 1 2 . 0 2 . 50 1 . 0 0 3 . 50 15 . 50
8 d 1 . 0 0 6.50 -- 2 . 50 1 . 50 6 . 50 2 . 0 0 1 . 0 0 3 .00 10.50
NH NH NH NH NH NH C . H j C . H j CH CHd d t t t t m m m m
6 i E 6 i Z 37b SM 6 i E 6 i Z 6 i E 2 2 6 i E 6 i/37
Sp e c t r u m taken after 8 days.
DCU/Me's
-175-
A p p e n d i x Five.
A. 5.1 18 -Crovm-6 + DCC + AcOH + N H 2 BU R e a c t i o n
C o n c e n t r a t i o n of 0.2M.
t\5 8 . 31 6 .29 3 .19 2 .71 1 . 97 0 . 8 8
A dd i ti o n of AcOH to reaction1 0 — — 13 . 0 •
25 1 . 0 — 13 . 0 — 1 . 0 —
40 1 . 0 — 12 . C -- 1 . 5 —
55 1 . 5 — 1 2 . 0 — 2 . 0 --
Addi t io n of NHaBu to reaction5 1 . 5 53.0 8 . 0 15.0 3 . 0 19.0
15 1 . 5 53 . 0 7 . 0 15 . 0 3 . 0 18.0
30 1 . 5 53 . 0 8 . 0 15 . 0 3 . 0 19.0
45 1 . 5 64 .0 8 . 5 17.0 4 . 0 2 1 . 0
60 1 . 5 53.0 8 . 0 16 . 0 3.0 18.0
2 0 h 1 . 7 41 . 3 6 . 7 13 . 3 5 . 3 17 . 3
8 d 5 . 0 48.0 4 . 0 14.0 14 . 0 2 1 . 0
NH NHa CH CHa CHa CHad s ro t s t
6 c Amine D C C / U Amine 6 c Amine
Spe c t r u m taken after 20 hours.
DCC/OCI
at a
-176-
A. 5. 2 DCC + AcOH + N H 2 BU Reaction at a Concentration of0 . 2M .
t \ S 8 . 27 3 . 92 3 . 46 3.31 3 .15 2 . 69 1 . 96
0 0 . 3 O'. 3 0 . 3 2 . 0 16 . 7 — 1.7
5 0 . 5 0.5 0 . 3 0 . 5 9 . 0 12 . 5 0 . 8
15 0 . 7 0.7 0 . 7 0 . 7 11 . 3 16 . 0 1 . 3
30 0. 5 0.5 0.5 0.3 9.3 13 . 7 1 . 3
45 0.5 0.5 0.5 0.5 8 . 0 11 . 5 1 . 5
60 0.5 0 . 5 0.5 0.3 7 . 5 11.5 1 . 5
80 0 . 7 0.7 0.7 0.7 1 0 . 0 15 . 3 2 . 0
1 2 0 1 . 3 1 . 0 1 . 0 0.5 1 1 . 0 2 1 . 0 3.0
165 1 . 5 1 . 5 1 . 5 1 . 0 1 1 . 0 2 2 . 0 4.5
2 1 0 1 . 2 1 . 2 1 . 2 0 . 8 1 0 . 0 17 . 6 4.0
7d 7.0 7.0 1 0 . 0 -- 4 . 0 2 1 . 0 19.0
NH CH CH CH CH CHa CH.d t m m m t s
6 c 6 c 6 c DCU D CC Amine 6 c
Sp e c t r u m taken after 165 minutes.
nh2ucu
OCC/OCU • Me'i r „
-177-
A . 5.3 NSu + DCC Reaction at a Concentration of 0.2M
t\5 10. 31 8 .31 5 . 38 3.19 3 .1 1 2 . 98 2 .73 2 . 65 2 . 58 2 .31
4 8 . 0 5 . 0 15 . 0 44 . 0
1 0 8 . 0 5 . 0 15 . 0 40 . 0
2 2 8 . 0 4 . 8 14 . 5 48 . 0
50 8 . 0 4 . 8 17 . 6 48.0
4h 8 . 0 — vis — 6 . 4 19.2 49. 6
17h 8 . 0 1 . 6 1 . 6 4.8 8 . 0 2 0 . 8 4 . 0 8 . 0 8 . 0 41 . 6
OHs
SM
NHt
30
NHd
DCU
CH*m
30
CHs
DCU
CHm
DCC/DCU
CH*t
30
CH*s
30
CH*s
30
CH*s
SM + DMSO
NSu OH signal used as internal stan da rd due to
o v e r la pp ing of the D MS O and NSu CHz signals.
Sp ect ru m taken after 17 hours.
CH*
CH-DCU
OCC/OCU
T
-178-
A. 5. 4 NSu + DCC + AcOH Reaction at a Concentration of
0 . 2M.
t\5 5 .51 3 . 30 3 .15 2 .76 2 .56 2 .30 1 . 8 6
5 0.5 5.0 27 . 0 0 . 5 74 . 0 1 . 0 34 . 0
2 0 1 . 5 5 . 0 27 . 0 2 . 5 74 . 0 1 . 5 34 . 0
30 2 . 0 7.0 27 . 0 4 . 0 72 . 0 2 . 0 34 . 0
45 2 . 5 8 . 0 28 . 0 4 . 5 71 . 0 3 . 0 34 . 0
225 4 . 5 1 2 . 0 19 . 0 16 . 0 50 . 0 1 1 . 0 30 . 0
NH CH CH CHa CHa CHa CHad ID m s s s s
DCU DCC/U DCC/U 31 NSu 31 AcOH
Spectrum taken after 225 minutes.
lOCC/U
DCU
OH,
'OCC/DClrr1-
-179-
A p p e n d i x Six.
lH NM E Data of the T r i p e p t i d e s S y n t h e s i s e d
Produc t NMR Data (5)
GlyGlyGlyOEt 8 . 75 NH 2 t Prod 4.21 CH* q Et SMC o mplex 8.47 NH t SM 4.08 CH* q Et P
8 . 13 NH* bd TEA 3 .83 C.H m Both(D M S 0 - d 6 ) 7 . 83 Nil, bd SM 3 . 06 CH* m TEA
7 .49 Ar d Tos 2 . 28 CH, s Tos7 . 11 Ar d Tos 1 . 2 2 CK, m E t/TEA
GlyGlyPheOMe 8 . 67 NH 2 1 Gly 4 . 48 C.H m Phe/PC omplex 8 .54 NH bd TEA 4 .28 C.H t Phe/SM
8.39 NH d Phe 4 .00 C.H m Gly(D M S 0 - d 6 ) 7 . 8 6 NH* bd SM/P 3 . 67 CH* s Est SM
7 . 47 Ar d Tos 3 . 58 CH* s Est P7.11 Ar d Tos 3 .53 CH, s 18C67 . 28 Ar m Phe 2 . 28 CH, s Tos
GlyGlyC 1 «0Me 9 . 44 NH t Gly 3 .69 CH, s Es tComplex 7 . 70 Ar d Tos 3.53 CH* s 18C6
7 . 01 Ar d Tos 2 . 2 1 CH, s Tos( C D C 1 3 ) 7 . 58 NH d C 1 4 1 . 65 CH* tn C 1 4
7 .31 NH* bd Gly 1 . 16 CH* m C 1 44 .28 3 . 8 8
C.HC.H
q(D
C 1 4Gly
0 . 77 CH, t C 1 4
G l y P h e C »«OMe 9 . 44 NH d C 1 4 3 . 87 C.H tn GlyCotnpl ex 8 . 0 0 NH d Phe 3 .73 CH, s Es t
7.79 Ar d Tos 3 . 56 CH* s 18C6(C D C 1 3 ) 7 . 1 0 Ar d Tos 3 .26 C p H a m Phe
7 . 27 Ar m Phe 2 . 95 C p H a m Phe4 . 6 6 C.H m Cl 4 P 2 .29 CH, s Tos4 . 56 C.H m Cx 4 S M 1.71 CH, tn C 1 44 . 37 C«H tn Phe P 1 .25 CH* m C 1 44 .29 C . H m Phe SM 0 . 97 C H , t C i *
GlyPheCioOMe 9 . 48 NH d C 1 0 3 .72 C H , s EstC o m pl e x S . 09 N H d Phe sJ O f C H * s 15C5
7 .78 Ar d Tos 3. 27 CpH * m Phe(CDC13) 7 . 10 Ar d Tos 2 . 97 CpH* tn Phe
7 . 27 Ar m Phe 2 . 30 CH, s Tos4 . 62 C.H m C 1 0 1 .77 CH, m C 1 04 .30 C.H m Phe 1 . 24 CH, m Cl o3 . 8 6 C.H m Gly 0 . 84 CH, t C 1 0
- 1 8 0 -
Appendix S e v e n .
Mass Spe ct ra l Data of Rel ev an t Products.
cpd Mass Spectral Data (m/z)
6 b 603 [^NHaCHaCONHCH a C O N R C O N H R 1+ 33 9 [NH a CH a CONHCH a C O N R C O N H R ] 214 [ N H a C H a C O N H C H a C O N R ] *
6 e 399 [(CH a )a SNHCHa C ON H C H a C O N R C O N H R ]* 27 4 [(CH a )a SNHCH a CO N H C H a C O N H R ]*
5a 193 [ ( C H a )a S N H C H a C O N H C H a C O O H ] * 148 [ ( C H a )a N H C H a C O N H C H a 1 +135 ( ( C H a )a S N H C H a C O N H a ]90 [ ( C H a )a S N H C H a ]+
5b 193 [ ( C H a )a S N H C H a C O N H C H a C O O H 1+ 177 [(CH a )a SNHCH a C ON H CH a C O H ] 133 [ ( C H a )a S N H C H a C O N H ] +90 [ ( C H a )a S N C H a ]*
7a 494 [ i N H a C H a C O N H C H a C O O N ( C O C H a )a 1 * 230 [NH a CH a CONHCH a C O O N (COCH a )a ]
7b as for 7 a .
8 a 290[ ( C H a )a S N H C H a C O N H C H a C O O N ( C O C H a )a ]*
8 b 2 47 [CH aSNHCH a CO NHCH a COONCOCH a CH a ]* 145 [NHaCHaCONHCHa C O O N ]*
| 285 [ ( CH a ) a SOC ( NHC a H i i ; =.NC<hi i J *
-101-
Appen d i x E i g h t .
S t r u c t u r e s of All N u m be re d Compounds.
DCC = / V n=C=N^ ^ DCU = / V-NHCNH
0t;P-PH= l8 -Crown-6 NSu = HON' 2
C-U-Ul! ^0
a X - S -0
©
R,=c6hu
©5 ©x >nh3chcooh> J Ich3
© 5 ©2 Cl 5 NH3CH3
3 X0^NHfcH2CH2CH2CH2CH2COOH
U X ^NH3CH2CONHCH2COOH
5 x0(ch3)2sNHCH2CONHCH2COOH
RCOR'NCONHR' a R= Cl SNH3(CH2)5
b R = X0 ^NHfcH2CONHCH2-
c R = CH3-
©5 ©~d R= X SNH3CH2-
e R = xe (CH3)2SNHCH2CONHCH2-
f R= X0 (CH3)2^IH C H 2-
g R= (CH3)3COCNHCH-0 ' CH3
0h R = (CH3)3COCNHCH2CONHCH2-
0i R= (CH3)3COCNHCH2-
©5 © .COCH,7 X ^NH3CH2CONHCH2COONCCO(i H 2
©, , © „ LOCH?8 X (CH3)2SNHCH2CONHCH2COONr ■ 2
C 0 C H o
9 X (CH3)2SNHCH3
0
10 h-h ' ^ h
h -c A R§-18 3-
X0 (CH3)2S®NHCH2CONHCH2CONHCH2CONHCH2COOH
X0 (CH3)2NHCONHCH2CONHCH2COOH
X0 > NH®CH2CONHCH2CONHCH2COOCH2CH3
Cl NHfcH2COOCH2CH3
Tos £ NHfcH2CONHCH2CONHCH2COOCH2CH3
Cl N H fcH C 00C H 3 CH,
oTos0 NH3CH2CONHCH2CONHCHCOOCH3
* CH,
Cl0 NH®CHCOOCH3 a n = 7(CH2)n b n =11CH
© STos ?NH3CH2CONHCH2CONHCHCOOCH3
(CH2)nCH3
Tos >NH3CH2CONHCHCOOHCH,
©Tos 5NH3CHoCONHCHCONHCHCOOCH.Zl C. * 1
CH2 (CH2)n . C H ^
u H .0C ^ r v
V °
'C(CH 3'3
23CF3C00e j NHfcHC0NHCH2C00H
CH,
00CH.
2 U CF3COO0• NH®CH2CONHCHCONHCHCOOCH3c h 2 ic h 2)7
c h 3
CF3COO0 > n h® chco n hch 2c o n h c h 2c o n h c h c o n h c h c o o c hc h . CH? (CH o )-7
“ V < HCH3 c ® 'NHCHCOOH
c h 3
oS ® rv\C "° r ^'C'r'3 CV0* A c w ,
H CH3
CH3^Nf c HCH3' © 'C l
9 H3 9h 3c c -o -c n h c h c o o h
c h 3 (ch2)7 c h 3
0 ou r- n 0 0 11H,C-Cn m h ,C-CH,h ^ c ,n o c n h c h 2c h 2c o n 0_ P
0 0
011X -C H ,
CH3COON <, 23 C-CH, 11 L
0
CH3NHCOCgH5
xe >nh®c h 2cooh
h 3c 's® n hch 2c o o h H 3 C Q
X- 1 8 6
0©J © /C-CHo
35 X i NH3CH2COONM ^0
0H 2C n © /C - C H 2
36 H ,C 'S=N. HCH^ 0 0 N 'C -C H
a R=CH3 . b R=H
i Y=CH3
ii y = c h 2- < Q ^ n o 2
iii Y= (CH2)17CH3
039 ((H3C)3CQCNHCH2C0NHCH2C0)20
w> : R'N=C-NHR'I0s® X®
j Y y iix 0
0 R37 ((H3C)3C0CNHCHC0)20
0 R38 (H3C)3COCNHCHCOOY
a R=CH(CH3)2
b Rr H2C
-187
A p pend i x N ine_.
Copies of the papers published, or a c c ep ted
publication , from this work.
for
-18 8-
J. ChEM SOC. fEKKIN TUANS. II 1987 323
The Use of Crown Ethers in Peptide Chemistry. Part 1. Syntheses of Amino Acid Complexes w ith the Cyclic Polyether 18-Crown-6 and their Oligomerisation in Dicyclohexylcarbodi-imide-containing Solutions
Paolo Mascagni,* Carolyn B. Hyde, M ario A. Charalambous. and Kevin J. WelhamD e p a rtm e n t o f P h a rm a c e u tic a l C hem istry , S c h o o l o f P ha rm acy. U n iv e rs ity o f L o n d o n , L o n d o n W C 1 N 1AX
The synthesis of amino acid complexes with the cyclic polyether 18-crow n-6 and their solubility properties in organic solvents are described. Oligo homo-amino acid peptides have been prepared using the crown ether complexes and dicyclohexylcarbodi-imide as coupling agent. The mechanism leading to the formation of the oligopeptides has been discussed and proved to involve the transferring of one N -H proton from the crown ether complex to the carbodi-imide nitrogen.
Since their discovery by Pedersen,1 crown ethers have found increasing application in the fields o f organic, inorganic, and analytical chemistry.1 Their most striking characteristic is the capacity to form siablc complexes w ith inorganic salts, which then become soluble in various organic solvents including nonpolar ones.1,1 This ability is not restricted to inorganic species only; charged amines have also been shown to enter the cavity formed by the donor atoms (N or O ) o f crown-type ligands.1'1 In the latter case H-bonds as well as ion-dipolc interactions have been considered responsible for the stability of such complexes.1 These observations led some authors4 to exploit the complcxation ability o f crown ethers for selective acylation o f secondary amines in the presence o f primary ones.
The use o f crown ethers as protecting groups suggested by this experiment is, in our view, extendable to other classes or compounds such as amino acids; thus peptide synthesis, both in solution and solid phase, implies selective masking o f the latter at the amino group. Should this approach be feasible, in situ protection could be performed which would result in a substantial shortening o f the overall synthetic process.
Recent results seem to substantiate this approach.1 Thus, following the original idea o f Cram and his co-workers,4 crown ethers bearing thiofunctioos were prepared and shown to function as etuyme model in the synthesis o f peptides.4
We report here the synthesis o f amino acid oligomers as part o f a wider study on the applications o f crown-type ligands to the field o f peptide and macromolecular synthesis.
The mechanism o f oligomerisation is proposed by analysing the behaviour towards dicydohcxylcarbodi-im ide (DCC) o f several amine and amino acid complexes w ith crown ether.
DCC is commonly used as coupling agent in peptide synthesis and its mode o f action is shown in Scheme I.1
E xperim enta ll8-Crown-6 was purchased from Aldrich Chemical and used w ithout further purification. M.p.s are uncorrccted. One- and two-dimensional n.m.r. spectra were taken on a Varian X L -300 instrument; chemical shifts refer to internal letramcthylsilanc as standard.
Fast-atom bombardment mass spectrometry was performed on a VG Analytical ZA B -IF double-focusing mass spectrometer. The samples were applied to the probe tip in a thioglyccrol matrix and bombarded by 8 kcV Xenon atoms. Full scan spectra were recorded in co. 7 s.
Alanine Hydrochloride Crown Ether Complex ( la ).— Alanine ( I equiv.; I g, 1.12 x Id*4 mol) was dissolved in water (5 m l) and HCI (1.1 equiv.; 0.4502 g. 1.06 ml) was added. The solution was stirred for 0.5 h and then lyophilised to give alanine hydrochloride in 100% yield. The hydrochloride ( I equiv.; I g. 7.96 mmol) was suspended in chloroform (5— 10 ml) and 18- crown-6 ( I equiv.) added. The solution was stirred until it became cleat, then the solvent was evaporated off to give a powder in 97% yield, m.p. 149— 150 “C (Found. C. 46.3; H, 8.3; N. 3.5. C , jH j jC IN O , requires C, 46.2; H, 8.27; N , 3.6%); M * (less C l" ) 354; S fC D C lj) 11 ( I H. br), 7.11 (3 H. br). 4.42 ( I H. mX 3.59 (30 H .s), and 1.5(3 H .J8 .8 Hz).
Alanine Tosylote 18-0-0x71-6 Complex (lb ).— The tosylate complex was made in a sim ilar way, but tolucnc-p-sulphonic
, 2 . NH,-CH{R4)-COORsR -NH-CH(R )- C O O H * R - N » C * N - R ► R'-NH-CHIR1)-CO — 2---- — -------► di-paptid*« DCU0s 1 s R -NH-C »N-R
R'-NH-CH(RJ)-C00H
DCU ♦ [r'-NH-CHIR1) - COLOo0CU»RJ-N-C-N-R>
H H
NH,-CHfRM - C00R*
-189-
324 J. C H EM . SOC. PE R K IN TRANS. I I 1987
. <fM’X* | NH,-CH-COOH <•)
Cl'| NHj-(CHj) j- COOH(J)
a , X « Clb . X » TsO"
c* fn* T"1X' I NH,-[CH-CO-NH^-CH-COOH O)-(C)
X
{
(3 ) n»1 (A) n • 2 (5 ) n - 3 («> n»5
X * c r , T * o *
« Crown ether
acid was used The complex was recrystallised from ethanol- ethyl acctalc mixtures in 77% yield, m.p. 123— 125 °C (Found: C, 50.0; H. 7.5; N , 28. C „ H „ N O , ,S requires C, 50.3; H, 7.5; N. 27%); 6{CDC1,) 7.8 (2 H, d, J 8.3 HzX 7.27 (3 H, brX 7.12 (2 H, d. J 8.3 HzX 4.25 (1 H, brX 3 65 (34 H, brX 2.31 (3 H. «X and 1.6(3 H .d , 76.9 HzX
b-Aminohexanoic Acid Hydrochloride Complex (2X— The synthesis was identical to that o f ( la and h) and gave 88% o f the complex. imp. 105— 107 *C [F o u n d C, 50.0; H, 8.8; N , 3.3. C l t H „ C lN O t requires C, 50.0; H. 8.9; N, 3.2%X 5(01X 3,) 9.76 ( I H, brX 686 (3 H, brX 3.40 (29 H. brX 273 (2 H, mX 253 (2 H. t. J 8 HzX 1.67 (2 H, mX 135 (2 H. mX end 1.43 (2 H. mX
Alanine Dipeptide Complex (3).— This had m.p. 192— 194 *Q M * (lessQ ‘ )4 2 5 ,5 ([, H .]D M S O ) 8.71 ( I H .d . 78.5 H z ).8.1 (3 H. brX 4.26 (1 H. mX 3.83 ( I H. ra). 3.52 (25 H, brX 1-38 (3 H. d J 7.1 HzX end 1-33 (3 H . d 3 7.0 HzX
Alanine Tripeptide Complex (4).— This has M * (less C l ' ) 496; S(CDC1,) 10.37 ( I H. d 79.4 HzX 10.04 (1 H. d. J 7.5 HzX 7.15 (3 H. br). 4.51 ( I H. m), 4.46 (1 H, m), 3.7 (26 H. brX 3.35 (1 H, m), and 1.57 (9 H, three partially overlapping dX
Alanine Tetrapeplide Complex (5).—This has M * 0 *“ O ' ) 567; S tf 'H .J D M S O ) 8.72 (1 H. d J 7.7 HzX 8.51 (1 H. d J 8.7 Hz), 8.27 ( I H .d J 9.6 Hz). 4.37 ( I H .m ). 4.27(1 H ,m X4 19(1 H. m). 3.85 ( I H. brX 3.55 (27 H. brX and 1.45— 1.22 (12 H, partially overlapping dX
Reaction between Vihmeier Reagent* and Alanine Hydrochloride Complex (la ).— The Vilsmcier reagent was prepared by adding PCI, (3 g) in small portions to an excess o f D M F at 0 °C with stirring.* A precipitate formed which was the Vilsmeicr reagent. The complex ( la ) (100 mg) was dissolved in C D O , (3 ml) and the solution cooled to — 40 *C. An aliquot portion o f Vilsmcier reagent was filtered, washed w ith diethyl ciher, and immediately transferred, w ith the aid o f a glass spatula, to the C D O , solution. I t was not possible to transfer known quantities o f the Vilsmcier reagent since the latter is extremely hygroscopic and decomposes very rapidly in the presence o f moisture. The C D C I, solution was allowed to equilibrate for 1 h at - 40 °C before a sample was taken out for n.m.r. analysis.
Results(a) Alanine Hydrochloride Complex with 18-Crown-6.—
Suspensions o f alanine hydrochloride salt in chloroform readily
turned in to clear solutions upon addition o f one equivalent o f the cyclic polyethcr 18-crow n-6. The crystals obtained after solvent removal and recrystallisation from the appropriate solvent were shown by mass spectrometry, *H n xn.r, and elemental analysis to be those o f the amino acid salt complex w ith the crown ether, ( la ).
The reactivity towards DCC o f the amino acid thus blocked was tested in actton ilrile and chloroform.
When one equivalent o f D CC was added to a solution o f ( la ) (100 mg) in acctonitrilc a large precipitate readily formed. This was isolated (33 mg) and shown to be dicyclohexylurca (D C U ) by comparison w ith an authentic sample. From the reaction mixture after an overnight o f stirring at room temperature alanine dipeptide complex (2) (IS mg) was isolated. Analysis o f the mother liquor revealed the following, (i) Based on the lack o f i.r. absorption at 2 120 c m '1, it was concluded that virtua lly a ll D CC had been consumed, (ii) The pH o f the solution was 29,* identical to that o f the m ixture p rio r to addition o f DCC. ( i ii)
- Solid (35 mg) was isolated after extraction w ith water and lyophilisalion. FAB mass Spectrometry and n.m.r. showed that this solid contained ra. 80% by weight o f the alanine tetrapeptide (5). (iv) Small amounts o f peptides o f different length were seen in the spectrum o f the reaction residue, (v) The dipcptide and the tetrapeptide thus isolated accounted for ra. 95% o f the in itia l amino acid.
When the reaction was repeated in chloroform solutions different results were obtained. This is illustrated by the follow ing example. Complex ( la ) (50 mg) was dissolved in C D O , (0.5 mIX The solution was then treated w ith one equivalent o f DCC and the reaction followed by 1H n.m.r. over a period o f three weeks. Representative spectra o f this lime- dependent experiment are illustrated in Figure 1.
The appearance o f doublets in the 6 7— 11 range confirmed the formation o f peptide bonds. Subsequent to the addition o f DCC and vigorous shaking o f the n.m.r. sample, the spectrum (Figure IA ) had a signal at 6 r a 9.7 which further analysis showed to be the dimeric species o f the alanine complex. Three other doublets o f lesser intensity were seen between & 7 and I I . Occasional runs in the follow ing five days indicated that the composition o f the m ixture was still changing (Figure IB and C). No further changes were seen in the shape o f the spectrum after this time (Figure IDX The reaction was assumed to have gone to completion. Separation o f dicyclohexylurca and treatment o f the o ily residue w ith (i) water and (ii) acctonitrilc yielded alanine tripcplide complex (10 mg) w ith crown ether (4). The analysis o f the remaining peptides was carried out by n.m.r. and mass spectrometry. A two-dimensional correlation spectrum o f the reaction mixture revealed a number o f couplings between the N H and a-protons, consistent w ith the presence o f several oligomeric species o f alanine. FAB mass spectra o f the reaction residue were more d ifficu lt to interpret. Thus small amounts o f by-products, and in particular d icydo- hexylurea, appeared to discriminate against the more abundant peptides. However upon repetition o f the reaction some peptides were positively identified: they were the tri- [(4X M * less O ' , 496], letra- [(5X Af * less Q ' , 567], and, hcxa-peptide [(6); M * less C l ' , 709] o f alanine complex w ith crown ether.
(b) Alanine Toiylate Complex with IS-Crown-6.—The complex o f alanine tosylate w ith the crown ether, ( lb ), was prepared in a fashion identical to that described for the corresponding hydrochloride complex. The effects caused by the substitution o f the counteranion on the reactivity o f the amino acid complexes were investigated in the follow ing manner
* A portion of the solution was diluted with water (2 vol.) and the pF measured using a pH-mcicr.
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J CHEM. SOC PERKIN TRANS II 1987 325
1 I I I I I I10 J *
Figure I. 'H N.m.r spectre of * ruction of (In) in DCC containing CDO, solution: A. after addition of I equivalent of DCC; B, after I b from addition of DCC: C, after 24 h from addition of DCC; D, after S days from beginning of reaction
The complex ( la or b) (100 mg) was dissolved in acctonitrilc (5 m l) containing 1 equivalent of DCC. After 0.5 h dicyclohexylurca was collected by filtra tion, dried, and weighed. The clear solution thus obtained was stirred for a further 24 h at room temperature, after which time the solvent was evaporated; Ihe o ily material thus obtained was taken up in chloroform and extracted w ith water. The water extracts were then lyophiliscd and checked by n.m.r for their amino acid content. The chloroform layers were also checked and shown not to contain any unchanged amino acid or derivatives. Following this procedure we were able to conclude that, although the two complexes produced oligomeric species in virtually the same relative ratio, the tosylate was more stable in the presence o f DCC. Thus the n.m.r. spectra of the water extracts showed, the presence o f di- and tri-peptide in 2; I ratio, w ith unquanlifiable traces o f higher peptides, and ca. 25% o f unchanged ( lb ) as compared to only 5% o f Ihe monomeric hydrochloride complex.* Furthermore, ca. 35% of the expected urea was obtained from the reaction involving the tosylate compound, whereas 58% urea was recovered from the hydrochloride complex reaction.
(c) U-M elhylam inr Hydrochloride Complex with I S - C V o h v i - 6.— When inorganic o r organic salts are solubilised in organic
“The rale of oligomerisation appeared to be different a hen the solutions wric vigorously stirred The reaction earned out in CDCJ, wat slower when compared uilh that of identical mixtures stirred in a flask with the aid of magnetic bars
solvents, (he counteranion is present in solution as an active, ‘naked* anion which is not' solvated. Because o f this characteristic the electron-donating ab ility o f Ihe anion is increased, »>. the nudeophilic ily and basicity o f the anion are enhanced.
Fearing that the complex counteranion in its highly activated state may participate in the reaction mechanism, we in vestigated the behaviour in chloroform o f dicyclohcxylcar- bodi-imidc-containing solutions o f ( I ) A’-melhylamine hydrochloride complex w ith crown ether, (2) the latter and benzoic acid, and (3) benzoic anhydride plus the methylaminc complex. Here is a summary o f the results obtained from Ihe three reactions.
When equimolar amounts o f the Af-methylaminc hydrochloride complex and DCC were dissolved in chloroform no dicyclohexylurca precipitated from Ihe solution even after 24 h stirring at room temperature, indicating that ihe two reagents were stable in solution in Ihe presence o f one another. That the “naked' chloro ion was not reacting in this condition was also confirmed by the i.r. spectra taken o f the reaction mixture. Thus the intensity o f the D CC absorption at 2 120 cm*1 d id not change upon addition o f the methylaminc complex, nor during the 24 h period o f s tirring at room temperature.
Surprisingly the addition o f benzoic acid to a chloroform solution o f A'-melhylaminc hydrochloride complex and D CC did not result in the complete conversion o f benzoic acid in to the amide derivative. Thus after 48 h o f vigorous stirring at room temperature the solvent was removed and the *H n m.r. spectrum o f Ihe o ily product thus obtained showed the presence of A'-melhylbcnzamide and benzoic anhydride (formed during the reaction) in 1:14 ratio.
Identical results were obtained upon treatment o f chloroform solutions o f the complex and D CC with benzoic anhydride. The latter is the condensation product o f two molecules o f benzoic acid. Such a condensation is often carried out w ith the aid o f DC C and in peptide chemistry the amino acid anhydride is regarded as a possible active intermediate o f Ihe coupling step mediated by DC C (Scheme I).
Following the experiments w ith Af-mcthylaminc it became apparent that the polymerisation process seen in the ease o f the alanine complexes takes place >io an intermediate involving the ammo group, the carboxylic group, and D C C and that the former two groups must belong tp the same molecule.
(d) (r-Aminohcxanoic Acid Complex with IE-CroHn-6— In order to confirm the latter conclusions and to establish i f the number o f covalent bonds separating the amino and carboxylic moieties plays any role at all in the reactivity o f these amino acid complexes, the behaviour of 6-aminohcxanoic acid complex w ith crown ether was investigated in the reaction conditions which yield oligomerisation The complex (2) was prepared from suspensions o f ihe amino acid salt and crown ether and shown to be homogeneous by elemental analysis and 'H n.m.r. Complex (2) and DCC in equimolar amounts were thoroughly mixed in chloroform or acctonitrilc and the solutions were stirred for 24 h. O nly traces o f precipitated dicyclohexylurca were collected at the end Typical amounts corresponded to 10% of the urea expected from complete conversion o f DCC. The amino acid components o f the reaction mixture were then extracted in watct and aftct lyophilisation the extracts were checked for 6-aminohcxanoic acid oligomers. The n.m.r. spectrum revealed that the water extracts contained the majority o f the amino acid molecules s till in the intact form; only 5% of the latter had been convened in to the dipeptide.
DiscussionThe coupling reaction between the amino and carboxylic groups to form the amide bond requires the presence in solution of the amino component as a free base. In order to explain the
-191-
326
:'(NVi-fmv
CHIR) - COOH 5P
OCC
CH(fl)- CO 3 = I 0r '-n ^c-n h -h1
I. CHEM . SOC. PE R K IN TRANS. I I 1987 =£ X ” NHj.CH(RI - C O O H ♦ |
H
X ' H - N - C H ( R ) - C Q * I I I <0 I
1NV CH(R)-CO-NH-CH(R)-CO H
(S)
h -n-\_-NH-R
NH,-CH(R -CO
n *c -n h r'N=C-NHR
HjO treatment
O C U *X' I N H , - C H ( R )-CO-NH-CH(R)-CO-NH-CH(R)-CO*9-^ oligopeptides ♦ OCU » HX( * I treatment
| *C ro w n e ther
\ * i'n «c -n h r ' R'X*
M »v /? PCI, , , _____J ► N»C ► N *CM . / V , \
„ / R C O O H r/ r
Me ClCf (9)
Jjcr^NHj-CH-COOH
CH, c* I CI’|NHr CH - c 'M.v. > 110)/N'c\Me/
c r
Scheme 3.
mechanism o f polymerisation o f the amino acid complexes shown here, it is necessary to find out which is Ihe mechanism that produces the free amino end, out o f the R N H , ' sale The results seem lo indicaie lhat an intramolecular rearrangement is involved in ihe latter mechanism. Furthermore, the data relative to ihe methylaminc complex rule out the presence in solution o f the equilibrium N H ,* N H , + HC l w ith DCC and, or traces o f water acting as catalyst. Thus, should the contrary apply, a complete con version o f benzoic acid (or benzoic anhydride) into the corresponding amide would take place. A possible reaction mechanism that takes in to account the experimental data is shown in Scheme 2 where the amino acid complex and the amino acid complex adduct w ith DCC are in equilibrium with the corresponding uncomplexed ion pairs. Because o f the close
proxim ity o f the N H ,* lo Ihe DCC nitrogen in the adducts (7) and (7 i), a hydrogen-bond bridge can be formed between these two centres. This results in a redistribution o f the positive charge, which is no longer concentrated on the amino acid nitrogen, and consequently a weakening o f ihe electrostatic interaction between the countcnn ion and N H ,* . The equ ilibrium between (7a) and the new species (8), in which the chloro atom forms an electrostatic interaction w ith the DCC nitrogen, provides the necessary free amino group for Ihe coupling reaction. Supporting evidence for this hypothesis came from the reaction between ( la ) and the Vitsmeier reagent* (9). The la tter is known to form adducts10 o f the type o f those between carboxylic acids and DCC (Scheme 3). Furthermore, the presence of the positive charge on the C»N * moiety would
- 1 9 2 -
I. CHtM SOC PfflklN TUANS II 1987 327
rpH,
i { 3Filurr 2. ‘H N.m.r. spectrum of a mixture of (la) and Viltmcicr reagent in CDCI,
prevent hydrogen bunding and hence polymerisation, w ith ihe assumption lhal ihe proposed mechanism holds. And indeed addition o f alanine hydrochloride complex lo C D C lj solutions containing freshly prepared* Vilsmcier reagent produced the seamed adduct (10) but no polymerisation look plaoe as indicated by n.m.r In Figure 2 t h e ‘ H n.m.r. spectrum o f Ihe latter solution after stirring o te rn igh l at room temperature shows tsso sets o f peaks attributed lo the free amino acid complex and to Ihe adduct (10). No traces o f polymeric form could be detected in the spectrum.
In the light o f these conclusions the experimental results presented above are readily explained Thus the lim ited reactivity of the 6-aminohcxanoic acid complex towards oligomerisation follows the almost tota l lack of hydrogen bonding between the nitrogen on DCC and the amino group in the complex. The spacer group (C H j) . introduced in the amino acid prevents close contact between the acceptor and donor of the hydrogen-bond thus stabilising the adduct (7c).
Identical reasons apply to the A'-mrihylam inc case. Here the lack of covalent bonding between the amino and Ihe carboxylic groups does not allow the DCC to come close to the N H j* and hence deprotonation o f the amine cannot take place.
On account of its larger size, ‘naked' tosylate ion should display greater stability than that o f the smaller chloride ion in
solvents of low polarity This characteristic should make the tosylate ton in ( lb ) less readily available for the formation o f the species (Hj thus explaining the presence of larger amounts of unchanged monomer found in the reaction mixtures of the tosylate complex.
ConclusionsW ith ihe objective of establishing the scope and generality o f Ihe use o f crown ethers as non-covalcnl block ing groups in peptide synthesis, we have investigated the mode o f action o f amino acid-crown ether complexes in DCC-containing solutions. The results presented indicate that oligomerisation o f a-amino acid complexes takes place in solution and prooeeds via the cyclic intermediate (7a) A hydrogen bond between Ihe amino group nitrogen and the DCC nitrogen ultimately causes the ‘deprolection’ o f the former and its coupling lo the carboxylaic o f a second amino acid unit Upon repetition o f these same steps oligomerisation is achieved.
because of Ihe nature o f their mode of action, the reactivity of these complexes is achieved at the price o f selectivity. Thus, using the experimental conditions here employed, it seems difficult lo contro l the reaction and hence produce selective coupling between amino acids o f dtflercnt Linds On the other hand the elucidation of the reaction mechanism dearly indicates that coupling reagents, having structural characteristics different from those o f DCC, can be used, in principle, for the selective formation o f peptide bonds.
Keferencex1 C. J Pedersen. J. Am C hrm . Soc.. I967,19, 2495.2 M. Hiraoks, ‘Crown Compounds,' Elsevier, Amslcrdani. 1982.3 C. J. Pedersen, J. Am. C hrm . Six'.. 1967, §9, 7017.4 A G. H Bareli and } . C. A Lana. J C h rm S a t , C hrm . Com m un.. 1978.471.
5 5 Sasaki. M. Shionoya, and K. Koga. J. Am . C hrm Soc, 19b5,107, >371.6 Y. Chao and D. i Cram. J. Am. C hrm . Sue., 1976, 9*. 1025.
7 M. bodansrk). Y. S. Maushcr. and M A Ondcili, 'Peptide Synthesis.' Wiley Inicrscience, New York. 1976.
£ A. Vijxnicicr and A. Haack. B r t. D t.\ch. C hrm . C n , 1927. 60. 119.9 D. R. Hepburn and H K Hudson. C hrm . In J (i jm d o n). 1974. 664 10 T. Fujisawa, T- Mori, S. Tsugc. and T. Saio. T n ra h rd ro n L t ’l l , 1983. 24. 1543.
R rc tii t d 22nd M ay 1986, Paprr 6/993
-193-
The U6e of Crown Ethers in Peptide Chemistry. Part 2.
Syntheses of Dipeptide Complexes with Cyclic Polyether l8-Crown-6
and their Derivatisation with DMSO.
*Carolyn B. Hyde, Kevin J. Welham, Paolo Mascagni
Department of Pharmaceutical Chemistry, School of Pharmacy, 29-39
Brunswick Sq, WC1N 1AX London, UK
*) To whom correspondence should be addressed to
-194-
Introduction
In a previous communication^ we have described the synthesis and
characterisation of amino acid complexes with Crown Ethers. The
objective of the research was to study their behaviour in organic
solvents in the presence of those coupling reagents commonly
employed in peptide synthesis. Using dicyclohexylcarbodiimide
(DCC) in acetonitrile or chloroform we have found that the
alanine derivative (1) reacts to form oligomeric 6pecies. The
mechanism of the reaction has been elucidated and shown to
involve an intermediate in which the carboxyl and NH^+ groups are
simultaneously linked to DCC via a covalent and an hydrogen bond
respectively (2). Thus activation of the carboxylic group and
deprotection of the NH^+ can occur, providing the basis for the
formation of peptide linkages.
In this paper we have gone on to explore the role of solvent and
reactant concentrations in order to find a method for controlling
the formation of oligomeric species. We have synthesised
glycylglycine complexes with 18-crown-6 (3) and studied their
reactivity with DCC in dimethylsulphoxide (DMSO) solutions.
Both solvent and substrate were selected in order to avoid the
destabilising effects of the hydrogen bond on the activated
carboxyl - DCC adduct (2). Thus the replacement of the amino acid
with a di peptide displaces the from the site it occupies in
structure (2). Furthermore, the use of DMSO, a solvent known for
its donor properties, should favour solvent - solute hydrogen
bonds over intramolecular ones.
-195-
All the reactions discussed in this paper have been studied by
^H-NMR with the signal of uncompletely deuteraced DMSO used as
internal standard. Repetition of the reactions on a preparative
scale afforded the products for their chemical and physical
characterisation.
Experimental
All reactions were followad by ^H-NMR on a Varian X1.300
Spectrometer. DMSO-d^ was supplied by Aldrich and stored over
type 5A molecular sieve in an airtight container. DMSO was
distilled under vacuum (31°C; 0.5mmHg) and kept in the dark over
type 5A molecular sieve. DCC was distilled under vacuum (92°C;
0.3maHg) and kept in a desiccator. All solvents used on the HP1.C
were of HPLC grade and filtered before use.
Synthesis of Tos Glycylglycine Complex (3a)_2lo a solution of p-toluenesulphouic acid (7.2g, 3.8x10 moles)
-2in H^O/EtOH (50 ml, 1:1), glycylglycine (5g, 3.8x10 moles) was
added. After one hour of stirring at room temperature the solvent
was removed, the dipeptide salt suspended in 50 ml EtOH
containing one equivalent of 18-crovn-6 (10.0 g) and the
suspension warmed until' it became clear. The solution was then
allowed to cool to room temperature and EtOAc added dropwise
until it became cloudy. On standing, the complex crystallised out
(20g, 93%), A similar method was used for the synthesis of (3b).
From 5g of glycylglycine dipeptide we obtained 13.Ig (80%) of the
crystallised complex.
An example of the procedure for a reaction followed by ^H-NMR is
as follows.
-196-
One equivalent of the purified dipeptlde complex (3a, 57mg,-it
1x10 moles) in 0,5 ml dry DMSO-d, was treated with oneoequivalent of freshly distilled dicyclohexylcorbodiimide (DCC,
20.6 mg). The solution was quickly transferred to an NMR tube and
spectra taken every 5 minutes for the first hour.
An identical method was used when one equivalent of N-hydroxy
succinimide (NSu, 115mg) was added to the initial reaction
solution.
The larger scale reactions were done in DMSO on the bench with no
stirring of the solutions in an attempt to duplicate the
conditions used for the NMR analysis. An example of this is as
follows :-3
The dipeptide complex (3a, 1.14g, 2x10 mole) was dissolved in
dry DMSO (10ml) and one equivalent of DCC added (412.6mg). After
18 hours at room temperature the solvent was removed in vacuo and
the residue treated with chloroform which precipitated DCU (278
mg, 62%). Upon repetition of this treatment virtually all DCU
could be recovered.
After removal of the solvent the oily residue was treated with
acetonitrile. Upon standing at room temperature a gummy material
formed; this was separated from the supernatant, dried and shown
by NMR and mass spectrometry to be the DMSO-dipeptide adduct
(5a) (566 mg, 78%) (see table I). Further purification of the
product was not possible due to its extreme instability.
3
- 197 -
Results
1) Tos-Glycylglycine Complex 3a (c-0.02M)
1x10 ** moles of complex (3a) were dissolved In 0.5 ml of DMSO-d^
containing one equivalent of DCC. The solution was quickly
transferred to an NMR tube and the reectlcn monitored at 300 MHz.
Within the first twenty hours there was no detectable consumption
of DCC and only after twenty-five hours two signals of identical
intensity, a triplet 8.60 and a doublet at 6-8.38, indicated
the presence in solution of a new species. The ratio of the
latter to the starting material was 3:5 and did not vary during
che following 70 hours.
The reaction was then repeated on a larger scale and the product
isolated by HPLC. A base peak at m/z 214 in the MS spectrum and
the presence of only one, intense nOe at the N-C-N carbon of DCC
upon irradiation of the NH doublet at <5-8.38 .indicated that the
structure of the product was chat of the N-acyl urea derivative
(4).
DC'J and N-acyl urea accounted for 90“ of the DCC used in the
reaction.
Repetition of the latter in the presence of one equivalent NSu
(see text below) did not afford any dipeptide ester and identical
results to those described above were obtained. We therefore
concluded that, in the conditions used for the reaction,
activation of the carboxylic group by DCC did not occur.
2) Tos-Glycylglycine complex 3a. (c-0.2M)
In an attempt to promote activation of the dipeptide by DCC we
4
-198-
ir.creased tenfold the reagents concentrations.
Precipitation of DCU immediately followed the addition of one-4equivalent of DCC to a DMSO-d, solution containing 1x10 mulesb
of (3a), However the first dipeptide product formed only after 20
minutes, as Indicated by the appearance of two triplets 8t & m 8,38 and 6.47. The concentration of this new product increased in
the following hour, although at a rate slower than that of DCC
consumption. The formation of the product ceased as the last of
the DCC wa6 consumed and resumed upon addition of a second
equivalent of the coupling reagent. When this was also consumed,
there was about 202 of the starting dipeptide left in solution
the rest having been converted into the diketopiperazine
derivative (17, less than 10%) and a major product of unknown
structure (about 70%).
The latter was isolated upon repetition of the reaccion on a
preparative scale, and shown by KMR and Hass Spectrometry to be
the dipeptide - DMS0 adduct (3a).
Ir. order to evaluate the role, if any, of the complex counter-ion
in the formation of (5a), we repeated the above reactions with
the tosylate ion substituted for the chloride ion in the glycyl-
glycine complex.
3) KCl-Glycvlglycine complex 3b (c»0.02M)
At a complex concentration of 0.02M, there was repetition of the
results obtained with (3a), n a m e l y :
1) ho activati or. of the carboxylic group occurred,
2) DCC was not consumed during the first 2 hours,
i) N-acyl urea derivative was the only product detected in
5
-199-
solution, and
A) Conversion to the N-acyl urea was over after 20 hours.
A) HCl-glycylglycine complex 3b (c»0.2M)
In this reaction, despite careful drying of tcch DMSC-d^. cr.d
complex, the spectrum of 3b showed presence of water at & “3.A5. Unlike the tosylate case, addition cf cr.e equivalent of DCC did
cot produce any dipeptide derivative, despite hycracicr. of DCC to
DCl) during the first 1« hours.
As the last of the DCC was consumed, two triplets of identical
intensity appeared at 6 - 8.88 end 7.AO. This new compound was
later identified as the sane dipeptide-DMSO adduct (5b) seen in
the case of the tosylate complex.
There was no further change in the spectrum until a second
equivalent of DCC was added. This Induced precipitation of DCU,
this time paralleled by a decrease of the starting peptide
complex and an increase in the intensity of (5b). At the end of
the reaction, (5b) accounted for about 20% of the starting
material. Other products of the reaction were the
diketopiperazine (17), the N-acyl urea derivative (A) and the
tetrapeptide-DMSO adduct (15).
In an attempt to trap the activated carboxylic group which we
believed was involved in the synthesis of (5a,b), the reactions
uere repeated with one equivalent of the nucleophile N-hydroxy
succinimide (NSu).
5) Tos-Glycylglycine complex 3a with 1 eq. NSu (c=0.02M)
6
-200-
The reactants appeared to be stable towards each other over the
entire reaction period. There was no formation of active ester
(within the NMR detection Units) and only N-acyl urea was being
formed at a rate which was similar to that observed in absence of
NSu (see Results part 1).
6) Tos-Clycyiciycir.e comclex 3a with 1 eo. NSu (c “ 0.2M)
Within the first 5 dilutes of addition cf DCC to a DMSO-d^
solution containing one equivalent of the complex (3a) and one
equivalent of NSu, a triplet and a singlet appeared at & m 9.00 and 6 - 2.71 respectively. The intensity of these signals conti
nued to Increase during the following 45 minutes, during which
time DCU was also forming at an identical rate.
After one hour, two triplets at ^ “ 8.39 and 6 - 6.38 appeared in
the NMR spectrum and their intensity increased steadily during
the next sixty minutes. Finally a third dipeptide derivative
began to form after an acditior.al hour cf reaction (triplets at
5 - 8.75 and 6.39).
The three products of the reaction were later isolated and
identified by Mass Spectrometry as 1) the succinimioe ester of
the complex (13a, m/z 494, 18’ ); 2) the dipeptide-DKSO adduct
(5a, m/z 193, 172) and 3) the succinimioe ester of adduct (5a),
(Ha, n/z 290, 152).
After sixteen hours there was still 372 of starting material
ief L i n solution.
7
-201-
7) HC1 Clycylglyclne complex 3b with 1 eg. NSu (c c 0.02M)
This reaction was similar co that with the tosylate complex
(Results, part 5), except for a slow formation of DCU during the
first 1 0 hours.
8 ) HCl-Glycylglycine complex 3b with 1 eq. KSu (c ■ 0.2M)
In the reaction of DCC with complex (2b) in the presence of one
equivalent NSu (C, 0.2M), the results were similar to those
obtained with the Tos complex (3a). One exception was the
formation of DCU which proceeded at a rate faster than that of
NSu ester (13b).
As the intensity of the ester signals began to steady, two other
products appeared in the spectrum. These were later identified as
the dipeptide-DMSO adduct free acid (5b) and its succinimide
ester derivative (14b).
When two equivalents of NSu were used the reaction gave the same
products, though the active esters were produced to a greater
extent.
9)HC1 Methylamine complex 16.
To elucidate the part played by the carboxylic group in the
reaction mechanism leading to the formation of adducts (5a,b), a
series of reactions were carried out using the crown ether
complex of methylamine hydrochloride (16).
Addition of one equivalent of DCC to a 0.2M solution of (16> in
DMSO-d^, did not induce formation of CCU or methylamine
complex derivatives. However, within ten minutes of the addition
8
-202-
of one equivalent of acetic acid a quartet at & “6.83 and a
doublet at 6 ■ 2.67 appeared in the NMR spectrum. After 26 hours,
this was the major component in the reaction solution and later
was shown to be the DMSO - methylamine adduct (18).
Similar results were obtained with catalytic amounts of acetic
acid, though the formation cf (18) occurred at e slower rate.
Pi scussion
One of the objectives of this study was to find experimental
conditions, under which activation of the carboxylic group of the
amino acid or peptide complexes with Crown Ether would occur
without polymerisation^. Since this phenomenon appeared to be
dependent on the apolar nature of the reaction solvents, the use
of DMSO was thought to prevent the formation of these oligomers.
And indeed in the reactions at higher reactants concentration
the oligomerisation process was largely suppressed, although
the peptide complexes proved to be un-stable in these conditions
due to a reaction between the amino group and the solvent DMSC.
However, when the reactions were conducted in the presence of a
nucleophile (NSu), there was formation of esters, showing that
activation of the carboxylic group had been achieved.
It was felt then, that the elucidation of the mechanisms involved
in these reactions could be used to design those experimental
conditions under which peptide-bond formation, without side
reactions like those encouncerd in these previous studies, is
possible using amino acid protected with'crown compounds.
9
-203-
2It Is postulated chac amino acids react in solution with DCC to
form the highly reactive O-acyl lsourea (6 ). This then either
condenses with a second molecule of amino acid to . form the
carboxy anhydride (7) or, in the case of N-acyl amino acids,
rearranges to the cyclic oxazolone (8 ). All these compounds are
highly reactive towards nucleophiles, whereas the N-acyl urea
(4), which is thoughc to derive from (6 ), is quite stable.
When we attempted a mechanistic explanation of the pher.citer.a seen
in the results section, the following facts were also considered:
1) Peptide or amino acid complexes of Che type described here arc
indefinitely stable in DMSO solution**;
2) At low concentration, activation of the peptide carboxyl group
does not take place as indicated by the lack of any of the
products characteristic of this process;
3) At higher peptide concentration the carboxyl group reacts
with DCC to yield an activated species, as shown by the formacion
of the succinimide ester;
4) The formation of the DMSO adduct (5a,b) is catalysed by the
carboxyl group;
5) DMSO does not react with DCC in the presence of etcher
catalytic or larger amounts of 18-Crown-6/;
6 ) Finally there is ho DCU precipitation from DMSO solutionsQ
containing DCC, 18-Crovn-6 and dry HC1 .
When these observations were combined wich those from the
literature^’ for the mechanism of carboxyl activation by DCC in
polar solvent, the results presented in this paper were readily
explained.
10
-204-
3Thus De Tar and co-workers have demonstrated chat DCC and
carboxylic acids react in solution to form Ionic pairs of the
type shown In structure (9). Here, a second molecule of the acid
component forms an hydrogen bond with the ionic pair thus
facilitating the formation of the reactive anhydride.
In our case, at a peptide concentration of 0 .2 M, the formation of
structure (9) is followed by the reaction with one of the
nucleophiles present in solution. These are K^O, DM£0, -XH0 and
NSu. The different pathways are discussed separately hereafter.
1) H^O: the water is present as an Impurity, either from the
highly hygroscopic solvent or from the peptide complex. In
solution it is presumably solvating the counter-ion, and this
solvation will be greater for the smaller chloride ion than for
the relatively stable tosyiate. The transport of water towards
protoneted DCC will be more efficient in the case of the choride
ion, hence explaining the higher rate of DCU formation seen for
this ion.
2) -NH,: this is available from one of the acid - base equilibria
sr.own in scnane 1 , ar.c car. react to t c m the terTsceptr.de when
water has been consumed.
3) DMSO: the activation of DMSO by DCC in the presence of
carboxlic acids has been shown"". Here the DMSO - DCC adduct (19)
reacts with -NH^ to form the product (5) (see scheme 1).
4) NSu: an alternative pathway is offered to the activated DCC -
COOH complex by the presence in solution of the nucleophile NSu.
The reaction which leads to the formation of the ester is faster
than any of the others described above. Water is therefore 6 till
1 1
-205-
present ir. solution and available to hydrolyse the newly formed
ester bond. An excess of NSu acts upon this equilibrium to yield
more ester.
At lower reactants concentration the protonation of DCC by the
carboxylic group does not take place as Indicated by the absence
cf any of the above reactions. Presumably, stabilisation of the
carboxylic group by DMSO prevents any reaction with the coupling
agent. The formation of the N-acyl urea derivative must therefore
be froc an independent mechanism, and we believe that (4) is
formed by nucleophilic attack of the DCC nitrogen on the
carboxylic C“0.
Cor.c lusions
lr. this paper we have described the reactions that the dipeptide
complexes (3a,b) undertake when solubilised in DMSO solutions
containing DCC. A summary of the different pathways which operate
at highar peptide cc.'.centraricr.s is shewn in schemes 1 end 2. Two
acid - base equilibria, where the base is DCC ar.d the acid
conp-r.er.es are ar.d COCK, exist simultaneously ir. solution.
Depending cr. the presence of a nucleophile such as NSu the ionic
pair (1 1 ) is "activated" to the complex (1 2 ) which in turn
becomes the peptide ester (13). In the absence of NSu only the
pathway leading to the DMSO-peptide derivative operates. As a
corollary cf this study, we have found that activation of the
carboxyl group to the O-acyl isourea does not occur in DMSO
solution at a peptide concentration up to 0 .2 K.
Finally, although by using DMSO we have*been able to avoid the
1 2
-206-
polymerisation that occurres in CHC1„ or MeCS, the reactionjbetween the peptide amino group and DMSO, defied one of the
objectives of this 6 tudy, that is protection of amino acids vlLh
crown ethers during peptide synthesis.
However, the replacement of DMSO with a solvent of equal
6 olvating properties, but less prone to react with DCC, should,
in principle, prevent this, and other, unwanted "side reactions".
Thus in preliminary experiments conducted using N,N-
dioethylformamide as solvent, we have found that the synthesis of
small peptides is possible starting from the amino acid complexes
This study is currently under development and in particular we
are trying to optimise the experimental conditions so that a
method for stepwise synthesis using a non-covalent blocking group
can be proposed.
-207-
References
1) P. Mascagni, C, B. Hyde, M. A. Charalaabous, K. J. Welhaa,
J. Chem. Soc., Perkin Trans. 2, 1937 323-327.
2) D. S. Kemp, The Pepcides, Vol. 1, Chapter 7, 315-383.
3) a) D. F. DeTar, R. Silverscein, J. Am. Chem. Soc., 1966 8 8
1013-1019
b) Z. F. Detar, R. Silverscein, J. An. Chen. See., 1966 8 8
1020-1023
c) D. F. DeTar, R. Silverstein, F. F. Rogers, Jr., J. Am.
Chea. Soc., 1966 38 102A- 1030.
<0 J. P. Taa, W. F. C. Runales, B. W. Erickson, R. B. Merrifield,
Tec. Leccers, 1977 4001.
5) a) K. E. Pfitzner, J. G. Moffatc, J. Am. Chem. Soc., 1965 87
5661-5570.
b) A. K. Fenselau, J. G. More act, J. An. Chea. 3oc., 1966 8 8
1762-1765.
6 ) Anchors observations.
7) The authors tried this reaction to ensure that activation of
the DMSO was noc by 18-crown-6, as shown by D. Marji, J. Chea.
Soc, Chem. Coomun. 1987 7-8.
8 ) This reaction was done to ensure that the activation of DMSO
by iS-crown- 6 was noc acid catalysed.
9) ?. Mascagni, C.B. Hyde, Manuscript in preparation
14
-208-
Table 1. Nuclear Magnetic Resonance and Mass Spectral Data.
c pd Diagnostic NMR Data (f) Mass Spectral Data (m/z)
A a 8.60 (t, 1H, NH cpx) 8.38 (d, !H, NH dec) A. 3 5 (d, 2H , C*.H 2 1 s )
603 [£ N K 3 C H 2 C0N H C H 2 C0NRC0NKR]+ 3 3 9 [ N H 3 CH 2 C ONK C K 2 CONRCCNKR.] +2 1 A [ NKj'CK 2 CONH CH 2 CONR )’+
A b not available 399 [ ( C H )iSNHCHiCONKCEoCONHCONKR]" 27 A [ ( CE 3 ) 2 8N3CH 2CONK CH 2 C0NHR ] "'■
5a 6.38 (t , 1H , NH p e p t ) 6 . A 7 (t, 1H, NH-S) 3.7 0 (m, AH, C » H 2 's)
193 [ ( CE 3 ) -SNHCK^CGNb’CK'. COOH] 'r 1 A 8 [(CH 3 ) 2 N H C H 2 C O N H C E 2I +135 ((CH 3 )2 SNHCH 2 C O N H 2 )+90 [(CH 3 )2 S NH C H 2 1+
5b 8 . 8 8 (t, 1H, N H p e p t ) 7 . AO (t, 1H, NH-S)
193 [ (CH 3 )2 SNHCH 2 C O N K C H ’5COOH] + 177 [(CH 3 )2 SNHCH 2 C O N H C H 2 COH]+ 133 [(CH 3 )2 S NH C H 2 C0NH]+ “90 [(CH 3 )2 SNCH2 )+
13a 9.00 (t , 1H, NH est) A. 2 5 (d, 2H, Co.H2 ’s) 2.71 (s, AH, CH 2 ’s)
A 9 A [f N K 3 CH 2 C O N H C H 2 COON(COCH'>)2 ] + 2 30 [ N H 3 CH 2 C ON H C H 2 C O C N ( C O C H 2 )2 ]’r
13b 9.A3 (t, 1H, NH est) A. 36 (d, 2 H , C y H i 1 s) 2.75 (s. AH, CH 2 T s )
as for 13a.
1 Aa 8.73 (t, 1H, NH-S) 6.39 (t, 1H, NHpept) A . 23 (d, 2H, Cfc.H 2 ' s ) 2.70 ( s , ‘tr., CH 2 ' s )
290I(CH 3 ) 2 SNHCH 2 C O N H C H 2 CO O N ( C O C H 2 )2 ]+
1 Ab 9.10 (t, 1H , NH-S) 7.22 (t, 1H, NHpept) A . 2 7 ( d , 2H , C* H 2 ' s ) 2.75 (s, AH, CH 2 ' s )
2 a 7 ! C H 3 S N K C H - C C N H C H ^ C O O N C O C E ' C H ' ! ~ 1 A 5 [ N H 2 C H 2 C O N H C H 2 C o 6 N ] i '
-209-
fici.b)
(2 )
( 3 Q. b)
(£ iy ii)
(5 a,b)
(6)
(7 i . i i )
(8 a,b)
X e > N H ? C H C O O H
CH3 I - 18-Crown-6CH3 <
NH,CHC-0^ ?R'-'N=CNHR'
0R'NH-C-NR'
COR
X ^ (H3C)2SNHCH2CONHCH2COOH
R'NH-C=NR'I0
0=C-R"
(RC0)20
h 9c - c =oV \
CH2n h 3©
T " © / .
-21-0-
(9 i. R N H - C z N R
©O U O HOC R -' 11V o
R
(15 a.b)
(16)
e ©X (H3C)SN HC H2CO(NHCH?CO)9OH
C|0 <NH?CH3
(17)
(18)
R if Os . N ©
C c- l - l
H' a ' N 'C''0h H©, ©
C l ( H 3 C ) 2 S N H C H 3
//
U
b
R‘
X - Cl
CtH-j*,
©
©? ©A
© <C,r - ~ ~ C h 9NH CO CH - NH 5(CHt U X* L sj
-211-
S c h e m e One
x° O H ' c h ; c o n h c h 7c o o h - r xN hC’c h x o n h c h x o o h . -( C- sj L L
(3 a,b) DCC
0 0X NH3CH2CONHCH2COO
(© H.r- - c (s' < ' iX' '3J2/ XJ
R'N = C- NR'
®N H 3 C H 2 C O N H C H 2 C 0 0
R N - C ~ N H R
Y© 6 X i© S(CHO -
(IS)
X (Cr i-OS ^ _w L I 0 ;1 < ©
R HN-C-NHR'
N Pi 2 e r~i 2 C/ o i \ \J CJ(10)
, , ©
X0 (H.3C)2S=NHCH2CONHCH2CQOK
jj (5a, b)
X0,(H,C)oS0 N H C H X O N H C H 9COOH
-212-
Scheme Two
xe?Nh®CH2CONHCH2COOH ± x eNK?CH2CONHCR2COOK *(3a,b)
(12)
X x o c c
GS © 0X i NH3CH2CONHCH9COO
NSu
©R N -C-NHR
(11)
©X S NH 3CH 2C ONHC H 2C 0 0
HON© ,
R N = C = NHR
- H 2 0
0 11X - C H
X - C H110
0
X ’ $n h 3c h 2c o n h c h 2c o o n 'r _ r u
O I \ '
(13)
n \ DCC,DMSO (3a,b) + NSu o
p > g C-CHX (H3C),S NHCH2CONHCH2COON' '
C “ C H
S c h e m e T h r e e
RCOOH + R N - C - N R
a
v
f p p ® D'm l j-/-' - k;D*l u l l - r\ i s|r, - - N hJ\j *
0 - H H - 0I pm IH X ® H
n H\ /0
r 'n h c =n r ‘
H H
-H©
0K NHC NHR"
X© 0 ©
Cl , Tos
-21 A-
THE USE OF CROWN. ETHERS IN PEPTIDE CHEMISTRY: PART 3 SYNTHESIS OF AN ENKEPHALIN DERIVATIVE
USING I8 -CROUN- 6 AS A NON-COVALENT AMINO PROTECTING GROUP
Carolyn B. Hyde, Paolo Mascagni*
Department of Pharmaceutical Chemistry, The School of Pharmacy, University of London, 29- 39 Brunswick. Sq., UC1N 1AX London, UK
Summary: The efficacy of amino acid protection with crown ethers is demonstrated by the solution synthesis of an enkephalin pentapeptide derivative. Extraction of the organic phase with a KC1 solution after each coupling steps Is used for the deprotection of the peptide Intermediate.
The use of crown Compounds for the protection of the amino group of amino acids offers, In principle, some advantages over the more commonly used groups such as t-boc and fmoc.Thus the non-covalent nature of the Interaction between the crown ether and the ammonium Ion and their large affinity for Inorganic lons^ can provide the basis for a rapid but mild protection and deprotection scheme.In order to te6 t this hypothesis we have studied the behaviour of amino acid and peptide complexes with 18-crown-6 in various organic solvents and In the conditions usually employed for peptide synthesis^*^.When the alanine complex 1 was reacted with equivalent amounts of 1,3-dicyclohexyl- carbodilmide (DCC) In either chloroform of acetonitrile, it was found that the hydrogen bond In the 0 -acyl derivative 2 induced, first deprotection of the amino group and subsequently oligomerisation of the amino acid (scheme 1). To favour solute-solvent Interactions and thus avoid the amino group deprotection, DMSO was hence used as the reaction solvent. Furthermore the alanine complex was replaced with Che dlpepclde complex 3 in an attempt to separate the amino group from the DCC nitrogen in the 0-acyl derivative2. Under these new conditions Che oligomerisation process resulted Inhibited; however complex reacied with the solvent to form the DMSO-peptide dehydration product A.Further progress toward the initial aim of this study was made possible by the elucidation of the mechanism leading to A and In this communication we show that selective coupling is achieved in good yields by using dipeptide complexes and dimethylformamide (DMF) as reaction solvent. The synthesis of the ( a-ami node c’anoyl]-5-enkephalin derivative 12 is described as an example of the successful application of this scheme.
-215-
1 X * ^ H jN-CH(CH3 )-COOH & C L ' ^ H jN-CHj -COO-CKj-CHj
1 tos ' ( +h 3n-ch2-conh-ch2-cooh „conh-ch2cooh2 CF,COO" \ H,N-CH
♦ - 3 I " ch,-c,h.-o-ch,-c,h«a (CHj)2SNH-CH2-CONH-CH2-COOH .X Z 6 4
2 TOs’ ^ +HjN-CH2-CONH-CH-COOH £ h 2N-CH2-CONH-CH2-CONH-CH2-COO”CH2-CHjCH,I i .C6Hft CL” . HjN-CH-COO-CHj 2 n ■ 11
(CH2)n Ifl b - 7CH,
11 TFA_ - HjN-CH2-CONH-CH-CONH-CH-COO-CHjch2 (ch2)7C6Hj CH,
1 2 tfa“ . H,N-CH-CONH-CH2-CONH-CH2-CONH-CH-CONH-CH-COO-CHjch2 ch2 (CH2)7c6h4 c6h 5 ch,O-Bj
CROWN ETHER X • CL. TOS
The dipepcide complexes 3,5 and 7 were prepared according to procedures described in the earlier work **2. Before use they were re-crystallised and shown to be homogeneous by elemental analysis and ^H-NMR. To optimise the reaction conditions the synthesis of the glycine tripeptide 8 was performed and It was found that the use of one-to-two fold excess complex improved the yields to about 85%. Reactions were typically carried out as follows: Cly-OEt 6 (140 mg, lmmol) and triethylamine (0.14 ml, 1 mmol) were dissolved in DM? (2 ml) and mixed with a DMF solution (3 ml) containing one equivalent each of complex 3 and DCC. After 24 hrs dlcyclohexylurea (DCU) and solvent were removed; the resulting oily material dissolved in chloroform and washed with dil. HC1, H 2O and dll. NaOH. To remove the crown protective group a final extraction with a saturated KC1 solution (pH 8 ) was performed. Evaporation of the solvent afforded the glycine tripeptide 8 whose degree of purity and chemical composition were determined by ^H-NMR and FAB mass spectrometry. Similar results were obtained when the fatty-amino acid esters 9 and 10 were used in place of the glycine ester 6 .The synthesis of the enkephalin derivative 12 was performed as follows. Trlpeptlde 11 was prepared in 82% yield from the Gly-Phe complex $ and the ester 10 , using an HOBT-DCC mediated coupling. After the extraction and deprotection procedures described above, 11 wa6 purified by semi-preparative reverse phase HPLC and its chemical composition
-216-
SCHEME 1
£+HjNCCH.DCC _ 4 i 3
NCHCOOH ------------ X • HjNCHCO^ ♦CH, H.% 0
^ RN-C-NMR
2DIPEPTIDE ♦ DCU -------| H2° oligopeptides ♦ DCU
^ 3 CROWN ether
ascertained by NMR and FAB mass spectrometry^. Purified trlpeptlde ester (27 mg, O.OS mmoles) and trlethylamlne (7.1 ul, 1 eq) were then dissolved In chilled DMF (2 ml) and added to a second chilled DMF solution containing two equivalents each of HOBT (15.6 mg), DCC (21 mg) and the Tyr-Gly complex 7 (50.8 mg). The reaction was allowed to proceed at r.t. for 24 hrs before DCU and solvent were removed. Deprctectlon with a saturated KC1 solution and purification by reverse phase HPLC afforded the O-benzyl pentapeptlde 12 in about 50Z yield^.
TH3H jNCHCO
0RHN-C-NHR
References and notes
1. M.Hiraoka, "Crown compounds: their characteristics and applications", Elsevier Publ.s, Amsterdam, 1982.
2. P.Mascagni, C.B.Hyde, M.Charalambous and K.J.Welham, J.Chem.Soc., Perkin trans.ll, 323- 327, (1987)
3. C.B.Hyde, K.J.Welham and P.Mascagni, J.Chem.Soc.,, Perkin trans. II, in press.4. FAB mass spectra were run on a VCZAB-SE spectrometer:
M*- (less counter-ion), expected for 11 , 406; M+ found, 406.1H-NMR spectra were run on a Bruker AM-500 spectroraeier; using CDCI3 as solvent and TMS as internal standard 11 gave the following 5 values:
- 2 1 7 -
7.95 (d,lH, NH-Phe); 7.67 (d,lH, NH-aminodecanoyl); 7.74 (br,l-2H, NH 3+-Cly);7.18 (m,5H, Ar-Phe); 4.85 (a,lH, H a-aminodecanoyl); 4.33 (m,lH, Ha-Phe); 3.98 (d,lH,Ha -Gly); 3.65 (d,lH, Ha -Gly); 3.63 (s,3H, COOMe); 2.85 (m,2H, Hp-Phe); 1.55 (br,14H, CH2 1s-aminodecanoyl); 0.88 (t,3H, CH3-aminodecanoyl).
5. Mf calculated for 12, 716; M* found, 716.^-NMR (DMSO-D6 ; 8.73 (br.lH, NH-Phe); 8.48 (d,lH, NH-aminodecanoyl); 8.18 (s,2H, NH3+- Tyr); 7.77-6.96 (Ar-Phe, Tyr, O-Bz plus NH-Gly); 5.51 (t.lH, NH-Gly); 5.07 (a,2H, CH2- OBz); 4.63 (m,1.3H, Ha-aminodecanoyl); 4.22 (m,lH, Ha-Phe); 3.99 (m,lH, Ha-Tyr); 3.86-3.65 (dd's,4H, Ha -Cly's); 3.62 (e,3H, COOMe); 3.04-2.77 (m,4H, Hp-Tyr, Phe); 1.58-1.51 (m,4H, Hp ,jf -aminodecanoyl); 1.24 (br,10H, CH2 '6-aminodecanoyl); 0.84 (t,3H CH3-aminodecanoyl).
-218-
R E F E R E N C E S
A 1 m y J . , G a r w o o d D. C. , C r a m L) . J . ,
J. Am. C h e m . S o c . 18 7 0 92 4 32 1
A n d e r s o n G. W . , M c G r e g o r A. C . ,
J . A m . C h e m . S o c . 1 9 5 7 7 9 6 180
A n d e r s o n G. W . , Z i m m e r m a n J. E . , C a l l a h a n F . ,
J . Am. C h e m . S o c . 1 9 6 3 85 3 0 3 9
A n d e r s o n G. \V . , Z i m m e r m a n 3 . F. , Cal 1 a h a n F . ,
J. Am. C h e m . S o c . 1 9 6 4 8 6 183 9
B a r re 1 1 A . G . M . , L a n a J . C . A . ,
j . C h e m . S o c . C h e m . C o m m a n . 197 8 471
B a r 1 s c h R . A . , M i n t z E . A . , Fa r 1 m a n 11 . M . ,
J . A m . C h e m . S o c . 197 1 96 4 2 4 9
B el 1 e a u B . , M a 1 eK G . ,
J . A m . C h e m . S o c .
B e n - l s h a i 1) . , B e r g e r A. ,
J . O r g . C h e m .
B e n o i t o n N . L ., C h e n F . M . F .,
C u n a d . J . C h e m .
B e r g m a n n M . , Z e r v a s L . ,
B e r . d t s c h . C h e m . G e s . 1 9 3 2 6 5 1 1 9 2
B o d a n s z k y M .,
N a l u r e I 9 5 5 17 5 6 8 5
B o d a n s z k y M . ,A n n . N . Y . A c a d . S c i . 1 9 6 0 8 8 65 5
B o d a n s z k y M . , F u n k K. W . , F i n k M. L .,
J. O r g . C h e m . 1 9 7 3 38 3 5 6 5
1 9 68 9 0 16 5 1
19 5 2 17 15 6 4
1 981 5 9 3 8 4
4 3 3 0
6 18 3
184 2
3 7 2
4 2 52
16 5 2
15 70
3 8 9
120 1
6 6 1
3 5 7 0
-219 -
B o d a n s z k y M. , R o n d o M . , Y a n g L i n C. , S i g l e r G. F. ,
J. O r g . C h e m . 197 4 3 9 4 4 4 - 44 7
B o d a n s z k y M. , K l a u s t i e r Y. S. , O n d e t t i M. A. ,
P e p t i d e S y n t h e s i s
W i l e y 1 9 7 6 13 - 17
B o i s s o n n a s R. A . ,
H e 1 v . C h i m . A c t a 19 5 1 3 4 8 7 4 - 8 7 9
B r a n d s L r o m A . , G u s t a v i i K . ,
A c t a C h e m . S c a n d . 1 9 6 9 23 12 1 5 - 12 18
B r a t by 1). M. , C o y l e S. , G r e g s o n H. P. , H a r d y G. W . ,
Y o u n g G . T .,
J . C h e m . S o c . P e r k i n T r a n s .J 19 79 190 1 - 1 9 0 7
G a m b l e It. , G a r n e r R. , Y o u n g G. T. ,
N a t u r e 1 9 6 8 2 1 7 2 4 7 - 2 4 8
G a m b l e R . . G a r n e r R . , Y o u n g G. T . ,
J . C h e m . S o c . ( C ) 1 9 6 9 1 9 1 1 - 1 9 1 6
C a r p i n o L . A . ,
J . Am. C h e m . S o c . 1 9 5 7 79 9 8 - 1 01
C u r p i n o L . . A.,
J . A m C h e m . S o c . 19 5 7 7 9 4 4 2 7 - 4 4 31
C a r p i n o L . A . , H a n G. Y. ,
J . Am. C h e m . S o c . 1 9 7 0 9 2 5 74 8 - 5 74 9
C a r p i n o L . A . , H a n G. Y. ,
J. O r g . C h e m . 197 2 3 7 3 4 0 4 - 3 40 9
C a s t i g l i o n e - M o r e l 1 i A. M. , L e l j F. , P a s t u r e A. ,
S a l v a d o r i S. , T a i l o r e d i T. . T o m a t i s R. , T r i v e i l o n e F. ,
T e m u s s i P . A . ,
.1 . M e d . C h e m . 1. 9 8 7 30 2 06 7 - 2 07 3
-220-
C h a o Y ., C r a m D .J .,
J . A m . C h e m . S o c . 197 6 9B 10 15
C h a o Y . , W e i s m u n G. R . , S o g a h G. 1). Y. , C r a m D . .J .
J . Am. C h e m . S o c . 1 9 7 9 101 4 9 4 8
C h o r e v M. , K l u u s n e r Y. F. ,
C h e m . C o m m o n . 1 9 7 6 5 9 6
C r a m I) . J .
A n g e w . C h e m . Int. Ed. E n g l . 1 9 8 6 2 5 103 9
C r a m 1). J . , T r u e b l o o d K. N. ,
C h a p t e r 2, H o s t - G u e s t C h e m i s t r y 1
T o p i c s in C u r r e n t C h e m i s t r y 98.
Ed. B o s c h k e F. L . ,
S p r i n g e r V e r l a g 1 9 8 1 4 3 -
C u i' L i u s 1 . ,
J . P r a k L . C h e m . 1 881 21 2 3 9
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