Design of Organophosphorus Reagents for Peptide Synthesis

41 Design of Organophosphorus Reagents for Peptide Synthesis R. RAMAGE, Β. ATRASH, and M. J. PARROTT

Downloaded by UNIV OF SYDNEY on July 25, 2013 | http://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch041

Department of Chemistry, The University of Manchester Institute of Science and Technology, Sackville Street, Manchester M60 1QD, England

Two crucial aspects of the stepwise synthesis of peptides are the temporary protection of α-amino functions and activation of carboxylic acids to enable facile formation of amide bonds.The amino protecting groups should be stable,except under specific mild cleavage conditions,and must not lead to diminished stereo chemical integrity of the protected α-amino acid during activation. Criteria for the choice of activation procedure adopted for the carboxylic acid function are no less stringent requiring rapid, highly efficient amide formation during the repetitive steps leading to the synthesis of polypeptides. One of the most successful classes of amino protecting groups is that based on the t-butyl urethane which may be cleaved by mild acid.Structural variation gives rise to groups susceptible to deprotection over a range of acid conditions.A disadvantage of this type of protection is the formation of carbenium ions during the deprotection process which can react with side chain functionality of cysteine,methionine,tryptophan or tyrosine leading to alkylated products.Although this can be mitigated by use of scavengers it was thought desirable to design another series of protecting groups which have the same propensity towards acid cleavage,but which occasion no deleterious side reactions during deprotection.lt was decided to investigate for this purpose the utility of the remarkable acid lability of the P-N bond in phosphinamidates.—Careful mechanistic researches have led to results which would suggest that acid-catalysed solvolysis of phosphinamidates can proceed via trigonal bipyramidal intermediates thus producing no reactive intermediates capable of entering side reactions.In order to maximise the effect of substituents it was judged that phosphinamidates would be capable of a wider range of reactivity towards acid hydrolysis than phosphoramidates which would also suffer from the disadvantages of offering two modes of fragmentation of the trigonal bipyramidal intermediate during solvolysis.Preliminary worklshowed that the Pt^PO.NHR group is more acid-labile than BuO.CONHR,therefore a series of protected amino acids were prepared using the readily accessible Ph^PO.Cl t

0097-6156/81/0171-0199$05.00/0 ©

1981

American Chemical Society

In Phosphorus Chemistry; Quin, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

200

PHOSPHORUS

CHEMISTRY

and these have now been i n c o r p o r a t e d i n t o a programme aimed towards the s y n t h e s i s of prohormones.Recent researches d i r e c t e d to the a p p l i c a t i o n o f phosphinamidates i n t h i s area have i n v o l v e d the mechanistic study of the a c i d - c a t a l y s e d methanolysis o f a s e r i e s o f phosphinamidates d e r i v e d from 3-phenylethyl amine i n c o r p o r a t i n g s u b s t i t u e n t s on phosphorus which were s e l e c t e d i n order to d e f i n e the optimum reagent f o r use i n peptide s y n t h e s i s . R P(0).NHCH CH Ph HCl/MeOH ^ ( Q ) ^ H^CH^H^h C l "


2

2

2

>

+

K i n e t i c r e s u l t s f o r a s e r i e s o f such r e a c t i o n s are given i n Table 1 and show an i n t e r e s t i n g combination of s t e r i c and e l e c t r o n i c e f f e c t s o f the s u b s t i t u e n t R.From X-ray d i f f r a c t i o n data on Ph P(0).N(Me)CH CH Ph i t could be seen that the geometry o f s u b s t i t u e n t s a t the n i t r o g e n atom i s non-planar and that only one phenyl r i n g was o r i e n t e d to allow i n t e r a c t i o n with the P=0 bond. Comparison of r a t e data i n Table 1 f o r the phosphinamidates R2=Ph ,R =Me and R =Me/Ph shows the l a t t e r to have the optimum balance o f s t e r i c e f f e c t (Me) and e l e c t r o n i c e f f e c t (Ph)for f a c i l e h y d r o l y s i s . T h e r a p i d onset of s t e r i c r e t a r d a t i o n may be seen from comparison of the r a t e s o f hydroLysis of dimethylphosphinamidates with the higher d i a l k y l analogues.Unfortunately the dimethyl s e r i e s proved too hygroscopic to be u s e f u l i n peptide s y n t h e s i s , however the diethylphosphinamides show promise f o r s i d e - c h a i n amino p r o t e c t i o n which r e q u i r e s r e l a t i v e l y g r e a t e r a c i d s t a b i l i t y . C a r b o x y l i c mixed anhydrides are very important f o r the r a p i d s y n t h e s i s o f peptides by the stepwise procedure,however the use o f c a r b o x y l i c mixed anhydrides,e.g.those d e r i v e d from p i v a l i c a c i d and a p r o t e c t e d amino a c i d ( 1 ) , s u f f e r s from two disadvantages. F i r s t l y , r e g i o s p e c i f i c i t y o f a t t a c k a t the d e s i r e d c a r b o x y l f u n c t i o n i s l a r g e l y determined by s t e r i c e f f e c t s and w i l l not be 100% f o r a l l c o u p l i n g reactions.Secondly,such mixed anhydrides have a propensity towards d i s p r o p o r t i o n a t i o n to symmetric anhydrides which i s h i g h l y u n d e s i r a b l e i n terms of r e a c t i o n e f f i c i e n c y . T h i s l a t t e r process can be depressed by o p e r a t i o n o f the r e a c t i o n at -15 °C, but with the concurrent decrease i n r e a c t i o n r a t e and,on l a r g e s c a l e manufacture,increased c o s t s . 2

2

2

2

2

2

2

2

Ç X.NH.CH.CO.O.COBut (I) K

J

2

f-

X.NH. CH. CO. 0. Ρ (0)R R1 r

(2) K

o

1

R R -> X.NH.CH.C0NH.OH.COOMe

l

J

H NCHC00Me R P(0)0H (3) With these c o n s i d e r a t i o n s i n mind i t was decided to i n v e s t i g a t e p h o s p h i n i c - c a r b o x y l i c mixed anhydrides i n peptide methodology^.Mechanistic c o n s i d e r a t i o n o f the r e a c t a n t s (2) and (3) and products shown above would suggest r e g i o s p e c i f i c nucleop h i l i c a t t a c k by the amine component due to the formation o f an amide bond with concomitant generation o f a new P-0 bond.As i n the study of phosophinamidates d i s c u s s e d above,a s e r i e s of phosphinic acids were s e l e c t e d f o r p r e p a r a t i o n o f the mixed anhydrides (2) because of the i n t i m a t e s t e r i c and e l e c t r o n i c ?

?


In Phosphorus Chemistry; Quin, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981. 5

Table

1

2

2

Me,Ph

q-p

P h

Me

n-Bu

2

88. 0

22

52.8

32

36. 0

too

f a s jt

to

measure? by HPLC

94.7

17

68. 7

21

53.6

28

41. 0

153.5

520.0

4

326. 0

6

201 . 0

8

145. 0

13

27.0

75

15. 4

111

10.4

169

6. 8

26.1

78

14. 8

118

9.7

169

6. 8

2

45

8.0

°

197

37

5. 9

ο

287

4.0

373

3. 1

(PhCH )

2

k

k

2

30 k

ο k

25

2

E t

R

ο

8

12

2

43

44

144

\

Rate Constants (s" χ 10 ) and H a l f - l i v e s (min) f o r Acid C a t a l y s e d M e t h a n o l y s i s of Phosphinamidates R P(0)NHCH C H / h

1



202

PHOSPHORUS

Table Rate

Constants

2 1

(1.mol" .s"

Disproportionation Diphenylphosphinic-Amino

Amino Acid Z-Gly Z-Ala Z-Leu Z-Phe Z-Val Z-Ply

0

°

3.3 1.4 0.5 1.4 11.7 —

30

CHEMISTRY

°

15.5 22.0 7.4 6.7 43.0 52.2

1

χ

5

10 )

f o r the

of Acid

40

Anhydrides

°

49.2 62. 4 18.1 26.0 67.3 135.0

50

138 113 40 75 109 248

°

Time 10% (min) 74 143 390 170 17 56


41.

RAMAGE E TAL.

Organophosphorus

Reagents for Peptide Synthesis

o\°


Ο

Φ

C

•Η

ε

οο ο ο t n ro r o οο Η Η OJ

σ>

h

ro

«Ή

Design of Organophosphorus Reagents for Peptide Synthesis

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