An improved method for anchoring of 9-fluorenylmethoxycarbonyl-amino acids to 4-alkoxybenzyl alcohol resins.

Tetrahedron Letters,Vol.28,No.49,pp 6147-6150,1987 Printed in Great Britain

0040-4039187 $3.00 + .oo Pergamon Journals Ltd.

AN IAPROVED METHOD FOR ANCHORING OF 9-FLUORENYLMETHOXYCARBONYL-AMINO ACIDS TO I-ALKOXYBENZYL ALCOHOL RESINS. Peter Sieber Pharmaceuticals Division, CIBA-GEIGY Ltd, CH-4002 Basel, Switzerland Abstract: Esterification of Fmoc-amino acids to 4-alkoxybenzyl alcohol polystyrene by 2,6-dichlorobenzoyl chloride represents a convenient method. It is free of the two side-reactions observed with dicyclohexylcarbodiimide/ 4-dimethylaminopyridine, viz. racemisation and dipeptide formation. Anchoring

of the C-terminal amino acid onto I-alkoxybenzyl alcohol resin' has generally been accomplished by dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP)2. However, this method suffers from two problems: I. The use of DMAP to catalyze the coupling of urethane-protected amino acids has been shown to promote racemization 3 . II. In the course of anchoring Fmoc-amino acids

dipeptide

decrease

these

formation

can occur due to the basic character of DMAP 4 . To

side reactions various modifications were suggested5, but all

have

the disadvantage that only low substitution levels (ca. 0.2 mmol/g) are obtained. In

an

effort

esterification

to

overcome these drawbacks we tested some other methods for the attachment of Fmoc-amino acids to 4-alkoxyben-

syl alcohol polystyrene, the Wang resin. We chose Fmoc-Asp(OBut)-OH and FmocPhe-OH for esterification because these amino acids are known to racemize very easily.

The results are summarized in table 1. For comparison the amino acids

were also anchored with DCC/DMAP2 (entries 1 and 10). Most of the tested methods suffered from low functionalization and/or partial racemization. The D-asstrinpartic acid content was low with Geiger's method', but even under more gent

conditions

acceptable

the loading capacity could not be improved (entries 6-8). An

functionalization and low racemization resulted with the Mitsunobu

reactionll (entry 13), but TLC analysis of Fmoc-Phe-OH after cleavage from the resin

with

trifluoroacetic acid (TFA)/1,2-dichloroethane (DCE) 1:l showed an

unknown lipophilic by-product. Yamaguchi 14 has published

a method for the synthesis of macrocyclic lac2,4,6-trichloroin which he applied a mixed anhydride produced from 15 modified the method by using the commercially availbenzoyl chloride. Reese able 2,6-dichlorbensoyl chloride for the esterification of a thymidine derivatones,

tive. The results we obtained with this approach are summarized in table 2. In a typical experiment, 10 g resin (0.74 mm01 hydroxymethyl groups/g) and 5.7 g Fmoc-Phe-OH (2 eq) are shaken in 50 ml DMF at r.t. for 15 minutes. 2 ml pyri6147

6148

Table 1. Esterification of Fmoc-amino acids to 4-alkoxybenzyl alcohol polystyrene by different methods (a,b). Entry

8 9 10 11 12 13 14 15 16

Fmoc-aa

Method

Catalyst eq

Asp(OBut)

DCC OTcp OTcp OTcp 50°C OTcp PPA PPA PPA 50°C (COC1)2+DMF DCC OTcp OTcp in CH Cl Ph P/EtOOC&=&-CooEt Bog-Cl Piv-Cl PhOP(0)C12

DMAP 0.1 Imidazole 3 HOBt 0.1 HOBt 0.1 HOBt 0.1, PY 5 --PY 5 PY 5 PY 5 DMAP 0.1 DMAP 0.1 DMAP 0.1 ----PY 3 PY 6

Phe

Ref. 2 7 a 9 10 2 11 12 13

Subst.c %D-aad 0.50 0.07 0.13 0.26 0.17 0.31 0.33 0.27 0.3 0.53 0.34 0.24 0.45 0.21 0.36 0.3

4.2 9.3 1.1 2.9 1.6 0.4 0.8 0.8 12.0 7.3 1.3 0.8 0.3 0.3 0.4 0.4

a. Standard conditions: 0.5g 4-alkoxybenzyl alcohol polystyrene (0.74 mmol/g), 2 eq- Fmoc-amino acid in 5 ml DMF, 15-20 h r.t. (unless otherwise indicated). b. Abbreviations: aa = amino acid. OTcp = 2,4,5-trichlorophenylester. HOBt = 1-hydroxybenzotriazole. Py = pyridine. PPA = n-propylphosphonic anhydride. chloride. Piv-Cl = BOP-Cl = bis(2-oxo-3-oxazolidinyl)phosphorodiamidic Pivaloyl chloride. c = mmol/g. The extent of functionalization of the resin was measured by UV determination of the 9-fluorenylmethylpiperidine after cleavage of the Fmoc-group with piperidine. d. The Fmoc-free amino acid was cleaved from the resin with6TFA/DCE 1:l and analyzed by GC on a "Chirasil-Val" column after derivatization . Table 2. Fmoc-amino benzoyl chloride. Entry

7 8 9

10 11 12

acid

Fmoc-amino acid Asp(OtBu) Arg(Mtr) Glu(OtBu) Gly Ile Leu Lys(Boc) Met Phe Ser(tBu) Thr(tBu) Tyr(tBu)

I-alkoxybenzyl esters prepared with 2,6-dichloroSubstitution mmol/g 0.49

0.36 0.45 0.41 0.41 0.46 0.42 0.45 0.47 0.45 0.42 0.47

% D-amino acid a 0.5 < 1.0 b 0.3 0.4 0.1 0.3 0.7 0.3 0.3 0.4 0.3

a: The data given have not been corrected with the D-enantiomer content of the starting Fmoc-amino acids (O.l-0.5%). b: Determined by HPLC as Leu-Arg after derivatization with Boc-Leu-OSu and cleavage with TFA. dine (3.3 eq) and 2.1 ml 2,6-dichlorobenzoyl chloride (2 eq) are added successively and the suspension is shaken for 15-20 h. After washing, any remaining hydroxyl groups of the resin are benzoylated with 3 ml benzoyl chloride and 3 ml pyridine in 80 ml DCE for 2 h 16 . If desired, a higher loading level can easily be obtained, e.g. by starting from a higher substituted resin (2.07 mmol hydroxymethyl groups/g). The reac-

6149

tion with 1 eq Fmoc-Phe-OH, 1.1 eq 2,6-dichlorobenzoyl chloride and 2 eq pyridine

for

18

h resulted in 0.77 mmol Phe/g resin, containing 0.2% D-Phe (not

included in the tables). As already mentioned, another problem of the esterification to the resin is the

formation of dipeptides due to a premature cleavage of the Fmoc-group un-

der

basic

produce

conditions. Esterification with the DCC/DMAP method is reported to

up

to

9% dipeptide17. No dipeptide could be detected by HPLC in the

glycine-resin prepared by the 2,6-dichlorobenzoyl chloride procedure. Histidine

is

especially

different

pathways'8.

Mitsunobu

reaction

In

prone the

(table

to

racemization, which can take place by

esterification of Fmoc-His(Trt)-OH1g only the

3, entry 3) produced a low level of racemization,

but TLC analysis of the cleaved Fmoc-His-OH showed, as in the case of Phe, an unknown lipophilic by-product. Racemization takes also place with the n-substituted N-1m -tert.butyloxymethyl(Bum) derivative2' (entries 4 and 5). The could be solved by using Trt-His(Trt)-OH (entry 7). The Na-Trt group cleaved selectively with 0.1 N HCl in DCE-MeOH 95:5 without affecting the NIm -Trt group 19 .

problem can be

Table 3. Esterification of histidine- and cysteine derivatives to rl-alkoxybenzyl alcohol polystyrene by different methods. Entry 1 2 3 4 5 6 7 8 1: 11

% D-aa

subst. mmol/g

Amino acid

Method

Fmoc-His(Trt) do. do. Fmoc- His(Bum) do. Trt-His(Trt) do. Fmoc-Cys(Acm) do. do. Fmoc-Cys(Trt)

DCC/DMAP dichlorobenzoyl Mitsunobu DCC/DMAP dichlorobenzoyl DCC/DMAP dichlorobenzoyl DCC/DMAP dichlorobenzoyl Mitsunobu dichlorobenzoyl

15.4 27

0.37 0.40 0.39 0.33 0.12 0.5 0.31 0.48 0.48 0.39 0.41

chloride chloride chloride 5 eq chloride chloride

::; 2.2 1.8
Cysteine is a further troublesome amino acid. The acetamidomethyl (Acm) deesterified with DCC/DMAP under mild conditions, e.g. with 5 eq rivative was amino

acid

only

0.02

eq DMAP for 1 h (table 3, entry 8). Even so 2.9%

acid is produced. The dichlorobenzoyl chloride (entry 9) and the Mit-

D-amino sunobu

and

(entry

10) methods resulted both in lower racemization. TLC analysis the three products showed several by-products. For comparison we also analyzed a commercially available Fmoc-Cys(Acm)-resin and found 4.2% D-Cys and similar by-products as in our samples. Racemization was also found with the

of

11). These

S-Trt derivative(entry of

the

most

We have be

performed

With

the

zation, of

critical shown

histidine

of

with

the

other could

derivate

to

Fmoc-amino

procedure

formation

Trt-His(Trt)-OH

again

respect of

one-pot

cysteine,

no dipeptide the

acids

esterification

by a simple

exception and

amino that

results

with amino

indicate

that

is

one

racemization. acids

to

the

Wang resin

2,6-dichlorobenzoyl acids

showed

be detected. should

cysteine

be used.

For

very

the

can

chloride. low

racemi-

esterification

6150

Acknowledgement. I thank Mrs. J. Seeberger for her excellent technical assistance, Mr. H. Miiller and Mr. G. Farrugio for the GC analysis. References and Notes. 1. G. Barany and R.B. Merrifield, in "The Peptides," eds. E. Gross and J. Meienhofer, Academic Press, New York, 1980, vol. 2, p.53-54. 2. see ref. 1, p.65. 3. E. Atherton, N.L. Benoiton, E. Brown, R.C. Sheppard, and B.J. Williams, J. Chem. Sot., Chem. Commun., 1981, 336. 4. E. Atherton, C.J. Logan, and R.C. Sheppard, J. Chem. Sot., Perkin Trans. I, 1981, 538. 5. a: see ref. 3; b: J.W. van Nispen, J.P. Polderdijk, and H.M. Greven, Reel. Trav. Chim. Pays-Bas, 104, 99 (1985), and ref. cited therein. 6. H. Frank, G.J. Nicholson, and E. Bayer, J. Chromatogr. Sci., 15, 174 (1977). 7. M. Bodanszky and D.T. Fagan, Int. J. Peptide Protein Res., lo, 375 (1977). 8. Y.S. Klausner and M. Chorev, J. Chem. Sot., Chem. Commun., 1975, 973. 9. H. Wissmann, W. KGnig, V. Teetz, and R. Geiger, in "Peptidesl980," Proc. 16th Europ. Peptide Symp., ed. K. Brunfeldt, Scriptor, Copenhagen, 1981, p. 174. 10. P.A. Stadler, Helv. Chim. Acta, 61, 1675 (1978). 11. 0. Mitsunobu, Synthesis, 1981, 1. 12. J. Diago-Meseguer, A.L. Palomo-Co11, J.R. Fernandez-Lizarbe, and A. Zugaza-Bilbao, Synthesis, 1980, 547. 13. H.-J. Liu, W.H. Chang, and S.P. Lee, Tetrahedron Lett., 1978, 4461. 14. J. Inanaga, K. Hirata, H. Saecki, T. Katsuki, and M. Yamaguchi, Bull. Chem. Sot. Japan, 52, 1989 (1979). 15. J.M. Brown, C. Christodoulou, C.B. Reese, and G. Sindona, J. Chem. Sot., Perkin Trans. I, 1984, 1785. 16. A control experiment showed that 2,6-dichlorobenzoyl chloride reacts very slowly with the Wang resin in the presence of pyridine. 17. E. Pedroso, A. Grandas, M.A. Saralegui, E. Giralt, C. Granier, and J. van Rietschoten, Tetrahedron 2, 1183 (1982). 18. J.H. Jones, W.I. Ramage, and M.J. Witty, Int. J. Peptide Protein Res., 15, 301 (1980). 19. P. Sieber and B. Riniker, submitted for publication to Tetrahedron Lett. 20. R. Colombo, F. Colombo, and J.H. Jones, J. Chem. Sot., Chem. Commun., 1984, 292. (Received in Germany 3 September 1987)