Indian Journal of Chemistry Vo\. 38B, April 1 999, pp. 4 1 8-423

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Convenient and simple synthesis of N- { [(9H-fluoren-9-yl)methoxy]carbonyl } (Fmoc) protected �-amino acids (=homo-a-amino acids) employing Fmoc-a

amino acids and dicyc1ohexylcarbodiimide(DCC) mixtures

K Ananda & V V Suresh Babu*

Department of Studies in Otemistry, Central College Campus, Bangalore University, Banga10re 560 00 1 , India

Received 29 October 1998; accepted (revised) 13 January J 999

A simple approach for the homologation of a-amino acids to �-amino acids by the Arndt-Eistert method employing Fmoc-a-amino acid and N, N'-dicyc!ohexylcarbodiimide (DCC) mixture for the acylation of diazomethane, synthesizing the key intermediates Fmoc-a-amino acyldiazomethanes as crystalline solids is described.

�-Amino acids (�-substituted �-amino acids), though less abundant than a-amino acids, are components of natural peptides ' ,2. They are also useful chiral building blocks for the synthesis of �-Iactam antibioticsJ,4. �-Peptides, i .e. oligomers of �-amino acids, containing minimum six units are found to form surprisingly stable helices5.8. The greater structural variability of possible �-amino acids leads to an even greater multitute of possible �-peptide primary and secondary structures9• 14. The �-hexapeptide, (H-�HVal-�-HAla-�-HLeuh-OH and its dimer, i.e" the corresponding �-dodecapeptide, are found to have the 3 1 -helical secondary structure until now. Furthermore, �-peptides carrying the common proteinaceous side chains of amino acids such as Ala, Val, Leu, Phe, Lys have been shown to be stable to common a-peptidases for at least two days ' 5,

The homologation of a-amino acids is the most important single route for the asymmetric synthesis of �-homoamino acids. The Arndt-Eistert reaction for the synthesis of homologous optically active �-amino acids from their a-amino acid counterparts is based on the findings that the Wolff rearrangement of diazoketones containing a chiral center next to the carbonyl group occurred with retention of configuration 1 6. 1 8 . The Wolff rearrangement of adiazoketones can be accomplished thermally, photochemically, by metal ion (Ag+) catalysis or promoted by ultrasound'9.

The acylation of diazoalkanes with acid chlorides, or less commonly, acid anhydrides is a familiar procedure for the preparation of a-diazoketones, Due to the inherent problems associated with the use of the

Boc- and Z-amino acid chlorides, the mixed anhydride method using isobutyloxyc.arbonyl chloride or ethyl chlorocarbonate was employed2,5. ' 9.2 1 . The same method was extended for the synthesis of Fmoca-amino acid diazoketones also2o,2 1 . In this case, the reaction results in low yield because of the eminent sensitivity of the Fmoc group towards basic conditions22. As Fmoc-a-amino acid chlorides are now known to be optically pure, crystalline and shelfstable, they have been used as acylating agents for the synthesis of Fmoc-a-amino acid diazoketones by us2J

as well as by Leggio et aL. 22. Recently, we have also developed alternative routes for the synthesis of aamino acyldiazomethanes employing Fmoc-a-amino acid pentafluorophenyl esters as acylating agents24. The present paper describes the synthesis of Fmoc-�amino acids using an equimolar mixture of Fmoc-aamino acid and N,N' -dicycJohexylcarbodiimide . (DCC) for the preparation of Fmoc-a-amino acid diazoketones, which are then converted to the corresponding �-amino acids by Wolff rearrangement (Scheme I).

The use of DCC and diisopropylcarbodiimide (DPC) as reagents for the formation of the peptide bond was a major event in the history of peptide synthesis25. The novel feature of coupling agents is that they could be added to the mixture of the carboxyl and the amine components. Hence activation and coupling proceed concurrently avoiding the necessity of the isolation of the activated species of the carboxyl component. DCC was also employed earlier for the acylation of diazomethane and diazoethane by Hodson et al.26,

ANANDA et al. : SYNTHESIS OF Il-AMINO ACIDS 419

RX· · · ·HOH _________ . Rx· · ·H � . CHN1 FmC!lC a FmGIC O

I

R

c) CH:zPh

d) CH (CH3h

e)

Q

F R)

"'" �

Table I---Characterization data of compounds 2a-j Sl. Compd 2 Yield m.p. Rr value [a]2S 0 Mol. fonnula Fond (Calc.) % 1 H NMR (0. ppm) No. (%) °C RrA R,B (c= l . C H N

CHCll)

a Fmoc-Ala-DAM 90 1 10- 12 0.59 0.85 -32.0 CI9H17N303 68. 1 0 5.28 12.48 L32 (3H. d). 4. 1 (2H, br), 4.5 (2H. d), 5.25 ( 1 10- 12)22 (68.05 5. 1 1 12.53) ( t H. s), 5.49 ( t H, br), 7.2-7.7 (8H, m).

b Fmoc-o-Ala- 85 1 1 2- 13 0.60 0.81 +32. 1 C1�HI7N303 68.25 5.09 1 2.27 1 .33 (3H. d) 4. 19 (2H. br) 4.42 (2H, d), 5.29 DAM (68.05 5 . 1 1 1 2.53) ( t H, s), 5.39 ( tH, br), 7.2-7.7 (8H, m).

c Fmoc-Phe-DAM 89 1 36-37 0.61 0.79 - 1 6.3 C2sH23N303 7 1 .89 5.57 1 0.21 2.6 (4H, m), 4.2 (2H, m), 4.5 (2H, d), 5.2 ( 1 33-35)12 (0.64) (_1 5.3)12 (72.69 5.60 10. 1 6) ( 1 H, s), 5.4 ( 1 H, br) 7.2-7.7 ( t 3H, m). Z

d Fmoc-Val-DAM 88 1 23-25 0.68 0.82 -23.0 C21H21N303 69.53 5.88 1 1 .62 0.9 (6H, d), 1 .75 ( t H, m), 4.25 ( t H, m), 4.45 0 ( 1 25-27)12 (0.69) (-43.3) (69.41 5.81 1 1 .56) (2H, m), 5.3 ( 1 H, s), 5.4 ( 1 H, d), 7.3-7.7 �

(c= 1 .05) (8H, m). ... (') e Fmoc-Pro-DAM 93 1 1 5- 17 0.60 0.8 1 -60.5 C21H2oN303 69.40 5.61 1 1 .48 2.0 (4H, br), 3.45 (2H, t), 4.2 (2H, m), 4.45 � (69.59 5.55 1 1 .59) (2H, d), 5.3 ( t H, br), 7.3-7.7 (8H, m). $: r Fmoc-Phg-DAM 9 1 1 48-49 0.65 0.80 -32.0 C24HI9N303 72.50 4.81 1 0.57 4.25 (2H, m), 4.4 (2H, d), 5 . 1 (1 H, s), 6.05 , tI)

(7 1 .92 4.56 10.69) ( 1 H, br), 7.2-7.8 ( 1 3H, m). trI (') g Fmoc-Leu-DAM 89 92-93 0.60 0.83 -42.5 C22H23N303 70.2 1 6.25 1 0.85 0.90 (6H, d), 1 .3- 1 .5 (3H, m), 4.2 ( 1 H, m), ,ttl

(90-9 1 )12 (0.70) (-48.4) 12 (70.01 6. 14 1 1 . 13) 4.4 - 4.7 (3H, m), 5.3 (2H, m), 7.3-7.9 (8H, � m). C! h Fmoc-Aib-DAM 85 141-42 0.71 0.85 C2oHI9N303 68.01 5.35 1 2.29 0.9 (6H, m), 4. 1 ( 1 H, m), 4.5 (2H, d), 5.2 r

(68.76 5.47 12.03) (1 H, s), 5.6 (1 H, br), 7.2-7.8 (8H, m). � Fmoc-Asp(OBu')- 80 7 1 -72 0.80 0.84 -26.3 C24H2SN30S 66.02 5.61 9.59 1 .4 (9H, s), 2.0 ( t H, m), 2.4 (2H, m), 4.3 '-C DAM (66. 1 8 5.78 9.65) (2H, d), 4.4 - 4.5 (2H, m), 5.4 ( 1 H, s), 5.5

( t H, s), 7.8 (8H, m). j Fmoc-Glu- 85 1 37-38 0.79 0.86 25.6 C2sH27N30S 66.86 5.79 9.30 1 .44 (9H, s), 1 .79 (1 H, m), 2. 1 (1 H, m), 2.4

(OBu')-DAM (66.80 6.05 9.35) (2H, m), 4.2 (2H, d), 4.3 (1 H, m), 4.5 (1 H, m), 5.4 ( t H, s). 5.6 ( I H. s). 7.8 (8H. m).

The abbreviations used for amino acids are in accordance with recommendations of IUP AC-IUP commission on Biochemical Nomenclature published in "Pure and Applied Chemistry' . 40. 1974. 3 14. Other abbreviations are: DAM-Diazornethane; Phg--Phenylglycine (a-amino phenylacetic acid). All the amino acids used. unless otherwise specified have L-configuration.

Table II-Characterization data of compounds 3a-j

SI Compd 3 Yield m.p. Rr value [a]2So Mol. Found (Calc .) % I H NMR formula (0, ppm)

No. '(%) eC) RrB RtC (c= l , C H N CHC�3)

a Frnoc-�-HAla 84 96-98 0.59 0.76 -21 .0 CI9HI9N04 70.30 5.72 4.52 1 . 10 (3H, d), 2.3 1 ( lH, d), 2.45 ( l H, d), > (96-98)22 (70. 14 5.89 4.30) 3.85 ( l H, m), 4. 1 -4.3 (3H, m), 7.3-7.9 Z > (9H, m). Z

b Frnoc-I)-�- 8 1 97-99 0.60 0.75 +21 .2 CI9HI9NO( 70.38 5.69 4.5 1 1 .08 (3H, d) 2.35 ( l H, d), 2.45 O H, d), 0 > HAla (7.0. 14 5.89 4.30) 3.85 O H, m) 4.2-4.28 (3H, m), 7.3-7.9 �

(9H, m). 1:1 !'-C Frnoc-J3-HPhe 80 1 10-12 0.62 0.75 -26.0 ClSH23NO( 74.81 5.78 3.49 2.43 O H, d), 2.52 (2H, m), 2.71 O H, d), . . til

( 1 25)12 (-19.3)12 (75.05 5.58 3 .70) 3.6 ( l H, m), 4. 1 O H, m) 4.2 (2H, m), -< 7.3-7.8 ( l4H, m).

� d FmOc-�-HVal 80 153-54 0.59 0.77 -36.2 C2IH23NO( 71 .24 6.38 3.78 0.85 (6H, d), 1 .75 ( l H, m), 2.30 ( l H, d), tTl ( 1 58)12 (-20.2)1

2 -(70. 14 5.89 4.20) 2.45 ( l H, d), 3.75 ( l H, m), 4.30 (3H, m), til en (c=1 .03) 7.3-7.8 (9H, m). � e Frnoc-�-HPro 79 176-78 0.61 0.71 -30. 1 C2IH2INO. 72. 16 6 . 19 3.63 2.0 (4H, br), 3.45 (2H, t). 4.2 (2H. m).

(7 1 .78 6.02 3.98) 4.45 (2H. d). 7.3-7.7 (8H. m). �

f Frnoc-J3-HPhg 85 1 84 0.60 0.78 -22.0 C24H21N04 74.40 5.45 3.61 2.5 (2H. d). 4.25 (2H. m). 4.4 (2H. d). � (74.58 5.49 3.99) 5.85 ( l H. br). 7.2-7.8 ( 13H. m).

-Z I Frnoc-�-HLeu 79 99 0.65 0.73 -12.0 C22H2SNO. 71 .75 6.95 3.74 0.85 (6H. d). 1 . 1 (2H. m). 1 .4 ( l H. m). 0

(106)12 (0.50) (-22.2)12 (7 1 .91 6.86 3.8 1 ) 2.35 (2H. m). 3.80 ( lH. m). 4.2 (3H. m). � (c= 1 . l 2) 7.3-7.9 (9H. m). 6 til h Frnoc-J3-HAib 83 1 08- 10 0.60 0.79 C21H23N04 70.58 6·.04 4.58 0.85 (6H. m). 2.45 (I H. d). 3.85 (I H. m).

(70.78 6.22 4. 12) 4. 1 (3H.m). 7.3-7.9 (9H. m). Frnoc-�- 80 82-83 0.68 0.76 +0.3 C24H27N06 67.39 6.41 3 . 19 .1 .43 (9H. s). 2.67 (4H. m). 4.2 - 4.4 (4H. HAsp (OBut) (c=1 .9,MeOH) (67.74 6.39 3.29) m). 6.5 ( I H. br). 7.2-7.8 (8H. m).

j Finoc-J3-H 80 58-60 0.69 0.75 -1 1 .4 C2sH29N06 68.28 6.70 3.20 1 .4 (9H. s). 2.4 (4H. m). 3.8-4.4 (4H. m). Glu (OBut) (68.32 6.65 3. 19) 5.6 ( l H. D). 7.2-7.8 (8H. m).

t; -

422 INDIAN J CHEM, SEC 8, APRIL 1999

Unlike in the mixed anhydride and pentafluorophenyl ester methods, the acylation reaction was carried out without using an organic base. The possible deprotection of Fmoc group during acylation step was avoided and all Fmoc-a-amino acid diazoketones were isolated in good yield. The Wolff rearrangement of diazoketones was also carried out without the use of a base.

Thus the acylation of diazomethane was carried out using Fmoc-a-amino acids directly without any activation step. This route makes a base for the synthesis of Fmoc-�-amino acids in large scale. They will be useful for the synthesis of �-peptides by the solid phase method using Fmoc chemistry which is in progress. However, this method is not useful for the synthesis of Fmoc-�-HGln and Fmoc-�-HAsn, because the corresponding a-amino acids are known to dehydrade under these conditions.

Experimental Section The melting points were determined using a Leitz

Wetzlar melting point apparatus and are uncorrected. Optical rotations were measured with an automatic AA- I O polarimeter (Optical Activity, U.K.). IR spectra were recorded on a Nicolet model Impact 4000 Ff-IR spectrometer (KBr pellets, 3 cm·1 resolution) and IH NMR spectra on a Brucker ACF 200 MHz spectrometer using TMS as an internal standard. Elemental analyses were done using Perkin Elmer Analyser and the samples were dried for 24 hr under vacuum before analysis. Reverse Phase HPLC was carried out using a Waters LC-3000 system consisting of a 484 tunable absorbance UV detector and a MiJlipore 745 data module. TLC was carried on precoated silica gel plates using solvent systems; (i) CHCI3 : methanol : acetic acid (40:2: 1, v/v), (ii) ethyl acetate : hexane (35 :65, v/v), and (iii) CHCI3: methanol (9: I , v/v) and Rr values are designated as RrA, RrB and RtC respectively. The diazomethane solution in dry THF was prepared using N-methyl-Nnitroso-toluene-p-sulphonamide as reported earlier27.

Fmoc-a-aminoacyldiazomethanes 2a-j: General procedure. To an ice cold solution of Fmoc-a-amino acid la-j ( I O mmoles) in dl1' THF (25 mL), was added a solution of DCC ( 1 0.5 mmoles) in dry THF (5 mL) and the mixture stirred for 30 min. Then, a saturated diazomethane solution in dry THF ( 100 mL) was added. It was then stirred for about 1 hr at O°C and kept in the refrigerator overnight. The predpitated DCU was filtered off and the filtrate was

evaporated to an oil. It was further redissolved in ether and cooled, the precipitated oeu filtered again and the solvent was evaporated. The resulting oily residue was precipitated using ethyl acetateln-hexane to get the compounds 2a-j in good yield.

Fmoc-�-homoamino acids 3a-j : General procedure. A solution of 2a-j ( 1 mmole) in 1 ,4-dioxane ( 10 mL) and water (5 mL) was treated with silver benzoate ( 10 mg, 0.4 mmole). The reaction mixture was refluxed at 70°C for 4-5 hr and then filtered. The solvent was evaporated under reduced pressure. The residue was dissolved in saturated aqueous Na2C03 (20 mL) and stirred for 30 min. The solution was washed with ether (2 x 30 mL). The aqueous layer was acidffied to pH 2 and extracted with ethyl acetate (3 x 25 mL). The combined organic layer was washed with water (2 x 20 mL), dried over Na2S04 and evaporated to get the corresponding �homoamino acids 3a-j in good yield.

Acknowledgement We thank Dr K M Sivanandaiah for useful

discussions. Our thanks are also due to the Chairman, Dept. of Chemistry, Central College, Bangalore. This work is partly . supported by the UGC-DRS programme, Govt. of India.

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