Indian Journal of Chemistry Vol. 41B, August 2002, pp 1733- 1735

Note Fmoc-amino acid azides in peptide

synthesis

Ganga- Ramu Vasanthakumar, Kuppanna Ananda & Vommina V Suresh Babu*

Department of Studies in Chemistry, Central College Campus, Bangalore University, Bangalore 560 001, India

Received 2 October 2000; accepted (revised) 3 December 2001

Fmoc-amino acid azides can be prepared from the corresponding acid chlorides and sodium azide. All the compounds made have been obtained as solids in good yield and purity. They are found to be shelf stable at room temperature for longer periods. Their storage at room temperature does not lead to the formation of isocyanates. Employing them as coupling agents, the synthesis of a few dipeptides is described.

The acid azide method, introduced by Theodor Curtius l nearly a century ago, is currently used not only for the stepwise coupling of troublesome amino acids such as serine or histidine, but also for fragment condensation, where it shows real merit. It is the most suitable method for segment condensation because little racemization occurs and side-chain protection can be kept at a minimum

2. It is also used in

preparation of polyamino acids3, in peptide cycliza­tions4 and is employed for the introduction of alkyl­oxycarbonyl groups [t-butyloxycarbonyl (Boc) and 9-fluorenyl-methoxycarbonyl (Fmoc) groups] into amino acids. Its utility in peptide synthesis is fully explored by Hirschmann et al.5•6 and Yajima et ae·8

in their syntheses of bovine ribonuclease A. The side reactions9 that generally occur in the azide

process are the Curtius rearrangement at room temperature producing a urea derivative and hydrolysis of azides giving an amide. In addition, side reactions that arise from specific amino acid residues and from protecting groups have also been identified. An additional difficulty in this procedure is the slow formation of the peptide bond. The coupling via azides can require considerable time, even several days, particularly if the reaction is carried out at low temperatures (below -20°C) to avoid Curtius rearrangement. Since Boc- and Z-amino acid azides rearrange rapidly at room temperaturelO to the corresponding isocyanates, their formation is immediately followed by the coupling process. Time

is an important factor in process involving not entirely stable intermediates. All these difficulties make this method not a simple one. Such difficulties notwithstanding, it remains a classical contribution in general I I and for the synthesis of gem-diamines 12 in particular.

The hydrazides of Boc- and Z-amino acids and peptides, from which the azides were made have been routinely obtained by the hydrazinolysis of a protected amino acid or peptide ester 13 • Their production from the corresponding acyl chlorides is stated as of no interest in the present context by Jones3 and Meienhoffer4 in their authoritative reviews on the azide method. Since the base labile Fmoc group is unstable during hydrazinolysis, Fmoc-amino acid azides have not been prepared so far to the best of our knowledge

2-4.9. 1 I . 1 2. 14- 1 8. A revised view of acid chlorides in peptide chemistry is being brought about by the discovery of the stability of Fmoc-amino acid chlorides I9-

23. This short communication deals with the utility of such acid chloride derivatives for the synthesis of Fmoc-amino acid azides.

o I I Acetone Fmoc-NH-CHR-C-CI + NaN3 -----i .. �

O oC o "

Fm oc-NH-CH R-C-N3

It is now found that Fmoc-amino acid azides can be prepared easily. The Fmoc-amino acid chloride was treated with sodium azide in acetone at O°c. TLC analysis, carried out on precoated silica gel G plates using the solvent systems ethyl acetate- n­hexane (35:65, v/v) and CHCh-methanol-acetic acid (40:2: 1 , v/v/v) , indicated that the azide formation was complete in about 1 5 min. In majority of the cases, the compounds separated as white solids. It was filtered, washed with water and the crude azide was recrysta-llized using CH2Ch-n-hexane. All the derivatives made have been obtained quantitatively as crystalline solids. The azides made along with their physical properties are given in the Table I. The IR spectra clearly showed the characteristic stretching vibrational frequency of the azide group at around 2138-2148 cm- I . The azides are found to be completely free from the corresponding Fmoc-amino

1734 INDIAN J CHEM., SEC B, AUGUST 2002

Table I�haracterization data of Fmoc-amino acid azides

SI. No. Compd Yield m.p. Rr Value* [alii Calc. %(Found)

(%) °C Rr A RrB (c = 1 , CHCI3) C H N

Fmoc-Gly-N3 90 1 32 0.61 0.69 63.34 4.37 17.38 (63.01 4. 19 17.03)

2 Fmoc-Ala-N3 93 162 0.62 0.68 - 16 64.27 4.78 16.66 (63.99 4.6 1 16.52)

3 Fmoc-Leu-NJ 92 123 0.76 0.68 +7 66.65 5.85 14.80 (66.20 5.70 14.6 1 )

4 Fmoc-Phe-NJ 94 175 0.61 0.75 +3 69.88 4.88 13 .58 (69.50 4.68 1 3.39)

5 Fmoc-L-Phg-NJ 90 125-26 0.63 0.76 - I I 69.33 4.54 14.06 (69.01 4.32 1 3.90)

6 Fmoc-D-Phg-NJ 89 127 0.63 0.76 +I I 69.33 4.54 14.06 (69.08 4.41 13.95)

*TLC analysis carried out using ethyl acetate-n-hexane (35:65, v/v) and CHCh-methanol-acetic acid (40:2: I, v/v/v) and the Rr values are designated as RrA & RrB respectively.

Table II--Physical properties of Fmoc-dipeptide esters*

SI. No. Dipeptide Yield m.p. Rr Value** [alii (%) °C

1 Fmoc-Gly-Phe-OMe 78 1 3 1 - 34 0.61 + 16 (c=0.5, MeOH) 2 Fmoc-Ala-Leu-OMe 74 128-30 0.8 1 -28.6 (c= l , CHCIJ) 3 Fmoc-Leu-Ala-OMe 76 162-63 0.64 - 30 (c=l , CHCh) 4 Fmoc-Phe-Phg-OMe 80 158-60 0.84 +23.6 (c=l , CHCIJ)

5 Fmoc-L-Phg# -Phe-OMe 72 193 0.89 + 22.6 (c=0.5, DMF)

6 Fmoc-D-Phg -Phe-OMe 73 192 0.9 1 -2 1 .3 (c=O.5, DMF)

* All the peptides fully characterized by I H NMR **TLC analysis carried out using CHCI3-methanol-acetic acid (40:2: I , v/v/v) N Phg, 2-amino-2-phenylacetic acid (phenylglycine)

acid isocyanates. It was revealed by the absence of the peak at around 2250 cm· 1 which is characteristic stretching frequency for isocyanates. Upon storage at room temperature on shelf for a few months, no changes in the IR spectrum were noticed. Similarly no change in their melting points was also observed. Employing them as coupling agents, the synthesis of a few dipeptides was carried out (Table II). A typical coupling procedure is given in experimental section. The coupling is found to be free from racemization. The IH NMR spectra of Carpino' s model diastereomeric dipeptides

24 Fmoc-L-Phg-Phe­

OMe [8 3 .58 (s, OCH3)] and Fmoc-D-Phg-Phe-OMe, [8 3 .64 (s, OCH3)] synthesized by this method, clearly showed that coupling reactions are free from racemization. The utility of these azides for the synthesis of �-casomorphin is in progress.

Conclusions Fmoc-amino acid azides can be prepared easily

from their corresponding acid chlorides. They can be isolated as crystalline solids, stored and used as coupling agents as and when required. They are found to be stable at room temperature for longer periods. Unlike Boc- and Z-amino acid azides, these compounds at room temperature have not rearranged to their corresponding isocyanates.

Experimental Section Preparation of Fmoc-amino acid azides :

General procedure. Fmoc-amino acid chloride ( l mmole) was dissolved in acetone (3 mL) and cooled to ODC in an ice-salt bath. NaN3 (0.098 g, 1 .5 mmoles) in water ( l mL) was added to the solution and stirred at ODC for about 15 min. Fmoc-amino acid azide gets

NOTES 1735

precipitated out. It was filtered, washed with water and dried. The crude product was recrystallized from CH2Clz - n-hexane. The physical constants are listed in the Table I.

General procedure for coupling To a stirred solution of Fmoc-amino acid azide ( 1

mmole) i n CH2Clz ( 5 mL), a solution o f C-protected amino acid ester ( 1 . 1 mmoles) in CH2Clz (3 mL) was added and stirred for about 1 8 hr at room temperature. During coupling, the pH of the reaction mixture was maintained at 7.5 by periodic addition of collidine. After completion of the reaction, it was washed with IN HCI (3 x 10 mL), 10% NaHC03 (3 x 10 mL), water (3 x 10 mL) and brine, dried over anhydrous Na2S04 and evaporated in vacuo. The resulting peptide was recrystallized using CH2Clz-n­hexane.

Acknowledgement The research work was supported by grants from

the Dept. of Science and Technology, Govt. of India. Authors thank Prof. K M Sivanandaiah for useful discussions. One of the authors (KA) thanks CSIR, New Delhi for the grant of a senior research fellowship. Author (GRVK) thanks KSVN Trust, Bangalore, for their kind help.

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