Rapid and efficient synthesis of the pentapeptide of elastin protein and peptides containing highly hindered α , α -dialkyl amino acids employing Fmoc-amino acid chlorides under microwave irradiation in the solution phase

A rapid and efficient synthesis of peptides in solution employing Fmoc-amino acid chlorides under microwave irradiation is described. A comparison study of the microwave assisted method with those of conventional peptide synthesis using acid chlorides and various coupling additives has been performed. It has been found that, in general, the formation of a peptide bond, employing Fmoc-amino acid chloride and zinc dust or TBDMS-OBt under microwave irradiation is complete in 30-45 seconds with 90% yield of pure isolated peptide. Employing zinc dust as a coupling additive, the synthesis of several dipeptides, the pentapeptide fragment Fmoc-Val-Pro-Gly-Val-Gly-OBzl, of elastin and the difficult highly hindered couplings of α , α - dialkylamino acids are reported.


Introduction
Synthesis of peptides employing the shelf-stable 9-fluorenylmethoxycarbonyl (Fmoc) amino acid chlorides makes couplings of even very hindered or weakly nucleophilic systems possible where other methods give only poor results. 1,2The trapping of the liberated hydrogen chloride during coupling has been accomplished by using a two-phase system with a mild inorganic base in the aqueous layer. 3Improvements in the coupling in homogeneous system by the use of coupling additives like AgCN, 4,5 potassium salt of 1-hydroxybenzotriazole 6,7 and 1-hydroxy-7-azabenzotriazole 8 along with Fmoc-amino acid chloride circumvented the formation of oxazol-5(4H)-one but also resulted in elimination of the premature deblocking of Fmoc group during prolonged coupling reactions.1-(t-Butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) 9 has been found to be advantageous due to its solubility in CH 2 Cl 2 /CHCl 3 and the separation of the side product TBDMS-Cl is also simple.Zinc 10 and indium 11 mediated coupling is an alternative reaction pathway for the mediation of coupling by the acid chloride method.Some of the advancements led to complete coupling in about 30 min.][14] The utility of microwave irradiation for the synthesis of several building blocks [15][16][17][18][19] useful in the synthesis of peptides and peptidomimetics like benzyl esters, conversion of Fmoc-/Boc-αaminodiazoketones to β-amino acids by Wolff rearrangement, Fmoc-/Z-/Boc-α-amino acids to Fmoc-/Z-/Boc-5-oxazolidinones and Fmoc-α-amino acid azides to the corresponding isocyanates has been demonstrated by us.The solid phase peptide synthesis employing Fmoc-amino acids and symmetric anhydride or preformed benzotriazole esters or PyBOP for coupling under microwave irradiation enhancing the coupling efficiency has been reported. 20,21Recently, the solid phase synthesis of β-peptides containing trans-2-aminocyclohexane carboxylic acid was realized efficiently by microwave irradiation.A comparison of microwave over conventional heating showed that, on treatment with microwave irradiation, the purity of peptide raised to 81%, whereas the conventional method resulted in only 21 % pure compound. 22The synthesis of dipeptides containing Z-/Boc-α-aminoisobutyric acid (Aib) in solution employing PyBOP/HOBt and HBTU/HOBt was also explored. 23

Results and Discussion
In this study, the effect of microwave irradiation on the formation of the peptide bond employing Fmoc-amino acid chlorides has been explored.The microwave reaction was carried out in a LG MS 194A microwave oven producing microwave radiation with a frequency of 2450 MHz.The microwave oven was a 1200 W oven and the reaction was specifically carried out at 60% of the total power output, which would correspond to an average power of 720 W. For the reactions carried out using a bi-phasic system, the reaction mixture was CHCl 3 -10% NaHCO 3 .The synthesis in a homogeneous system in the presence of several hydrogen chloride acceptors was also performed.In the case of the coupling additive TBDMS-OBt, a solution of Fmoc-amino acid chloride in CH 2 Cl 2 and TBDMS-OBt was used.The zinc, bismuth and indium metal mediated synthesis was carried out using a suspension of metallic powder and Fmoc-amino acid chloride in CH 2 Cl 2 .The completion of the coupling was confirmed by TLC using the solvent systems a) chloroform: methanol: acetic acid (40:2:1) and b) ethyl acetate: n-hexane (35:65).A simple work-up of the reaction mixture and recrystallization using a suitable solvent gave the peptide.The duration of the coupling as well as the yield of Fmoc-Phe-Leu-OBzl using various coupling additives in both homogeneous and bi-phasic systems along with the reported data are furnished in the Table 1.It has been found that the coupling mediated by zinc dust and TBDMS-OBt is complete within 30 to 45 sec.The isolated yield of the pure peptide was 90 %.The method was subjected to a racemization test and, as demonstrated by the 1 H NMR, and HPLC analysis of the diastereomeric dipeptides, Fmoc-L-Phe-and D-Phe-Ala-OMe, made by employing zinc dust as well as the TBDMS-OBt method, revealed that the method is completely free from racemization.Also, the scale-up of the synthesis of Fmoc-Phe-Leu-OBzl by this method using a 20 mmolar quantity resulted in isolation of 10.8 g (92 %) of the pure peptide.Employing a rapid deprotection condition using tris(2-aminoethyl)amine (TAEA), the present method was extended towards the synthesis of Fmoc-Val-Pro-Gly-Val-Gly-OBzl, 24 a repeating pentapeptide fragment of the protein elastin.Thus, all four of the couplings involved in its synthesis, namely the di, tri, tetra and peptapeptide couplings, have been accomplished under microwave irradiation.The final pure pentapeptide was obtained in 67% yield (Table 2).The 1 H NMR, HPLC (Fig 2 ) and mass spectrum (Fig 3) of the pentapeptide were found to be satisfactory.Encouraged by these results, we have extended the similar reaction conditions for the incorporation of α,α -dialkylamino acids.The formation of a peptide bond between α,αdialkylamino acids, both acyclic and cyclic, under normal conditions employing even efficient coupling agents like PyBOP, HBTU etc., needs several hours with poor yields.Even under microwave irradiation, the synthesis of Boc-/Z-Aib-Aib-OMe using HBTU/HOBt required 30 min.It is now found that the coupling of Fmoc-Aib-Cl to Aib-OBzl hydrochloride, mediated by activated zinc dust under microwave irradiation, has been found to be complete within 90 sec.with 84% yield.Similar results have been obtained in the coupling of several other peptides possessing cyclic as well as acyclic α,α -dialkylamino acids, and these are summarized in Table 3.

Conclusions
The synthesis of peptides employing Fmoc-amino acid chlorides using a mild microwave irradiation technique in the solution phase has been described.The method achieves the purpose of high speed and efficient coupling of peptides, including those of highly hindered α,αdialkylamino acids.Thus, a facile method to produce peptides in high yield makes this yet another useful and attractive procedure employing the microwave technique in organic synthesis.

Experimental Section
General Procedures.All the solvents were freshly distilled prior to use.Melting points were determined by the capillary method and are uncorrected.TLC analysis was carried out on precoated silica gel-GF 254 plates purchased from Merck containing 13% CaSO 4 as binding agent, using solvent systems a) chloroform : methanol : acetic acid (40:2:1) and b) ethyl acetate : nhexane (35:65).IR spectra were recorded on a Nicolet model impact 400D FT-IR spectrometer.
The HPLC analysis was carried on Shimadzu CLASS-VP V6.1 analytical HPLC using a Merck RP-18, 250 x 4.0 mm column, with detection at 215 nm.Optical rotations were determined using automatic AA-10 polarimeter (Optical Activity, U. K.).Elemental analyses were carried out on a Perkin-Elmer MODEL 240 analyzer. 1H NMR spectra were recorded using a Bruker AMX 400 MHz spectrometer.Mass spectra were obtained using a Kratos PCKompact SEQ V1.2.2 spectrometer.

Figure 1 .
Figure 1.Synthesis of peptides employing zinc dust

Table 2 .
List of peptides made by the zinc dust mediated coupling microwave irradiation method