Synthesis, Characterization and Antimicrobial activity of protected dipeptides and their deprotected analogs

Introduction

Peptides are the constituents of proteins. They are biologically active chemical compounds formed by amino acid units linked together by amide linkage. They are useful pharmaceutical agents which are used for the treatment of arthritis,diabetes,  cardiovascular diseases,immune diseases, growth problems and many more.Peptides are smaller in size_­, less immunogenic, highly active and more stable at room temperature than proteins and antibodies. Also they have low manufacturing cost.

Peptides are widely used as pharmaceutical agents due to the diversity of their constituents and their minimal toxicity.  The 20 naturally occurring amino acids found in proteins offer a tremendous number of possible combinations for a peptide sequence¹. Structural modifications of synthetic peptides also provide diversity in their pharmaceutical design. Cyclic peptides offers enhanced stability and retain biological activity².  We have described a simple three step procedure for the synthesis of dipeptides from component amino acids and checked their antimicrobial activity.

Materials and Method

Materials

BOC- protected amino acid and Thionyl Chloride used for synthesis of dipeptides were obtained from Qualikems fine chemicals private limited (New Delhi). Amino acids like L-Tyrosine, L-Leucine, L-Proline,and solvents like Dichloromethane (DCM), Methanol, Diethylether, Tetrahydrofuran (THF) and other reagents like N-Methylmorpholine (NMM), Dimethyl for mamide (DMF), Trifluoroacetic acid (TFA) were purchased from Loba Chemie Pvt Ltd (Mumbai). Coupling reagent Isobutylchloroformate (IBCF) was obtained from Spectrochem Pvt Ltd (Mumbai) and Luria Bertani (LB) agar was taken from Himedia Laboratories Pvt Ltd (Mumbai). The fungal species like Aspergillus fumigatus (NCIM-902),Pencillium chrysogenum (NCIM-738) and bacterial species; E.Coli (NCIM- 2563) and Salmonella typhimurium (NCIM-2501) used in the process are the products of NCIM, National Chemical Laboratory (Pune).

Scheme

Step1. Carboxyl group protection of L-amino acids with methyl ester

(Where amino acids taken are Tyrosine, Leucine, and Proline)

Step2. Coupling of protected amino acids to form protected dipeptide:

Step3. Deprotection of protected dipeptides:

Method

Protection of Carboxyl Group of L-Amino Acids Using Methanol and Thionyl Chloride³:

50 ml Methanol was taken in 250 mL round bottom flask under the ice cold conditions followed by the addition of 24 mmole of Thionyl chloridein a drop wise manner along with continuous stirring. After that 12 mmole of L-amino acid (Tyrosine, Leucine and Proline)was added pinch wise. The contents of the flask were then refluxed for 4 hours. The progress of reaction was monitored time and again by TLC on readymade silica plates (Merck, UV active _max­₂₅₄­­­­_nm) and the solvent was evaporated under reduced pressure. The residue was obtained as hydrochloride salt and was dried in vacuum desiccator using P₂O₅.

Coupling of Methyl Ester Protected L-Amino Acid with BOC-Protected L-Amino Acids^4,5

7.5 mmole of BOC-Phenylalanine was dissolved in 20 mL of Tetrahydrofuran in a round bottom flask followed by the addition of 7.6mmole of N-Methylmorpholine. At the same time, 9mmole of methyl ester of amino acid (L- Leucine, L-Prolineand L-Tyrosine) was dissolved in minimum amount of Dimethylformamidein another round bottom flask and 9.2 mmole of N- Methylmorpholine was also added to it so as to neutralise the salt under ice cold conditions. Coupling reagent Isobutylchloroformate was then added at -15^oC to first flask with vigorous stirring and contents of second flask were added to that. The reaction mixture was allowed to stir for 2 hours. The filtrate was evaporated under reduced pressure and the oily residue left was dissolved in ethyl acetate. It was then washed with 10% aqueous sodium bicarbonate, citric acid solution and finally with brine. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. Residue was then dried in vacuum desiccator using P₂O₅. The crude product was purified over silica gel column using Methanol : Chloroform as solvent system to get pure compound.

Deprotection of Protected Dipeptides^{6, 7}:

BOC-Protected dipeptides were dissolved in Dichloromethane in a round bottom flask having a guard tube attached to it. About 1mL of Trifluoroacetic acid was added in the flask. The contents of the flask were allowed to stand for 1 hour. Solvent was then evaporated under reduced pressure and the oily residue obtained was triturated in diethylether to get solid residue which was then stored in desiccator.

Antimicrobial Activity

The antimicrobial activity of protected and deprotected dipeptides was checked in vitro by Kirby-Bauer disc diffusion method⁸. In this method the antimicrobial activity of test compounds was examined by measuring the diameter of zone of inhibition.⁹The antimicrobial activity was checked against two fungal strainsAspergillus fumigatus(NCIM-902), Pencillium chrysogenum(NCIM-738) and two Gram-negativeE.Coli(NCIM-2563), Salmonella typhimurium(NCIM-2501) bacterial strains.LB plates were  streaked by spread plate method and discs of 1mm  Whatsmann filter paper were soaked in 250 ppm, 500 ppm, and 1000 ppm of dipeptides (both protected and deprotected), along with Ciprofloxacin(1µg/disc) as a standard and solvent solution as control and placed in the centre of the inoculated petriplates. The plates were incubated at 37^oC and results were interpreted after 24 hrs¹⁰.The results of antimicrobial activity of protected and deprotected dipeptides are shown in table 1 and 2.

Results and Discussion

¹H NMR; 400MHz CDCl₃( δppm);

1. BOC-PheLeu-OMe:0.874-0.915(m,9H,CH₂CH(CH₃)₂), 1.33(s,9H,(CH₃)₃CO),1.49-1.57 (m,2H,NHCH₂CH₂CH(CH₃)₃),2.77-2.83(m,1H,HNCHCH₂C₆H₅) , 3.64(s,3H,OCH₃), 4.26-4.30  (m,2H,CH₂C₆H₅), 7.18-7.23 (m,5H,C₆H₅ ) FTIR-

8400S; v(C=O) peak 1688 cm^{– 1},v( N-H) peak 3065 cm^-1

2. BOC-PheTyr-OMe:1.33-1.39(m,9H,(CH₃)₃CO), 2.54-2.66(m,2H,CH₂C₆H₅),2.76-2.92(m,2H,CH₂C₆H₄OH), 3.63 (s,3H,OCH₃ ), 4.26-4.28 (m,1H,NHCHCH₂C₆H₅ ),  4.57-4.59 (m,1H,NHCHCH₂C₆H₄OH ),  6.68-6.70 (m, 5H,C₆H₅), 7.16-7.29 (m,4H,C₆H₄OH )  FTIR-8400S; v(C=O) peak 1688cm^-1,v( N-H) peak 3063 cm^-1

3. BOC-PhePro-OMe: 1.27-1.47 (m,9H,(CH₃)₃CO ), 1.89-2.18 (m,2H,CH₂C₆H₅ ), 2.93-3.46 (m,4H,NCH(CH₂)₂ ),3.57-3.63 (m, 1H,NCH(CH₂)₃),  3.70(s, 3H,OCH₃), 4.67-5.03 (m,1H,NHCOCHCH₂C₆H₅), 7.20-7.28  (m,5H,C₆H₅ ) FTIR-8400S; v(C=O) peak 1642 cm^-1,  v( N-H) peak 3431 cm^-1

4.  PheLeu OMe: v(C=O) peak 1672cm^-1,  v( N-H) peak 3289 cm^-1

5.  PheTyr-OMe: v(C=O) peak 1671cm^-1,  v( N-H) peak 3178 cm^-1

6.  PhePro-OMe: v(C=O) peak 1699cm^-1,  v( N-H) peak 3144 cm^-1

Antimicrobial Activity

Table 1.  Antimicrobial activity of protected dipeptides:

S. No.	Compound name	Concentration	Aspergillusfumigatus(NCIM902)	Pencilliumchrysogenum(NCIM738)	E.coli(NCIM2563)	Salmonellatyphimurium(NCIM2501)	CH3OH	Ciprofloxacin(standard)
1	BOC-PheLeu-OMe	250ppm	2mm	X	1mm	X	X	20mm
		500ppm	3mm	X	2mm	X	X	24mm
		1000ppm	5mm	3mm	3mm	1mm	X	24mm
2	BOC-PheTyr-OMe	250ppm	1mm	2mm	X	X	X	18mm
		500ppm	1mm	3mm	X	X	X	18mm
		1000ppm	4mm	8mm	4mm	2mm	X	20mm
3	BOC-PhePro-OMe	250ppm	3mm	X	3mm	3mm	X	22mm
		500ppm	7mm	X	3mm	6mm	X	20mm
		1000ppm	11mm	2mm	8mm	10mm	X	20mm

 

Table 2.  Antimicrobial activity of deprotected dipeptides:

S. No.	Compound name	Concentration	Aspergillusfumigatus(NCIM902)	Pencilliumchrysogenum(NCIM738)	E.coli(NCIM2563)	Salmonellatyphimurium(NCIM2501)	CH3OH	Ciprofloxacin(standard)
1	PheLeu-OMe	250ppm	1mm	3mm	2mm	X	X	22mm
		500ppm	2mm	5mm	4mm	X	X	26mm
		1000ppm	4mm	8mm	7mm	X	X	24mm
2	PheTyr-OMe	250ppm	3mm	2mm	X	X	X	18mm
		500ppm	4mm	4mm	2mm	X	X	19mm
		1000ppm	5mm	6mm	3mm	X	X	20mm
3	PhePro-OMe	250ppm	3mm	2mm	4mm	X	X	22mm
		500ppm	4mm	1mm	5mm	X	X	25mm
		1000ppm	4mm	5mm	6mm	X	X	27mm

 

Figure1: The disk represents the antimicrobial activity of PheLeu-OMe with their zone of inhibition at three concentrations of 250, 500 and 1000 ppm in comparison to standard cipro floxac in (± 30 mm) and solvent (Methanol) control Click here to View figure

 

The antimicrobial activity of protected and deprotected dipeptides against fungal and bacterial strains is shown in table 1 and 2. Among protected dipeptides BOC-PhePro-OMe shows maximum potency against fungi Aspergillus fumigatus with inhibition zone diameter of 11mm at 1000 ppm (standard 20mm).This compound is not so effective against Pencillium chrysogenum. But it shows good antibacterial activity against gram- negative strains of E.coli(8mm) and Salmonella typhimurium (10 mm). This compound shows moderate potency against Aspergillus fumigatus, E.coli and Salmonella typhimurium at 250 and 500 ppm concentrations.

BOC-PheLeu-OMe is negligibly effective against Aspergillus fumigatus at all concentrations (250, 500 and 1000 ppm) with zonal diameter of 1mm, 2mm and 4mm respectively  while standard has diameter of 20mm-24 mm.

Protected dipeptide BOC-PheTyr-OMe show moderate activity against fungal strain Pencillium chrysogenum at 1000 ppm (8mm).It is not effective against Aspergillus fumigatus, E.coli and Salmonella typhimuriumat concentrations of 250, 500 and 1000 ppm.

Deprotected dipeptides also show antibacterial and antifungal activities against the four strains. All the three deprotected dipeptides PheLeu-OMe, PheTyr-OMe, PhePro-OMe show moderate antifungal activity against Aspergillus fumigatusat concentrations of 250 ppm, 500 ppm and 1000 ppm. They are not showing any activity against Salmonella typhimurium even at higher concentration of 1000 ppm.Deprotected dipeptidesPheLeu-OMe, PheTyr-OMe, PhePro-OMe are showing good antifungal activity against fungal strain  Pencillium chrysogenumat higher concentration (1000 ppm) with zonal diameter of 8mm, 6mm and 5mm (standard 22-27mm). They are also effective against gram-negative bacteria E.coliat 1000 ppm with inhibition zone diameter of 7mm, 3mm and 6mm respectively.

Conclusion

With the increase in pollution all over the world, various infectious diseases are emerging;also the microorganisms are be coming resistant to existing antibiotics. Therefore it is becoming a challenge for researchers to develop new methods for the treatment of these diseases and microorganisms¹¹.From the results, we found that these protected and deprotected dipeptides exhibit antimicrobial activities at different concentrations. Out of these compounds BOC-PhePro-OMe and PheLeu-OMe(fig 1) are showing good antimicrobial activity against biologically important pathogens so these can be used as potent therapeutic agents after their further investigations.

Acknowledgment

We are grateful to Dr. Joginder Singh for antimicrobial activity, Lovely Professional University for lab facility and SAIF, Panjab University Chandigarh for NMR facility.

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