Infrared, 1 H-NMR Spectral Studies of some Methyl 6- O -Myristoyl- α -D-Glucopyranoside Derivatives: Assessment of Antimicrobial Effects

. This study was carried out to regioselective myristoylatio n of methyl α -D-glucopyranoside ( 1 ) using the direct acylation method gave the corresponding methyl 6- O - myristoyl- α -D-glucopyranoside ( 2 ) in fair yield. A number of 2,3,4-tri- O -acyl derivatives ( 3 - 15 ) of this 6- O -substitution product using a wide variety of acylating agents were also prepared in order to obtain newer derivatives of synthetic and biological importance. The reaction conditions are reasonably simple and yields were very good. The structures of the title compounds ( 2 - 15 ) were established by using analytical, physicochemical techniques and spectroscopic data (IR and 1 H-NMR). All the synthesized compounds were employed as test chemicals for in vitro antimicrobial functionality test against Gram-positive Bacillus subtilis , Staphylococcus aureus , Gram-negative Escherichia coli , Pseudomonas aeruginosa bacteria and plant pathogenic fungi Aspergillus niger and Candida albicans . For comparative studies, antimicrobial activity of standard antibiotics, Ampicillin and Nystatin were also carried out against these microorganisms. The study revealed that the tested samples exhibited moderate to good antibacterial and antifungal activities. It was also observed that the test substances were more effective against fungal phytopathogens than those of the human bacterial strains. Encouragingly, a number of tested chemicals showed nearest antibacterial and antifungal activities with the standard antibiotics employed.


INTRODUCTION
Carbohydrates are key molecules in nature with multiple roles in biological processes. For a long time, carbohydrates have been very attractive field for scientists due to their immense importance in biological systems [1]. They are the source of the metabolic energy supply, but also for the fine-tuning of cell-cell interactions and other crucial processes [2,3]. As a consequence, the chemistry and biochemistry of carbohydrate derivatives is an essential part of biochemical and medicinal research. Owing to the many functional groups and the configurational variety, the number of possible carbohydrate derivatives is huge. Therefore, the synthesis of carbohydrate derivatives is complicated, generally requiring many steps, and a range of selectivity problems has to be solved. So, selective acylation is very important in the field of carbohydrate chemistry because of its usefulness for the synthesis of biologically active products. Protection of a particular functional group of an organic compound is not only necessary for the modification of properties of the remaining functional groups but also for the synthesis of newer derivatives of great importance. Various methods for acylation of carbohydrates and nucleosides have so far been developed and  LTD, 2015 employed successfully [4][5][6][7][8]. Of these, the direct acylation method is considered as one of the most effective [8,9] for selective acylation of carbohydrates.
Microbial food contamination problems have been the cause of much public concern over the last few decades because of an increase in the number of infections and diseases originating from the consumption of spoiled food [10]. Antibacterial and antifungal agents are necessary for food preservation, especially for food processors, because bacterial and fungal growth are important causes of food spoilage. For this reason, many investigators have focused their research efforts on finding new efficient, low toxicity and environmentally friendly antibacterial and antifungal agents.
It was found from the literature survey that a large number of biologically active compounds also possess aromatic, heteroaromatic and acyl substituents [11]. The benzene, substituted benzene and also nitrogen, sulphur and halogen containing substituents are known to enhance the biological activity of the parent compound [12]. It also known that if an active nucleus is linked to another active nucleus, the resulting molecule may possess greater potential for biological activity [11]. Results of an ongoing our research project on selective acylation of carbohydrates [13][14][15][16][17] and also evaluation of microbial activities [18][19][20][21] reveal that in many cases the combination of two or more heteroaromatic nuclei and acyl groups enhances the biological activity manifold than its parent nucleus [22,23].

Materials and methods
In this present work, all chemicals used were purchased from Sigma-Aldrich and Merck Company unless reported otherwise and used without further purifications. Infrared spectra were recorded on a FTIR spectrophotometer (SHIMADZU) using the CHCl 3 technique at the Department of Chemistry, University of Chittagong, Bangladesh. 1 H-NMR (400 MHz) spectra were recorded using CDCl 3 as a solvent with TMS as the internal standard at the Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Dhaka, Bangladesh. Evaporations were carried out under reduced pressure using vacuum rotary evaporator (Germany). Melting points were determined using an electro-thermal melting point apparatus (England) and are uncorrected. Thin layer chromatography was performed on GF 254 and spots were detected by spraying the plates with 1% H 2 SO 4 . Column chromatography was carried out with silica gel G 60 (100-200 mesh).
International Letters of Chemistry, Physics and Astronomy Vol. 58 123

Reaction of methyl-α-D-glucopyranoside (1) with myristoyl chloride
A suspension of methyl α-D-glucopyranoside (1) (200 mg, 1.04 mmol) in dry pyridine (3 ml) was cooled to 0 0 C (maintained by ice and common salt) whereupon myristoyl chloride (0.3 ml, 1.1 molar eq.) was added to it. The mixture was stirred at this temperature for 5-6 hrs and then allowed to stand overnight. The progress of the reaction was checked by t.l.c (methanol-chloroform, 1:12) which indicated full conversion of the starting material into a single product (R f = 0.50). A few pieces of ice were added to the flask and then extracted the product mixture with chloroform (3×10 ml).

General procedure for the synthesis of methyl 6-O-myristoyl-α-D-glucopyranoside derivatives (3-15)
To a rapidly stirred and cooled (-5 0 C) solution of the triol (2) (78 mg, 0.19 mmol), in dry C 5 H 5 N (3 ml) was added acetic anhydride (0.1 ml, 5 molar eq.). The reaction mixture was stirred at -5 0 C temperature for 8 hrs and then stirred overnight at room temperature. The progress of the reaction was monitored by t.l.c (ethyl acetate-n-Hexane, 1:2) which indicated the complete conversion of the starting material into faster moving product (R f =0.52). Work-up as described earlier and purification by silica gel column chromatography (with ethyl acetate-n-hexane, 1:2 as eluant) afforded the acetyl derivative (3).

Antibacterial activity assay
The in vitro antibacterial spectrum of the synthesized chemicals were done by disc diffusion method [24] with little modification [25]. Sterilized paper discs of 4 mm in diameter and Petri dishes of 150 mm in diameter were used throughout the experiment. The autoclaved Mueller-Hinton agar medium, cooled to 45°C, was poured into sterilized Petri dishes to a depth of 3 to 4 mm and after solidification of the agar medium the plates were transferred to an incubator at 37°C for 15 to 20 minutes to dry off the moisture that developed on the agar surface. The plates were inoculated with the standard bacterial suspensions (as McFarland 0.5 standard) followed by spread plate method and allowed to dry for three to five minutes. Dried and sterilized filter paper discs were treated separately with 50 µg dry weight/disc from 2% solution (in CHCl 3 ) of each test chemical using a micropipette, dried in air under aseptic condition and were placed at equidistance in a circle on the seeded plate. A control plate was also maintained in each case without any test chemical. These plates were kept for 4-6 hours at low temperature (4-6°C) and the test chemicals diffused from disc to the surrounding medium. The plates were then incubated at 35±2°C for 24 hours to allow maximum growth of the microorganisms. The antibacterial activity of the test agent was determined by measuring the mean diameter of zone of inhibitions (in millimeter). Each experiment was repeated thrice. All the results were compared with the standard antibacterial antibiotic Ampicillin (20µg/disc, BEXIMCO Pharm. Bangladesh Ltd).

Antifungal activity assay
The antifungal activities of the D-glucopyranoside derivatives (scheme-1) were investigated by food poisoned technique [26]. Two percent solution of the test chemical (in CHCl 3 ) was mixed with sterilized melted Saburaud agar medium to obtain the desired concentration (2%) and this was poured in sterilized Petri dishes. At the center of each plate, 5 days old fungal mycelial block (4 mm in diameter) was inoculated and incubated at 27˚C. A control set was also maintained in each experiment. Linear mycelial growth of fungus was measured after 3-5 days of incubation. The percentage inhibition of radial mycelial growth of the test fungus was calculated as follows: International Letters of Chemistry, Physics and Astronomy Vol. 58

Characterization and chemistry
The main objective of the piece of work reported in this dissertation was to perform regioselective myristoylation of methyl a-D-glucopyranoside (1) using the direct acylation method (scheme 1). The resulting myristoylation product was converted to a number of derivatives using a series of acylating agents (scheme 2). The structure of the main acylation product and their derivatives were ascertained by analyzing their IR and 1 H-NMR spectra. In continuation of a project going on in our laboratory of Carbohydrate and Protein Chemistry, we intended to prepare a series of D-glucose derivatives for use as test chemicals for biological evaluation. Keeping this objective in mind, we thus prepared a set of derivatives containing a wide variety of substituents in a single molecular framework (Table 2 and 3). All the acylation products thus prepared were employed as test chemicals for antibacterial activity studies against a number of Gram-positive and Gramnegative human pathogenic bacteria. The antifungal activities of these derivatives were also performed against a number of phytopathogenic fungi. The selection of a wide variety of acylating agents was deliberate with the aim of finding biologically active agents.
Our initial effort was to prepare the methyl 6-O-myristoyl-α-D-glucopyranoside (2). Thus, treatment of methyl a-D-glucopyranoside (1) with myristoyl chloride as acylating agent in dry pyridine at -5 0 C and after usual work-up, compound 2 was obtained in high yields. This compound was sufficiently pure for use in the next stages. However, an analytical sample was prepared by recrystallisation from chloroform-hexane. Its IR spectrum showed absorption bands at 1702 cm -1 (for -CO stretching) and 3335-3510 cm-1 (br) (for -OH stretching). In its 1 H-NMR spectrum, a twoproton multiplet at δ 2.34 {CH 3 (CH 2 ) 11 CH 2 CO-}, a twenty-two proton multiplet at δ 1.24 {CH 3 (CH 2 ) 11 CH 2 CO-} and a three-proton multiplet at δ 0.86 {CH 3 (CH 2 ) 12  The structure of the myristoyl derivative (2) was further ascertained by its conversion to and identification of its acetyl derivative (3). Thus, reaction of compound 2 with an excess of acetic anhydride in pyridine, followed by usual work-up procedure and silica gel column chromatographic purification, provided the acetyl derivative (3). The IR spectrum of this compound showed the absorption peaks at 1742, 1705 and 1680 cm -1 due to carbonyl (-CO) stretching. The introduction of three acetyl groups in the molecule was demonstrated by the appearance of three three-proton singlets at δ 2.07, δ 2.00 and 1.98 in its 1 H-NMR spectrum. The C-2 proton resonated at δ 5.04 (as dd, J = 3.6 and 10.0 Hz) and shifted downfield from the precursor triol (2) (δ 3.72); C-3 proton resonated at δ 4.92 (as t, J = 9.6 Hz) and shifted downfield from the precursor triol (2) (δ 4.10); also, C-4 proton resonated downfield to δ 4.86 (as t, J = 9.7 Hz) as compared to the precursor compound 2 (δ 3.94), thereby suggesting the attachment of the acetyl groups at positions 2, 3 and 4. By complete analysis of the IR and 1 H-NMR spectra, the structure of the triacetate was ascertained as methyl 2,3,4-tri-O-acetyl-6-O-myristoyl-α-D-glucopyranoside (3).
Pivaloylation of compound 2 by direct method using pivaloyl chloride in dry pyridine, furnished the pivaloyl derivative (10). The IR spectrum of compound 10 showed peaks at 1752 cm -1 due to carbonyl stretching. In its 1 H-NMR spectrum, a twenty seven-proton singlet at δ 1.23 {3×(CH 3 ) 3 CCO-} corresponded to the presence of three pivaloyl groups in the molecule. The introduction of the pivaloyl groups at positions 2, 3 and 4 were demonstrated by downfield shift of C-2 to δ 5.05, C-3 to δ 4.92 and C-4 to δ 4.85 from their precursor triol δ values (2). Complete
For derivatization, we then used benzenesulfonyl chloride as acylating agent for this purpose. Thus treatment of compound (2) with 5 molar equivalent of benzenesulfonyl chloride in pyridine, followed by usual work-up and column chromatography, we obtained compound 12 as needless. IR spectrum of this compound displayed absorption bands at 1762 cm -1 (-CO stretching) and 1365 cm -1 (-SO 2 stretching). In its 1 H-NMR spectrum, the peaks at δ 7.85 (6H, m), δ 7.63 (3H, m) and δ 7.52 (6H, m) corresponded the protons of three phenyl groups. The downfield shift of C-2 to δ 5.48, C-3 to δ 5.05 and C-4 to δ 4.86 from their precursor compound (2) ascertained the attachment of benzenesulfonyl groups at positions 2, 3 and 4. Complete analysis of the IR and 1 H-NMR spectra, led us to assign its structure as methyl 2,3,4-tri-O-benzenesulfonyl-6-O-myristoyl-α-D-glucopyranoside (12). 4-Chlorobenzoylation of compound (2) by direct method using 4-chlorobenzoyl chloride in dry C 6 H 5 N and after similar work-up and purification techniques, the product (13) was isolated in 77.5% yield. The IR spectrum of this compound showed absorption band at 1746 cm -1 for carbonyl stretching. In its 1 H-NMR spectrum, the two six-aromatic proton multiplet at δ 7.92 (as m), δ 7.67 (as m) are characteristic of p-substituted benzoyl groups. The deshielding of C-2, C-3 and C-4 protons from their usual values (compound 2) and the resonance of other protons in their anticipated positions confirmed the structure of this compound as methyl 2,3,4-tri-O-(4-chlorobenzoyl)-6-Omyristoyl-α-D-glucopyranoside (13).
In a similar way, the myristoyl derivative (2) was converted to compound 14 by reaction with with 4-t-butylbenzoyl chloride in anhydrous pyridine and after usual work-up and chromatographic purification, it yielded 4-t-butylbenzoate (14). The IR spectrum of this compound indicated absorption bands at 1662 cm -1 corresponding to carbonyl (-CO) stretching. Its 1 H-NMR spectrum displayed the following characteristic aromatic two six-proton multiplet peaks at δ 8.02 (as m, Ar-H), δ 7.49 (as m, Ar-H) and a twenty seven-proton singlet at δ 1.33 {as s, 3×(CH 3 ) 3 C-} which corresponded to the presence of three 4-t-butylbenzoyl groups in the compound. The deshielding of C-2, C-3 and C-4 protons to δ 5.40, δ 5.08 and δ 4.83 from their usual values confirmed the attachment of three 4-t-butylbenzoyl groups at these positions. By complete analysis of the IR and 1 H-NMR spectrum led us to establish its structure as methyl 2,3,4-tri-O-(4-tbutylbenzoyl)-6-O-myristoyl-α-D-glucopyranoside (14).
Finally, we have carried out cinnamoylation of 2 with an excess of cinnamoyl chloride in pyridine as same work-up and purification techniques, we isolated compound (15) as needles. IR spectrum showed absorption bands at 1705 cm -1 (for -CO stretching) and 1631 cm-1 (for -CH=CHstretching). In the 1 H-NMR spectrum three one-proton doublets at δ 7.67, 7.50, 7.36 (3×1H, 3×d, J = 16.0 Hz, 3×PhCH=CHCO-) and also three one-proton doublets at δ 6.46, 6.36, 6.30 (3×1H, 3×d, J = 16.1 Hz, 3×PhCH=CHCO-) due to the presence of three cinnamoyl groups in the molecule. In addition a six-proton multiplet at δ 7.60 (as m, Ar-H) and a nine-proton multiplet at δ 7.25 (as, m, Ar-H) due to the three aromatic ring protons. The downfield shift of C-2, C-3 and C-4 to δ 5.35 (as dd), δ 5.01 (as t) and 4.88 (as t) from their usual values in the precursor compound 2 and the resonances of other protons in their anticipated positions, showed the presence of the cinnamoyl group at positions 2, 3 and 4. The rest of the IR and 1 H-NMR spectrum was in accord with the structure of this compound assigned as methyl 2,3,4-tri-O-cinnamoyl-6-O-myristoyl-α-Dglucopyranoside (15).
Thus, a series of acylated methyl α-D-glucopyranoside derivatives (scheme 1 & 2) were prepared using a wide variety of acylating agents. These acyl chlorides were chosen so as to contain probable biologically prone atoms or groups in order to find biologically active carbohydrate derivatives. All the acylation products thus prepared were employed as test chemicals for determining their antibacterial and antifungal activities against a number of human and plant pathogens.

Assessment of antimicrobial effects
The antibacterial results of the test chemicals and the standard antibiotic, Ampicillin against Gram-positive bacteria and Gram-negative bacteria are presented in Fig. 2. From the result we observed that compound 7 and 8 were very sensitive towards all of both Gram-positive and Gram-negative bacterial organisms. In case of 7 B. subtilis (16 mm), S. aureus (14 mm), E. coli (10 mm), P. aeruginosa (08 mm) and in case of 8 B. subtilis (12 mm), S. aureus (12 mm), E. coli (14 mm), P. aeruginosa (16 mm) were found very sensitive (Fig. 3). The inhibition (15 mm) of the growth of P. aeruginosa by 14 was remarkable. Compounds 3, 5, 9, 10, 11 and 12 were quite insensitive towards any of the Gram-positive or Gram-negative bacteria. So the test compounds 7 and 8 were exhibited highest potential antibacterial power against all tested microorganisms.
International Letters of Chemistry, Physics and Astronomy Vol. 58  Table 4. Antifungal activity of the tested compounds with standard antibiotic, Nystatin.
Compound no.
% of fungal mycelial growth inhibition a Compound no.

Aspergillus niger Candida albicans Aspergillus niger Candida albicans
The results of the in vitro antifungal studies of the test chemicals and the standard antibiotic, Nystatin is furnished in Table 4. From the results we found that compound 7 and 8 were very sensitive towards the mycelial growth of all the fungal test organisms. It was observed that the decanoyl derivative (7) showed the highest inhibition (62.00%) against Aspergillus niger which was comparable to the standard antibiotic, Nystatin (66.41%) (Fig. 4). Also the lauroyl derivative (8) showed maximum inhibition (60.00%) against Candida albicans which is also very close to that of standard antibiotic, Nystatin (63.10%). Again Aspergillus niger in case of 2, 6, 12 and Candida albicans in case of 6, 12 were found very sensitive. Rest of the test chemicals showed their antifungal activities by different degrees.

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ILCPA Volume 58 From the results discussed above it is clear that the presence of some particular groups in the test compounds enhanced their sensitivities towards the growth of bacteria and fungi. The presence of decanoyl and lauroyl group made the test chemicals very effective towards the growth of both Gram-positive and Gram-negative bacteria. The test chemicals containing octanoyl, decanoyl, lauroyl and benzenesulphonyl group were found to show very high antifungal activity which was in accordance with our previous work [27][28][29][30]. So these compounds may be targeted for future studies for their usage as broad spectrum antibiotics.

CONCLUSION
In the present study, we have described the regioselective synthesis of fourteen methyl α-Dglucopyranoside derivatives with various acyl groups containing different carbon chain length. This direct acylation method demonstrates a very simple and efficient method for the total synthesis and methyl 6-O-myristoyl-2,3,4-tri-O-octanoyl-α-D-glucopyranoside (6) was found to be encouraging in terms of high selectivity and excellent yield as 95.57%. Thus, a good number of test compounds reported herein exhibited promising antimicrobial effects. This is the first report regarding the effectiveness of the selected compounds against the selected pathogens.